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Cellular and Subcellular Nanotechnology 2013

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Cellular and Subcellular Nanotechnology 2013

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Methods in

Molecular Biology 991

Volkmar Weissig
Tamer Elbayoumi
Mark Olsen Editors

Cellular and
Subcellular
Nanotechnology
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY™

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:


http://www.springer.com/series/7651
Cellular and Subcellular
Nanotechnology

Methods and Protocols

Edited by

Volkmar Weissig, Tamer Elbayoumi, and Mark Olsen


Department of Pharmaceutical Sciences, Midwestern University College of Pharmacy, Glendale, AZ, USA
Editors
Volkmar Weissig Tamer Elbayoumi
Department of Pharmaceutical Sciences Department of Pharmaceutical Sciences
Midwestern University College of Pharmacy Midwestern University College of Pharmacy
Glendale, AZ, USA Glendale, AZ, USA

Mark Olsen
Department of Pharmaceutical Sciences
Midwestern University College of Pharmacy
Glendale, AZ, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)


ISBN 978-1-62703-335-0 ISBN 978-1-62703-336-7 (eBook)
DOI 10.1007/978-1-62703-336-7
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2013933992

© Springer Science+Business Media New York 2013


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Preface

“While early ideas about the impact of nanotechnology on healthcare focused on fanciful
ideas involving small submarines and cancer-zapping robots, current advances have been
enabled by advances in imaging, control over materials and an increased understanding of
how biology works at the nanoscale” (Tim Harper, CEO Cientifica).
This book is dedicated to showcase the most recent advances that have been made in
utilizing the enormous potential of nanotechnology for probing, imaging, and manipulat-
ing life on a cellular and subcellular level. All chapters were written by leading experts in
their particular fields. Daniel Moyano and Vincent Rotello describe a novel “chemical nose”
approach, i.e., nanoparticle-based sensor arrays for the differentiation of biomolecules
through pattern recognition that utilizes functionalized gold nanoparticles as receptors and
Green Fluorescent Protein as transducer. This new strategy allows the identification of cel-
lular signatures in early stages of cancer without previous knowledge of specific receptors or
ligands. Sunaina Surana and Yamuna Krishnan demonstrate the utility of an externally
introduced, pH-triggered DNA nanomachine inside the multicellular eukaryote
Caenorhabditis elegans. This nanomachine uses FRET to effectively map spatiotemporal
pH changes associated with endocytosis in coelomocytes of wild type as well as mutant
worms. Syed K. Sohaebuddin and Liping Tang describe a method which allows the assess-
ment of lysosomal membrane integrity upon exposure to various nanoparticles. The elec-
tron microscopic visualization of 1–2 nm gold nanoparticles, which are used as nano
markers, allows Valeriy Lukyanenko and Vadim Salnikov to determine the precise localiza-
tion of a variety of nano-objects within a cell. The same author also describes a saponin-
based method for membrane permeabilization allowing the delivery of particles up to
20 nm in size to the perinuclear and perimitochondrial space of cardiomyocytes.
Howard Gendelman’s laboratory provides in two chapters protocols for the isolation of
subcellular compartments containing sequestered nanoparticles. Indriati Pfeiffer and
Michael Zäch describe the use of nanostructured SiO2 surfaces prepared by the colloidal
lithography technique to scrutinize the formation of suspended lipid bilayers from a solu-
tion of nano liposomes. These authors employ atomic force microscopy (AFM) and quartz
crystal microbalance with dissipation monitoring (QCM-D) to characterize nanostructure
fabrication and lipid bilayer assembly on the nanostructured surface. QCM-D is also being
utilized by Rickard Frost and Sofia Svedham to monitor the interaction of nanoparticles
with lipid membranes in real time. The authors demonstrate how the outcome of such
analysis provides information on the adsorption process (importantly kinetics and adsorbed
amounts) as well as on the integrity of both the nanoparticles and the lipid membrane upon
interaction. A protocol for studying the interactions of nanoparticles with proteins is pro-
vided by Lennart Treuel and Marcelina Malissek. These authors describe a procedure to
study the adsorption of proteins onto nanoparticle surfaces based on circular dichroism
(CD) spectroscopy. Jerry Chang and Sandra Rosenthal describe the principles, methodolo-
gies, and experimental protocols for quantum dot-based single-molecule imaging.

v
vi Preface

Ben Zhong Tang and his colleagues describe the fabrication of fluorescent silica nanopar-
ticles (FSNPs) containing aggregation-induced emission (AIE) luminogens. By employing
surfactant-free sol–gel reaction the authors are able to generate FSNPs with uniform size
and high surface charge and colloidal stability. Simon C.W. Richardson group applies single
cell imaging technology for studying the intracellular trafficking of both biological and
synthetic macromolecules and they demonstrate the possibility of temporally dissecting
novel and default trafficking of both macromolecular “drugs” and macromolecular drug
delivery systems. Irene Canton and Giuseppe Battaglia describe a polymersomes-mediated
delivery of fluorescent probes for targeted and long-term imaging in live cell microscopy.
Junghae Suh and colleagues explain in their chapter one of the most complicated aspects of
real-time particle tracking, i.e., the mean square displacement (MSD) calculation, in a sim-
ple manner designed for the novice particle tracker. By providing comprehensive instruc-
tions needed to perform particle tracking experiments, their chapter will enable researchers
to gain new insight into the intracellular dynamics of nanocarriers, potentially leading to
the development of more effective and intelligent therapeutic delivery vectors. Mi-Sook
and Song Her provide a direct method for quantifying cellular transduction of PTD in vitro
and in vivo using bioluminescence imaging. Their methodology exploits noninvasive tech-
niques to create an environment suitable for the real-time imaging of PTD transduction
and appears therefore as a promising tool for studying the mechanism of PTD transduction
and the in vivo application of new therapeutic candidates. Achim Göpferich group describes
a procedure for monitoring the intracellular route of polyplexes based on the use of labeling
PEI and pLL with a reduction-sensitive fluorescent dye. Katye M. Fichter and Tania Q. Vu
describe the use of single nanoparticle quantum dot (QD) probes to quantitatively investi-
gate the complex endocytic trafficking pathways that receptors undergo following ligand
activation. The use of cell-penetrating peptides (CPPs) to facilitate the cellular internaliza-
tion of quantum dots (QDs) is described by Yue-Wern Huang and colleagues. Their
approach is based on simple noncovalent interactions between CPPs and QDs. Lo and
Wang describe the use of peptide-based carriers for the intracellular delivery of biologically
active proteins as well as methods for the qualitative and quantitative evaluation of their
delivery efficiency. Jan van Hest’s laboratory presents a novel strategy for the preparation of
gold nanoparticles exhibiting a stimuli-responsive behavior, which is based on the use of a
ligand consisting of only a single repeat of the elastin-based pentapeptide VPGVG. The
authors provide protocols for the solid-phase peptide synthesis of thiol-terminated VPGVG
ligand and for the preparation of gold nanoparticles covered with the pentapeptide through
a ligand-exchange reaction. Jae Sam Lee and Ching-Hsuam Tung have developed an
improved CPP-based cellular delivery vector, named lipo-oligoarginine peptide (LOAP),
by conjugating an oligoarginine peptide with a fatty acid moiety. The prepared LOAPs
were further stabilized by introducing different combinations of D-Arg residues into the
peptide backbone, and were systematically evaluated for their membrane penetrating prop-
erties and metabolic stabilities in cells. Andrea Alessandrini and Paolo Facci describe the use
of electrochemical scanning tunneling microscopy (ECSTM) and spectroscopy (ECSTS)
for studying the electron transport through single redox molecules with the aim of under-
standing the transport mechanisms ruling the flow of electrons via a single molecule placed
in a nanometer-sized gap between two electrodes, while elucidating the role of the redox
density of states brought about by the molecule. Yamuna Krishnan’s group has constructed
an icosahedron from DNA using a modular self-assembly strategy. They describe a method
to determine the functionality of DNA polyhedra as nanocapsules by encapsulating differ-
ent cargo such as gold nanoparticles and functional biomolecules like FITC dextran from
Preface vii

solution within DNA icosahedra. The use of polymer-gold nanorods assemblies for the
delivery of plasmid DNA into mammalian cells is described by Kaushal Rege’s laboratory.
Puiyan Lee and Kenneth K.Y. Wong describe a technique for the synthesis of a novel lipo-
philic nano carrier for the incorporation of hydrophobic and toxic potent cancer drugs,
such as gold (III) porphyrin. Tamer Elbayoumi’s laboratory provides protocols for prepar-
ing mitochondria-targeted nanoemulsions loaded with tocopherol and Cyclosporine A
which are able to protect cardiac muscle mitochondria from doxorubicin-induced oxidative
stress. Achim Weber and colleagues describe the production of uniform protein-binding
biofunctional fluorescent spherical silica core-shell nanoparticles. The authors characterize
their novel nanoparticle system including its surface functionalization via microelectropho-
resis, dynamic light scattering (DLS) and a colorimetric detection of the amount of nano-
particle-attached protein via a bicinchoninic acid (BCA) assay. Such fluorescently spiked
nanoparticle cores with biofunctional shells for molecular recognition reactions may be
used as imaging tools or reporter systems. Neskovic and her colleagues describe the assess-
ment of genotoxic properties of purified single wall carbon nanotubes (SWCNT), multiwall
carbon nanotubes (MWCNT), and amide functionalized purified SWCNT using cultured
human lymphocytes and human fibroblasts. Dusica Maysinger’s group has developed a
suitable fractionation method for field flow fractionation, an analytical technique that allows
the separation of nano and microparticles over a wide size range. The authors present asym-
metrical flow field-flow fractionation (AF4) conditions that have proven their reliability for
the analysis of quantum dots and other nanoparticles in the 5–50 nm size range. Maxwell
B. Zeigler and Daniel T. Chiu give detailed steps necessary to perform laser surgery upon
single adherent mammalian cells, where individual organelles are extracted from the cells by
optical tweezers and the cells are monitored post-surgery to check their viability. Yaron R.
Silberberg and Andrew E. Pelling describe a method to quantify the intracellular mechani-
cal response to an extracellular mechanical perturbation, specifically the displacement of
mitochondria. A combined fluorescent-atomic force microscope (AFM) was used to simul-
taneously produce well-defined nanomechanical stimulation to a living cell while optically
recording the real-time displacement of fluorescently labeled mitochondria.
We are extremely grateful to all authors for having spent parts of their valuable time to
contribute to this book. It is our hopes that together we have succeeded in providing an
essential source of know-how and at the same time a source of inspiration to all investiga-
tors who are as fascinated as we are about the potential of applying nanotechnology to all
areas of biomedical sciences. Last but not least we would like to thank John Walker, the
series editor of “Methods in Molecular Biology” for having invited us to assemble this book
and above all for his unlimited guidance and help throughout the whole process.

Glendale, AZ, USA Volkmar Weissig


Tamer Elbayoumi
Mark Olsen
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1 Nanoparticle-GFP “Chemical Nose” Sensor for Cancer


Cell Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Daniel F. Moyano and Vincent M. Rotello
2 A Method to Map Spatiotemporal pH Changes in a Multicellular
Living Organism Using a DNA Nanosensor . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Sunaina Surana and Yamuna Krishnan
3 A Simple Method to Visualize and Assess the Integrity
of Lysosomal Membrane in Mammalian Cells Using a Fluorescent Dye . . . . . . 25
Syed K. Sohaebuddin and Liping Tang
4 Gold Nanoparticle as a Marker for Precise Localization
of Nano-objects Within Intracellular Sub-domains. . . . . . . . . . . . . . . . . . . . . . 33
Valeriy Lukyanenko and Vadim Salnikov
5 Immunoisolation of Nanoparticles Containing
Endocytic Vesicles for Drug Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Ari Nowacek, Irena Kadiu, JoEllyn McMillan,
and Howard E. Gendelman
6 Methods for Isolation and Identification of
Nanoparticle-Containing Subcellular Compartments . . . . . . . . . . . . . . . . . . . . 47
Ari Nowacek, Irena Kadiu, JoEllyn McMillan,
and Howard E. Gendelman
7 Permeabilization of Cell Membrane for Delivery
of Nano-objects to Cellular Sub-domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Valeriy Lukyanenko
8 A Method to Encapsulate Molecular Cargo
Within DNA Icosahedra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Dhiraj Bhatia, Saikat Chakraborty, Shabana Mehtab,
and Yamuna Krishnan
9 Delivery of Plasmid DNA to Mammalian Cells Using
Polymer–Gold Nanorod Assemblies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
James Ramos, Huang-Chiao Huang, and Kaushal Rege
10 Lipophilic-Formulated Gold Porphyrin Nanoparticles for Chemotherapy . . . . 93
Puiyan Lee and Kenneth K.Y. Wong
11 Mitochondria-Specific Nano-Emulsified Therapy for Myocardial
Protection Against Doxorubicin-Induced Cardiotoxicity . . . . . . . . . . . . . . . . . 99
Amy Faulk, Volkmar Weissig, and Tamer Elbayoumi

ix
x Contents

12 Formation of Pit-Spanning Phospholipid Bilayers


on Nanostructured Silicon Dioxide Surfaces for Studying
Biological Membrane Events. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Indriati Pfeiffer and Michael Zäch
13 Characterization of Nanoparticle–Lipid Membrane
Interactions Using QCM-D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Rickard Frost and Sofia Svedhem
14 Single-Cell Nanosurgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Maxwell B. Zeigler and Daniel T. Chiu
15 Single Quantum Dot Imaging in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . 149
Jerry C. Chang and Sandra J. Rosenthal
16 Fabrication of Fluorescent Silica Nanoparticles
with Aggregation-Induced Emission Luminogens for Cell Imaging . . . . . . . . . 163
Sijie Chen, Jacky W.Y. Lam, and Ben Zhong Tang
17 Monitoring the Degradation of Reduction-Sensitive
Gene Carriers with Fluorescence Spectroscopy and Flow Cytometry . . . . . . . . 171
Constantin Hozsa, Miriam Breunig, and Achim Göpferich
18 Quantification of Intracellular Mitochondrial
Displacements in Response to Nanomechanical Forces . . . . . . . . . . . . . . . . . . 185
Yaron R. Silberberg and Andrew E. Pelling
19 Imaging Select Mammalian Organelles Using Fluorescent
Microscopy: Application to Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Paul D.R. Dyer, Arun K. Kotha, Marie W. Pettit,
and Simon C.W. Richardson
20 Real-Time Particle Tracking for Studying Intracellular
Trafficking of Pharmaceutical Nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Feiran Huang, Erin Watson, Christopher Dempsey, and Junghae Suh
21 Interactions of Nanoparticles with Proteins: Determination
of Equilibrium Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Lennart Treuel and Marcelina Malissek
22 Tracing the Endocytic Pathways and Trafficking Kinetics
of Cell Signaling Receptors Using Single QD Nanoparticles . . . . . . . . . . . . . . 237
Katye M. Fichter and Tania Q. Vu
23 Cellular Internalization of Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Yue-Wern Huang, Han-Jung Lee, Betty Revon Liu,
Huey-Jenn Chiang, and Chi-Heng Wu
24 Electrochemical Scanning Tunneling Microscopy and Spectroscopy
for Single-Molecule Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Andrea Alessandrini and Paolo Facci
25 Intracellular Delivery of Biologically Active Proteins
with Peptide-Based Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Seong Loong Lo and Shu Wang
Contents xi

26 Lipo-oligoarginine-Based Intracellular Delivery . . . . . . . . . . . . . . . . . . . . . . . . 281


Jae Sam Lee and Ching-Hsuan Tung
27 Fluorescent Spherical Monodisperse Silica Core-Shell
Nanoparticles with a Protein-Binding Biofunctional Shell . . . . . . . . . . . . . . . . 293
Achim Weber, Marion Herz, and Günter E.M. Tovar
28 Direct Quantification of PTD Transduction
Using Real-Time Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Mi-Sook Lee and Song Her
29 Genotoxic Assessment of Carbon Nanotubes. . . . . . . . . . . . . . . . . . . . . . . . . . 315
Olivera Nešković, Gordana Joksić, Ana Valenta-Šobot,
Jelena Cvetićanin, Djordje Trpkov, Andreja Leskovac, and Sandra Petrović
30 Separation Science: Principles and Applications
for the Analysis of Bionanoparticles by Asymmetrical
Flow Field-Flow Fractionation (AF4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Alexandre Moquin, Françoise M. Winnik, and Dusica Maysinger
31 Polymersomes-Mediated Delivery of Fluorescent Probes
for Targeted and Long-Term Imaging in Live Cell Microscopy . . . . . . . . . . . . 343
Irene Canton and Giuseppe Battaglia
32 Protocol for the Preparation of Stimuli-Responsive
Gold Nanoparticles Capped with Elastin-Based Pentapeptides . . . . . . . . . . . . . 353
Vincent Lemieux, P. Hans H.M. Adams, and Jan C.M. van Hest

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Contributors

P. HANS H.M. ADAMS • Department of Organic Chemistry, Institute for Molecules


and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands
ANDREA ALESSANDRINI • CNR-NANO-S3, and Physics Department,
University of Modena and Reggio Emilia, Modena, Italy
GIUSEPPE BATTAGLIA • Department of Biomedical Science, The Krebs Institute,
The University of Sheffield, Sheffield, UK
DHIRAJ BHATIA • National Centre for Biological Sciences, Tata Institute
of Fundamental Research, Bangalore, India
MIRIAM BREUNIG • Lehrstuhl für Pharmazeutische Technologie,
Universität Regensburg, Regensburg, Germany
IRENE CANTON • Department of Biomedical Science, The Krebs Institute,
The University of Sheffield, Sheffield, UK
SAIKAT CHAKRABORTY • National Centre for Biological Sciences,
Tata Institute of Fundamental Research, Bangalore, India
JERRY C. CHANG • Department of Chemistry, Vanderbilt University,
Nashville, TN, USA
SIJIE CHEN • Division of Biomedical Engineering, Hong Kong University
of Science and Technology, Kowloon, Hong Kong, China
HUEY-JENN CHIANG • Institute of Biotechnology, National Dong Hwa University,
Hualien, Taiwan
DANIEL T. CHIU • Department of Chemistry, University of Washington,
Seattle, WA, USA
JELENA CVETIĆANIN • Institute of Nuclear Sciences “Vinča”, University of Belgrade,
Belgrade, Serbia
CHRISTOPHER DEMPSEY • Department of Bioengineering, Rice University,
Houston, TX, USA
PAUL D.R. DYER • School of Science, University of Greenwich, Kent, UK
TAMER ELBAYOUMI • Department of Pharmaceutical Sciences,
Midwestern University College of Pharmacy, Glendale, AZ, USA
PAOLO FACCI • CNR-NANO-S3, Modena, Italy
AMY FAULK • Department of Pharmaceutical Sciences, Midwestern University
College of Pharmacy, Glendale, AZ, USA
KATYE M. FICHTER • Department of Biomedical Engineering, Oregon Health
and Science University, Portland, OR, USA
RICKARD FROST • Department of Applied Physics, Chalmers University of Technology,
Göteborg, Sweden
HOWARD E. GENDELMAN • Department of Pharmacology and Experimental
Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA
ACHIM GÖPFERICH • Lehrstuhl für Pharmazeutische Technologie,
Universität Regensburg, Regensburg, Germany

xiii
xiv Contributors

SONG HER • Division of Bio-Imaging, Chuncheon Center, Korea Basic Science


Institute, Chuncheon, Republic of Korea
MARION HERZ • Fraunhofer Institute for Interfacial Engineering and Biotechnology
IGB, Stuttgart, Germany
CONSTANTIN HOZSA • Lehrstuhl für Pharmazeutische Technologie,
Universität Regensburg, Regensburg, Germany
FEIRAN HUANG • Department of Bioengineering, Rice University, Houston, TX, USA
HUANG-CHIAO HUANG • Wellman Center for Photomedicine, Massachusetts
General Hospital and Harvard Medical School, Boston, MA, USA
YUE-WERN HUANG • Department of Biological Sciences, Missouri University
of Science and Technology, Rolla, MO, USA
GORDANA JOKSIĆ • Institute of Nuclear Sciences “Vinča”, University of Belgrade,
Belgrade, Serbia
IRENA KADIU • Department of Pharmacology and Experimental Neuroscience,
University of Nebraska Medical Center, Omaha, NE, USA
ARUN K. KOTHA • School of Science, University of Greenwich, Kent, UK
YAMUNA KRISHNAN • National Centre for Biological Sciences,
Tata Institute of Fundamental Research, Bangalore, India
JACKY W.Y. LAM • Department of Chemistry, Hong Kong University of Science
and Technology, Kowloon, Hong Kong, China
HAN-JUNG LEE • Department of Natural Resources and Environmental Studies,
National Dong Hwa University, Hualian, Taiwan
JAE SAM LEE • Department of Radiology, Methodist Hospital Research Institute,
Weill Cornell Medical College, Houston, TX, USA
MI-SOOK LEE • Division of Bio-Imaging, Chuncheon Center, Korea Basic Science
Institute, Chuncheon, Republic of Korea
PUIYAN LEE • Department of Surgery, Li Ka Shing Faculty of Medicine,
The University of Hong Kong, Pokfulam, Hong Kong
VINCENT LEMIEUX • Department of Organic Chemistry, Institute for Molecules
and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands
ANDREJA LESKOVAC • Institute of Nuclear Sciences “Vinča”, University of Belgrade,
Belgrade, Serbia
BETTY REVON LIU • Department of Natural Resources and Environmental Studies,
National Dong Hwa University, Hualian, Taiwan
SEONG LOONG LO • Department of Biological Sciences, National University
of Singapore, Singapore
VALERIY LUKYANENKO • Department of Medicine, Johns Hopkins School of Medicine,
Baltimore, MD, USA
MARCELINA MALISSEK • Physical Chemistry, University of Duisburg-Essen,
Essen, Germany
DUSICA MAYSINGER • Department of Pharmacology and Therapeutics,
Faculty of Medicine, McGill University, Montreal, Canada
JOELLYN MCMILLAN • Department of Pharmacology and Experimental
Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA
SHABANA MEHTAB • National Centre for Biological Sciences, Tata Institute
of Fundamental Research, Bangalore, India
Contributors xv

ALEXANDRE MOQUIN • Faculty of Pharmacy, and Department of Pharmacology


& Therapeutics, Faculty of Medicine, Université de Montréal and McGill University,
Montreal, QC, Canada
DANIEL F. MOYANOA • Department of Chemistry, University of Massachusetts, Amherst,
MA, USA
OLIVERA NEŠKOVIĆ • Institute of Nuclear Sciences “Vinča”, University of Belgrade,
Belgrade, Serbia
ARI NOWACEK • Department of Pharmacology and Experimental Neuroscience,
University of Nebraska Medical Center, Omaha, NE, USA
ANDREW E. PELLING • Department of Physics, Department of Biology,
Institute for Science, Society and Policy, University of Ottawa, Ottawa, Canada
SANDRA PETROVIĆ • Institute of Nuclear Sciences “Vinča”, University of Belgrade,
Belgrade, Serbia
MARIE W. PETTIT • School of Science, University of Greenwich, Kent, UK
INDRIATI PFEIFFER • Department of Cell biology and Genetics, Erasmus Medical Center,
Rotterdam, Netherlands
JAMES RAMOS • School of Biological and Health Systems & Chemical Engineering,
Center for the Convergence of Physical Science and Cancer Biology,
Arizona State University, Tempe, AZ, USA
KAUSHAL REGE • School of Biological and Health Systems & Chemical Engineering,
Center for the Convergence of Physical Science and Cancer Biology,
Arizona State University, Tempe, AZ, USA
SIMON C.W. RICHARDSON • University of Greenwich, School of Science, Kent, UK
SANDRA J. ROSENTHAL • Department of Chemistry, Department of Pharmacology,
Department of Chemical and Biomolecular Engineering, Department of Physics
and Astronomy, Institute of Nanoscale Science and Engineering,
Vanderbilt University, Nashville, TN, USA
VINCENT M. ROTELLO • Department of Chemistry, University of Massachusetts,
Amherst, MA, USA
VADIM SALNIKOV • Kazan Institute of Biochemistry and Biophysics, Kazan Scientific
Centre Russian Academy of Sciences, Kazan, Russia
YARON R. SILBERBERG • Biomedical Research Institute (BMRI), National Institute
of Advanced Industrial Science and Technology (AIST), Kyoto University,
Kyoto, Japan
SYED K. SOHAEBUDDIN • Department of Bioengineering, University of Texas
at Arlington, Arlington, TX, USA
JUNGHAE SUH • Department of Bioengineering, Rice University, Houston, TX, USA
SUNAINA SURANA • National Centre for Biological Sciences, Tata Institute
of Fundamental Research, Bangalore, India
SOFIA SVEDHEM • Department of Applied Physics, Chalmers University of Technology,
Göteborg, Sweden
BEN ZHONG TANG • Department of Chemistry, Hong Kong University of Science
and Technology, Kowloon, Hong Kong, China
LIPING TANG • Department of Bioengineering, University of Texas at Arlington,
Arlington, TX, USA
xvi Contributors

GÜNTER E.M. TOVAR • Institute for Interfacial Engineering IGVT, University


Stuttgart, Stuttgart, Germany; Fraunhofer Institute for Interfacial Engineering
and Biotechnology IGB, Stuttgart, Germany
LENNART TREUEL • Institute of Applied Physics and Center for Functional
Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Karlsruhe,
Germany; Physical Chemistry, University of Duisburg-Essen, Essen, Germany
DJORDJE TRPKOV • Institute of Nuclear Sciences “Vinča”, University of Belgrade,
Belgrade, Serbia
CHING-HSUAN TUNG • Department of Radiology, Methodist Hospital Research
Institute, Weill Cornell Medical College, Houston, TX, USA
ANA VALENTA-ŠOBOT • Institute of Nuclear Sciences “Vinča”, University of Belgrade,
Belgrade, Serbia
JAN C.M. VAN HEST • Department of Organic Chemistry, Institute for Molecules
and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands
TANIA Q. VU • Department of Biomedical Engineering, Oregon Health and Science
University, Portland, OR, USA
SHU WANG • Institute of Bioengineering and Nanotechnology, Singapore;
Department of Biological Sciences, National University of Singapore, Singapore
ERIN WATSON • Department of Bioengineering, Rice University, Houston, TX, USA
ACHIM WEBER • Fraunhofer Institute for Interfacial Engineering and Biotechnology
IGB, Stuttgart, Germany; Institute for Interfacial Engineering IGVT, University
Stuttgart, Stuttgart, Germany
VOLKMAR WEISSIG • Department of Pharmaceutical Sciences, Midwestern University
College of Pharmacy, Glendale, AZ, USA
FRANÇOISE M. WINNIK • Faculty of Pharmacy and Department of Chemistry,
Université de Montréal, Montreal, QC, Canada
KENNETH K.Y. WONG • Department of Surgery, Li Ka Shing Faculty of Medicine,
The University of Hong Kong, Pokfulam, Hong Kong
CHI-HENG WU • Department of Biological Sciences, Missouri University of Science
and Technology, Rolla, MO, USA
MICHAEL ZÄCH • Department of Applied Physics, Chalmers University of Technology,
Gothenburg, Sweden
MAXWELL B. ZEIGLER • Department of Chemistry, University of Washington,
Seattle, WA, USA
Chapter 1

Nanoparticle-GFP “Chemical Nose” Sensor for Cancer


Cell Identification
Daniel F. Moyano and Vincent M. Rotello

Abstract
Nanoparticle-based sensor arrays have been used to distinguish a wide range of bio-related molecules
through pattern recognition. This “chemical nose” approach uses nanoparticles as receptors to selectively
identify the analytes, while a transducer reports the binding through a readable signal (fluorescence). Here
we describe a procedure that uses functionalized gold nanoparticles as receptors and green fluorescent
protein (GFP) as the transducer to identify and differentiate cell state (normal, cancerous, and metastatic),
an important tool in early diagnosis and treatment of tumors.

Key words Sensor, Chemical nose, Gold nanoparticle, GFP, Fluorescence, LDA

1 Introduction

Antibody-based sensing techniques are an important tool in the


early detection of cancer (1). These techniques employ specific rec-
ognition to identify the analytes, targeting different biomarkers of
each cell state (2). However this approach also limits the applicabil-
ity of this method due to constrains in the availability of specific
markers (3). As an alternative to this methodology, the “chemical
nose” approach utilizes multiple selective receptors that generate a
unique response pattern for each analyte, allowing its classification
(4). The identification is achieved by taking advantage of differen-
tial interactions between the analytes and the receptors. In the sen-
sor approach described here, gold nanoparticles (AuNPs) are used
as the receptors, controlling the nature of the interaction by tuning
the chemical properties at the nanoparticle surface (5). AuNP–
analyte interactions are then transduced by a fluorescent probe
(GFP), initially quenched when bound to the AuNP, and then dis-
placed from the AuNP surface upon the addition of the analyte,
with concomitant restoration of fluorescence. This strategy has been
successfully applied to identify and differentiate a variety of bio-
constructs, from proteins (6) to bacteria (7) and cancer cells (8).

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_1, © Springer Science+Business Media New York 2013

1
2 Daniel F. Moyano and Vincent M. Rotello

Here we report the procedure to apply this methodology to the


identification of cancer cells, independent of their origin (isogenic),
and differentiating among a diversity of cell states (normal, cancer-
ous, and metastatic). This strategy allows a versatile identification
of cellular signatures in early stages of cancer, a major hurdle in
cancer therapy, without previous knowledge of specific receptors
or ligands.

2 Materials

1. Phosphate buffer (PB) solution: 5 mM phosphate, pH 7.4.


Mix 3.87 mL of 1.0 M Na2HPO4 with 1.13 mL of 1.0 M
NaH2PO4 solution. Complete to a final volume of 1.0 L using
type I ultrapure water (see Note 1). Adjust pH if necessary and
store at room temperature (25°C).
2. Functionalized gold nanoparticles solution: three cationic and
one neutral gold nanoparticles (Fig. 1) are synthesized by place
exchange reaction (9), from pentanethiol-capped 2 nm diameter
gold nanoclusters, and using the desired ligands (synthesized as
shown in (10)). Prepare 200 mL of a 40 mM stock solution for
each nanoparticle in type I ultrapure water, calculating the AuNP
concentration by its absorbance at 506 nm (e506 = 4.9 × 105 M−1 cm−1
for 2 nm diameter gold core) according to the reported meth-
odology (11). Store at 4°C (see Note 2).
3. Green fluorescent protein solution: GFP is expressed according
to reported procedure (12). Prepare 400 mL of a GFP stock
solution of 250 mM in 5 mM PB, calculating the protein con-
centration by its absorbance at 488 nm (e488 = 5.6 × 104 M−1 cm−1)
(see Note 3).

Fig. 1 Chemical structure of the gold nanoparticles, featuring h-bond, p-stacking, and hydrophobic groups at
the surface
Nanoparticle-GFP “Chemical Nose” Sensor for Cancer Cell Identification 3

4. Cell suspension: Grow cancer, metastatic, and normal state


cells (analytes) according to the provider procedures in T75/
T25 flasks. Remove the media, wash the cells with DBPS buf-
fer, add 2 mL of trypsin EDTA 1× (per container), and collect
the cells in serum-containing media. Centrifuge to obtain cell
pellets and suspend them in DMEM non-serum medium.
Count the cells and dilute the suspension with DMEM to have
a final concentration of ~200 cells/mL (see Note 4).
5. 96-well 300 mL black polystyrene plates with dark flat bottom.
6. 50 mL PVC pipette basins, 50 mL PP Falcon tubes, and 7 mL
glass vials.

3 Methods

All procedures are done at room temperature.

3.1 Determination 1. Prepare 40 mL of 150 nM GFP solution in a Falcon tube


of Optimal Sensor using 5 mM PB, starting from the initial GFP stock solution
Conditions (see Note 5).
2. In a 7 mL glass vial, pour 10 mL of one of the nanoparticles stock
solutions. Complete the solution to 2 mL using the 150 nM
GFP solution. Mix gently and allow this AuNP–GFP complex
solution to reach equilibrium for 15 min (see Note 6).
3. During the equilibration time, add sequentially decreasing
values of the 150 nM GFP solution into a 96-well plate, accord-
ing to the scheme (Fig. 2).

Fig. 2 Sequential volumes of the GFP and AuNP–GFP solutions to be used in the 96-well plate for titration
purposes
4 Daniel F. Moyano and Vincent M. Rotello

Fig. 3 Cationic-AuNP titration curve featuring the position of the optimal AuNP/
GFP ratio to use in the sensing procedure

4. After the 15 min, add sequentially increasing values of the


AuNP–GFP complex solution to each well according to the
scheme (Fig. 2), to complete a total volume of 200 mL per well.
Each nanoparticle has to be titrated by triplicate (see Note 7).
5. Mix gently with the pipette tip to avoid bubbles and leave the
system reach equilibrium for another 15 min.
6. After the final equilibration time, take fluorescent intensity
measurements at 510 nm, using an excitation wavelength of
475 nm.
7. Plot the normalized fluorescence intensity against the nano-
particle concentration in each well (see Note 8).
8. Select the ideal AuNP/GFP ratio for sensing purposes (Fig. 3).
This is the ratio to be used in Subheading 3.2 (see Note 9).
9. Repeat the same procedure using the four different nanopar-
ticles. The AuNP/GFP optimal ratio may differ among all
nanoparticles (see Note 10).

3.2 Sensing 1. Prepare 40 mL of 150 nM GFP solution in Falcon tube using


Procedure 5 mM PB and starting from the initial GFP stock solution
(see Note 5).
2. In a 7 mL vial, pour the amount of the nanoparticle stock solu-
tion necessary to reach the optimal AuNP/GFP ratio (150 nM
of GFP), completing to 2 mL using the 150 nM GFP solution.
Mix gently and wait for 15 min. Repeat this step for the other
nanoparticles (see Notes 6 and 10).
3. After the equilibrium time, add 200 mL of each AuNP/GFP
solution in the 96-well plate according to the scheme in Fig. 4
(see Note 11). Six replicates are done for each specific
nanoparticle.
Nanoparticle-GFP “Chemical Nose” Sensor for Cancer Cell Identification 5

Fig. 4 Input scheme to use the different AuNP–GFP complexes against one cell
analyte. The output of this experiment generates the fingerprint for the given
analyte

4. Record the fluorescent intensity at 510 nm, using an excitation


wavelength of 475 nm. This is the initial intensity (Ii).
5. Add 25 mL of the cell suspension (~5,000 cells) in each well
(see Note 12). Mix with the pipette tip gently and leave 30 min
for incubation time (see Note 6).
6. After the incubation time, record the fluorescent intensity at
510 nm, using an excitation wavelength of 475 nm. This is the
final intensity (If).
7. Calculate the log(If/Ii) for each point, generating the average
and the standard deviation with the six replicates, to build up
the fingerprint of each analyte (see Note 13).
8. Using linear discriminant analysis software (like SYSTAT 11),
use all log(If/Ii) values (analytes x replicates x nanoparticles) as
an input to generate the canonical score plots and the jackknife
classification percentage for cell identification (see Notes 14
and 15).

4 Notes

1. The 1.0 M solution of NaH2PO4 has to be fresh each time that


the buffer is prepared to avoid later crystallization problems of
this solution that can alter the final concentration.
6 Daniel F. Moyano and Vincent M. Rotello

2. After place exchange reaction, nanoparticles are purified by


dialysis using a 10,000 MWCO tubing membrane. The nano-
particles are then lyophilized, dispersed in type I ultrapure
water, and passed through a 0.22 mm membrane filter. TEM,
MS, NMR, and DLS techniques are used to control the quality
of the nanoparticles according to literature (10).
3. Continuous freezing cycles of GFP can denature the protein,
affecting the reproducibility. To avoid this problem, prepare the
stock solution and divide it in different containers, store them
at −78°C in dark (aluminum foil), and take out one container
each time that the solution is needed. No glycerol or DTT is
used given the stability of GFP in PB at low concentrations.
4. If no recommendations are given, grow human- and mouse-
type cells in Dulbecco’s Modified Eagle’s Medium (DMEM,
4.0 g/L glucose) supplemented with 10% fetal bovine serum
and 1% antibiotics (100 U/mL penicillin and 100 mg/mL
streptomycin) in T75 or T25 flasks and under humidified
atmosphere of 5% CO2 at 37°C. The cells should be main-
tained in the mentioned conditions and subcultured once every
4 days.
5. Maintain the 150 nM GFP solution in an ice bath and cover
the Falcon tube with aluminum foil while performing the
experiments to avoid photobleaching and denaturation.
Homogenize the solution by mixing gently.
6. During the equilibrium times, the system should be maintained
in dark conditions (covering the plate/vial with aluminum foil).
7. Given that each nanoparticle can be titrated (with its three rep-
licates) in 48 wells, 2 different nanoparticles can be analyzed
per plate, optimizing the time performance.
8. The normalized fluorescence is obtained by
Ic / In
|| I || = ,
I co / I no
where Ic is the intensity of a given point of a cationic nanopar-
ticle, In is the intensity of the same point of the neutral (con-
trol) nanoparticle, and Ico and Ino are the measurements in the
absence of nanoparticles in the solution (first point). This is
done to eliminate the effect of the gold core natural
absorption.
9. Choose a point in the curve at the bottom of the plot, after the
principal quenching (initial slope) but before reaching the hor-
izontal tendency. Points in this zone have the biggest DI after
the addition of the analyte due to its sensitive nature in the
competitive binding (13). The control nanoparticle does not
have a titration point; in this case, use a concentration similar
to the cationic nanoparticles.
Nanoparticle-GFP “Chemical Nose” Sensor for Cancer Cell Identification 7

10. The total time used to analyze each nanoparticle should be


consistent in all the cases to avoid reproducibility problems.
Use the same amount of time when preparing as well as mixing
the solutions for each one of the nanoparticles.
11. We find that the minimum amount of cationic nanoparticles as
predictors to have a good percentage of identification (>90%)
is three, featuring functional groups for h-bond, p-stacking,
and hydrophobic interactions (14). However more nanoparti-
cles can be used to increase identification accuracy.
12. Four different cells can be run simultaneously in the same
plate, optimizing the analysis process (Fig. 4).
13. We find that six is a reasonable number of replicates to have
good representation in the canonical graphs with a 95% of
confidence (no overlapping).
14. LDA generates canonical factors to minimize the training
matrix. The best two canonical factors are the ones used to
generate the 2D score plots.
15. In the case of an unknown test sample, see Subheading 3.2 with
the unidentified cell line. Using Mahalanobis distance analysis,
compare the results with the previously generated matrix for
proper identification. This analysis is done using SYSTAT as
described above.

Acknowledgment

This work was supported by the NIH (GM077173).

References
1. Haab BB (2006) Applications of antibody array 7. Phillips RL, Miranda OR, You CC et al (2008)
platforms. Curr Opin Biotechnol 17:415–421 Rapid and efficient identification of bacteria
2. Kingsmore SF (2006) Multiplexed protein using gold-nanoparticle-poly(para-phenylene-
measurement: technologies and applications of ethynylene) constructs. Angew Chem Int Ed
protein and antibody arrays. Nat Rev Drug 47:2590–2594
Discov 5:310–321 8. Bajaj A, Rana S, Miranda OR et al (2010) Cell
3. Klee EW (2008) Data mining for biomarker surface-based differentiation of cell types and
development: a review of tissue specificity anal- cancer states using a gold nanoparticle-GFP
ysis. Clin Lab Med 28:127–143 based sensing array. Chem Sci 1:134–138
4. Albert KJ, Lewis NS, Schauer CL et al (2010) 9. Templeton AC, Wuelfing WP, Murray RW
Cross-reactive chemical sensor arrays. Chem (2000) Monolayer-protected cluster molecules.
Rev 100:2595–2626 Acc Chem Res 33:27–36
5. Moyano DF, Rotello VM (2011) Nano meets 10. De M, Rana S, Akpinar H et al (2009) Sensing
biology: structure and function at the nanopar- of proteins in human serum using conjugates
ticle interface. Langmuir 27:10376–10385 of nanoparticles and green fluorescent protein.
6. You CC, Miranda OR, Gider B et al (2007) Nat Chem 1:461–465
Detection and identification of proteins using 11. Liu X, Atwater M, Wang J et al (2007)
nanoparticle–fluorescent polymer ‘chemical Extinction coefficient of gold nanoparticles
nose’ sensors. Nat Nanotechnol 2:318–323 with different sizes and different capping
8 Daniel F. Moyano and Vincent M. Rotello

ligands. Colloid Surface B Biointerfaces mer complexes for protein sensing. Faraday
58:3–7 Discuss 152:33–42
12. De M, Rana S, Rotello VM (2009) Nickel-ion- 14. Bajaj A, Miranda OR, Kim IB et al (2009)
mediated control of the stoichiometry of his- Detection and differentiation of normal, can-
tagged protein/nanoparticle interactions. cerous, and metastatic cells using nanoparticle-
Macromol Biosci 9:174–178 polymer sensor arrays. Proc Natl Acad Sci USA
13. Moyano DF, Rana S, Bunz UHF et al 106:10912–10916
(2011) Gold nanoparticle-polymer/biopoly-
Chapter 2

A Method to Map Spatiotemporal pH Changes


in a Multicellular Living Organism Using a DNA Nanosensor
Sunaina Surana and Yamuna Krishnan

Abstract
Environmental pH has a determining role in the structure of biomolecules, thus playing an important role
in regulating cellular activities. Eukaryotic cells must, therefore, strive to stringently regulate pH in various
intracellular organelles so as to confer normal functioning at the level of whole organism. Several pH-
sensitive probes have been reported, each of which can be used to map the pH dependence of an in vivo
process. However, these probes suffer from some inherent drawbacks. Here we demonstrate the utility of
an externally introduced, pH-triggered DNA nanomachine inside the multicellular eukaryote Caenorhabditis
elegans. The nanomachine uses FRET to effectively map spatiotemporal pH changes associated with endo-
cytosis in coelomocytes of wild type as well as mutant worms, in a variety of genetic backgrounds. It shows
highest dynamic range in the pH regime 5.3–6.6 and has a half-life of ~8 h, thus positioning it well to
interrogate a variety of pH-correlated biological phenomena in vivo.

Key words I-switch, pH sensor, Caenorhabditis elegans, Coelomocytes

1 Introduction
Protons have a determining role in the charge and, in turn, struc-
ture of biomolecules. Hence, proton concentration plays an impor-
tant role in regulating cellular and, in turn, organismal activities.
Eukaryotic cells, thus, have stringently controlled pH in their vari-
ous intracellular organelles (1). Perturbation of cytoplasmic and
organellar pH has been shown to lead to defects in receptor-
mediated endocytosis, intracellular targeting of newly synthesized
lysosomal proteins, calcium homeostasis, protein processing and
sorting, and degradation of neurotransmitters (2). In vivo, these
cellular defects are manifested in terms of tumor metastasis (1),
growth defects (2), neurodegeneration (3), lysosomal storage dis-
orders (4), defects in embryogenesis, spermatogenesis, and excre-
tion (5), to name a few. Hence, pH is an important correlate of
biological processes in vivo.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_2, © Springer Science+Business Media New York 2013

9
10 Sunaina Surana and Yamuna Krishnan

Due to the molecular and cellular complexity encountered in


the environment of a whole organism, there are only few reports of
pH-responsive probes in vivo. Most pH measurements in organ-
isms have relied on genetically encodable, pH-sensitive GFP vari-
ants termed pHluorins, which can be fused to a specific protein
and, thus, used to mark a pathway of interest (6). pHluorins enable
ratiometric imaging, which helps to cull differences in protein
expression. These properties have led to its use in a variety of sys-
tems, right from bacteria to mice. In bacterial systems like
Escherichia coli and Listeria, this probe has been used to measure
stress response to optical tweezers (7). On the other hand, Dittman
et al. have elegantly used pHluorins to identify genes regulating
abundance of vesicular SNAREs (soluble NSF attachment protein
receptors) at Caenorhabditis elegans cholinergic motor neuron syn-
apses (8). In another report, Poskanzer et al. have used synap-
topHluorins to study the dynamics of resting pools of synaptic
vesicles in the Drosophila neuromuscular junction (9). Despite such
promising applications, pHluorins suffer from some issues such as
(1) fixed wavelength, which limits its use in the background of
GFP-expressing transgenics; (2) simultaneous tracking of two
pathways is not possible; (3) quenching of its fluorescence at acidic
pH values that leads to difficulties in visualization in highly acidic
compartments; and (4) its fixed pH sensitive regime given that
many physiological processes often operate at pH regimes beyond
the pH sensitivity of pHluorins.
Less widely used pH-sensitive probes in vivo are fluorescein
and its derivatives fluorescein isothiocyanate (FITC), 2¢,7¢ bis
(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and
carboxyfluorescein (10). Rare examples are the use of BCECF to
measure the pH of interstitial space in neoplastic tumors of the rab-
bit ear (11) and carboxyfluorescein to study intracellular pH in the
bacterial species Lactobacillus delbrueckii and Listeria innocua (12).
However, their inability to be targeted specifically to a pathway of
interest, fixed wavelength, and suboptimal photophysical proper-
ties has precluded their wider applicability in whole organisms.
Thus, there is a need for a pH probe that combines robust
fluorescence properties, targeting, and the ability to manipulate
wavelengths as well as pH regimes. Here, we describe the func-
tioning of a rationally designed pH-sensitive DNA nanomachine,
called the I-switch (13), in vivo. This FRET-based pH sensor uti-
lizes bright and stable fluorophores and can be used at different
wavelengths, positioning it well in the context of fluorescent-
protein-expressing transgenics. It functions autonomously and
reversibly in the organismal milieu, making it amenable to a variety
of mutant backgrounds. The structure and charge of the DNA
backbone also makes it facile for targeting. It has highest dynamic
range in the pH regime 5.3–6.6, enabling it to map spatiotemporal
pH changes occurring during endosomal maturation (14).
A Method to Map Spatiotemporal pH Changes in a Multicellular Living Organism… 11

Importantly, due to its rational design, it is possible to change its


dynamic range as well as pH-sensitive regime, positioning it well to
track pH changes in other physiological processes in vivo.

2 Materials

2.1 Oligonucleotides 1. All oligonucleotides (obtained from MWG Biotech, Germany,


and Sample or IBA GmbH, Germany) are high-performance liquid chro-
Preparation matography (HPLC) purified and lyophilized (Table 1).
Oligonucleotides are dissolved in Milli-Q (MQ) water to pre-
pare 200 μM stocks, aliquoted, and all aliquots stored at
−20°C, until further use. Fluorescently labeled oligonucle-
otides are subjected to ethanol precipitation prior to use to
remove any traces of free dye.
2. Ethanol, absolute.
3. 3.0 M potassium acetate solution: 2.94 g CH3COOK dissolved
in 10 mL MQ water and pH adjusted to 5.2.
4. Phosphate buffer – 100 mM KH2PO4: 1.36 g KH2PO4 dis-
solved in 10 mL MQ water. 100 mM K2HPO4: 1.74 g K2HPO4
dissolved in 10 mL MQ water. Each potassium phosphate solu-
tion is diluted to 10 mM. Add 10 mM KH2PO4 and 10 mM
K2HPO4 to obtain a buffer of pH 5.5.
5. 3.0 M potassium chloride solution: 2.23 g KCl dissolved in
10 mL MQ water.
6. Heat block and water bath.

2.2 Fluorescence 1. FluoroMax-4 instrument (Horiba Jobin Yvon, Japan), equipped


Spectroscopy with a mercury–Xe lamp as the light source.
2. Clamping buffer – 20× salt solution: 3.57 g KCl, 0.116 g
NaCl, 0.044 g CaCl2, and 0.081 g MgCl2 dissolved in 20 mL
MQ water. 20× HEPES solution: 1.9 g HEPES dissolved in
20 mL MQ water.

Table 1
Oligonucleotide sequences used for the I-switch

Sequence
5¢-CCCCAACCCCAATACATTTTACGCCTGGTGCC-3¢
5¢-CCGACCGCAGGATCCTATAAAACCCCAACCCC-3¢
5¢-TTATAGGATCCTGCGGTCGGAGGCACCAGGCGTAAAATGTA-3¢
5¢-Alexa 488-CCCCAACCCCAATACATTTTACGCCTGGTGCC-3¢
5¢-CCGACCGCAGGATCCTATAAAACCCCAACCCC-Alexa 647-3¢
12 Sunaina Surana and Yamuna Krishnan

3. 10 mL of 1 N HCl and 1 N NaOH solutions.


4. Prior to use, clamping buffers of desired pH values (5.0, 5.5,
6.0, 6.5, and 7.0) are made by mixing the salt and HEPES
solutions and diluting to make 1× solution. The pH is then
adjusted by using 1 N HCl or 1 N NaOH.
5. Samples are diluted to 100 nM using clamping buffers of
various pH.
6. 1 cm quartz cuvette.

2.3 C. elegans 1. C. elegans is grown at 22°C on nematode growth medium


Maintenance (NGM) containing a lawn of OP50 bacteria.
and Strains 2. All strains have been obtained from Caenorhabditis Genetics
Center (University of Minnesota, USA).
3. Wild-type strain: C. elegans isolate from Bristol (strain N2).
4. Mutant strains: rme-1(b1045), rme-4(b1001), rme-5(b1013),
and rme-6(b1014).
5. Transgenic strains: arIs37 [pmyo-3::ssGFP], cdIs131
[pcc1::GFP::RAB-5 + unc-119(+) + myo-2::GFP], cdIs66
[pcc1::GFP::RAB-7 + unc-119(+) + myo-2::GFP], and pwIs50
[lmp-1::GFP + Cb-unc-119(+)].
6. BOD incubator at 22°C for maintenance of nematode stocks.

2.4 Coelomocyte 1. 5 μM I-switch sample containing acceptor label only (IA647)


Labeling and pH and both donor and acceptor labels (IA488/A647) is diluted to the
Clamping desired concentration (see Subheading 3.1) in 1× medium 1.
2. 10× medium 1: 4.37 g NaCl, 0.18 g KCl, 0.055 g CaCl2, 0.1 g
MgCl2, and 0.95 g HEPES dissolved in 50 mL MQ water and
pH adjusted to 7.3. This is filter-sterilized using 0.22 μm
membrane filter.
3. TE2000-S inverted microscope, equipped with a 40×, 0.75
NA objective (Nikon, Japan), and microinjection setup
(Narishige, Japan).
4. Borosilicate glass capillaries.
5. 2.0% agarose pads, made on 22 × 50 mm glass coverslips.
6. 2.0% agarose: 0.2 g agarose dissolved in 10 mL MQ water.
7. Halocarbon oil.
8. 10 mM nigericin: 1.0 mg nigericin (Sigma-Aldrich, USA) is
dissolved in 133 μL of absolute ethanol. 10 μL aliquots are
made and stored at −20°C.
9. 10 mM monensin: 1.0 mg monensin (Sigma-Aldrich, USA) is
dissolved in 120 μL of absolute ethanol. 10 μL aliquots are
made and stored at −20°C.
A Method to Map Spatiotemporal pH Changes in a Multicellular Living Organism… 13

10. Clamping buffers: See Subheading 2.2 above. To each of these


buffers, 10 mM monensin and 10 mM nigericin are added to
obtain final concentration of 100 μM each.

2.5 Competition 1. Maleic anhydride.


Experiments 2. Bovine serum albumin (BSA).
3. BSA solution: 6 mg/mL solution in 0.1 M sodium carbonate
bicarbonate buffer (pH 9).
4. BSA solution: 6 mg/mL solution in 1× phosphate-buffered
saline (PBS) of pH 7.4.
5. 1× PBS: For 1 L, dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4,
and 0.24 g KH2PO4; adjust pH to 7.4; and bring to 1 L with
MQ water.
6. Dextran, 10 kDa MW.
7. Dextran solution: 3.2 mg/mL solution in 1× PBS of pH 7.4.
8. Dextran sulfate, 9–20 kDa MW.
9. Dextran sulfate solution: 4.4 mg/mL solution in 1× PBS of
pH 7.4.
10. Heparan sulfate, 30 kDa MW.
11. Heparan sulfate solution: 2.0 mg/mL solution in 1× PBS of
pH 7.4.
12. Unlabeled I-switch, diluted to 100 nM using 1× medium 1.

2.6 Fluorescence 1. Axiovert Apotome microscope (Zeiss, Bulgaria), equipped


Microscopy and with 40×, 1.3 NA objective, metal halide lamp (Zeiss, Bulgaria),
Image Analysis and filters suitable for each fluorophore.
2. TE2000-U inverted microscope (Nikon, Japan), equipped
with 60×, 1.4 NA objective, mercury arc illuminator (Nikon,
Japan), filters suitable for each fluorophore (Chroma, USA),
and Cascade II CCD camera (Photometrics, USA).
3. Image acquisition software: MetaMorph (Universal
Imaging, USA).
4. Fluoview 1000 confocal microscope (Olympus, Japan),
equipped with argon ion laser (Spectra-Physics, USA) for
488 nm excitation and He–Ne laser (Spectra-Physics, USA) for
633 nm excitation and a set of excitation, emission, and dichroic
filters suitable for each fluorophore (Olympus, Japan).
5. Image analysis software: ImageJ ver. 1.42 (NIH, freely avail-
able from website: http://rsbweb.nih.gov/ij/).
6. 40 mM sodium azide: 13 mg NaN3 dissolved in 5 mL M9
buffer.
14 Sunaina Surana and Yamuna Krishnan

7. 1× M9 buffer: 0.6 g Na2HPO4, 0.3 g KH2PO4, 0.5 g NaCl,


and 0.1 g NH4Cl dissolved in 100 mL MQ water and pH is
adjusted to 7.3. The buffer is filter-sterilized through a 0.22 μm
membrane filter.

3 Methods

We describe the use of a rationally designed synthetic DNA assem-


bly called the I-switch that functions as a pH sensor to map spa-
tiotemporal pH changes in a multicellular living organism. The
I-switch consists of two DNA duplexes connected to each other by
a flexible hinge and bearing cytosine-rich single-stranded over-
hangs at the duplex termini. Upon protonation, these C-rich over-
hangs base pair with each other to form an I-motif, causing a
structural transition at acidic pH (13, 14). At neutral pH, the
I-motif dissociates and entropic forces as well as electrostatic repul-
sion between the duplex arms cause the reversal of the structural
transition (Fig. 1a). This forms the molecular basis of a FRET-
based pH sensor using the I-switch.
Endocytosis is known to be accompanied by changes in pH as
vesicles mature from the early endosomes to the lysosomes, via the
late endosomes (15). These pH changes, measured in cultured
cells, are known to be in the regime of 6.0–6.2 in early endosomes
to ~5.5 in late endosomes and ~5.0 in lysosomes (16). Given that
this shows a good match with the pH-sensitive regime of the
I-switch, we describe its use in mapping spatiotemporal pH changes
associated with endosomal maturation in vivo. As an example, we
use it to map the same along the anionic ligand-binding receptor
(ALBR) pathway in the coelomocytes of wild-type C. elegans. We
show that this pH sensor can map variations in pH in different
genetic backgrounds such as mutants and RNAi knockdowns that
perturb pH homeostasis. In a model organism like C. elegans,
where pathways are elucidated primarily by genetics, this is of special
importance, since it demonstrates the utility and non-perturbative
nature of the I-switch in this model organism.

3.1 Sample 1. The I-switch is generally prepared at 5 μM concentration in a


Preparation volume of 50 μL. 1.25 μL of O1, O2, and O3 (each from a
200 μM stock) with 1.65 μL of 3 M KCl are mixed. The vol-
ume is made up to 50 μL by adding 10 mM potassium phos-
phate buffer of pH 5.5 (see Note 1). The solution is briefly
vortexed.
2. The solution is heated at 90°C for 5 min in a heat block and then
cooled to room temperature at a rate of 1°C per 3 min. Samples
are then equilibrated at 4°C overnight. Fluorescently labeled
I-switch is prepared in a similar manner with fluorophore-labeled
oligonucleotides. Samples are used within 7 days of annealing.
A Method to Map Spatiotemporal pH Changes in a Multicellular Living Organism… 15

a b

O1 O2

O3

c I647 d 120
GFP Merge
100

% Labeling
80
60
40
20
0

UL
ol

SA
PG

X
A

W
DE
BS
ntr

AS
XS
mB
HS
Co

DE
Fig. 1 I-switch marks endosomes of the anionic ligand-binding receptor (ALBR) pathway in coelomocytes of
Caenorhabditis elegans. (a) Schematic showing the principle of the I-switch. (b) Epifluorescence image of
wild-type C. elegans hermaphrodite microinjected with IA647. Arrowheads indicate labeled coelomocytes. Scale
bar: 50 μm. Inset: confocal image of IA647-labeled coelomocyte. Scale bar: 5 μm. (c) I-switch specifically marks
coelomocytes upon injection in the pseudocoelom of arIs37 hermaphrodites. Scale bar: 10 μm. Inset shows a
typical image of one such endosome, showing co-localization between GFP and IA647. (d) Competition assay
where IA647 is co-injected with 300 equivalents of various endocytic markers to establish mode of uptake of
I-switch in coelomocytes (HSPG: heparan sulfate proteoglycan, BSA: bovine serum albumin, mBSA: maleylated
bovine serum albumin, DEX: dextran, DEXSUL: dextran sulfate, ASW: Competing DNA). Error bars indicate
s.e.m. (n = 20 worms)

3. Prior to use, samples are diluted in 1× medium 1 and vortexed


to enable mixing. They are centrifuged at 9300 rcf for 20 min.

3.2 Microinjections 1. For coelomocyte labeling, I-switch made from Alexa 647-func-
and Coelomocyte tionalized O2 (IA647) is used. 5 μM stock solution of I-switch
Labeling sample is diluted to 100 nM using 1× medium 1.
2. One-day old hermaphrodites grown on NGM plates (+OP50)
are mounted on a 2% agarose pad containing a droplet of halo-
carbon oil.
3. Injections are performed, using borosilicate capillaries, at
50–55 psi in the dorsal side of the pseudocoelom, just opposite
the vulva.
4. Injected worms are released using 1× M9 buffer and trans-
ferred to NGM plates (+OP50). Plates are incubated at 22°C
for 1 h.
16 Sunaina Surana and Yamuna Krishnan

5. After 1 h, injected worms are mounted on a glass slide contain-


ing a 2.0% agarose pad, anesthetized using 40 mM NaN3 in
M9 buffer, and imaged.
6. Wild-type hermaphrodites, when injected with IA647 and imaged
on an Axiovert Apotome microscope, show bright puncta in
coelomocytes (Fig. 1b). Uptake is quantified by percentage of
coelomocytes labeled postinjection.
7. Confirmation that the I-switch marks coelomocytes in C. elegans
is obtained by injecting IA647 in the strain arIs37. Co-localization
between GFP and IA647 is performed by merging images taken
on an Olympus Fluoview confocal microscope (Fig. 1c).
8. For confirming the mode of endocytosis, competition experi-
ments are performed with an excess of anionic ligands, which
are known to bind ALBRs with high affinity. IA647 is mixed with
competitor ligands mBSA, dextran sulfate, heparan sulfate, and
unlabeled DNA in a 1:300 ratio, such that the final concentra-
tion of IA647 is 100 nM. As a control, injections with the neutral
molecules BSA and dextran at the same molar ratios are also
performed. Imaging is performed on an Axiovert Apotome
microscope, equipped with a 40×, 1.3 NA objective.
9. Uptake is quantified by percentage of coelomocytes labeled
postinjection; all values are normalized to uptake in hermaph-
rodites injected with IA647 alone (Fig. 1d).

3.3 pH Clamping 1. For pH measurements, doubly labeled I-switch (IA488/A647) is


used.
2. First, an in vitro pH calibration curve is generated by diluting
5 μM fluorescently labeled I-switch to 100 nM in clamping
buffer of the desired pH, ranging from pH 5.0 to 7.0. All
samples are vortexed and equilibrated for 30 min at room tem-
perature. The samples are excited at 488 nm, and emission is
collected between 500 and 700 nm with a bandwidth of 1 nm
(for excitation) and 10 nm (for emission) and spectral scan
speed of 1 nm/s.
3. Fluorescent intensities at 520 nm (D) and 665 nm (A) are
obtained, and then D is divided by A for every pH value to
generate an in vitro pH response curve.
4. For in vivo pH measurements, 5 μM stock solution of IA488/A647
is diluted to 500 nM using 1× medium 1.
5. The functionality of a sensor in vivo is determined by assessing
the fold change of its donor/acceptor (D/A) ratio in the
dynamic regime and comparing this to the in vitro fold change.
Good correspondences of the fold change values indicate the
sensor integrity in the given environment.
A Method to Map Spatiotemporal pH Changes in a Multicellular Living Organism… 17

6. One-day old wild-type hermaphrodites are injected with


IA488/A647, incubated at 22°C for 1 h, and then kept in a dish
containing 1 mL clamping buffer of the desired pH for 75 min.
This buffer contains the ionophores nigericin and monensin at
a final concentration of 100 μM each, which equilibrate the
intra-coelomocyte pH to that of the external buffer. Using this
method, the pH of coelomocytes is clamped at pH 5.0 in ten
worms and at pH 7.0 on ten different worms. Prior to soaking
in clamping buffer, the cuticle of the worms is perforated in
three regions (anterior, middle, and posterior) with a microin-
jection needle. After 75 min in the clamping buffer, the worms
are mounted on a glass slide using the same clamping buffer
(with nigericin and monensin) and the coelomocytes imaged
(see Note 2). This is done three times independently, on ten
worms each (see Note 3).
7. For each endocytic vesicle, fluorescence intensity at 520 nm
(D) is divided by the intensity at 665 nm (A). This gives the
D/A ratio for that vesicle. Cells clamped at pH 5.0 show a low
D/A ratio, while those clamped at pH 7.0 show elevated val-
ues (Fig. 2a, b).
8. Fold change is calculated by dividing the D/A ratio at the
higher end of the dynamic regime to that obtained at the lower
end (pH 7.0 and pH 5.0, respectively, in this case). This fold
change is then compared to the in vitro fold change in order to
assess the performance of the sensor (Fig. 2c).
9. pH is then clamped at intermediate values (5.0, 5.5, 6.0, 6.5,
and 7.0) to obtain the standard calibration curve (Fig. 2d).
This will now be used to calculate pH values from D/A ratios
in the system under study (see Note 4).

3.4 Spatiotemporal 1. In order to assess the pH changes occurring during endocytic


pH Mapping maturation as a function of time, temporal regimes of the resi-
dence times of the I-switch in different populations of vesicles
are determined. This is done by performing co-localization
studies, as a function of time, of IA647 in the GFP-expressing
transgenics cdIs131, cdIs66, and pwIs50. These strains express
GFP fusions of Rab-5 (early endosomal marker), Rab-7 (late
endosomal/lysosomal marker), and Lmp-1 (lysosomal
marker).
2. IA647 is diluted to 500 nM using 1× medium 1.
3. IA647 is injected in wild-type hermaphrodites and, after the req-
uisite time (from 5 to 60 min, at increments of 5 min for the
first 30 min), transferred to chilled NGM plates (+OP50), and
kept on ice. This method efficiently stops endocytosis and
trafficking of vesicles (see Note 5).
18 Sunaina Surana and Yamuna Krishnan

a pH 5 pH 7 b
25

No. of endosomes
20
Donor

15
10
5
0
0 40 80 120 160
Acceptor

D/A ratio
25

No. of endosomes
20
15
5 10
5
D/A

0
0 40 80 120 160
D/A ratio
7 d
1.2
Normalized D/A ratio

c 1.0
5
0.8
Fold change in

4
D/A ratio

0.6
3
0.4
2
1 0.2

0 0.0
o o 4.8 5.2 5.6 6.0 6.4 6.8 7.2
itr iv
Inv Inv pH

Fig. 2 In vivo characterization of the I-switch. (a) Donor channel (D), acceptor channel (A), and respective
pseudocolor D/A images of coelomocytes labeled with IA488/A647 and clamped at pH 5.0 and 7.0. Scale bar:
10 μm. (b) Histograms showing typical spread of D/A ratios of endosomes clamped at pH 5.0 (white bars) and
pH 7.0 (black bars) (n 3 25 endosomes). (c) In vitro and in vivo fold change in D/A ratios of IA488/A647 between pH
7.0 and pH 5.0. (d) pH calibration curve of IA488/A647 in vivo (black trace) and in vitro (gray trace) showing normal-
ized D/A ratios versus pH. Error bars indicate s.e.m. (n 3 50 endosomes)

4. Coelomocytes are imaged on the Fluoview 1000 confocal


microscope. Co-localization of GFP and IA647 is determined by
counting the numbers of IA647-positive puncta that co-localize
with GFP-positive puncta and expressing them as a percentage
of the total number of IA647-positive puncta. The time point
where the I-switch shows maximal co-localization with an
endocytic marker is chosen for pH measurements in that par-
ticular endocytic vesicle (Fig. 3a–d).
A Method to Map Spatiotemporal pH Changes in a Multicellular Living Organism… 19

5 mins 17 mins 60 mins d 100


a b c
80

% Colocalization
GFP

60

40

20
IA647

0
5 10 15 17 20 25 30 45 60
h Time (min)
15

No. of endosomes
Merge

10

0
0 10 20 30 40
5 mins 17 mins 60 mins D/A ratio
e f g No. of endosomes
25
20
Donor

15
10
5
0
0 10 20 30 40
Acceptor

D/A ratio
100
No. of endosomes

75
5 50
25
D/A

0
0 10 20 30 40
7 D/A ratio

Fig. 3 Spatiotemporal mapping of pH in coelomocytes. Confocal images showing co-localization of IA647 with
(a) GFP::RAB-5 at 5 min. (b) GFP::RAB-17 at 17 min. (c) LMP-1::GFP at 60 min. Scale bar: 5 μm. (d) Trafficking
of endocytosed IA647. Percentage co-localization of IA647 with GFP-tagged endosomal markers (RAB-5, black
circles; RAB-7, gray circles ; LMP-1, black open circles) at indicated times (n ~ 75 endosomes). (e–g)
Representative pseudocolor D/A images of IA488/A647-labeled coelomocytes in wild-type hermaphrodites at indi-
cated times. Scale bar: 5 μm. (h) Histograms of D/A ratios of maturing endosomes; early endosomes at 5 min
(black bars ), late endosomes at 17 min (gray bars), and lysosomes at 60 min (white bars) (n ~ 100
endosomes)

5. Now, to map the pH of a particular population of vesicles,


500 nM IA488/A647 is injected in wild-type hermaphrodites. After
the requisite time (chosen according to the co-localization
studies), the worms are transferred to chilled NGM (+OP50)
plates. The worms are anesthetized using 40 mM NaN3 in M9
buffer, and the coelomocytes are imaged.
20 Sunaina Surana and Yamuna Krishnan

3.5 Ratiometric 1. All ratiometric images are collected using a Nikon TE2000-U
Microscopy and Image epifluorescence microscope. Coelomocytes are located and
Analysis imaged so as to focus maximal number of puncta. Fluorescence
images of the cells are obtained by exciting Alexa 488 and col-
lecting emission using the 530 ± 15 nm emission filter. This
yields a donor image (D). The cells are then re-excited at
488 nm, and emission of the acceptor is acquired using a
710 ± 40 nm filter. This is the FRET image (A). A third image
is obtained by directly exciting the acceptor and collecting
emission at acceptor emission wavelength. This is the acceptor
image (I).
2. Autofluorescence of each image (D, A, and I) is calculated by
measuring mean pixel intensity over an adjacent cell-free area
in that image. This autofluorescence is subtracted from the
corresponding image, prior to all image processing.
3. Each endosome in the donor is selected by the ROI plug-in
within ImageJ program, and total and mean intensities in each
endosome are measured and recorded.
4. Each saved ROI is recalled, and total and mean intensities of
the corresponding vesicles in the FRET image are measured.
5. Dividing the mean intensity of each endosome in the donor
image by the corresponding intensity in the FRET image pro-
vides a donor/acceptor (D/A) ratio for that endosome. This is
repeated for all the cells imaged (each reading is obtained from
coelomocytes of ten worms) to obtain a spread of D/A ratios
for that time point (Fig. 3e–h). These values are then used to
calculate a mean D/A ratio for the corresponding time point.
6. The standard in vivo calibration curve is then used to convert
this D/A ratio to its corresponding pH value (Table 1).

3.6 pH Mapping 1. The I-switch is now used to measure pH in genetic back-


in Mutants grounds that perturb the endocytic pathway (Table 2). Two
methods are chosen to induce this perturbation: one, a genetic
knockout in the rme-1 gene (this gene functions to recycle
internalized receptors to the plasma membrane) and, the other,
a knockdown of the VHA-8 protein (this protein is a compo-

Table 2
Mean endosomal pH (±s.e.m.) at various time points postinjection

Strain 5 min 17 min 60 min


Wild type 6.4 ± 0.12 6.0 ± 0.09 5.4 ± 0.03
rme-1 6.1 ± 0.06 6.0 ± 0.1 5.7 ± 0.05
vha-8 (RNAi) 5.7 ± 0.1 5.2 ± 0.07 6.0 ± 0.04
A Method to Map Spatiotemporal pH Changes in a Multicellular Living Organism… 21

a
No. of endosomes

20 25 80
15 20 60
15
10 40
10
5 5 20
0 0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30
b
No. of endosomes

40 60 60
30
40 40
20
20 20
10
0 0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30
D/A ratio D/A ratio D/A ratio

Fig. 4 pH measurements in various genetic backgrounds using the I-switch. (a, b) Histograms of D/A ratios of
endosomes undergoing progressive maturation, from early endosomes at 5 min (black bars) to late endo-
somes at 17 min (gray bars) to lysosomes at 60 min (white bars) in rme-1(b1045) and vha-8 (RNAi) hermaph-
rodites, respectively (n = 10 cells, 3 60 endosomes)

nent of the V-ATPase complex that maintains pH of


organelles).
2. Ten hermaphrodites of each strain are injected with 500 nM
IA488/A647. After the requisite time (according to the co-localiza-
tion studies in wild-type hermaphrodites), the worms are trans-
ferred to chilled NGM plates. The worms are anesthetized
using 40 mM NaN3 in M9 buffer and the coelomocytes are
imaged (Fig. 4a, b).
3. Imaging and analysis is performed as outlined in Subheading 3.5,
and pH values are calculated from the standard calibration
curve (Table 1).

3.7 Targeting 1. The native I-switch enters coelomocytes via the ALBR path-
the I-Switch way. It can be induced to enter other endocytic pathways by
to Other Pathways saturating the ALBRs with mBSA and tagging the I-switch
with the appropriate endocytic ligand. This can be done by
injecting the I-switch with mBSA at molar ratios greater than
1:500.

4 Notes

1. It is imperative that the I-switch sample is always annealed in


phosphate buffer of pH 5.5 to minimize variability.
2. Coelomocytes are present in the pseudocoelom, and accessibil-
ity of the cells to the clamping buffer is sometimes an issue.
22 Sunaina Surana and Yamuna Krishnan

Hence, there may be a few cells whose pH may not be efficiently


clamped. These cells are discarded from the final analysis.
3. Coelomocytes clamped at pH 5.0 are very distinctive and can
immediately be distinguished from those clamped at pH 7.0
due to their small sizes.
4. The in vitro and in vivo calibration curve is normalized by divid-
ing the D/A values at every pH by the D/A value at pH 7.0.
5. Worms should be grown and maintained at 22°C. Maintenance
at lower temperatures slows down all physiological processes,
including endocytosis, which may alter temporal regimes of
trafficking.

Acknowledgments

We thank Sandhya P. Koushika for inputs on experiments, Souvik


Modi for technical input, Central Imaging Facility at NCBS and
the Caenorhabditis Genetics Center (funded by NIH-NCRR) for
nematode strains, and DBT and the Nanoscience and Technology
Initiative of DST for funding. S.S. acknowledges the CSIR, and
Y.K. acknowledges the Innovative Young Biotechnologist Award
and Wellcome Trust–DBT India Alliance for fellowships.

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Chapter 3

A Simple Method to Visualize and Assess the Integrity


of Lysosomal Membrane in Mammalian Cells Using
a Fluorescent Dye
Syed K. Sohaebuddin and Liping Tang

Abstract
Fluorescent dyes have been used as “nanosensors” for visualization and determination of various processes
occurring inside a cell, or intracellular events, such as cell cycle progression and intracellular trafficking.
Here, we describe a novel use of acridine orange to visualize lysosomes and discriminate cells with healthy
lysosomes from cells with damaged lysosomes in two different types of mammalian cells: fibroblasts and
macrophages. This method allows assessment of lysosomal membrane integrity upon exposure to various
foreign particles, i.e., engineered nanoparticles. The uniqueness of this method enables investigators to
acquire fluorescent images with a dye that is susceptible to photo-bleaching under UV light. These acquired
images bolster the quantitative data, providing a visual representation of the cell morphology as well as
assess its nucleus and lysosomes.

Key words Lysosomal membrane permeability, Lysosomes, Acridine orange, Nanoparticles, Carbon
nanotubes, Lysosomal membrane damage

1 Introduction

The use of fluorescent dyes or enzymes with specificity to a par-


ticular target, such as a cellular receptor or an intracellular struc-
ture, are invaluable in investigating etiology of diseases and thus
developing promising treatments for those diseases (1). Fluorescent
dyes and enzymes have also been used to examine the mechanisms
of various cellular processes such as endocytosis, exocytosis, and
cell death (2). Furthermore, the morphology of intracellular struc-
tures such as the nucleus, mitochondria, lysosomes, and actin can
be examined using fluorescent dyes (3). These dyes are able to
fluoresce due to the presence of a functional group which will
absorb energy at a specific wavelength and reemit energy at a dif-
ferent wavelength (4). This causes that molecule to fluoresce,
enabling us to detect the presence/absence of that molecule in the
experimental samples (4, 5). The amount of fluorescence can be

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_3, © Springer Science+Business Media New York 2013

25
26 Syed K. Sohaebuddin and Liping Tang

detected and quantified through the magnitude of fluorescence.


Such quantitative analysis is performed using either fluorescent
plate readers or flow cytometry (6). To bolster the quantitative
data, qualitative assessment can be performed which allows us to
observe the physical location(s) of our target in the experimental
sample as well as quantify their presence at a particular location(s)
in the same sample. The use of both quantitative and qualitative
analysis leads to lucid understanding of experimental results and
observed phenomenon.
There are, however, some dyes which are not compatible for
qualitative assessment mainly because irradiation of the dyes to cer-
tain wavelengths of light causes damage to the cell’s internal organ-
elles. Damaged organelles may leak the dye to other cellular
components such as the cytoplasm, leading to false qualitative results
(7). Acridine orange is one of these dyes which, when irradiated with
intense blue light, causes damage to the lysosome’s membrane (8).
Acridine orange is a weak base metachromatic dye capable of cross-
ing plasma membranes and staining nucleic acids and lysosomes. At
low concentrations, it can differentiate lysosomes (reddish-orange
granules) from other cellular components (diffuse green) (9).
Acridine orange molecules become protonated under acidic condi-
tions and hence get trapped within lysosomes. Accumulation of acri-
dine orange molecules in lysosomes leads to a shift in excitation from
green = 530 nm to red = 620 nm (10). When pH of the lysosomes
rises or if their membranes are damaged, acridine orange molecules
become deprotonated, and these molecules can then cross back into
the cytoplasm. This shifts the emission back from red to green (11).
Therefore, extended exposure (>1 min) of acridine orange loaded
cells to blue light leads to lysosomal membrane damage and a shift
in the lysosomes’ color from red to green.
Here, we have established a methodology to obtain visual rep-
resentation of cells loaded with acridine orange before acridine
orange molecules cause any disruption to the membrane of the
lysosomes.

2 Materials

Prepare culture media and PBS using distilled water and autoclave
the final solution to sterilize it. Prepare all nanomaterial solutions
fresh under sterile conditions and store overnight at 4°C before
performing the experiment. Prepare acridine orange solution fresh
under sterile conditions and store it overnight at 4°C before per-
forming the experiment. Follow waste disposal regulations when
disposing waste materials.

2.1 Cell Culture 1. Dulbecco’s Modified Eagle’s Medium.


Components 2. Calf serum.
Florescence Method to Assess Lysosomal Integrity 27

3. Penicillin/streptomycin: 10,000 U/mL penicillin (base),


10,000 mg/mL streptomycin (base).
4. Trypsin solution: 2.5 g/mL trypsin, 0.38 g/L EDTA·4Na in
Hank’s balanced salt solution without calcium and magnesium
salts (0.25% EDTA 1 mM).
5. 1× PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
1.47 mM KH2PO4, pH 7.4. Weigh 8 g of NaCl, 0.2 g of KCl,
1.44 g Na2HPO4, 0.24 g KH2PO4. Dissolve in 800 mL of dis-
tilled H2O. Adjust pH to 7.4. Adjust volume to 1 L with addi-
tional distilled H2O. Sterilize by autoclaving.
6. Sterile tubes, 75 mL cell culture flasks.
7. 6-well cell culture plate and tissue culture treated for cell cul-
ture of anchorage-dependent cells.
8. Hemacytometer and glass coverslips.
9. 6-well plates.

2.2 Mammalian 1. 3T3 fibroblasts (American Type Cell Culture, Manassas,


Cells VA, USA).
2. RAW 264.7 (macrophages) (American Type Cell Culture,
Manassas, VA, USA).

2.3 Nanomaterials 1. TiO2 (anatase, 5–10 nm in diameter).


(Sun Innovations, 2. SiO2 (30 nm in diameter).
Fremont, CA, USA)
3. Multiwalled carbon nanotube (MWCNT) (<8 nm in diameter,
0.5–2 mm length). Carbon nanotubes are allotropes of carbon
with a cylindrical structure, and MWCNT are aligned individ-
ual nanotubes held together by van der Waals forces.
4. Multiwalled carbon nanotube (MWCNT) (<20–30 nm in
diameter, 0.5–2 mm length).
5. Multiwalled carbon nanotube (MWCNT) (>50 nm in diame-
ter, 0.5–2 mm length).

2.4 Fluorescence 1. Acridine orange dye stock solution: Prepare stock acridine
Microscopy orange stain solution by weighing 5 mg of acridine orange
Components powder. Dissolve this in 5 mL of cell culture medium to obtain
a concentration of 1 mg/mL.
2. Leica DMLP microscope (40× lens magnification and FITC-
Texas Red dual excitation band fluorescence filter, required for
best imaging).
3. Nikon E500 camera (indicate minimum exposure time of
1/8 s required for image acquisition).
28 Syed K. Sohaebuddin and Liping Tang

3 Methods

3.1 Cell Culture 1. Warm DMEM, calf serum, and antibiotics to 37°C in a warm
Medium Preparation bath before use.
2. Sterilize the interior of laminar flow hood by spraying it with
70% ethanol (see Note 1).
3. Once they are warmed up, spray the containers with 70% etha-
nol and transfer them to a laminar flow hood along with sterile
pipettes, pipette tips, sterile tubes, cell culture flasks, 6-well
plates, and glass coverslips.
4. Sterilize all items mentioned in the above steps under UV light
in the laminar flow hood for 15 min.
5. Prepare the cell culture medium by mixing 1 mL of calf
serum and 100 mL of antibiotics for every 9 mL of DMEM
(see Note 2).

3.2 Culturing 1. Culture the mammalian cells until they are sub-confluent in
Mammalian Cells 75 mL cell culture flasks.
2. Remove the culture medium from the flasks, and rinse the cells
three times with approximately 5 mL of 1× PBS.
3. Add 3 mL of trypsin to each flask, enough to cover the surface
of the 75 mL cell culture flasks.
4. Place these flasks in 37°C incubator for 3 min.
5. Visualize the cells under microscope to determine the percent-
age of detached cells. Gently tap the cell culture flasks on your
palm to detach any loosely attached cells.
6. Spray these flasks with 70% ethanol and place them under the
laminar flow hood.
7. Add 3 mL of calf serum to each flask to deactivate trypsin.
Rinse the cells 2–3 times to collect all the cells. Pool the flasks
containing the same mammalian cells.
8. After transferring them to a sterile tube, centrifuge the cells at
200 – 400 ´ g for 10 min.
9. Discard the supernatant and resuspend the cell pellet in 1 mL
of cell culture medium. Place a glass coverslip on top of a
hemacytometer and pipette 10 mL of this solution into a slot of
hemacytometer.
10. Count the cells and calculate the density of the cell solution.
11. Place glass coverslips in the wells of 6-well plates, and seed
25,000 cells on each of the glass coverslips (Fig. 1). Add 3 mL
of cell culture medium to each well (see Note 3). Incubate the
well plates overnight in a 37°C incubator.
Florescence Method to Assess Lysosomal Integrity 29

Fig. 1 Illustration represents the seeding of cells on a coverslip in a well of a


6-well plate

3.3 Acridine 1. Prepare working acridine orange stain solution by mixing 5 mL


Orange Stain of acridine orange stock solution with 995 mL of cell culture
medium to obtain a final concentration of 5 mg/mL (see
Note 4).
2. Remove the cell culture medium from the 6-well plates. Rinse
the cells three times with 1 mL of 1× PBS.
3. Add 2 mL of acridine orange working solution to the cells in the
well plate and incubate this in a 37°C incubator for 15 min.
4. After 15 min, rinse the cells three times with 1× PBS to remove
any excess acridine orange stain.

3.4 Treatment 1. Prepare nanoparticle solutions at a concentration of 1 mg/


with Foreign Particles mL. Sonicate this solution to disperse the nanoparticles homo-
geneously in the solution.
2. Add 3 mL of cell culture medium in one of the 6-well plates;
this will be the control of the experiment.
3. Add 2.7 mL of cell culture medium to the rest of the wells in
the 6-well plate and add 300 mL of nanoparticle solution to
their respective wells to obtain a nanoparticle exposure con-
centration of 100 mg/mL. Incubate the cells in a 37°C incuba-
tor for 4 h.
4. After 4 h, remove the cell culture medium from the wells,
rinse the cells three times with 1 mL of 1× PBS, and add 3 mL
of 1× PBS to each well.

3.5 Imaging 1. Place the well plate under a microscope and using one area of
the Cells the control well, switch to the UV light (see Note 5). Under
dual FITC/TX-Red filter, bring the cells into focus.
2. Switch to a separate area in the same well and acquire an image.
If done properly, one should clearly see the cytoplasm stained
as diffuse green and lysosomes as reddish orange (Fig. 2).
30 Syed K. Sohaebuddin and Liping Tang

Fig. 2 Visualization of the lysosomes in (a) 3T3 fibroblasts and (b) RAW macrophages with and without expo-
sure to 100 mg/mL of nanomaterials for 4 h. The lysosomes (reddish orange) and cytoplasm (green) can be
clearly seen in control cells. 3T3 cells exposed to the TiO2 and SiO2 nanoparticles exhibit very little to no harm
to lysosomes. 3T3 cells exposed to MWCNT <8 nm exhibit severe damage to lysosomes shown by enhanced
green (cytoplasm) intensity and very low to none orange (lysosome) intensity. 3T3 cells exposed to MWCNT
20–30 nm and MWCNT >50 nm exhibit moderate damage to lysosomes. Exposure of nanomaterials to RAW
did not lead to any lysosomal membrane damage

3. Follow the same procedure to acquire images of cells in the


other wells of the 6-well plate. Cells with lysosomal damage
have enhanced green fluorescence in their cytoplasm, and their
lysosomes have yellowish to green fluorescence, depending on
the extent of lysosomal damage (Fig. 2).

4 Notes

1. To sterilize the interior of the laminar flow hood, cover the


work area surface with 70% ethanol and wipe it dry using tissue
paper.
2. Prepare 10 mL of cell culture medium for every 75 mL cell
culture flask.
3. Be careful not to allow cell solution to leave the glass coverslip
so that cells only attach and grow on the glass coverslip.
Initially, add only enough medium needed to keep the cells on
the glass coverslips. After 2 h of incubation in a 37°C incuba-
tor, the cells should have adhered to the glass coverslip. At this
time additional medium can be added.
4. Protect acridine orange solution from direct light.
5. Since acridine orange causes photooxidation of lysosomes
under blue light, it is always better to use one area of the sam-
ple to adjust parameters and settings that will allow acquisition
of high-quality images. Once these settings are established,
switch over to another area and acquire images immediately
and then move over to other areas and acquire more samples.
Florescence Method to Assess Lysosomal Integrity 31

Acknowledgment

This work was supported by NIH grant EB007271.

References
1. Brugger W, Mocklin W, Heinfeld S et al (1993) steady-state concentration of H2O2 is a conse-
Ex vivo expansion of enriched peripheral blood quence of lysosomal rupture. Biochem J 356:
CD34+ progenitor cells by stem cell factor, 549–555
interleukin-1 beta (IL-1 beta), IL-6, IL-3, 7. Zdolsek JM, Olsson MG, Brunk UT (1990)
interferon gamma, and erythropoietin. Blood Photooxidative damage to lysosomes of cul-
81:2579–2584 tured macrophages by acridine orange.
2. Li N, Zheng Y, Chen W et al (2007) Adaptor Photochem Photobiol 51:67–76
protein LAPF recruits phosphorylated p53 to 8. Zdolsek JM (1993) Acridine orange-mediated
lysosomes and triggers lysosomal destabilization photodamage to cultured cells. APMIS 101:
in apoptosis. Cancer Res 67:11176–11185 127–132
3. Moseley JB, Goode BL (2006) The yeast actin 9. Kobayashi Y, Vohimoto T, Nohara H et al
cytoskeleton: from cellular function to bio- (1999) Mechanism of apoptosis induced by a
chemical mechanism. Microbiol Mol Biol Rev lysosomotropic agent L-Leucyl-L-Leucine
70:605–645 methyl ester. Apoptosis 4:357–362
4. Tsien RY, Waggoner A (1995) Fluorophores 10. Lovelace MD, Cahill DM (2007) A rapid cell
for confocal microscopy. In: Pauley JB (ed) counting method utilizing acridine orange as a
Handbook of biological confocal microscopy. novel discriminating marker for both cultured
Pleum, New York, pp 267–274 astrocytes and microglia. J Neurosci Methods
5. Rietdrof J (2005) Microscopic techniques. In: 165:223–229
Rietdorf J (ed) Advances in biochemical engi- 11. Zareba M, Raciti MW, Henry MM et al (2006)
neering/biotechnology. Springer, Berlin, pp Oxidative stress in ARPE-19 cultures: do mel-
246–249 anosomes confer cryoprotection? Free Radic
6. Antunes F, Cadonas E, Brunk UT (2001) Biol Med 40:87–100
Apoptosis induced by exposure to a low
Chapter 4

Gold Nanoparticle as a Marker for Precise Localization


of Nano-objects Within Intracellular Sub-domains
Valeriy Lukyanenko and Vadim Salnikov

Abstract
Delivery of nano-objects to certain intracellular sub-domains is crucial for nanomedicine. Therefore delivery
of nano-object to desirable cellular compartment has to be confirmed. The most valuable confirmation of
the delivery comes from direct visualization of the nano-object. This visualization usually requires use of
microscope and corresponding probe which has to be conjugated with the nano-object. There are two
most popular methods of the visualization: confocal and electron microscopy. The former has significant
limitations due to diffraction limited resolution of confocal systems and three-dimensional convolution of
fluorescence. The latter should be significantly modified for needs of the visualization. Here we describe
the method for precise localization of nano-object within the cell using electron microscopy and 1–2 nm gold
particles as a nanomarker.

Key words Gold nanoparticle, Delivery of nano-objects, Intracellular sub-domains, Electron


microscopy

1 Introduction
Therapeutic delivery of genes and drugs to intracellular sub-domains
with nanocarriers requires the creation of reliable delivery systems
(1, 2). The methods available for monitoring the delivery of nano-
objects could be divided into retrieval of the products of the nano-
object delivery and visualization of nano-objects. The former is
especially important in the case of gene delivery when the level of
corresponding proteins clearly confirms the delivery. The latter,
visualization, has to show the location of nano-objects directly.
The direct visualization of the nano-object requires use of
microscope and corresponding probe which has to be conjugated
with the nano-object. There are two most popular methods of the
visualization: confocal and electron microscopy. They require cor-
respondingly fluorescent or electron-dense probe to be tagged to
nano-objects. Unfortunately, confocal microscopy could be used

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_4, © Springer Science+Business Media New York 2013

33
34 Valeriy Lukyanenko and Vadim Salnikov

mostly for in vitro experiments. Also, the precise localization of


nano-objects with confocal microscopy is significantly complicated
due to diffraction limited resolution of confocal systems and three-
dimensional convolution of fluorescence (3). This along with fold-
ing of cellular membranes and clamping of nanoparticles makes
practically impossible precise localization of nano-objects within
structures smaller than 0.5 μm. For example, diameter of isolated
mitochondria is about 1 μm, and mitochondrial inner membrane
makes multiple invaginations. It is clear that confocal microscopy
cannot resolve between fluorescent dot located inside mitochondrial
matrix and another dot located in the mitochondrial intermem-
brane space.
Hence, for purposes of localization of nanoparticles more accu-
rate confocal microscopy methods, such as Förster resonance energy
transfer (FRET) or more accurate systems, as superresolution struc-
tured illumination microscopy (SSIM) or electron microscopy
(EM), should be used. However, both FRET and SSIM has some
limitations of confocal microscopy and cannot be used for localiza-
tion of nanoparticles in tissue. The EM allows studying of tissue
fragments after in vivo experiments and precise localization of
electron-dense objects >10 nm in diameter within ultrathin sections
of the tissue (50–90 nm).
To make this localization of nanogold particles more precise,
we slightly modified the usual procedure of EM preparation (4, 5).
Namely, we employed water-soluble resin for cell polymerization
and silver enhancement within ultrathin sections (4, 5). Figure 1
shows the major steps of the method. The silver enhancement
significantly increases size of gold nanoparticles and makes their
distribution obvious. In addition, the silver enhancement is more
effective closer to the surface of the slice; therefore, the size of silver
grains allows deciding about location of the gold sol within the
ultrathin section. Our approach allows precise localization of 1–2
nm gold particles that could be tagged to any nano-object.
Here we describe the method for precise localization of
nano-object within the cell using the EM. The method could be
useful for electronopaque nano-objects and nanoparticles tagged
to electron-dense marker, such as gold sols.

2 Materials
1. LR White resin.
2. Silver Enhancing Kit (Ted Pella, Inc., Redding, CA).
3. Gold nanoparticles. To prevent aggregation of the gold particles
in experimental solution and their binding to cell proteins, the
nanoparticles should be pretreated (coated) with polyvinylpyr-
rolidone (PVP). For that we incubated gold sols in 1% PVP
(MW 10,000) for 10 min with gentle agitation (see Note 1 ).
Precise Localization of Nano-objects Within the Cell 35

Fig. 1 Simplified schematic representation of a method for localization of PVP-


coated gold nanoparticles within cells (reproduced from ref. 1 with permission
from Future Medicine)

4. Ultramicrotome.
5. Transmission electron microscope.
6. Cell culture. Freshly isolated cells should be plated on coverslips
coated with laminin.
7. 0.1 M Na+-cacodylate buffer:
(a) Prepare 0.2 M stock solution of sodium cacodylate in
double distilled water (21.4 g/500 ml).
(b) Add 27 ml of 0.2 M HCl per 500 ml cacodylate stock
solution.
(c) Add double distilled water to a final volume of 1 L.
8. Fixation buffer: 6 ml of 25% glutaraldehyde in 19 ml of 0.1 M
Na+-cacodylate buffer.
9. Rinse buffer: Na+-cacodylate buffer supplemented with 0.2 M
RNAase-free sucrose in 500 ml of 0.1 M Na+-cacodylate buffer.
10. Postfix buffer: 1% osmium tetroxide in the 0.1 M Na+-cacodylate
buffer.
11. 2% aqueous uranyl acetate solution.
12. Lead citrate solution (Reynold’s lead citrate stain):
(a) 50 ml lead solution: 0.19 M Pb(NO3)2 in double distilled
boiled (30 min, CO2-free) and filtered H2O.
(b) 50 ml of 0.28 M tribasic sodium citrate solution in double
distilled boiled (30 min, CO2-free) and filtered H2O. Add
one drop of the lead solution.
36 Valeriy Lukyanenko and Vadim Salnikov

(c) 50 ml of freshly made 1 N sodium hydroxide solution in


double distilled boiled (30 min, CO2-free) and filtered
H2O.
(d) Lead citrate solution: mix 21 ml lead solution and 21 ml
lead citrate solution and shake vigorously for 2 min (solu-
tion will be a milky white); then in 30 min of gentle shak-
ing, add 8 ml 1 N NaOH (the solution should be clear).
(e) Store in syringes (needle down into the rubber cork, with-
out air) at 4°C.

3 Methods
Carry out all procedures at room temperature unless otherwise
specified.
In our experiments we used primary culture of rat ventricular
myocytes (single freshly isolated cells); however, any monolayer
cell culture of any confluency could be used.
1. Fix cells or small pieces of tissue (~1 mm3) in 2 ml (per sample
unit) 6% glutaraldehyde in 0.1 M Na+-cacodylate buffer
(pH = 7.4), for 20 min. Rinse two times with the Na+-cacodylate
buffer supplemented with 0.1 M sucrose. Postfix the cells with
1% osmium in Na+-cacodylate buffer for 1 h.
2. Stain samples en bloc with 1% uranyl acetate in 25% ethanol for
1 h. Dehydrate cells in ethanol and acetone step by step as
shown:
(a) Increase ethanol concentration by moving the cells from
one solution to another. Amounts of ethanol in water
solution: 30, 40, 50, 60, 70, 80, 90% (every step is 10 min,
1 time), and 100% (10 min, 3 times).
(b) Acetone: 100%—10 min, 3 times.
3. Embed the cells in increasing concentrations of LR White
resin. Proportions of LR White to acetone (use 50 mm glass
Petri dishes): (1) 1 to 3, (2) 2 to 3, (3) 3 to 1, and (4) fresh
resin. Every step is 12 h.
To embed cultured cells (on coverslips) in LR White resin
for the final step, use a 1.5-ml tube with cap cut off. Fill the
tube with the resin; cover (seal) the tube with the cover slip, so
that cells face the resin; and tightly bind the construction with
parafilm, scotch tape, and foil to prevent the resin from exposure
to air.
4. For resin polymerization, put the tubes upside down into
thermostat (+60°C) for 24 h.
5. Remove the bandage from the tube. To separate embedded
cells from coverslip, dip the coverslip into liquid nitrogen for
Precise Localization of Nano-objects Within the Cell 37

Fig. 2 Distribution of gold nanoparticles in permeabilized cardiomyocytes. Representative electron


micrographs show the distribution of the nanoparticles in the cytoplasm along Z lines (a), but not in the
nucleus or mitochondria (b). M mitochondrion; Z line; N nucleus; arrows—T-tubules; black ovals show nano-
particles located deeper inside the ultrathin section and therefore having smaller diameters after silver
enhancement (reproduced from ref. 4 with permission from Cell press)

about 5 s. Now your cells are on the top of polymerized resin,


and you can see them with binocular microscope.
6. Sharpen the block for ultramicrotome cutting.
7. Obtain ultrathin sections with ultramicrotome. Most valuable
slices are 85–90 nm thick (they have a light gold color). Collect
the slices on formvar-coated nickel grids.
8. Perform silver enhancement with Silver Enhancing Kit (follow
instructions from Ted Pella, Inc.) (see Note 2).
9. After drying, stain slices for 15 min with 2% aqueous uranyl
acetate and then for 2 min with lead citrate.
10. Dry them. Now the slices are ready for electron microscopy.
11. Store images in tiff format (see Notes 3 and 4). Representative
micrographs (Fig. 2) show the typical distribution of nanoparti-
cles within ventricular cardiomyocytes.

4 Notes
1. To prevent aggregation of gold nanoparticles and their binding
to proteins, after conjugation, gold sols have to be coated with
polyvinylpyrrolidone (PVP) or polyethylene glycol PEG (Fig. 3).
38 Valeriy Lukyanenko and Vadim Salnikov

Fig. 3 Stabilizing the effect of polymer polyvinylpyrrolidone (PVP) on gold nanoparticles. Electron micrographs
(without silver enhancement) show 10 nm gold sols (a) in water, (b) in 150 mM potassium aspartate solution
(pH 7.2), and (c) in particles pretreated with 1% PVP and in aspartic acid solution. The suspensions were air-
dried on formvar-coated grids. No staining. (d) The absorption spectra for colloidal gold (peak at 520 nm) and
mixtures of gold and 150 mM potassium aspartate and/or 5% PVP. OD optical density (depends on gold par-
ticles concentration); dashed lines show the absorbance maximum for colloidal gold (523 nm). (e) The absorp-
tion spectra for 10 nm colloidal gold stabilized with 5% PVP before and after adding 1% bovine serum albumin
(BSA) to the cuvette (reproduced from ref. 4 with permission from Cell press)

For that, coating the sols has to be pretreated for 10 min in 1%


PVP10 (neutral, MW = 10,000). To measure the size of the
PVP-coated nanoparticles, we employed dynamic light scatter-
ing (Protein Solutions Ltd., England). These measurements
showed that 1% PVP increased the diameter of the gold parti-
cles, adding about 2 nm to their original size (4).
2. The size of the silver grains depends on the time of exposure
and the accessibility (i.e., depth of position within the section)
of the gold. We applied the solution for 8 min.
3. To find real silver grains on the EM micrograph, reduce contrast
by 70–80% (until less dense cell structures vanish) with Adobe
Precise Localization of Nano-objects Within the Cell 39

Photoshop (Adobe Systems Incorporated, San Jose, CA). Then


mark them and retune the image contrast to normal.
4. To calculate the number of particles, we recommend using Image
J 1.31v (National Institutes of Health, Bethesda, USA).

References
1. Lukyanenko V (2007) Delivery of nano-objects in cardiac muscle cells. Biophys J 71:
to functional sub-domains of healthy and failing 2942–2957
ventricular myocytes. Nanomedicine 2: 4. Parfenov AS, Salnikov V, Lederer WJ, Lukyanenko
831–846 V (2006) Aqueous diffusion pathways as a part
2. Lukyanenko V (2010) Therapeutic nano-object of the ventricular cell ultrastructure. Biophys J
delivery to sub-domains of cardiac myocytes. In: 90:1107–1119
Weissig V, D’Souza GGM (eds) Organelle- 5. Salnikov VV, Lukyanenko YO, Frederick CA,
specific pharmaceutical nanotechnology. Artech Lederer WJ, Lukyanenko V (2007) Probing the
House, Norwood MA, pp 433–448 outer mitochondrial membrane in cardiac mito-
3. Pratusevich VR, Balke CW (1996) Factors chondria with nanoparticles. Biophys J 92:
shaping the confocal image of the calcium spark 1058–1071
Chapter 5

Immunoisolation of Nanoparticles Containing Endocytic


Vesicles for Drug Quantitation
Ari Nowacek, Irena Kadiu, JoEllyn McMillan,
and Howard E. Gendelman

Abstract
Cell-mediated nanoparticle delivery has recently emerged as an efficacious method of delivering therapeutic
agents across physiological barriers. Use of cells as nanodelivery vehicles requires accurate assessment of
their loading capacity and identification of intracellular compartments where nanoparticles are seques-
tered. This is of great interest since specific endocytic trafficking routes can ultimately influence the mode
of nanoparticle release and their efficacy and function. Here, we describe a technique that allows for the
isolation of individual populations of nanoparticle-containing endosomes for subsequent quantitative anal-
ysis and more accurate description of where nanoparticles are stored on a subcellular level.

Key words Nanoparticles, Immunoisolation, Endosomes, Subcellular trafficking, Macrophage

1 Introduction

For over 20 years, nanoparticles (NP) have been researched for


their use in drug delivery (1, 2). Drug-loaded NP have the poten-
tial to increase efficacy, reduce toxicity, and improve clinical out-
comes of diseases. These nanoparticles tend to be designed to
deliver drugs or other therapeutic compounds, such as protein or
DNA, to specific cell populations. One of the important questions
when researching drug-carrying nanoparticles is determining pre-
cisely how much drug the target cells are able to take up. Generally,
this question is not difficult to answer. However, of even greater
importance than how much drug the target cells are able to pick up
is where within the cells are the nanoparticles being trafficked and
stored.

Ari Nowacek and Irena Kadiu have contributed equally.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_5, © Springer Science+Business Media New York 2013

41
42 Ari Nowacek et al.

2 Materials

Prepare all solutions using ultrapure water (prepared by purifying


deionized water to attain a sensitivity of 18 MW cm at 25°C) and
analytical grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Diligently follow all
waste disposal regulations when disposing waste materials.

2.1 Conjugation 1. PureProteome Protein A and Protein G Paramagnetic Beads


of Magnetic Beads (Millipore).
to Antibodies 2. Antibodies to endosome surface markers of interest (see Note 1).
3. Bovine serum albumin fraction V (10%).
4. Sterile 1× phosphate-buffered saline (PBS).
5. Microcentrifuge tubes (1.7 mL).
6. Microcentrifuge tube tumbler rotator.
7. Magnetic separator rack.
8. Refrigerated tabletop centrifuge.

2.2 Cellular 1. Cells in culture (see Note 2).


Treatment 2. Cell incubator.
Components
3. Serum-free DMEM (or other appropriate serum-free culture
medium).
4. Nanoparticles (see Note 3).
5. Sterile PBS.

2.3 Homogenization 1. Homogenization buffer: 10 mM HEPES–KOH, pH 7.2,


of Nanoparticle- 250 mM sucrose, 1 mM EDTA, and 1 mM Mg(OAc)2.
Loaded Cells 2. Cell scrapers.
3. Dounce homogenizer (7 mL).
4. 15 mL centrifuge tubes.
5. Refrigerated centrifuge.

2.4 Isolation 1. Homogenate from nanoparticle-treated cells (from


of Nanoparticle- Subheading 2.3).
Containing Endosomes 2. Magnetic beads with attached antibodies (from
Subheading 2.1).
3. Magnetic separator rack.
4. Sterile PBS.
5. Refrigerated tabletop centrifuge.
Immunoisolation of Nanoparticles Containing Endocytic Vesicles… 43

2.5 Quantification of 1. HPLC-grade methanol.


Drug Content by HPLC 2. Sonicator disruptor with probe tip.
3. Refrigerated tabletop centrifuge.
4. 0.5 mL microcentrifuge tubes.
5. HPLC autoinjector vials with low-volume inserts.

3 Methods

3.1 Conjugate 1. In a 1.7-mL microcentrifuge tube, combine 1 mL of 10%


Antibodies bovine serum albumin in PBS with 20 mL of magnetic bead
to Magnetic Beads slurry and 20 mg of antibody of interest (see Note 1).
2. Place tubes on a microcentrifuge tube tumbler rotator and
rotate at 15 rpm for 12 h at 4°C.
3. Place tubes in the magnetic separator rack for up to 1 h at 4°C.
Remove supernatant and resuspend antibody-bead conjugates
in sterile PBS. Repeat wash cycle two more times. Finally,
resuspend antibody-bead conjugates in 500 mL of sterile PBS
and use within 24 h.

3.2 Treat Cells 1. Wash cells three times for 10 min with 37°C serum-free
with Nanoparticles medium to remove residual serum protein.
2. Add nanoparticles in sterile serum-free DMEM (or other appro-
priate cell medium) to cells at desired concentration (Fig. 1a).
3. Incubate at 37°C to allow cells to take up nanoparticles (see
Note 4).
4. Once maximum nanoparticle uptake has been reached, wash
the cells three times with 37°C sterile PBS to remove any nano-
particles that have not been taken up. Keep cells in PBS and
immediately begin homogenization.

3.3 Homogenization 1. Remove PBS and add enough homogenization buffer to cover
of Nanoparticle- the bottom of each well or flask being used.
Loaded Cells 2. Detach cells from bottom of well or flask using cell lifter.
3. Add scraped cells to Dounce homogenizer and grind cells with
15 strokes (Fig. 1b) (see Note 5).
4. Add entire volume to a 15 mL centrifuge tube and remove
nuclei and unbroken cells by centrifuging at 400 × g for 10 min
at 4°C (Fig. 1c).
5. Remove supernatant, place in 1.7 mL microcentrifuge tubes,
and use for immune isolation of endocytic compartments.

3.4 Isolate 1. In a 1.7-mL microcentrifuge tube, combine 1 mL of the


Nanoparticle- cellular homogenate from Subheading 3.3, step 5, and the
Containing Endosomes entire volume (500 mL) of one antibody-bead combination
44 Ari Nowacek et al.

a b c
Endocytic vesicles
Nanoparticles with nanoparticles

Centrifuge
PBS 24 h
10 min
Wash
400 x g

Load cells Rupture cells Remove nuclei


with nanoparticles and cellular debris

d e f
0.30
HPLC
0.25

0.20
30 min Drug

AU
0.15
Wash 3X analysis
0.10

0.05

0.00
Paramagnetic 0.00 5.00 10.00 15.00
Protein A/G-Ab Minutes

Fig. 1 Schematic diagram of immunoisolation of nanoparticle-containing endosomes. Cells are first treated
with nanoparticles (a). After maximum nanoparticle endocytosis, cells are ruptured in homogenization buffer
using a Dounce tissue homogenizer (b). Cell homogenate is then slowly centrifuged in order to remove nuclei,
organelles, and cellular debris (c). Following enrichment, the endosome fraction is exposed to protein A/G
paramagnetic beads conjugated to antibodies and allowed to bind with the endosomes carrying the surface
markers of interest. (d) The endosome population bound to the beads is isolated by magnetic separation (e).
The isolated fraction undergoes several washes with sterile cold PBS prior to drug quantitation (f)

from Subheading 3.1, step 3 (Fig. 1d). Be sure to include one


tube with blank beads (i.e., fresh beads with no antibody con-
jugate) as a control.
2. Place tubes on a microcentrifuge tube tumbler rotator and
rotate at 15 rpm for 18–24 h at 4°C.
3. Place tubes on microcentrifuge tube magnetic separator and
allow for magnetic separation for 1 h at 4°C (Fig. 1e). The
solution should become clear as the beads form a layer in the
tube wall facing the magnet.
4. Carefully remove the solution without disturbing the beads.
Add 1 mL of cold sterile PBS and resuspend the beads (see
Note 6). Repeat this wash cycle two more times. Endosomes
are now ready for quantitative analysis (store at 4°C until ready
for analysis).
Immunoisolation of Nanoparticles Containing Endocytic Vesicles… 45

3.5 Quantification 1. Take samples from Subheading 3.4, step 4, and centrifuge at
of Drug Content 10,000 × g for 10 min at 4°C. Remove the supernatant and add
of Nanoparticle- 400 mL of 100% methanol (see Note 7).
Containing Endosomes 2. Sonicate solution with sonicator probe for 3 s at 20% amplitude.
by HPLC 3. Centrifuge solutions at 20,000 × g for 10 min at 4°C. Remove
supernatant and add to a clean 0.5 mL microcentrifuge tube.
4. Transfer 70 mL to an HPLC autosampler vial and inject three
20 mL aliquots onto the HPLC for drug quantitation (see
Notes 8 and 9).
5. Determine drug content by comparing peak area of drug in sam-
ple to peak areas of known concentrations of drug standards.

4 Notes

1. There are hundreds of potential endosomal markers to choose


from, which can be very overwhelming. When first performing
this experiment, it may be wise to select an antibody to isolate
each of the major endosomal compartments, for example, early
endosomes (early endosome antigen-1), recycling endosomes
(Rab-11), late endosomes (Rab-7), and lysosomes (lysosome-
associated membrane protein-1). In this way one can find out
which major endosomal compartment the nanoparticles are
trafficked to. Afterwards, a more thorough search of that
specific endosomal compartment can be performed.
2. This protocol has been used primarily with primary human
monocytes and human monocyte-derived macrophages.
However, any cells that will take up the nanoparticle being
tested could be used. Adjust the protocol appropriately to
accommodate for cell type being used. It is suggested to use at
least 10 × 106 cells in order to isolate enough nanoparticle-
containing endosomes for drug analysis.
3. This protocol has been used only for crystalline antiretroviral
nanoparticles coated in lipophilic surfactants (3). For this
method drug-containing nanoparticles are being used because
the final step of the protocol involves quantitation of drug levels
by HPLC. However, this protocol could easily be used to isolate
nanoparticle-containing endosomes for any type of nanoparti-
cle. Although, if the nanoparticle contains a nondrug therapeu-
tic compound (such as protein or DNA), a method other than
HPLC will be necessary to quantify the amount of therapeutic
material contained within each endosomal compartment.
4. Both the amount of nanoparticles to be added to serum-free
DMEM (or other appropriate cell medium) and the duration
of treatment must be determined ahead of time by previous
experiments. It is suggested to allow the cells enough time for
maximum nanoparticle uptake before harvesting for endosomal
46 Ari Nowacek et al.

isolation in order to ensure that sufficient material will be


available.
5. When using the Dounce homogenizer, press and pull the piston
hard enough to keep the solution flowing past the glass rod at a
steady rate. Only light force is necessary to effectively homoge-
nize the cells and excess force could break the homogenizer.
6. Be very careful not to disturb the beads when removing the
solution otherwise endosomes will be lost during the wash pro-
cess. Place the pipette tip on the wall of the tube opposite from
the side with the beads just beneath the surface and remove the
solution from the top down. If the beads are disturbed, place
the magnetic rack back in the refrigerator for about 10 min to
allow the beads to gather at the magnet again.
7. An internal standard of known quantity may be added to the
methanol (or other extraction solvent) prior to addition to the
samples. Acetonitrile extraction of drug may also be used. To
concentrate samples prior to HPLC, sample extracts can be
evaporated to dryness using a SpeedVac concentrator and
resuspended in methanol or mobile phase.
8. Low-volume inserts for autosampler vials are available for many
1–4 mL vial types. Vial types appropriate for your system will
be identified in the autoinjector user manual. Glass inserts are
preferred because of their inertness to solvents used for drug
extraction. Add enough volume to the autosampler vials to
provide for at least two injections of sample onto the HPLC
system, although three injections are preferred.
9. Depending on the drug of interest, HPLC with UV/Vis or
mass spectrometry detection may be used.

Acknowledgments

The work was supported by the National Institutes of Health


grants 1P01 DA028555, 2R01 NS034239, 2R37 NS36126, P01
NS31492, P20RR 15635, P01MH64570, and P01 NS43985 (to
H.E.G.) and a research grant from Baxter Healthcare. The authors
thank Ms. Robin Taylor for critical reading of the manuscript and
outstanding graphic and literary support.

References

1. Speiser PP (1991) Nanoparticles and lipo- 3. Kadiu I, Nowacek A, McMillan J, Gendelman


somes: a state of the art. Methods Find Exp HE (2011) Macrophage endocytic trafficking
Clin Pharmacol 13:337–342 of antiretroviral nanoparticles. Nanomedicine
2. Douglas SJ, Davis SS, Illum L (1987) (Lond) 6:975–994
Nanoparticles in drug delivery. Crit Rev Ther
Drug Carrier Syst 3:233–261
Chapter 6

Methods for Isolation and Identification of Nanoparticle-


Containing Subcellular Compartments
Ari Nowacek, Irena Kadiu, JoEllyn McMillan, and Howard E. Gendelman

Abstract
Nanoparticle-based drug delivery systems have considerable potential for improvement of drug stability,
bioavailability, and reduced dosing frequency. Important technological advantages of nanoparticles include
high carrier capacity across biological membranes and controlled drug release. Ultimately, success of nano-
delivery systems depends on toxicologic issues associated with the understanding of the fate of nanocarriers
and their polymeric constituents within the targeted cells. Here we describe a method for determining
subcellular distribution of nanoparticles by isolation and identification of organelles that come in direct
contact with these structures.

Key words Macrophage, Nanoparticle, Drug delivery, Endosome, Subcellular trafficking

1 Introduction

A wide variety of nanoparticles have been developed for the cellular


delivery of various therapeutic compounds and the potential clinical
benefits of these particles are great (1, 2). However, very little is
known about the subcellular distribution of nanoparticles in the
targeted cells. This information is necessary if we are to explain
how nanoparticles function on a subcellular level and to identify
any potential sources of cellular toxicity. In order to accomplish
this, a method must be used that can simultaneously allow for the
isolation and subsequent identification of proteins that interact
with a nanoparticle while it is in a cell. Here, we demonstrate that
the proteins that come into contact with a nanoparticle can be
individually labeled, isolated, and then identified by liquid chroma-
tography–mass spectrometry (LC-MS/MS). This relatively simple
method involves four basic steps: (1) labeling of the nanoparticles

Equal contributions were made by Ari Nowacek and Irena Kadiu.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_6, © Springer Science+Business Media New York 2013

47
48 Ari Nowacek et al.

with a visible dye, (2) treatment of cells with the nanoparticles,


(3) isolation of nanoparticle-laden subcellular compartments on a
sucrose gradient, and (4) identification of the proteomes of subcel-
lular compartments by LC/MS-MS. This method provides the
user with a broad view of the subcellular distribution of nanopar-
ticles within the same experiment. It is appropriate for use by
researchers who do not know the fate of their nanoformulations
within the targeted cells or their mechanisms of release. It can also
be used successfully to identify the subcellular trafficking pathways
of crystalline antiretroviral nanoparticles in human monocyte-derived
macrophages (3). Alternative approaches such as immunostaining
and confocal imaging of every cellular organelle and internalized
nanoparticles as well as measurement of their fluorescence overlap
are time consuming and costly.

2 Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water to attain a sensitivity of 18 MΩ cm at 25°C) and
analytical grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Diligently follow all
waste disposal regulations when disposing waste materials.

2.1 Components 1. Crystalline nanoparticles (see Note 1).


to Label Nanoparticles 2. Coomassie Brilliant Blue R250 (CBB) (see Note 2).
3. Sterile 1× phosphate buffered saline (PBS).
4. 0.5 or 1.7 mL microcentrifuge tubes.
5. Microcentrifuge tube tumbler rotator.
6. Table-top refrigerated centrifuge that can reach 20,000 × g.
7. Sonicator with probe.
8. Method to measure nanoparticle size and charge (see Note 3).

2.2 Cellular 1. Cells in vitro (see Note 4).


Treatment Components 2. Cell incubator.
3. Serum-free DMEM (or other appropriate serum-free cell cul-
ture medium).
4. Labeled nanoparticles.
5. Sterile PBS.

2.3 Homogenization 1. Homogenization buffer: 100 mM sucrose, 10 mM imidazole,


of Nanoparticle- pH 7.4.
Loaded Cells 2. Dounce homogenizer (7 mL).
3. 15 mL centrifuge tubes.
4. Refrigerated centrifuge.
Endocytic Sorting of Antiretroviral Nanoparticles 49

2.4 Enrichment 1. Homogenization buffer: 100 mM sucrose, 10 mM imidazole,


of Nanoparticle-Laden pH 7.4.
Compartments 2. Sucrose solutions: 10, 20, 35, and 60% weight/volume,
10 mM imidazole, pH 7.4 in sterile water.
3. Transparent ultracentrifuge tubes (12 mL; Beckman-Coulter).
4. Refrigerated ultracentrifuge that can reach 100,000 × g and
swinging bucket.
5. 3 mL syringe with an 18-gauge needle.
6. Sterile PBS.

2.5 Sample 1. Lysis buffer: 30 mM Tris-Cl, 7 M urea, 2 M thiourea, 4%


Processing for 1D (w/v) 3-((3-cholamidopropyl)dimethylammonio)-1-propane-
Electrophoresis and sulfonate, 20 mM dithiothreitol, 1× protease inhibitor cock-
Mass Spectrometry tail, pH 8.5 (Sigma-Aldrich) (see Note 11).
Analysis 2. ReadyPrep™ 2D Cleanup Kit (Bio-Rad Laboratories, Inc.).
3. 2D Quant kit (GE Healthcare).
4. Bis–Tris 4–12% and 7% Tris-Glycine gels (Invitrogen).
5. Fixation buffer: 10% methanol and 7% acetic acid in distilled–
deionized water.
6. Colloidal coomassie (GE healthcare).
7. Destaining buffer: 20% methanol and 10% acetic acid in dis-
tilled–deionized water.
8. Single edge razor blades.
9. Sterile glass autosampler vials (Thermo-Fisher Scientific).
10. Vacuum concentrator centrifuge (SpeedVac) with cooling trap.
11. In-Gel Tryptic Digestion Kit (Thermo-Fisher Scientific).
12. μC18 ZipTip® pipette tips (Millipore; see Note 12).
13. Resuspension buffer: 0.5% trifluoroacetic acid (TFA; Sigma-
Aldrich).
14. Wetting solution: 100% acetonitrile (ACN; Thermo-Fisher
Scientific).
15. Equilibration/wash solution: 0.1% TFA.
16. Elution solution: 50% ACN, 0.1% TFA.

3 Methods
3.1 Label 1. Combine nanoparticles (see Note 1) with 0.01% (weight/
Nanoparticles with volume) of CBB (see Note 2) in sterile PBS. Mix on a micro-
Coomassie Brilliant centrifuge tube tumbler rotator at 15 rpm for 12 h at room
Blue R250 temperature (see Note 5).
50 Ari Nowacek et al.

2. Centrifuge the mixture at 20,000 × g for 5 min to pellet the


particles and then remove the supernatant with a pipette. Add
sterile PBS to the tube and briefly resuspend the particles by
sonicating them at 20% amplitude for 1–3 s with a sonicator
probe (see Note 6). This will remove the excess dye. Repeat
the PBS wash and sonication cycle five times or until no more
dye is visible in the supernatant (Fig. 1a).
3. Store labeled particles in sterile PBS at 4°C until ready for use.

3.2 Treat Cells with 1. Wash cells three times for 10 min with 37°C sterile PBS to
CBB-Labeled remove residual serum protein.
Nanoparticles 2. Add nanoparticles in sterile PBS to cells at desired
concentration.
3. Incubate at 37°C to allow cells to take up nanoparticles. The
cells will visibly become blue as they take up the labeled nano-
particles (Fig. 1b) (see Note 7).
4. Once the cells have taken up the nanoparticles, wash the cells
three times with 37°C sterile PBS to remove any residual non-
internalized nanoparticles. Keep cells in PBS and immediately
begin homogenization.

3.3 Homogenization 1. Remove PBS and add 6 mL of homogenization buffer to each


of Nanoparticle- flask if working in a T75 culture flask. Adjust the buffer volume
Loaded Cells to the minimum necessary for covering the dish surface if
working with other culture systems.
2. Detach cells from bottom of flask using a cell lifter.
3. Add entire volume to Dounce homogenizer and grind cells
with 15 strokes (see Note 8).
4. Add entire volume to a 15 mL centrifuge tube and remove
cellular debris and nuclei by centrifuging at 500 × g for 10 min
at 4°C.
5. Remove supernatant and centrifuge for 1 h at 100,000 × g at
4°C. Resuspend the pellet in 3 mL of 10% sucrose, 10 mM
imidazole, pH 7.4, solution and store on ice.

3.4 Enrichment 1. Take sucrose solutions and set up sucrose gradient in 12 mL


of Nanoparticle-Laden thin-walled ultracentrifuge tube. Take 3 mL of 60% sucrose
Compartments solution and place it in the bottom of the tube followed by
layering 3 mL each of 35 and 20% sucrose solutions one on top
of the next in the order given (see Note 9).
2. Carefully add the supernatant from the last step of
Subheading 3.3 to the top of the sucrose gradient and centri-
fuge at 100,000 × g at 4°C for 1 h.
3. Using a 3 mL syringe with an 18-gauge needle, carefully per-
forate the tube at the end of the blue sucrose band and aspirate
Endocytic Sorting of Antiretroviral Nanoparticles 51

Brilliant Blue R-250 MDM loaded with Isolation of subcellular


labeled nanoparticles labeled RTV-NP RTV-NP+ compartments

Protein identification
In-gel trypsin digest
with LC/MS

Fig. 1 Representative images of processes for nanoparticle staining, cell treatment, endosome enrichment and
protein processing and identification. Crystalline nanoparticles that have been labeled with CBB with all
unbound dye washed away (a). Human monocyte-derived macrophages after being treated with CBB-labeled
nanoparticles. Note the cells have developed a purple color after ingesting the labeled nanoparticles (b).
Enriched endosomes containing proteins stained by CBB-labeled nanoparticles are seen as bands on a sucrose
gradient after being centrifuged at 100,000 × g for 1 h at 4°C (c). One lane of a gel showing labeled proteins
separated by molecular weight (d). Chromatogram of protein fractionation followed by identification using
LC/MS-MS (e)

the solution until the color disappears (Fig. 1c). Transfer each
band to a separate ultracentrifuge tube (see Note 10).
4. Pellet the nanoparticle-enriched subcellular compartments by
centrifuging at 100,000 × g at 4°C for 1 h. Remove superna-
tant and wash pellet with PBS and subsequent centrifugation
at 100,000 × g at 4°C for 1 h to remove residual sucrose.
52 Ari Nowacek et al.

3.5 Sample 1. Solubilize enriched subcellular compartments in lysis buffer by


Processing for 1D resuspending the pellet and pipetting five times (see Note 11).
Electrophoresis and 2. Precipitate proteins using a ReadyPrep™ 2D Cleanup Kit (GE
Mass Spectrometry Healthcare) per manufacturer’s instructions.
Analysis
3. Quantify protein using a 2D Quant kit (GE Healthcare) per
the manufacturer’s instructions.
4. Run samples on Bis–Tris 4–12% and 7% Tris-Glycine gels
(Invitrogen) to separate low and high molecular weight
proteins.
5. Incubate gels in fixation buffer for 1 h at room temperature
followed by staining with colloidal coomassie for 24 h at room
temperature (Fig. 1d).
6. Destain gels by rinsing with destaining solution until solution
is light blue or clear.
7. Manually excise the bands using a razor blade and place each
band in a separate glass autosampler vial. Excise each gel band
in several pieces to increase surface contact (see Note 12).
8. Perform in-gel tryptic digestion using the In-Gel Tryptic
Digestion Kit per the manufacturer’s instructions.

3.6 Peptide 1. Resuspend the extracted peptides from the in-gel tryptic digestion
Purification and procedure in 30 μL of resuspension buffer and vortex vigor-
Concentration for ously for 5 min.
Mass Spectrometry 2. Wet Zip-Tip pipette tip by aspirating and releasing wetting
Analysis and Protein solution three times.
Identification 3. Equilibrate tip by pipetting and discarding equilibration/wash
solution three times.
4. Bind peptides to the zip tip by pipetting 15 times inside the
sample tube then discard sample.
5. Wash tip three times (and discard fluid) in equilibration/wash
solution.
6. Elute the peptides into a vial insert by pipetting 10 μL at a time
of the elution solution (100 μL). Keep vials on ice until all
samples are zip tipped.
7. Freeze samples briefly at −80°C then SpeedVac to dryness.
8. Resuspend peptides with the appropriate volume of 0.1%
formic acid and analyze by LC-MS/MS (see Note 13).

4 Notes
1. This protocol has been used only for crystalline antiretroviral
nanoparticles coated in lipophilic surfactants such as polox-
amer-188 (P188), 1,2-distearoyl-phosphatidyl ethanolamine-
methyl-polyethyleneglycol-2000 (mPEG2000-DSPE), and
Endocytic Sorting of Antiretroviral Nanoparticles 53

1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (3).


We suggest using rigid nanoparticles that have a lipophilic
coating because the dye will easily label the particle without
disrupting its structure. If other types of nanocarriers are used,
it is highly encouraged that the nanoparticles are re-character-
ized after labeling to ensure that they have not been altered.
2. This protocol has been used only with Brilliant Blue R250.
However, the goal is to coat the particles with a visible dye that
will label the proteins that the nanoparticles come into contact
with. Thus, other dyes that label proteins and are readily seen
in the visible light spectrum, such as bromophenol blue, could
also be used. It is unknown how using Brilliant Blue R250
(or other such dyes) to label different types of nanoparticles
will affect their physical properties. Thus, it is highly encour-
aged that the nanoparticles be re-characterized after labeling
to ensure that they have not been altered.
3. There are a number of methods for measuring nanoparticle
size and charge. Companies such as Horiba, Malvern, TSI, and
many others offer equipment that will simultaneously measure
both the size and charge of nanoparticles. No one method or
machine is preferred for this protocol.
4. This protocol has been used primarily with primary human
monocytes and human monocyte-derived macrophages.
However, any cells that will take up the nanoparticle being
tested could be used. Adjust the protocol appropriately to
accommodate for the cell type being used. It is suggested to
use at least 100 × 106 cells in order to purify enough protein for
proteomic analysis.
5. Typically the labeling procedure can be carried out in a 0.5 mL
microcentrifuge tube. When using this small of a volume, using
just a few grains (about 1–3 grains) of Brilliant Blue R250 dye
will be enough to sufficiently label the particles without alter-
ing their physical characteristics. If too much dye is used, there
is a risk of nanoparticle aggregation. It does not take much dye
to label the particles so use less dye rather than more.
6. The purpose of sonication is to resuspend the particles so that
they can be efficiently washed. However, it is possible to over-
heat or dissolve the particles with too much sonication.
Therefore, a brief sonication (10 s) to resuspend the particles
is all that is necessary.
7. The investigators will need to adjust the cell-nanoparticle expo-
sure time based on endocytic activity of the targeted cells and
the size and coating of their nanoformulations. It is suggested
to allow for maximal nanoparticle uptake since this will allow for
a better identification of nanoparticle-laden compartments.
8. When using the Dounce homogenizer, press and pull the
piston hard enough to keep the solution flowing past at a
54 Ari Nowacek et al.

steady rate. Excess force is not necessary and could break the
homogenizer.
9. When forming the sucrose gradient, carefully add each subse-
quent layer of sucrose by gently pouring it down the side of the
tube. This will help to prevent mixing of the layers and will
increase the intensity of the protein bands that form during
centrifugation.
10. Do not disturb the contents of the ultracentrifuge tube when
removing it from the centrifuge. The fractions need to be
collected within 15 min after centrifugation; otherwise the
bands will diffuse. When collecting the fractions (2–4 blue bands
representing nanoparticle-laden compartments), use separate
needles and syringes for each band. Start from the top band.
Insert the needle-syringe at the bottom of band line facing up
and aspirate until very little blue is left. Do not remove the
syringe after band aspiration. Insert a new needle-syringe at
the level of the next lower band and repeat.
11. Use nitrile gloves when handling the gel and the in-gel tryptic
digestion solutions. Latex can interfere with downstream mass
spectrometry analysis. Use a clean surface and equipment and
avoid contact of gloves with skin or hair during the processing
of gel bands. Dust and shedding epithelial cells can be a major
contaminant of the samples, and it compromises the mass spec-
trometry analysis.
12. HPLC-grade reagents and HPLC-grade water should be used
to make solutions used for in-gel tryptic digestion and peptide
extraction (zip-tipping).
13. The volume necessary for resuspension of samples depends on
the method used for mass spectrometry analysis and the instru-
ment configuration (nanospray or electrospray). Typically,
peptides from in-gel tryptic digestion are resuspended in
4–8 μL of 0.1% formic acid and are run in a nanospray
configuration.

Acknowledgments

The work was supported by the National Institutes of Health


grants 1P01 DA028555, 2R01 NS034239, 2R37 NS36126, P01
NS31492, P20RR 15635, P01MH64570, and P01 NS43985
(to H.E.G.) and a research grant from Baxter Healthcare. The
authors thank Ms. Robin Taylor for critical reading of the manu-
script and outstanding graphic and literary support.
Endocytic Sorting of Antiretroviral Nanoparticles 55

References
1. Nowacek A, Gendelman HE (2009) NanoART, 3. Kadiu I, Nowacek A, McMillan J, Gendelman HE
neuroAIDS and CNS drug delivery. (2011) Macrophage endocytic trafficking of anti-
Nanomedicine (Lond) 4:557–574 retroviral nanoparticles. Nanomedicine (Lond)
2. Nowacek A, Kosloski LM, Gendelman HE 6:975–994
(2009) Neurodegenerative disorders and nano-
formulated drug development. Nanomedicine
(Lond) 4:541–555
Chapter 7

Permeabilization of Cell Membrane for Delivery


of Nano-objects to Cellular Sub-domains
Valeriy Lukyanenko

Abstract
Delivery of nano-objects to specific cellular sub-domains is a challenging but intriguing task. There are two
major barriers on the way of a nano-object to its intracellular target: (1) the cell membrane and (2) the
intracellular barriers. The former is a common issue for all nanomedicine and a matter of very intense
research. The latter is the primary problem for targeted delivery of nano-objects to specific cellular sub-
domains and can be studied more easily using permeabilized cells. Membrane permeabilization for nano-
medical research requires (1) perforation of the outer membrane, (2) development of a solution that
will keep cellular sub-domains in the functional state, and (3) modification of the perimembrane cytoskel-
eton. We developed a very successful model of saponin membrane permeabilization of cardiomyocytes.
This allowed us to deliver particles up to 20 nm in size to perinuclear and perimitochondrial space. Here
we describe the method.

Key words Saponin permeabilization, Perimembrane cytoskeleton, Gold nanoparticle, Delivery of


nano-objects, Intracellular sub-domains, Electron microscopy

1 Introduction

Successful delivery of genes and drugs to intracellular sub-domains


depends on solving two major problems: transport of nano-objects
through both cellular and intracellular membranes (1, 2). While
the former is a common problem for nanomedicine (and a matter
of intensive research with multiple very promising results), the
intracellular pathways for delivery of nano-objects to certain cellular
organelles remain to be elucidated (1). This research could be
significantly facilitated with careful (without damage of cellular
sub-domains) permeabilization of cell membrane.
Excluding the development of a specific protocol, membrane
permeabilization for nanomedical research could be divided into
three separate tasks:
1. Cell membrane permeabilization without permeabilization or
damage of intracellular membranes.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_7, © Springer Science+Business Media New York 2013

57
58 Valeriy Lukyanenko

2. Development of “intracellular” solution that will keep cellular


organelles functional and maintain the intracellular distances
unchanged (for instance, will prevent mitochondrial swelling).
3. Modification of the cytoskeleton along the cell membrane to
allow particles up to 20 nm to enter the cell.
In our experiments, we used rat ventricular myocytes obtained
by enzymatic dissociation (3). Spatio-temporal characteristics and
the frequency of elementary Ca2+ release events (Ca2+ sparks) are
very sensitive to any mistake in permeabilization. For example, high
[Ca2+] in the intracellular solution results in an almost immediate
increase in amplitude and frequency of Ca2+ sparks (4), while per-
meabilization of the endoplasmic reticulum (SR in muscle cells)
membrane will abolish the sparks. In our experiments, we monitored
the Ca2+ sparks, the amount of Ca2+ in the SR and contraction of the
myocytes with confocal microscopy (4, 5). Intracellular distances in
intact and permeabilized cardiomyocytes were measured with cali-
brated gold nanoparticles using electron microscopy (6, 7).
There are many methods of cell membrane permeabilization,
from mechanical skinning to making membrane holes only for
special ions with corresponding ionophores. Gentle saponin per-
meabilization allows the removal of the outer cell membrane with-
out damaging intracellular membranes.
Figure 1 shows saponin permeabilization in cardiac myocytes.
The upper panel (a) presents the same myocyte before and after
permeabilization. Note that the myocyte after permeabilization has
the same shape and size. The middle panel (b) confirms the permea-
bilization. The myocyte was preloaded with membrane-permeable
form of Ca2+-sensing fluorescent dye. Therefore, before permeabi-
lization (b a), Ca2+ sparks (local event) and waves (global Ca2+
release events) could be seen very well. Within 1 min after permea-
bilization (b b), the fluorescent dye left the cell and the sparks
disappeared. The addition to the bathing solution of the same but
membrane-impermeable Ca2+-sensing fluorescent dye (b c, d) again
allows seeing the Ca2+ release events, which have the same velocity,
frequency, and spatio-temporal properties (c). This demonstrates
that intracellular membranes were not damaged with our method
of saponin permeabilization.
This method of membrane permeabilization is suitable for
nano-objects as well. Figure 2 shows saponin permeabilization of
the cardiac cell transfected with green fluorescent protein (GFP),
which is a 4.2 nm long cylinder with a cylindrical diameter of 2.4 nm
(8). The cell was exposed to 0.01% saponin for 30 s. Multiple GFP-
filled membrane blebs indicate gentle permeabilization. Therefore,
GFP takes much longer to leave the cell after permeabilization (7),
and GFP stays within cellular sub-domains (including nuclei; N)
even after 5 min. This reminds us that membrane is not the only
barrier for free diffusion of nano-objects inside the cell.
Permeabilization of Cell Membrane 59

Fig. 1 Saponin permeabilization of cardiomyocytes has no effect on Ca2+ release from sarcoplasmic reticulum.
(a) Images of a cardiac myocyte obtained in transmitted light before and after permeabilization with saponin.
(b) Line scan images of fluorescence in a portion of the same cell preloaded with fluo-3 AM measured before
permeabilization (a), after permeabilization in an internal solution with no dye (b), and after addition to the
internal solution 30 mM fluo-3 potassium salt in the presence of 0.1 (c) or 0.5 mM EGTA (d) (pCa 7). Calibration
bars: horizontal 10 mm, vertical 0.4 s, the pseudo scale bar represents changes in units of absolute fluorescence.
(c) Surface plots of averaged Ca2+ sparks measured before permeabilization (a) and after permeabilization
(reproduced from ref. 5 with permission from Wiley-Blackwell)

Fig. 2 Light permeabilization. Before the permeabilization, the cardiomyocytes were transfected with GFP.
N nucleus, PNM perinuclear mitochondria (reproduced from ref. 7 with permission from Cell press)
60 Valeriy Lukyanenko

Our experiments showed that the cytoskeleton of permeabilized


myocytes does not allow nano-objects ³11 nm in diameter to enter
the cell. We should note that this problem pertains only to permea-
bilized cells because of a lack of transmembrane transporting
mechanisms. In the case of saponin-permeabilized cells, the mesh
of actin filaments located along the cell membrane creates an addi-
tional barrier for nano-objects. However, short pretreatment of
intact cells with cytochalasin D significantly reduces the mesh
integrity and allows particles up to 20 nm to diffuse inside of the
myocytes (6). Electron micrographs in Fig. 3 show the effect of
40 mM cytochalasin D on the distribution of nanoparticles in
permeabilized ventricular myocytes. Note that the silver grains are
only markers for the location of calibrated gold nanoparticles
(the deeper in the slice the particle was located, the smaller the
silver grain produced with the silver enhancement procedure).
Here we describe the method of saponin permeabilization of
the cell membrane. The method is shown to be useful for the deliv-
ery of nano-objects to perinuclear and perimitochondrial space.

2 Materials

1. Tyrode solution: 140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2,


1 mM CaCl2, 10 mM Hepes, 0.25 mM NaH2PO4, 5.6 mM
glucose, pH 7.3 (6).
2. The permeabilization solution: 100 mM K+ aspartate (see Note
1), 20 mM KCl, 3 mM MgATP, 0.81 mM MgCl2
([Mg2+]free = ~1 mM), 0.5 mM EGTA, 0.114 mM CaCl2
([Ca2+]free = ~100 nM), 20 mM Hepes, 3 mM glutamic acid,
and 3 mM malic acid, pH 7.2 (see Note 2) (6).
3. 1% saponin in permeabilization solution.
4. The solution for permeabilized cells: 100 mM K+ aspartate (see
Note 1), 20 mM KCl, 3 mM Mg ATP, 0.81 mM MgCl2
([Mg2+]free = ~1 mM), 0.1 mM EGTA, 0.03 mM CaCl2

Fig. 3 Remodeling of cytoskeleton with cytochalasin D allows particles up to


20 nm to diffuse inside of cells. Representative micrographs show the distribu-
tion of the nanoparticles before (a) and after (b) partial ablation of the cytoskel-
eton (20 min pretreatment with 40 mM cytochalasin D). (c) Graphs representing
the density of nanoparticles in intact ventricular cells before (gray) and after (dark
gray, only for particles ³11 nm) 20 min of pretreatment with 40 mM cytochalasin
D. Asterisks indicate data that are statistically different from the corresponding
control. M mitochondrion, Z Z line. Arrows on (b) indicate T-tubules and black
ovals show nanoparticles located deeper inside the ultrathin section, which
therefore have smaller diameters after silver enhancement (reproduced from ref. 6
with permission from Cell press)
Permeabilization of Cell Membrane 61

Silver grains

Z Z M

M 1 mm

b
Z

1 µm

c 6 Control
Cytochalasin D
5
Density, particles/µm3

4
*
3

0
3 6 11 16 21
Size of gold particles, nm
62 Valeriy Lukyanenko

([Ca2+]free = ~60 nM), 20 mM Hepes, 3 mM glutamic acid,


3 mM malic acid, 10 mM phosphocreatine, 5 U/ml creatine
phosphokinase, and 1% polyvinylpyrrolidone (PVP10; MW
10,000), pH 7.2 (see Note 2) (7).
5. Cells have to be attached to the bottom. To do that, we coated
cover slips with laminin as recommended by the manufac-
turer (Molecular Probes; Invitrogen) and allowed cells 30 min
to attach.

3 Methods

Carry out all procedures at room temperature.


In our experiments, we used Tyrode solution and primary cul-
ture of rat ventricular myocytes (single freshly isolated cells).
However, any monolayer cell culture (any confluency) and corre-
sponding media could be used.
1. Pretreat cells 20 min with 40 mM cytochalasin D in 1 ml of
Tyrode solution.
2. Replace the Tyrode solution with 1 ml of permeabilization
solution for 1 min (see Notes 3 and 4).
3. Replace the permeabilization solution with 1 ml of the same
(permeabilization) solution containing 0.01% saponin for
30–60 s.
4. Replace the permeabilization solution with 1 ml of the solu-
tion for permeabilized cells containing nanoparticles.
To visualize cells, we used C-Apochromat 63×/1.2 W corr
objective. Under the mentioned conditions, intracellular organ-
elles (such as mitochondria and sarcoplasmic reticulum) of cardiac
myocytes remain functional at room temperature for at least 2 h
(see Note 5 ).

4 Notes

1. For both permeabilization solution and the solution for per-


meabilized cells, we used DL-aspartic acid potassium salt.
2. During the preparation of the solution for permeabilized cells,
pay attention to the pH. The pH is very important for Ca2+
buffering power of EGTA.
3. All steps of the saponin permeabilization have to be performed
under visual control. We used an inverted microscope equipped
with at least ×40 objective.
4. The moment of permeabilization is seen (within a minute) as a
sharp reduction in cell shining (cells become gray; Fig. 1a).
Permeabilization of Cell Membrane 63

After that, the permeabilization solution should be immedi-


ately replaced by the solution for permeabilized cells.
5. We found that entry of 3-nm particles into VDAC pore (located
in the outer mitochondrial membrane) is significantly restricted
in permeabilized cardiomyocytes in comparison to isolated
mitochondria (7).

References
1. Lukyanenko V (2007) Delivery of nano-objects 5. Lukyanenko V, Györke S (1999) Ca2+ sparks
to functional sub-domains of healthy and failing and Ca2+ waves in saponin-permeabilized car-
ventricular myocytes. Nanomedicine 2:831–846 diac myocytes. J Physiol 521:575–585
2. Lukyanenko V (2010) Therapeutic nano-object 6. Parfenov AS, Salnikov V, Lederer WJ,
delivery to sub-domains of cardiac myocytes. Lukyanenko V (2006) Aqueous diffusion path-
In: Weissig V, D’Souza GGM (eds) Organelle- ways as a part of the ventricular cell ultrastruc-
specific pharmaceutical nanotechnology. Artech ture. Biophys J 90:1107–1119
House, Norwood, MA, pp 433–448 7. Salnikov VV, Lukyanenko YO, Frederick CA,
3. Györke S, Lukyanenko V, Györke I (1997) Lederer WJ, Lukyanenko V (2007) Probing
Dual effects of tetracaine on spontaneous the outer mitochondrial membrane in cardiac
calcium release in rat ventricular myocytes. mitochondria with nanoparticles. Biophys J
J Physiol 500:297–309 92:1058–1071
4. Lukyanenko V, Viatchenko-Karpinski S, 8. Yang F, Moss LG, Phillips GN Jr (1996)
Smirnov A, Wiesner TF, Györke S (2001) The molecular structure of green fluorescent
Dynamic regulation of the SR Ca2+ content by protein. Nat Biotechnol 14:1246–1251
lumenal Ca2+-sensitive leak through RyRs in rat
ventricular myocytes. Biophys J 81:785–798
Chapter 8

A Method to Encapsulate Molecular Cargo


Within DNA Icosahedra
Dhiraj Bhatia, Saikat Chakraborty, Shabana Mehtab,
and Yamuna Krishnan

Abstract
DNA self-assembly has yielded various polyhedra based on platonic solids. DNA polyhedra can act as
nanocapsules by entrapping various molecular entities from solution and could possibly find use in targeted
delivery within living systems. A key requirement for encapsulation is that the polyhedron should have
maximal encapsulation volume while maintaining minimum pore size. It is well known that platonic solids
possess maximal encapsulation volumes. We therefore constructed an icosahedron from DNA using a
modular self-assembly strategy. We describe a method to determine the functionality of DNA polyhedra as
nanocapsules by encapsulating different cargo such as gold nanoparticles and functional biomolecules like
FITC dextran from solution within DNA icosahedra.

Key words DNA icosahedron, Polyhedra, Nanocapsules, Encapsulation, Gold nanoparticles,


FITC dextran

1 Introduction

Encapsulation of a range of molecular cargo inside a given biomo-


lecular host is highly challenging. This is due to the large size and
sensitive nature of the cargo, and retention of functionality of cargo
post encapsulation (1). DNA has been shown to be capable of
assembling into various polyhedral structures using one pot assembly
(2), modular self-assembly (3), and origami based approaches (4).
Encapsulation is an attractive property of DNA polyhedra and we
describe methods to characterize DNA encapsulated cargo using
single molecule methods as well as bulk biophysical methods. Here
we describe a detailed method to study encapsulation characteris-
tics of DNA icosahedra. These methods can be generalized for
encapsulation of various entities like biomacromolecules and func-
tional nanoparticles, etc.
We outline first a strategy to assemble the molecular host, the
DNA icosahedron in high yields using a modular approach (5).

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_8, © Springer Science+Business Media New York 2013

65
66 Dhiraj Bhatia et al.

This assembly involves a step-wise association of different modules


(5-way junctions) having programmable overhangs into two half
or hemi-icosahedra. These two hemi-icosahedra then self-assemble
into icosahedral DNA nanocapsules that enclose a hollow cavity.
We describe the use of complementary hemi-icosahedra to encap-
sulate two types of (1) inorganic cargo such as gold nanoparticles
(GNPs) and (2) biomacromolecules such as fluorescently labeled
dextran FD10 inside DNA icosahedra (5, 6).
This method of cargo encapsulation inside DNA polyhedra is
advantageous because (a) It is not limited to molecules that need
to undergo molecular recognition with the host scaffold. This
affords the following advantages: (1) Larger varieties of molecules
may be encapsulated provided they have a size compatibility with
the polyhedron. (2) The size of the polyhedron can also be easily
altered to encapsulate differently sized molecules. (3) Guest mol-
ecules do not need to undergo a chemical reaction for encapsula-
tion. (b) The DNA scaffold is amenable to site specific chemical
modifications using multiple orthogonal chemistries. This affords
the following advantages: (1) The ability to uniformly functional-
ize DNA polyhedra in a precisely tunable manner, with multiple
tags in bulk. (2) Greater homogeneity of functionalized DNA
polyhedra carrying cargo internally, and carrying surface displayed
tags for targeting. We describe in detail how one may characterize
such loaded DNA polyhedra both at the single molecule level and
using bulk biophysics.

2 Materials

2.1 Oligonucleotide 1. Prepare 1 mM stocks in Milli-Q (MQ) water (Millipore, USA)


Sample Preparation of all oligonucleotides shown in Table 1. Oligonucleotides are
obtained from Sigma, HPLC purified and lyophilized.
2. Ethanol, absolute from Merck.
3. 3.0 M Potassium chloride solution: 2.23 g KCl dissolved in
10 mL MQ water.
4. Phosphate buffers: 100 mM (10×) NaH2PO4: 1.2 g NaH2PO4
dissolved in 100 mL MQ water. 100 mM (10×) Na2HPO4:
1.42 g Na2HPO4 dissolved in 100 mL MQ water. The phos-
phate buffer (10 mM, pH 6) is prepared by mixing appropriate
amounts of Na2HPO4 and NaH2PO4.
5. Magnesium chloride (10 mM): 0.203 g MgCl2 dissolved in
100 mL MQ water. Sodium chloride (1 M): 5.84 g NaCl dis-
solved in 100 mL MQ water.
6. Heat Block.
7. Adenosine triphosphate (ATP) stock, 100 mM.
A Method to Encapsulate Molecular Cargo Within DNA Icosahedra 67

Table 1
Oligonucleotide sequences for icosahedron

Name Sequence
V1 5¢-GCCTGGTGCCACCGGTGACGTTCCGC-3¢
V2 5¢-GCCTGGTGCCCCGCGTCCTCACCGGT-3¢
V3 5¢-GCCTGGTGCCGCCACGCTTTGGACGCGG-3¢
V4 5¢-GCCTGGTGCCGCGAGTGCAAAGCGTGGC-3¢
V5 5¢-GCCTGGTGCCGCGGAACGAAGCACTCGC-3¢
U1 5¢-CATCAGTCGCACCGGTGACGTTCCGC-3¢
U2 5¢-TTATAGGACTCCGCGTCCTCACCGGT-3¢
U3 5¢-TTATAGGACTGCCACGCTTTGGACGCGG-3¢
U4 5¢-GCGACTGATGGCGAGTGCAAAGCGTGGC-3¢
U5 5¢-GGCACCAGGCGCGGAACGAAGCACTCGC-3¢
L1 5¢-CATCAGTCGCACCGGTGACGTTCCGC-3¢
L2 5¢-AGTCCTATAACCGCGTCCTCACCGGT-3¢
L3 5¢-AGTCCTATAAGCCACGCTTTGGACGCGG-3¢
L4 5¢-GCGACTGATGGCGAGTGCAAAGCGTGGC-3¢
L5 5¢-GGCACCAGGCGCGGAACGAAGCACTCGC-3¢

8. T4 Polynucleotide kinase (10 U/mL) and associated buffer—


500 mM Tris–HCl, pH 7.6 at 25°C, 100 mM MgCl2, 50 mM
DTT, 1 mM EDTA, 1 mM Spermidine.

2.2 Gel 1. 50× Tris-Acetate-EDTA (TAE) buffer: 24.2 g Tris buffer,


Electrophoresis 5.71 mL glacial acetic acid and 10 mL of 0.5 M EDTA (pH 8.0)
dissolved in 100 mL of MQ water.
2. 10× Tris-Boric Acid–EDTA (TBE) buffer: 5.4 g Tris base,
2.75 g Boric acid and 20 mL of 0.5 M EDTA (pH 8.0)
dissolved in 100 mL of MQ water.
3. 40% polyacrylamide stock: 29.9 g Acrylamide, 0.8 g N,N¢-
Bis-methylene acrylamide dissolved in 100 mL of MQ water.
4. 0.8% Agarose gel: 0.8 g agarose powder (Bangalore Genei,
India) dissolved in 98 mL MQ water and 2 mL 50× TAE
buffer.
5. 10 and 15% Polyacrylamide gel: Different percentages of poly-
acrylamide gels can be made from 40% stock in 0.1% Ammonium
persulfate and 20 mL Tetramethylethylenediamine (TEMED).
68 Dhiraj Bhatia et al.

6. Ammonium persulfate, TEMED, and Ethidium bromide


(EtBr) (Sigma, USA).
7. All gels are run in a cold room at 4°C and visualized by EtBr
staining under UV illuminator.

2.3 Ligation: 1. 5.5 g Cyanogen bromide (BrCN) and 3.2 g Imidazole are
Preparation of dissolved in 25 mL and 50 mL of dry benzene respectively.
N-Cyano Imidazole 2. A solution of 5.5 g BrCN is added drop wise with stirring to a
solution of 3.2 g imidazole in 50 mL Benzene.
3. The reaction mixture is warmed to 50°C during the addition
and for 5 min after the addition is done.
4. The reaction mixture is cooled at 4°C for 8 h. This may be
preferably left overnight.
5. The resultant yellow solid is filtered through Whatman filter
paper and the supernatant solution is collected.
6. The filtrate is concentrated to dryness under reduced pressure.
7. A white crystalline solid remains which is collected and purified
by sublimation. The sublimate is pure N-Cyano imidazole that
is aliquoted in eppendorf tubes and stored at −20°C (7).

2.4 Gold 1. Auric chloride, Tri-sodium citrate, Tannic Acid, Potassium


Nanoparticles bicarbonate.
2. 2 mg Auric chloride is taken in 16 mL MQ water in a round
bottom flask. Heat up to 60°C in an oil bath with constant
stirring. This is solution A.
3. In three different eppendorfs the following solutions are taken:
20 mg Trisodium citrate in 2 mL MQ water, 20 mg tannic acid
in 2 mL MQ water, and 6.9 mg Potassium carbonate in 2 mL
MQ water.
4. Now 0.8 mL citrate, 1 mL tannic acid and 1 mL Potassium
carbonate and 1.2 mL water are mixed together to form 4 mL
of solution B.
5. Solution B is also heated to 60°C.
6. Solution B is added to solution A with constant stirring. The
color changes from yellow to wine red.
7. The temperature is increased to 100°C and the solution is
refluxed for 30 min.
8. This protocol gives homogeneous gold nanoparticles of 5 nm
size. The size of GNPs can be changed by changing the
amounts of citrate and tannic acids keeping the amount of
Auric chloride constant.
9. The sizes of gold nanoparticles may be checked by transmission
electron microscopy (TEM) or dynamic light scattering (DLS).
10. Using this protocol, GNPs from 2 to 15 nm can be made (8).
A Method to Encapsulate Molecular Cargo Within DNA Icosahedra 69

2.5 Transmission 1. 400 mesh carbon coated and glow discharged grids
Electron Microscopy (Ted Pella, USA).
2. 1% Uranyl acetate (Ted Pella, USA).
3. Transmission electron microscope—JEOL 100 CX II operating
at acceleration voltage 80 kV; Tecnai 12 Biotwin, FEI,
Netherlands operating at acceleration voltage 120 kV.
4. Images are acquired using side-mount 1,024 × 768 pixel reso-
lution CCD camera.

2.6 Size Exclusion 1. Biosep-SEC-S3000 (Phenomenex) of dimensions


Chromatography 300 × 4.6 mm, 5 mm bead size and 29 nm pore size.
2. Shimadzu HPLC system equipped with a temperature control-
ler, a photodiode array detector, fraction collector, and auto-
injector (Shimadzu, Japan).
3. Acetonitrile (HPLC grade), Degassed MQ water.

2.7 Fluorometer 1. Fluorolog 3 L instrument (Horiba Jobin Yvon, Japan) having


and Anisotropy Setup the polarizing angle fixed (90°).
2. g factor calibrated using Fluorescein (pH 7, 50 nM) as stan-
dard (r = 0.018).
3. The sample is excited at 488 nm and emission is collected at
515 nm with the slit widths adjusted accordingly.

2.8 Dynamic Light 1. DynaPro-99 unit (Protein Solutions, USA) operating at 25°C.
Scattering 2. Buffers and samples are first filtered through 0.02 mm filters
and 0.22 mM filters, respectively and spun at 9300 rcf for
10 min prior to use.
3. Experimental settings used an acquisition time of 3 s; S/N
threshold of 2.5 and sensitivity of 70%.
4. Samples were illuminated with 829.4 nm laser and scattering
intensity at 90° was measured.
5. Fluctuations greater than 15% in the scattering intensity are
excluded from the analysis.
6. The DynaLS software (Protein Solutions, USA) is used to
resolve acquisitions into well-defined Gaussian distributions of
hydrodynamic radii.

2.9 Quenchers 1. Quenchers used: Iodide (0.5 nm), Amino TEMPO (1 nm),
Nanogold (1.5 nm), Gold nanoparticles (GNPs) (2, 3, 4,
and 5 nm), TEMPO-Dextran 1 kDa (2.5 nm).
2. TEMPO Dextran (1 kDa) is obtained by coupling Dextran
(10 mg) to carboxy TEMPO (50 mg) using dicyclohexylcar-
bodiimide (20 mg) in dry DMSO at 20°C for 8 h and purified
by SEC-HPLC.
3. All GNPs are synthesized by the procedure given in
Subheading 2.4 and characterized by TEM.
70 Dhiraj Bhatia et al.

2.10 Lifetime 1. Frequency domain Fluorolog Tau 3 (Horiba Jobin Yvon,


Measurements Japan) operating at 25°C and at 10 MHz frequency.
2. The S and T channels are calibrated using glycogen as a
standard.
3. For each sample, the frequency and modulation are spanned
from 10 to 150 MHz using 7–10 intermediate frequency
readings.
4. The data is fitted using the associated software and only read-
ings showing c2 value less than 1.2 are selected for analysis.

3 Methods
DNA icosahedra are constructed from three distinct five way junction
(5WJ) components V, U and L, with programmable overhangs
(Fig. 1a). Each 5WJ module V, U and L are constructed from
equimolar ratios of the respective five phosphorylated single
strands. V forms a 1:5 complex with L to give VL5 (Fig. 1b). The
complementary module VU5 is similarly synthesized from compo-
nents V and U in a 1:5 ratio. At this stage, contiguously hybridized
strands in VU5 and/or VL5 are chemically ligated with N-Cyano
imidazole (NCI), to enhance stability. The two different hemi-
icosahedra, VU5 and VL5, each have ten identical overhangs where
the overhangs in VL5 are complementary to the ones in VU5. VU5
and VL5 complex with each other in a 1:1 ratio to yield the DNA
icosahedron and the contiguous termini are ligated again with NCI
(Fig. 1c).
In order to encapsulate cargo inside these DNA icosahedra,
the two halves VU5 and VL5 are annealed together in a 1:1 ratio in

Fig. 1 Construction and characterization of DNA icosahedron. (a) The icosahedron I (right ) is constructed from
two half icosahedra VU5 and VL5. Each half icosahedron (middle) is made from two types of 5WJs: V and U for
VU5 and V and L for VL5 (left ). Complementary overhangs are color coded. (b) 10% PAGE showing formation of
5WJ and half icosahedra. Lane 1: VL5; lane 2: VU5; lane 3: 5WJ V; lane 4: V1 oligonucleotide; lane 5: DNA
marker; (c) 0.8% Agarose gel showing the formation of the icosahedron from VU5 and VL5. Lane 1: ligated
icosahedron; lane 2: ligated VL5; lane 3: ligated VU5
A Method to Encapsulate Molecular Cargo Within DNA Icosahedra 71

Fig. 2 Encapsulation of molecular cargo like gold nanoparticles (GNPs) within DNA icosahedron. (a) General
schematic showing encapsulation of various molecular cargo inside DNA icosahedra. The two half icosahedra
(VU5 & VL5) are mixed in 1:1 ratio in presence of excess of desired cargo so that few molecules of cargo
are encapsulated within the icosahedron. These loaded icosahedra are purified from bulk of free molecules.
(b) A representative low-resolution TEM image shows the dense core of gold nanoparticles encapsulated
within DNA icosahedra. The inset shows representative high-resolution image in which the individual gold
nanoparticles can be seen to be present within the icosahedral cages. Scale bar: 50 nm. (c) Representative
TEM micrographs of platinum shadowed icosahedra showing hexagonal (top) and pentagonal (bottom)
symmetries. Corresponding theoretically calculated distances (in nm) are shown in left. Scale bar: 20 nm

presence of an aqueous solution of cargo like GNPs or FD10


(Fig. 2a). The complex, i.e., cargo loaded DNA icosahedron, is
separated from bulk, unencapsulated cargo using size separating
techniques like dialysis and/or gel electrophoresis. Electron dense
cargo such as GNPs inside DNA icosahedra may be characterized
by single molecule methods such as electron microscopy (TEM)
(Fig. 2b, c), while biomolecular cargo such as FD10 inside icosa-
hedra may be characterized by bulk biophysical methods such as
fluorescence spectroscopy (Fig. 3c, d).

3.1 Sample 1. To an eppendorf tube is added 2 mL oligonucleotide (from


Preparation 1 mM stock), 10 mL MQ water, 2 mL 10× T4 PNK
(Polynucleotide kinase) buffer (500 mM Tris–HCl, pH 7.6 at
3.1.1 Phosphorylation
25°C, 100 mM MgCl2, 50 mM DTT, 1 mM EDTA, 1 mM
of Oligonucleotides
Spermidine).
72 Dhiraj Bhatia et al.

a 1 2 3 800 40 60
100 3
50

Intensity (AU)

Intensity (AU)
I(AU)

Intensity (AU)
Intensity (AU)
400
80 30
2 40
60 0
0 5 10 15
Time (min) 20 30
40 1 20
20 10
0 10
0 0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Time (min) Time (min)

Fluorescence Intensity
b c 90 d 1.3
1.0
0.8 80 1.2

FD10
Intensity

70

τI τ
0.6

FD10 /
60 1.1
0.4
0.2 50 1.0
0.0
0.1 1 10 100 0 1 2 3 4 5 0 1 2 3 4 5
Mean RH (nm) Mean Size of Quencher (nm) Mean Size of Quencher (nm)

Fig. 3 Encapsulation of FD10 inside DNA icosahedron. (a) (Left ) Gel electrophoretic mobility shift assay for the
formation of IFD10. 0.8% agarose gel (1× TAE) showing association of FD10 with icosahedron: lane 1. FD10,
lane 2. 1:1 (VU5: VL5) + 2 mM FD10 post ligation, lane 3. Purified IFD10. Gel was visualized using 488 nm excita-
tion. (Middle) Size exclusion chromatogram (SEC-HPLC) of IFD10 complex post gel excision. SEC traces were
followed at 254 nm (grey ) and 488 nm (black ). Inset: SEC of standard, reference sample of unlabeled, unloaded
icosahedron I (retention time 8 min). (Right ) SEC trace of free FD10 was followed at 254 nm (black ) and
488 nm (grey ). (b) Dynamic light scattering (DLS) traces of free FD10 (black squares), the standard sample of
DNA icosahedra, I (open grey circles) and purified IFD10 complex (grey squares). (c) Fluorescence intensity-based
quenching assay for free FD10 (black squares) and IFD10 complex (grey squares). (d) Fluorescence lifetime
measurements of free FD10 (black squares) and IFD10 complex (grey squares) with the same quenchers. Mean
values of two independent experiments are presented, along with their s.d

2. 2 mL T4 PNK enzyme is added to this mixture. The concentration


of enzyme is 10 U/mL.
3. 4 mL of 1 mM ATP is added to the above solution, vortexed
for 1 min to mix the solution well and the tube is incubated at
37°C in a heat block for 1 h.
4. Post-incubation, the enzyme is deactivated by heating the
mixture to 75°C for 10 min.
5. The DNA is precipitated by addition of 80 mL absolute ethanol
and 20 mL 3 M potassium acetate, and incubated at −20°C for
8 h.
6. Post incubation at −20°C, the tubes are spun at 20800 rcf for
40 min at 4°C.
7. The DNA pellet is visible at the bottom of the tube. All super-
natant solution is carefully removed and discarded.
8. The pellet is washed with 100 mL of 70% ethanol and spun at
14 k for 2 min.
A Method to Encapsulate Molecular Cargo Within DNA Icosahedra 73

9. All ethanol is removed carefully and eppendorfs are air dried


for 15 min to remove any remaining ethanol.
10. The pellet is dissolved in 20 mL Nuclease free water and the
phosphorylated oligos are quantified by their UV absorbance
at 260 nm.

3.1.2 5-Way Junctions 1. The buffer used to make the 5WJ is 10 mM phosphate buffer
(pH 6), 1 mM MgCl2 and 100 mM NaCl.
2. From the stocks of phosphorylated oligos for V 5WJ, the oligos
V1:V2:V3:V4:V5 are mixed together in a 1:1:1:1:1 ratio in
50 mL of buffer containing 20 mM concentration of each oligo.
In a similar manner, 5WJ of U and L are made from oligos
U1–U5 and L1–L5, respectively.
3. Once all solutions are added, the eppendorfs are heated to 90°C
for 15 min. After 15 min, the sample is annealed from 90°C at
the rate of 1°C/3 min till room temperature, incubated at
room temperature for 2 h and then stored at 4°C for 48 h.
4. The individual 5WJ are characterized by 15% PAGE stained
with EtBr (see Subheading 2.2) (Fig. 1b).

3.1.3 Half Icosahedra 1. In an eppendorf tube, 50 mL of U 5WJ (20 mM) and 10 mL of


( VU5 and VL5 ) V 5WJ (20 mM) are mixed to form half icosahedron VU5.
2. The tube is heated in a heat block at 45°C for 4 h, and then the
temperature is decreased at the rate of 1°C/3 min till room
temperature (20°C), where the samples are incubated for 2 h
and then stored at 4°C for 72 h.
3. VL5 can be similarly made by mixing V and L 5WJs.
4. The half icosahedra are characterized by 15% PAGE stained
with EtBr as described in Subheading 2.2 (Fig. 1b).

3.1.4 Ligation 1. Half icosahedra VU5 or VL5 are ligated using NCI as described
below.
2. 50 mL of sample (VU5 or VL5, 3.33 mM) is taken in an eppen-
dorf tube.
3. To this 0.3 mg solid NCI and 2 mL NiCl2 (from a 1 M solution
of NiCl2) is added and incubated for 48 h.
4. After 48 h, step 3 is repeated and the tubes containing ligated
VU5 or VL5 are stored at 4°C for 72 h (see Note 1).

3.1.5 Icosahedron 1. 10 mL of VU5 and VL5 (3.33 mM) each are mixed in an eppen-
dorf to form icosahedra.
2. The tube is heated in a heating block at 45°C for 4 h, and the
temperature is decreased at a rate of 1°C/3 min till room
temperature (20°C) followed by incubation at 20°C for 2 h.
Then the sample is transferred to 4°C to equilibrate for 72 h.
74 Dhiraj Bhatia et al.

3. The half icosahedra and full icosahedra are characterized on


0.8% agarose gel (see Subheading 2.2) (Fig. 1c).
4. The icosahedron is then ligated as described in Subheading 3.1.4.

3.2 Encapsulation 1. Citrate capped GNPs of diameters 2, 3.5, 8 nm are prepared


of GNPs according to procedure given in Subheading 2.4.
2. 15 mL VU5 and VL5 (200 nM) were mixed with 30 mL solution
of GNPs (at 400 nM GNP concentration) of desired size in
10 mM phosphate buffer.
3. The tube is heated in a heating block at 45°C for 4 h. The
temperature is then decreased at the rate of 1°C/3 min till
room temperature (20°C), where it is incubated for 2 h and
then equilibrated for 72 h at 4°C.
4. Finally, the solution is ligated using NCI following the proce-
dure described earlier (see Subheading 3.1.4).

3.3 Purification 1. The DNA icosahedra loaded with GNPs (IGNP) are separated
from free GNPs in solution using dialysis.
2. 50 mL of the solution of GNPs and post-ligated icosahedron is
loaded in a dialysis membrane size: 3.4 × 15 cm, 50 kDa
MWCO, sealed from bottom using a plastic clip as provided by
the supplier.
3. This sample was further diluted to 1 mL with a buffer containing
10 mM phosphate, pH 6 and 100 mM NaCl (see Note 2).
4. The resultant solution is dialyzed against buffer containing
10 mM phosphate buffer, pH 6 and 100 mM NaCl for 24 h at
20°C, where the external buffer is changed every 6 h.
5. Post-dialysis, the sample is vacuum concentrated (Labconco
Centrivac Console) at 20°C and this solution containing loaded,
purified icosahedra is taken for further characterization.

3.4 Transmission 1. TEM is a good method to characterize icosahedra whether


Electron Microscopy they contain GNP cargo or whether investigating icosahedra
that have not been subjected to any encapsulation process.
2. For visualizing the icosahedral shell, 10 mL of icosahedron
solution (10 nM) is adsorbed on the carbon-coated, glow dis-
charged copper grid of 400 mesh size for 20 min.
3. The excess solution is wicked off using a Whatman filter paper.
4. The grid is stained by placing a drop of 1% uranyl acetate solution
for 2 s and immediately wicking off with a Whatman filter
paper.
5. The grid is then loaded onto a holder and visualized by TEM
using low beam current (4–8 nA).
6. Alternatively, the samples can also be visualized by platinum
shadowing instead of uranyl acetate staining.
A Method to Encapsulate Molecular Cargo Within DNA Icosahedra 75

7. For platinum shadowing, the samples are loaded on the grids


as described, and these grids are rotary platinum shadowed for
10 s in a vacuum evaporator. These icosahedra are visualized as
pentagonal and hexagonal particles (icosahedral symmetry) in
bright field EM (Fig. 2c).
8. For TEM characterization of icosahedra loaded with gold
nanoparticles, only uranyl acetate staining is used. The samples
are prepared as described in point 2 of this section.
9. At low magnification (50k×), particles with highly electron
dense core are seen. At high magnification (160k×), individual
gold nanoparticles can be seen present inside the DNA icosa-
hedra (Fig. 2b) (see Note 3).

3.5 Encapsulation 1. 5 mM stock of FD10 is prepared by dissolving 5 mg of FD10


of FITC Dextran, in 100 mL phosphate buffer, 10 mM, pH 7.
10 kDa ( FD10) 2. In an eppendorf tube, 15 mL of VU5 and VL5 (3.33 mM) and
20 mL of 5 mM FD10 are mixed. This will result in a final
concentration of FD10 of 2 mM. This concentration of FD10
was selected because at 2 mM we have one FD10 molecule per
1,000 nm3. This is the internal volume of a single DNA
icosahedron.
3. The tube is heated in a heating block at 45°C for 4 h and the
temperature is decreased at the rate of 1°C/3 min till 20°C
where it is incubated for 2 h and equilibrated for 72 h at 4°C.
4. Finally, the solution is ligated using NCI (see Subheading 3.1.4).

3.6 Purification The DNA icosahedron loaded with FD10 (IFD10) is separated from
free FD10 using a two-step purification—(a) gel electrophoresis
followed by (b) Size exclusion chromatography (SEC-HPLC).

3.6.1 Gel Electrophoresis 1. The ligated mixture of IFD10 (1 mM, DNA) is loaded on 0.8%
agarose gel (Fig. 3a left).
2. When resolved on gel, free FD10, being an unstructured poly-
mer, migrates as a smear along the lane.
3. The band corresponding to IFD10 (Fig. 3a) is excised and eluted
in 100 mM NaCl, 1 mM MgCl2 solution for 24 h at room
temperature (Fig. 3a, left).
4. This sample is vacuum concentrated and then subjected to a
second round of purification by size exclusion chromatog-
raphy (SEC).

3.6.2 Size Exclusion 1. The gel purified and vacuum concentrated IFD10 sample is sub-
Chromatography jected to SEC.
2. 50 mL solution of IFD10 (1 mM, DNA) is injected onto the
HPLC column which is pre-rinsed with degassed Acetonitrile/
water mixture.
76 Dhiraj Bhatia et al.

3. The elution is carried out at a flow rate of 0.5 mL/min and


absorbance at 488 nm (FITC absorbance) and 254 nm
(FITC + DNA absorbance) are monitored.
4. The plain icosahedron sample without any FD10 (i.e., I) is
used as reference for IFD10. The empty icosahedron elutes close
to 8 min in the chromatogram (Fig. 3a middle inset).
5. Free FD10, being a polydisperse, unstructured polymer, elutes
as a broad peak from 5 to 8 min (Fig. 3a, right).
6. IFD10 elutes at 8 min as a sharp peak showing absorbance both
at 488 nm as well as 254 nm indicating the formation of IFD10
complex.
7. The ratio of 254 and 488 nm can also be used to calculate the
number of FD10 molecules encapsulated per DNA icosahe-
dron on average (Fig. 3a, middle).
8. The eluted fractions are collected, vacuum-concentrated, and
adjusted to buffer conditions (see Subheading 3.1.2).

3.7 Dynamic Light 1. DLS has been used extensively to study particle sizes and also
Scattering to study the interactions between various biomolecules. DLS
can be used to study the sizes of DNA polyhedra and also it can
be used as a tool to predict the association of various cargo
molecules with DNA icosahedra.
2. Filter water and buffer (10 mM phosphate buffer with 1 mM
MgCl2 and 100 mM NaCl) through 0.02 mm Whatman syringe
filter paper. The DNA samples and FD10 should be filtered
through 0.22 mm Millipore filter.
3. 100 mL of 1 mM sample of DNA icosahedron, IFD10 complex
and 1 mM sample of FD10 in buffer above are used for
investigation.
4. All samples including water and buffers are spun at 9300 rcf
for 10 min using a centrifuge at room temperature.
5. The DLS cuvettes are washed rigorously with water and then
with buffer.
6. 50 mL of buffer in a cuvette is taken and the counts in DLS are
measured using 100% beam intensity. The average counts
should be less than 5 and stay constant for at least 2 min. This
indicates that the solution and cuvette are clean and free of
dust particles.
7. 50 mL of I is taken and the laser intensity is fixed at 70%, the
S/N ratio at 2.5, and the acquisition time at 3 s. The sample
readings are initiated by monitoring autocorrelation functions
and 40 continuous readings are taken.
8. Individual readings are checked by the shape of autocorrelation
function and only those readings that show sharp, straight ends
of the autocorrelation function are chosen for further analysis.
A Method to Encapsulate Molecular Cargo Within DNA Icosahedra 77

9. Then, the distribution is selected. In the distribution, all readings


below 1 nm are discarded since these are due to solvent
scattering.
10. All other readings are used to plot the distribution of intensity
as a function of hydrodynamic radius (RH) (Fig. 3b).

3.8 Quencher 1. Each quencher has an intrinsically different ability to collision-


Characterization ally quench fluorescence.
2. This is corrected for by using that concentration of the
quencher which results in a 50% decrease in fluorescence inten-
sity of the sample. This is obtained from the reciprocal of their
measured Stern–Volmer constants (9).
3. Quenchers of different sizes are selected based on literature
reports. These include Iodide (0.5 nm), Amino TEMPO
(1 nm), and Nanogold (1.5 nm). Quenchers in the regime
2–5 nm are all gold nanoparticles and are synthesized as
described in Subheading 2.4.
4. 400 mL of 50 nM solution of free FD10 is taken in a quartz
cuvette and its emission is scanned from 500 to 600 nm when
it is excited at 488 nm in a Fluorolog 3 L instrument. The
fluorescence intensity at 515 nm is taken as F0.
5. Then the quenchers are added in small aliquots. After each
addition of quencher, the solution is equilibrated for 2 min.
6. Then, the fluorescence value, F, at 515 nm is recorded. The
value of F obtained is further corrected for dilution by multi-
plying with the dilution factor.
7. After approximately ten such readings, the plot of F0/F is plot-
ted against the quencher concentration. This yields a straight
line whose y-intercept is 1.
8. The slope of the line gives the Stern–Volmer Constant, Ksv.
9. Thus, the concentration that is used in further experiments, to
observe 50% quenching will be (1/Ksv) M of the relevant
quencher.

3.9 Intensity Based 1. The IFD10 complex could have the FD10 externally associated,
Quenching or internally entrapped within the DNA Icosahedron.
2. In order to confirm the encapsulation of FD10 within the
DNA icosahedral cavity of I, free FD10 and IFD10 are subjected
to external quenchers of various sizes ranging from 0.5 to 5 nm
and their extents of quenching is studied (Fig. 3c).
3. 400 mL of 50 nM solution of free FD10 is taken in a quartz
cuvette and its emission is recorded from 500 to 600 nm
when it is excited at 488 nm in a Fluorolog 3 L instrument.
The fluorescence intensity at 515 nm is taken as F0. This value
is chosen for normalization for all other readings and is taken
as 100%.
78 Dhiraj Bhatia et al.

4. 1/Ksv amount of each quencher is added, equilibrated for


2 min and the emission at 515 nm is recorded. This value
should be half of the original F0 value (Fig. 3c).
5. The cuvette is cleaned after each reading rigorously with water
and ethanol to remove trace amounts of quenchers present on
the walls of the cuvette.
6. Then, 400 mL of 50 nM, pH 7, solution of IFD10 is taken in a
quartz cuvette and its emission is recorded from 500 to 600 nm
upon excitation at 488 nm. The fluorescence intensity at 515 nm
is taken as F0. This value is chosen for normalization.
7. 1/Ksv amount of each quencher is added to this cuvette, equil-
ibrated for 2 min and the emission at 515 nm is recorded.
8. In case of IFD10, only quenchers smaller than 2 nm, which can
diffuse through the pores of the DNA icosahedron, access the
fluorophore and quench it to 50%. Quenchers larger than 3 nm
cannot access the internal fluorophore and fluorescence from
FD10 is resistant to quenching.
9. From the plot of percentage fluorescence against size of the
quencher, the pore size of the icosahedron is evident.

3.10 Lifetime Based 1. Intensity quenching can be supported by similar studies of


Quenching fluorescence lifetimes.
2. 400 mL of 5 mM FD10, pH 7, is taken in a quartz cuvette. For
lifetime measurements, we need two cuvettes, one containing
a standard sample and other containing the sample of interest
(see Note 4).
3. Glycogen is used as the standard for instrument response fac-
tor (IRF) measurements.
4. For the standard sample the settings are maintained as, excita-
tion and emission at 488 nm, while for the desired sample exci-
tation and emission were 488 nm and 515 nm, respectively.
The instrument measures values for both standard and sample
at all operating frequencies.
5. A frequency range from 10 to 150 MHz is selected and ten
intermediate values are recorded with each value repeated in
quadruplicate.
6. The instrument measures the lifetime and modulation at each
frequency and gives the raw data.
7. The associated data analysis program allows one to change
component lifetimes.
8. FITC exhibits a two component lifetime and the average life
time is calculated from two lifetimes using formula:
<tavg> = (t1 f12 + t2 f22)/(t1 f1 + t2 f2)
where t1 and t2 are the lifetimes of two components and f1 and f2
are the respective fractions of the component (10).
A Method to Encapsulate Molecular Cargo Within DNA Icosahedra 79

9. For each average lifetime, the instrument gives a c2 value which


is called the goodness of fit parameter. The average lifetime for
which the c2 value is less than 1.2 is selected. Two such read-
ings are taken for each sample and the lifetime is presented as
mean lifetime with associated standard deviation.
10. The lifetimes of FD10 and IFD10 are first measured without any
quencher to investigate the effect of the DNA polyhedron on
the lifetime of the encapsulated FD10.
11. Free FD10 exhibits an average lifetime of 3.8 ns at pH 7. To
measure the lifetime quenching, the quencher is added in small
amounts and the life time is measured till it decreases to 2 ns
(this will correspond to the 1/Ksv amount of quencher).
12. This amount of quencher is added to the samples of IFD10 and
the lifetimes are determined.
13. The results from lifetime measurements give the same results
as those obtained from intensity based quenching and both
should be consistent with the pore size of the DNA capsule
(Fig. 3d).

4 Notes
1. Sometimes, post-addition to the sample to be ligated, NCI
forms a white precipitate. The precipitate formed is nickel
phosphate which solubilizes by the addition of dilute acid. So
in case of precipitate, 5 mL of acetic acid is added along with
10 mL water, the eppendorf is vortexed and then the sample
can be used further.
2. In samples containing GNPs, Mg2+ is avoided since it causes
aggregation of GNPs. Hence dialysis is performed against only
phosphate buffer and NaCl.
3. GNPs below 3 nm diameter cannot be encapsulated inside
DNA icosahedron as the effective pore size of the DNA icosa-
hedron is in the range of 2.5–3 nm.
4. Even though the FD10 is encapsulated within DNA icosahe-
dra in phosphate buffer of pH 6, for all the fluorescence
studies the pH should be adjusted to 7 since FITC is a pH
sensitive fluorophore and its fluorescence and lifetime are
maximal at pH 7.

Acknowledgments

We thank Dr. S.S. Indi and Dr. Atanu Basu at Department of


Microbiology and Cell Biology, IISc and NIV, Pune, respectively,
for providing electron microscopy facilities, Prof. Dipanker
Chatterji, MBU, IISc for use of the lifetime instrument. D.B.,
80 Dhiraj Bhatia et al.

S.M., and S.C. thank CSIR, Government of India (GoI) for


research fellowships. This work was funded by the Nano Science
and Technology Initiative, DST, GoI, and the Innovative Young
Biotechnologist Award, DBT (GoI) to Y.K.

References

1. Holliday BJ, Mirkin CA (2001) Strategies 6. Bhatia D, Surana S, Chakraborty S, Koushika


for the construction of supramolecular SP, Krishnan Y (2011) A synthetic, icosahedral
compounds through coordination chemistry. DNA-based host-cargo complex for functional
Angew Chem Int Ed Engl 40:2022–2043 in vivo imaging. Nat Commun 2:339.
2. Goodman RP, Schaap IA, Tardin CF, Erben doi:10.1038/ncomms1337
CM, Berry RM, Schmidt CF, Turberfield AJ 7. Ghodke HB, Krishnan R, Vignesh K, Kumar
(2005) Rapid chiral assembly of rigid DNA GV, Narayana C, Krishnan Y (2007) The
building blocks for molecular nanofabrication. I-tetraplex building block: rational design and
Science 310:1661–1665 controlled fabrication of robust 1D DNA scaf-
3. He Y, Ye T, Su M, Zhang C, Ribbe AE, folds via non-Watson Crick self-assembly.
Jiang W, Mao C (2008) Hierarchical self- Angew Chem Int Ed Engl 46:2646–2649
assembly of DNA into symmetric 8. Mirkin CA (2000) Programming the assembly
supramolecular polyhedra. Nature 452: of two- and three-dimensional architectures
198–202 with DNA and nanoscale inorganic building
4. Douglas SM, Dietz H, Liedl T, Högberg B, blocks. Inorg Chem 39:2258–2272
Graf F, Shih WM (2009) Self-assembly of 9. Lakowicz JR, Weber G (1973) Quenching of
DNA into nanoscale three-dimensional shapes. fluorescence by oxygen: probe for structural
Nature 459:414–418 fluctuations in macromolecules. Biochemistry
5. Bhatia D, Mehtab S, Krishnan R, Indi SS, Basu 12:4161–4170
A, Krishnan Y (2009) Icosahedral DNA nano- 10. Lakowicz JR (2006) Principles of fluorescence
capsules by modular assembly. Angew Chem spectroscopy, 3rd edn. Springer, New York,
Int Ed Engl 48:4134–4137 pp 353–380
Chapter 9

Delivery of Plasmid DNA to Mammalian Cells


Using Polymer–Gold Nanorod Assemblies
James Ramos, Huang-Chiao Huang, and Kaushal Rege

Abstract
Functionalized and surface-modified gold nanorods (GNRs) have emerged as promising vehicles for the
delivery of several therapeutic agents. Ease of functionalization, increased stability, biocompatibility, and
size-dependent plasmonic properties make gold nanorods attractive in sensing, imaging, and delivery to
different cellular types. Here, we demonstrate the use of polyelectrolyte-coated GNRs (PE-GNRs) for
delivering plasmid DNA to mammalian cells for transgene expression.

Key words Gold nanoparticle, Nonviral gene delivery, Cationic polymers, Polyelectrolytes, Stability

1 Introduction

Many novel nanomaterials, including gold nanoparticles, are


showing strong potential as nanocarriers for delivery of thera-
peutic agents (1–5). In particular, gold nanorods (GNRs) are
being increasingly investigated for biological sensing (6, 7), imaging
(8, 9), photothermal therapy (10, 11), and drug (12, 13) and
gene delivery (14–16), due to their unique optical properties and
facile methods of surface modification and functionalization (17).
Functionalized gold nanorods provide a high surface area ratio
which allows for high payload (e.g., plasmid DNA)-to-carrier
ratios. Furthermore, hydrophobicity and charge can be tuned
through polymer monolayer coverage of the gold nanorods,
which can result in improved cellular uptake as well as decreased
cytotoxicity (5). Specifically, functionalization of gold nanorods
with polymers further increases their potential for use in noninva-
sive therapeutic applications as it results in increased stability,
lower cytotoxicity, controllable surface properties, and the possi-
bility for further surface functionalization (e.g., with targeting

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_9, © Springer Science+Business Media New York 2013

81
82 James Ramos et al.

biomolecules) (18). Thus, several strategies have been pursued in


which gold nanoparticles have been functionalized with polymers
for use as a delivery vehicle (19–21).
Here, we demonstrate the generation of polyelectrolyte-
coated GNRs (PE-GNRs) based on candidates from a library of
cationic polymers recently synthesized in our laboratory (22–25).
These cationic polyelectrolytes engender high colloidal stabilities
of gold nanorods and also facilitate high loadings of plasmid
DNA on the nanorods by means of electrostatic interactions. In
vitro studies with human prostate cancer cell lines demonstrated
that subtoxic concentrations of PE-GNRs, loaded with exoge-
nous plasmid DNA, are capable of mediating transgene delivery
and expression.

2 Materials

Prepare all solutions using nanopure water (18.2 MΩ-cm, resistivity)


unless otherwise specified. Prepare and store all reagents at room
temperature unless otherwise specified. Diligently follow all waste
disposal regulations when disposing waste materials.

2.1 Polymer 1. 0.01× PBS: Prepare 500 mL of 10× phosphate-buffered saline


Synthesis (PBS) by mixing 40.9 g NaCl, 7.098 g Na2HPO4, and 1.006 g
KCl in 500 mL of water. Dilute desired amount to 0.01× PBS
and adjust the pH to 7.4 using 6N HCl and 3 M NaOH.
2. 20 mL glass scintillation vials.
3. 1,4-cyclohexanedimethanol Diglycidyl Ether.
4. 1,4-bis(3-aminopropyl)piperazine.

2.2 Gold Nanorod 1. Gold nanorod (GNR) synthesis technique was adapted from
Synthesis the seed-growth method described by El-Sayed et al. (26).
2. Gold(III) chloride trihydrate (HAuCl4⋅3H2O).
3. Cetyltrimethyl ammonium bromide (CTAB).
4. L-ascorbic acid.
5. Sodium borohydride.
6. Silver nitrate.

2.3 Generation of 1. Poly(styrene sulfonic acid) (PSS): Dissolve PSS in 0.01× PBS
Polyelectrolyte-Gold to a concentration of 10 mg/mL (see Note 1).
Nanorod Assemblies 2. Serum-free media: RPMI-1640 media supplemented with 1%
penicillin–streptomycin antibiotic.
3. Water bath sonicator.
Delivery of Plasmid DNA to Mammalian Cells… 83

2.4 Determination For the purpose of this discussion, we describe the determined
of Polyelectrolyte– cytotoxicity of the PE-GNRs towards PC3 and PC3-PSMA pros-
Gold Nanorod tate cancer cells.
Assembly Cytotoxicity
1. PC3 cells (ATCC, Manassas, VA).
2. PC3-PSMA cells (a generous gift from Dr. Michel Sadelain of
the Memorial Sloan Cancer Center (27)).
3. Tissue culture treated 24 well plates (Costar).
4. MTT Cell Proliferation Assay Kit (ATCC, Manassas, VA):
Includes MTT reagent and detergent reagent.
5. Serum-free media as described above.
6. Aluminum foil.

2.5 PE-GNR- For the purpose of this discussion, we describe the transfection and
Mediated Plasmid determination of transgene expression in PC3 and PC3-PSMA
DNA Delivery and cells using the firefly luciferase encoding pGL3 plasmid DNA and
Transgene Expression appropriate assays.
1. PC3 cells.
2. PC3-PSMA cells.
3. 96 well white flat bottom polystyrene assay plates.
4. Clear 96 well plates.
5. One 1.5 mL microcentrifuge tube for each treated well of the
24 well plate.
6. 1× PBS: Dilute previously described 10× PBS to 1× PBS by
adding nanopure water.
7. 1× lysis solution: Dilute 5× Cell Culture Lysis Reagent
(Promega, Madison, WI) to 1× Cell Culture Lysis Reagent
with water.
8. Luciferase Assay System (Promega, Madison, WI): Contains
luciferase assay substrate and luciferase assay buffer. One of
each will be needed for a single luciferase assay.
9. Pierce® BCA Protein Assay Kit (Thermo-Fisher Scientific,
Rockford, IL): Contains Pierce® BCA Protein Assay Reagent
A, Pierce® BCA Protein Assay Reagent B, and Albumin
standards.
10. Serum-free media as described above.
11. pGL3 plasmid DNA control vector (Promega, Madison, WI).

3 Methods

Carry out all procedures at room temperature unless otherwise


specified.
84 James Ramos et al.

3.1 Polymer Cationic polymers can be synthesized using the following described
Synthesis methods of ring opening of diglycidyl ethers by amines (22, 25).
For the purpose of this discussion, we will use the cationic polymer
synthesized via the polymerization of 1,4-cyclohexanedimethanol
Diglycidyl Ether (1,4C) and 1,4-bis(3-aminopropyl)piperazine
(1,4Bis) as an example.
1. In 20 mL glass scintillation vials, mix equimolar amounts of
1,4C (238.2 μL) and 1,4Bis (269.5 μL) and mix well.
2. Set scintillation vials aside for 12–16 h (see Note 2).
3. Weigh out and dissolve polymerized mixture in 0.01× PBS at a
concentration of 10 mg/mL (see Note 3). Adjust the pH to
7.4 and place solutions on a rotator overnight (see Note 4).
4. Check and adjust pH of polymer solutions next day to a value
of 7.4 (see Note 5).

3.2 Gold Nanorod 1. Prepare a gold “seed” solution by adding 5 mL of 0.5 mM


Synthesis HAuCl4⋅3H2O to 5 mL of 200 mM CTAB in a 15 mL poly-
propylene conical tube with gentle mixing by inversion for
2 min.
2. Prepare 0.6 mL of 0.01 M sodium borohydride and allow to
chill to 4°C, following which, immediately add this solution to
the gold seed dispersion (see Note 6).
3. Prepare a growth solution by mixing 5 mL of 1 mM
HAuCl4⋅3H2O with 5 mL of 200 mM CTAB solution contain-
ing 280 μL of 0.004 M silver nitrate in a 15 mL polypropylene
conical tubes.
4. Add 70 μL of 0.0788 M L-ascorbic acid to the growth solution
(see Note 7).
5. Add 12 μL seed solution to the growth solution and allow
to mix for 4 h under continuous stirring at 28°C, 150 rpm (see
Note 8).
6. Pellet the as-prepared GNRs, by centrifugation at 6,000 rcf for
10 min.
7. After sedimentation, remove the clear supernatant and re-dis-
perse GNR pellet to the initial volume with nanopure water.

3.3 Synthesis of 1. Adjust GNR optical density to 0.5 absorbance units (a.u.)
Polyelectrolyte–Gold (Fig. 1) in nanopure water and centrifuge 0.5 mL of GNR
Nanorod Assemblies dispersion in 1.5 mL microcentrifuge tubes at 6,000 rcf for
10 min (Microfuge 18 centrifuge, Beckman Coulter). Remove
excess CTAB surfactant.
2. Re-disperse GNR pellet in 100 μL of a poly(styrene sulfonate)
(PSS) solution (10 mg/mL in 0.01× PBS).
Delivery of Plasmid DNA to Mammalian Cells… 85

Fig. 1 Absorption spectrum of gold nanorods possessing a transverse band at ~520 nm and a longitudinal
band at ~750 nm

3. Remove excess CTAB 7. Remove excess PSS 11. Remove excess cationic polymer

4. Add PSS 8. Add 12. Resuspend in serum-free media


cationic
polymer

1. GNR

5. Sonication 9. Sonication
13. PE-GNR

2. Centrifugation 6. Centrifugation 10. Centrifugation

Fig. 2 Schematic of PE-GNR generation process

3. Immediately sonicate in a water bath sonicator for 30 min at


room temperature to allow for the formation of PSS-coated
GNRs (PSS-CTAB-GNRs).
4. Centrifuge PSS-CTAB-GNRs at 6,000 rcf for 10 min. Remove
excess PSS by removing the supernatant.
5. Re-disperse PSS-CTAB-GNRs pellet in 300 μL of nanopure
water.
6. Add 200 μL of cationic polymer to dispersion (see Note 9).
7. Immediately sonicate for 30 min to allow for the formation of
the cationic polymer (polyelectrolyte)-coated PSS-CTAB-
GNRs or PE-GNRs.
8. Centrifuge PE-GNRs at 6,000 rcf for 10 min. Remove excess
cationic polymer by removing the supernatant (Fig. 2).
9. Disperse PE-GNR in serum-free media and adjust the optical
density to 0.25 a.u. (see Note 10).
86 James Ramos et al.

3.4 Determination 1. Plate 50,000 cells per well in a tissue culture treated 24 well
of Cytotoxicity of plate and incubate overnight at 37°C and 5% CO2 to allow
Polyelectrolyte–Gold cells to attach.
Nanorod Assemblies 2. After overnight incubation, remove cell growth media and
replace with 500 μL of PE-GNR dispersions in serum-free
media set to different optical densities (see Note 11).
3. Allow to incubate for 6 h. Use untreated cells (i.e., those not
treated with PE-GNRs) as a live control and prepare a dead
control (i.e., treated with 5 μL of 30% hydrogen peroxide).
4. After 6 h, remove serum-free media from wells and replace
with fresh growth media.
5. Add 20 μL of MTT reagent to each well, wrap each plate in
aluminum foil, and incubate at 37°C for 4 h (see Note 12).
6. Following 4 h, add 150 μL detergent reagent to each well.
Wrap plates in aluminum foil and incubate for 4 h at room
temperature.
7. Following 4 h, mix solution in wells and transfer 150 μL to a
clear 96 well plate (see Note 13). Read absorbance and
570 nm.
8. Determine cell viability by reporting values as a percentage of
live and dead controls.
9. Determine a cutoff value for subtoxic treatment conditions of
PE-GNRs (see Note 14).

3.5 Determination 1. Plate 50,000 cells per well in a tissue culture treated 24 well
of PE-GNR-Mediated plate and incubate overnight at appropriate conditions to allow
Transgene Expression cells to attach.
2. Synthesize PE-GNRs (see Subheading 3.3, step 9) and aliquot
previously determined subtoxic doses of PE-GNRs into a well
of a 96 well plate. Add desired amounts of plasmid DNA
(see Note 15) and allow to co-incubate for 30 min (Fig. 3).
3. Remove cell growth media and replace with 500 μL minus the
PE-GNR treatment volume of serum-free media.
4. Add PE-GNR/DNA assemblies to wells and allow to incubate
for 6 h.
5. Following 6 h, remove serum-free media and replace with fresh
growth media. Incubate at 37°C and 5% CO2 conditions
for 48 h.
6. Following 48 h, remove growth media and place in a micro-
centrifuge tube (see Note 16).
7. Wash each well with 150 μL of 1× PBS. Remove PBS and place
into same microcentrifuge that growth media from that well
was placed in.
Delivery of Plasmid DNA to Mammalian Cells… 87

Fig. 3 Schematic of PE-GNR loaded with plasmid DNA

8. Add 150 μL of 1× Cell Culture Lysis Reagent. Incubate for


approximately 3 min at room temperature and check cells
under a microscope to ensure complete lysis.
9. Add growth media/PBS mixture in the microcentrifuge tubes
back to the corresponding wells in the 24 well plate.
10. Aliquot 50 μL of the cell lysate into four wells of the white
plate for luciferase assay. This will allow for four readings of the
cell lysate.
11. Aliquot 100 μL of the cell lysate back into the corresponding
microcentrifuge tube. Add 900 μL of water to each tube for
use in BCA assay.
12. Luciferase Assay:
(a) Add one full bottle (10 mL) Luciferase Assay Buffer to
Luciferase Assay Substrate (see Note 17) and immediately
add 100 μL to each well in the 96 well plate (see Note 18).
(b) Immediately read the luminescence in plate reader and
record relative light (or luminescence) units (RLU).
13. BCA Assay:
(a) Aliquot 10 μL from albumin standards and the microcen-
trifuge tubes to a well in a clear 96 well plate.
(b) Mix Pierce® BCA Protein Assay Reagent A and Pierce®
BCA Protein Assay Reagent B at a 50:1 ratio to make
working reagent (see Note 19).
(c) Add 190 μL of working reagent to each well and incubate
at 37°C for 30 min (see Note 20).
(d) Following 30 min incubation, read absorbance at 562 nm.
Using standards, back-calculate the concentration of each
sample in mg/mL.
88 James Ramos et al.

Fig. 4 Transgene (luciferase) expression in PC3 and PC3-PSMA cells with PE-GNR loaded with different
amounts of pGL3 plasmid DNA (ng). Luciferase expression, in relative luminescence units (RLU), was analyzed
48 h post transfection and normalized to total protein content (RLU/mg) (28)

14. Using calculated mg/mL for each sample, determine amount


of protein in each well used for luciferase assay.
15. Normalize the measured relative light units measured by divid-
ing by the calculated amount of protein in each well and report
values as RLU/mg.
16. We show an example of the transgene expression (Fig. 4).
Higher DNA amounts lead to higher transgene expression up
to a point at which a maximum is reached. Following that,
there is a slight decrease as more DNA is loaded. This is likely
due to shielding of the polymer’s positive charge by the nega-
tively charged plasmid DNA.

4 Notes

1. We find that preparing this fresh works best.


2. This mixture will be used following polymerization to make
polymer solutions. Depending on the monomers used, polym-
erization may take varying amounts of times.
3. Polymers should be viscous; if they have “hardened”
(cured) or remain “watery,” then it will be necessary to repeat
the process with a shorter or longer incubation time
respectively.
4. Polymer solutions will be basic. When dissolving polymers in
solution, they may appear milky or may not completely dis-
solve initially. Adjusting the pH may allow them to dissolve
Delivery of Plasmid DNA to Mammalian Cells… 89

further. Lowering polymer concentration may also help increase


solubility. Discard any polymers that do not dissolve.
5. Polymer solutions may oftentime become slightly more basic
following the initial pH adjustment.
6. Seed solution should become pale brown.
7. After introduction of the L-ascorbic acid, growth solution
should become colorless.
8. During incubation, solution color should change from color-
less to purple in between 20 min.
9. Check to make sure the dispersion is not milky pink in color.
This is typically indicative of undesirable aggregation due to
very high local concentrations of the cationic polymer, and the
process must be restarted. To avoid this, addition of more than
300 μL of water in the previous step can prevent this or removal
of more of the excess PSS.
10. It is best to disperse the PE-GNRs in half the volume of the
initial GNR sample (250 μL in this case) and slowly add serum-
free media until the desired optical density is reached.
11. Recommended PE-GNR dispersions in serum-free media opti-
cal densities are 0.0025, 0.005, 0.0075, 0.0125, 0.025,
0.0375, 0.05, and 0.125.
12. After 4 h incubation, metabolically active cells should become
a purple color following addition of the MTT reagent.
13. It is recommended that this step is done in triplicate for each
well.
14. Concentrations that result in >70% cell viability may be treated
as subtoxic.
15. Recommended amounts of pGL3 plasmid DNA range from
10 to 200 ng.
16. A microcentrifuge tube should be “assigned” for each treated
well. Do not dispose of any solution.
17. Luciferase assay reagents are stored at −20°C and need ade-
quate time for thawing. It is recommended that they are taken
out of storage at Subheading 3.5, step 6 and kept at room
temperature to thaw.
18. Once mixed, the luciferase substrate decays rapidly. It is rec-
ommended that these steps are carried out as fast as possible
(within 3–5 min if possible).
19. Working reagent should be green in color.
20. After 30 min incubation, the solutions should turn from green
to a purple color.
90 James Ramos et al.

Acknowledgments

This work was supported by the National Science Foundation


(Grant CBET-0829128) and National Institutes of Health (Grant
5R21CA133618-02).

References

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Chapter 10

Lipophilic-Formulated Gold Porphyrin Nanoparticles


for Chemotherapy
Puiyan Lee and Kenneth K.Y. Wong

Abstract
Lipophilic formulation is an invaluable technique for the delivery of cancer drugs. Incorporation of poorly
soluble and toxic compounds into a lipophilic carrier vehicle improves both the stability and compatibility
in blood and body fluids. Currently, although a large proportion of novel cancer drugs are poorly water
soluble, most existing drug carriers are only able to encapsulate hydrophilic drugs. As the ultimate goal of
drug delivery (in particular cancer drug delivery) is to achieve high therapeutic effect with minimal toxic-
ity, it would thus be beneficial to invest substantial efforts in the development of lipophilic carrier systems.
Here we describe our technique to synthesize a lipophilic carrier for hydrophobic and toxic potent cancer
drugs, such as gold(III) porphyrin.

Key words Lipophilic formulation, Gold porphyrin, Hydrophobic drugs, Carrier, Nanoparticles

1 Introduction
Research in various methods of drug delivery has been a hot topic
since early 1900s (1–3), as evident by the enormous number of
published articles in the literature. The idea of using drug delivery
system was first initiated with the aim of raising the drug concen-
tration in blood (1, 4).
With the increase in the mean age of the population, diseases
such as cancer, diabetes, and obesity have become the focus for
pharmaceutical companies to meet the demand of market. Cancer
is a common disease which kills 13% of the population worldwide
every year, according to statistics from World Health Organization
(WHO). Discovery of novel and effective cancer drugs represents
significant advances in academic or pharmaceutical research (5–7).
It is, however, a long way before a drug can be sold on the market.
Indeed, although many novel and potent cancer drugs are under-
going the clinical trial, only a few become pharmaceutical products
due to adverse side effect of the cancer drugs. Nonetheless, this has

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_10, © Springer Science+Business Media New York 2013

93
94 Puiyan Lee and Kenneth K.Y. Wong

not hampered the enthusiasm in researching various models of


cancer drug delivery systems. Considerable amounts of efforts and
money have been put in designing novel drug delivery systems
(8–11), with the ultimate aim to facilitate the effectiveness and
reduce the side effects of cancer drugs.

1.1 Drug Delivery A drug delivery system is necessary to overcome the challenges in
System and Drug clinical and pharmaceutical areas, assuming that a greatly effective
Encapsulation anticancer drug is available. The issues involving drug encapsula-
tion release and targeting need to be tackled so that the effect of
drug can be maximized in a site-specific manner. Drug encapsula-
tion is the most important issue in improving drug delivery.
Encapsulating a drug within a carrier stabilizes the drug and pro-
tects it against interaction with blood proteins, resulting in an
increase in the circulation time. Toxic cancer drugs shielded within
a carrier can further reduce the side effects to healthy tissues.
A carrier vehicle also acts as a platform for anchoring tumor-targeting
ligands to aid site-specific delivery. Since the therapeutic effect
depends on the drug availability and concentration, degradation of
the carrier has to be guaranteed for drug dissociation from the
vehicle carrier at the tumor site.
To meet these criteria, an ideal drug carrier would be inert but
biodegradable and biocompatible in the aqueous body environ-
ment. Among different carriers, which include polymers, lipid has
been considered as a more physiological option and is expected to
have higher biocompatibility. Lipid naturally becomes liposome
vesicle during self-assembly in an aqueous environment. The use of
liposome for drug delivery began in 1980 (12), with liposome-
encapsulating doxorubicin, Doxil®, being the first FDA-approved
liposomal chemotherapeutic agent in 1995 (13). Despite the suc-
cess of Doxil®, approximately 40% of other potent and effective
cancer drugs are hydrophobic (14). The encapsulation efficiency of
hydrophobic drug within liposome is not satisfactory because, in
most cases, the hydrophobicity of the drug during the self-assem-
bly process renders the highly hydrophobic drug in the vesicle
membrane, leaving it unencapsulated on the surface (15).
Here we describe the use of fatty acids to synthesize lipophilic
formulation for the encapsulation of hydrophobic cancer drugs,
such as the highly potent but hydrophobic nanometallic gold por-
phyrin. We were able to incorporate gold porphyrin into the lipo-
philic carrier, with >90% of the compound encapsulated in the
lipophilic carrier (16). The size of the final particle was around
101.94 ± 27.9 nm, as measured by transmission electron micros-
copy (TEM) (Fig. 1). We used direct light scattering (DLS) method
to measure the polydispersity index (PDI) of 0.32. This is impor-
tant as nanoparticles <200 nm have been suggested to be the most
effective in delivering drugs to tumor sites (17).
Lipophilic-Formulated Gold Porphyrin Nanoparticles for Chemotherapy 95

Fig. 1 Morphology and size of the gold porphyrin lipidic nanoparticle (GPNP) TEM
shows the spherical morphology

2 Materials
Prepare the nanoparticle emulsion with clean glassware. Thoroughly
clean the glassware and spatula with concentrated nitric acid fol-
lowed by ethanol. Make sure no residue of white fatty acid and
reddish gold porphyrin remain in the glassware. Furthermore, use
double distilled water during the entire synthesis to guarantee the
purified grade. Follow the organic disposal regulation for the dis-
posal of emulsion residue if there is disposal residue with gold
porphyrin.
1. Organic phase: fatty acid cetyl alcohol.
2. Polysorbate surfactant: Brij 78.
3. Double distilled (DDI) water.
4. 5 mL small beaker. Make sure the bottom is flat so the gold
porphyrin (or any other drug to be used) can be evenly distrib-
uted in the organic phase when the nanoparticle template is
formed.
5. Magnetic stir bar (<1 cm in length) so it can fit into the small
5 mL beaker. Rinse with ethanol before use.
6. Weigh balance (up to 0.001 mg in precision for accuracy).
96 Puiyan Lee and Kenneth K.Y. Wong

7. Gold(III) porphyrin 1a was manufactured and kindly provided


by the Department of Chemistry, HKU. The characterization
identified with UV–vis (CH2Cl2) λmax/nm (log): 409 (5.68),
521 (4.73). 1H NMR (CDCl3): δ 9.28 (s, 8H), 7.89 (m, 8H),
8.24 (d, 8H), and 7.89 (m, 4H). m/z = 809 (see Note 1).

3 Methods
Perform all the procedure at 60°C unless specified.
1. Turn on the hot plate and adjust to 60°C. Prewarm the clean
5 mL beaker on the hot plate for 5 min (see Note 7).
2. Add the cetyl alcohol pellet into the prewarmed 5 mL beaker.
Melt cetyl alcohol in the 5 mL beaker. When cetyl alcohol is
melted, you will notice that the white cetyl alcohol pellet
becomes clear solution.
3. Put 0.1 mg gold porphyrin into the melt organic phase cetyl
alcohol (the maximum drug loading to organic phase does not
exceed 1:1 ratio) (see Notes 1–3). Allow the gold porphyrin to
dissolve in the organic phase for 5 min. No need to stir. When
it is completely dissolved, you will notice mixture of the gold
porphyrin fatty acid turning to clear reddish color.
4. Add the surfactant Brij 78 in reddish gold porphyrin-fatty acid
platform. Stir the surfactant Brij 78 for 5 min with clean mag-
netic stir bar (see Notes 5–8). The mixture becomes clear red
again when Brij 78 is melted into the wax platform.
5. Nanoemulsion—emulsifying the organic platform with aque-
ous solution to form nanosized particles: Use 1 mL pipette to
add drop by drop 1 mL of DDI water into the fatty acid plat-
form. This is to make sure the fatty acid platform will not cool
down dramatically. Add a new drop promptly after the previous
drop blends well into platform.
6. Keep stirring the platform gently in water for 20 min on hot
plate (see Note 5).
7. Remove the beaker from the hot plate. Leave the beaker at
room temperature for 10 min to cool down the nanoparticle
solution (see Note 4).
8. Use 0.2 mm filter to sterilize the nanoparticles solution before
in vivo injection (see Note 9).
9. Store the nanoparticle solution at 4°C for later use (see Note 10).
10. The morphology of the encapsulated nanoparticles can be
observed using transmitting electron microscopy (TEM). The
size of nanoparticles can be measured using dynamic light scat-
tering (DLS).
Lipophilic-Formulated Gold Porphyrin Nanoparticles for Chemotherapy 97

4 Notes

1. Gold(III) porphyrin 1a was manufactured and kindly provided


by the Department of Chemistry, HKU (18, 19).
2. Wear a mask when weighing and transferring the gold porphy-
rin (or any other toxic compounds) to avoid exposure by inha-
lation. Clean the balance and cover the weigh boat with plastic
wrap to avoid gold porphyrin exposing to colleagues and lab-
mates.
3. The addition of gold porphyrin in the beaker should be per-
formed in the fume hood. The rest of synthesis is suggested to
be in the fume hood to avoid the exposure of the gold
porphyrin.
4. The surfactant has to be coated on the surface to be compatible
with the aqueous environment.
5. Carefully keep track of the reaction time and temperature to
control the size of the nanoparticles.
6. The stirring should be slow to avoid spillage from the beaker
for safety and quality control.
7. The platform mixture sometimes accumulates at the edge at the
bottom of the beaker and cannot be reached by the stir bar.
This is due to the uneven thickness of bottom surface. Avoid
loss of mixture with the help of spatula.
8. Make sure the bottom of the beaker is flat and even thickness.
This also helps easier stirring with the stir bar and forming bet-
ter emulsion.
9. Nanoparticles are best prepared fresh just before use.
10. The nanoparticle solution, if not in use, should be best stored
at 4°C. Discard it if you notice increased turbidity.

References
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8. Cinti C, Taranta M, Naldi I, Grimaldi S (2011) 14. Tang R, Ji W, Panus D, Palumbo RN, Wang C
Newly engineered magnetic erythrocytes for (2010) Block copolymer micelles with acid-
sustained and targeted delivery of anti-cancer labile ortho ester side-chains: synthesis, charac-
therapeutic compounds. PLoS One 6(2): terization, and enhanced drug delivery to
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189–194 Che CM, Wong KKY (2012) Enhancement of
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analog RC-160 on endocrine status and tumor effects of lipidic formulated gold-porphyrin
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Chapter 11

Mitochondria-Specific Nano-Emulsified Therapy


for Myocardial Protection Against Doxorubicin-Induced
Cardiotoxicity
Amy Faulk, Volkmar Weissig, and Tamer Elbayoumi

Abstract
The quinonoid anthracycline, doxorubicin (Adriamycin), is a widely used potent antineoplastic agent,
showing the broadest spectrum of antineoplastic activity against various types of solid carcinomas, hema-
tological malignancies, and soft tissue sarcomas. Unfortunately, the clinical use of doxorubicin is associated
with cumulative dose-limiting cardiac toxicity, manifested as cardiomyopathy and congestive heart failure,
in which mitochondrial damage is primarily implicated. Free radical formation at and inside mitochondria,
in particular the rise of reactive oxygen species (ROS), has long been hypothesized as the common mecha-
nism by which doxorubicin causes this severe cardiotoxicity. Concomitant with newly gained insights into
the central role of mitochondria in programmed cell death (apoptosis), irreversible destabilization of mito-
chondrial membrane permeability transition (mMPT), and disruption of mitochondrial Ca2+ homeostasis
have been strongly implicated in triggering myocardial apoptosis, due to accumulated doxorubicin dosing.
Hence, our current protocols show the development of mitochondria-targeted nanoemulsions (NEs),
based on previous work using nano-vesicle surface modification with mitochondriotropic triphenylphos-
phonium (TPP) ligands, which have successfully been demonstrated to target drug and DNA-loaded
liposomes to mitochondria in living mammalian cells. Our mitochondria-specific TPP-coated therapeutic
NEs are prepared using tocopherol oxygen scavengers and are highly loaded with mitochondria-stabilizing
therapeutics, namely, cyclosporine A (CsA). Our targeted nano-formulation, proposed as injectable adju-
vant therapy, is capable of reaching target affected mitochondria in sufficient therapeutic concentration, in
order to revert or at least limit oxidative and non-oxidative doxorubicin-induced mitochondrial damage,
manifested in affected cardiac muscle tissues, Based on several encouraging studies using in vitro model rat
cardiac muscle, H9C2 cardiomyocytes, and vascular media tunica media, A10, cell cultures, our proof-of
principal mitochondriotropic nano-therapy demonstrates strong potential to improve not only the cardiac
safety profile, through concurrent rescue administration of targeted nano-encapsulated FDA-approved
cyclosporine A (CSA), but also dosing range of the currently available potent adriamycin/doxorubicin-
based chemotherapy regimens.

Key words Anthracycline, Mitochondrial damage, Mitochondriotropic, Cyclosporine A, Cardio-


myocytes, Chaotropic effect, Apoptosis

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_11, © Springer Science+Business Media New York 2013

99
100 Amy Faulk et al.

1 Introduction

Doxorubicin (DOX) (Fig. 1) is an antitumor drug that is being


widely used in the treatment of a wide variety of hematological
malignancies and solid tumors (1). The clinical efficacy of this drug
is often compromised due to the development of a cumulative
dose-dependent cardiac toxicity, characterized by an irreversible
dilated cardiomyopathy and congestive heart failure (2, 3). The
risk of cardiotoxicity is proportional to the cumulative dose of dox-
orubicin received as follows (4): <400 mg/m2 = 0.14%, 550 mg/
m2 = 9%, 700 mg/m2 = 25%; for this reason, a total cumulative dose
of 550 mg/m2 is considered the therapeutic endpoint of doxoru-
bicin therapy (5).
Although several mechanisms have been suggested to explain
this cardiotoxicity, the exact mechanism and its metabolic conse-
quences remain unclear. Biochemical and physiological data favor
the hypothesis that DOX causes the formation of reactive free radi-
cals (ROS) that stimulate lipid peroxidation and alter cellular mem-
brane integrity (6).
The selective accumulation of DOX in mitochondria, coupled
with increased ROS generation, renders cardiomyocytes more vul-
nerable to DOX toxicity.
Mitochondria are the most redox-active organelles and indis-
pensable for cells to initiate or inhibit apoptosis, specifically, since
more than 90% of the ATP utilized by cardiomyocytes is produced
by mitochondrial respiration (7). Subsequently, any alterations in

Fig. 1 Simplified diagram of different mitochondrial MPT activators and inhibitors


Mitochondria-Specific Nano-Emulsified Therapy for Myocardial… 101

mitochondrial function, membrane structure, and the mitochondrial


redox state are most important in determining cell survival and cell
death (8, 9).
It was reported, at low concentrations (50 mM or less), doxo-
rubicin can catalyze a site-specific oxidative damage to the NADH
oxidation pathway. In contrast, tenfold higher doxorubicin con-
centrations (or more) are required for non-oxidative inactivation
of the electron transport chain, probably via binding to cardiolipin
and/or generalized membrane chaotropic effects. Mitochondria
from DOX-treated rats under chronic doxorubicin treatment
expressed a dose-dependent and irreversible decrease in calcium
loading capacity that correlated with the accumulation of DNA
adducts, histopathology, and clinically observed cardiomyopathy.
These findings indicated that this cyclosporine-sensitive altered
regulation of mitochondrial calcium homeostasis may be a critical
factor involved in the pathogenic pathway of the cumulative and
irreversible cardiomyopathy associated with long-term DOX can-
cer chemotherapy. At the same time, these mitochondria-perturba-
tion events are shown to be insensitive to antioxidant treatments,
which cannot reverse the precipitated cardiomyopathy signs at this
late stage of cumulative doxorubicin cancer therapy.
Many reports proposed the direct correction of these MPT-
dependent events in the cumulative doxorubicin toxicity via admin-
istration of MPT stabilizers, such as cyclosporine (CSA) (Fig. 1).
CSA is potentially able to prevent or at least delay doxorubicin-
induced cardiomyopathy first by directly inhibiting mMPT via
cycophilin binding and second by restoring the mitochondrial cal-
cium loading capacity. Restoring the intracellular calcium homeo-
stasis in turn eliminates calcium as a facilitator for mMPT thereby
also preventing or delaying cell death. The low-water solubility and
membranotropic nature of CSA result in or diminished plasma
concentration, as well as pronounced renal toxicity associated with
high systemic doses of CSA, and generally stand against its sufficient
intracellular localization. The main hurdle facing this approach is
the need for relatively high local concentration of these lipophilic
molecules into the mitochondria fraction of the cell, as well as the
means for direct delivery of these drugs into the target mitochon-
dria of affected cardiomyocytes.
We designed a nanocarrier system to mediate the selective
delivery of cyclosporine A (CSA) to and into mitochondria within
cardiomyocytes, which will prevent or at least delay doxorubicin-
induced cardiomyopathy thus allowing to increase the total cumu-
lative lifetime cap dose of DOX. We developed pharmaceutical
formulation of CSA into long-circulating and mitochondria-
targeted lipid-based nanocarrier system, namely, vegetable oil-based
nanoemulsions (NEs). The advantages of nanoemulsion platform
include an opportunity to solubilize highly hydrophobic compounds,
102 Amy Faulk et al.

Fig. 2 Schematic representation of the incorporation of cyclosporine A and vitamin E into mitochondriotropic
STTP-targeted lipidic nanocarriers

such as CSA (Fig. 2) and FK506 that serve as stabilizing ligands for
mMPT. Triphenylphosphonium cations (TPPs) have been used
previously to render long-circulating liposomes mitochondriotro-
pic (10–13). TPPs were conjugated with stearyl residues produc-
ing stearyl-triphenylphosphonium cations (STPP) in order to
facilitate the incorporation of TPPs into lipidic vesicles. The stearyl
residue literally anchors the cation into the lipid bilayer and suc-
cessfully onto the surface of nanoemulsion oil droplets. Based on
previous in vitro and in vivo reports, STPP-coupled lipidic nano-
carriers, such as liposomes and micelles, were capable of mitochon-
dria-targeted delivery of proapoptotic agents, resulting in
significantly increased anticancer activity of the corresponding
agents. Analogously, our recent in vitro studies, our mitochondria-
specific STPP-modified CSA-loaded nanoemulsions, as a targeted
pharmaceutical lipid-based nanocarrier system (Fig. 2), were also
Mitochondria-Specific Nano-Emulsified Therapy for Myocardial… 103

capable of delivering a sufficient amount of CSA to mitochondria


in cardiac myocytes in order to inhibit or delay doxorubicin-
induced muscle damage and death.
Our targeted nano-therapy is capable of intracellular delivery
of both therapeutic antioxidants and CSA, specifically and effec-
tively preserving cardiac muscle mitochondria, acting as an adju-
vant therapy to combat doxorubicin chemotherapy-induced cardiac
failure; such mitochondriotropic nano-therapy has the potential to
expand the eligible patient profile, effective dose range, and spec-
trum of malignancies, eligible for treatment with potent adriamy-
cin-based chemotherapy regimens. Moreover, our targeted
platform carries significant prospects for treating closely related
oxidative damage molecular mechanisms involved in ischemia-
reperfusion injury, congestive heart failure, cardiomyopathies, and
acute cardiogenic shock.

2 Materials

2.1 Preparation and 1. High omega-3 fatty acid-containing argan oil (Jedwards
Characterization of International, Quincy, MA).
Control and Targeted 2. Phosphate buffered saline (PBS, 200 mM), pH 5.8: dissolve
CSA-Loaded 137 mM NaCl, 2.7 mM KCl, 200 mM Na2HPO4, and 1.8 mM
Nanoemulsions of KH2PO4 in 800 mL of H2O, adjust pH to 5.8 using HCl,
then add H2O up to 1 L.
3. Doxorubicin HCl (DOX, LC Laboratories, Woburn, MA).
Prepare stock solution of 1.7 M of DOX per 1 mL of 1× PBS,
pH 5.8.
4. Chloroform (dry).
5. Ethanol (200% proof).
6. Cyclosporine A (CSA, LC Laboratories, Woburn, MA).
7. HEPES-buffered saline, pH 7.4.
8. Solutol HS-15 (Mutchler Inc.)
9. D-a-Tocopherol (Vitamin E, VE).
10. D-a-Tocopheryl polyethylene glycol 1000 succinate (TPGS,
Antares Health Products Inc, St. Charles, Illinois).
11. Double distilled (DDI) water.
12. 10 and 25 mL pear-shaped glass flasks that fit rotary evaporator
spout, for cosolvent evaporation.
13. Rotary evaporator (Labconco, Kansas City, MO).
14. Ultra-Turrax 10 homogenizer (IKA Works, Inc., Wilmington,
NC).
104 Amy Faulk et al.

15. Sonic probe dismembrator (Misonix XL-2000, Qsonica LLC,


Newtown, CT).
16. Nitrogen gas-operated LIPEX™ extruder (Northern Lipids
Inc., Burnaby, BC, CA).
17. LIPEX™—compatible polycarbonate filter disks size 100 and
200 nm (Northern Lipids Inc., Burnaby, BC, CA).
18. Weigh balance (up to 0.001 mg in precision for accuracy).
19. Malvern Zetasizer Nano ZS (Malvern Instruments,
Westborough, MA).

2.2 Cell Viability 1. One vial of 1 × 105 cells of model rat cardiomyocytes, H9C2
Assay (American Type Culture Collection, ATCC catalogue# CRL-
1446, Manassas, Virginia).
2. ATCC-formulated Dulbecco’s Modified Eagle’s Medium
(DMEM).
3. Fetal bovine serum (FBS added to growth media as 10%
vol/vol).
4. Complete serum-free medium (SFM).
5. Clinical centrifuge at 100–1,000 × g.
6. Cell culture plates 96-well opaque-walled plates compatible
with fluorometer, with clear.
7. Multichannel pipettor.
8. CellTiter-Blue® Cell Viability Assay (Promega, Madison, WI).
9. Fluorescence plate reader with excitation 530–570 nm and
emission 580–620 nm filter pair, Synergy 2 multi-mode
microplate reader (BioTek Instruments, Winooski, VT).

2.3 MitoPT JC-1 1. 15 mL polystyrene centrifuge tube (1 per sample).


Mitochondrial 2. Microfuge at 13,000 × g. Clinical centrifuge at 100–1,000 × g.
Polarization Assay
3. Pipette(s) capable of dispensing at 10 mL, 500 mL, and 1 mL.
4. Graduated cylinder.
5. Cell culture grade sterile dimethyl sulfoxide—DMSO.
6. Vortex mixer.
7. Hemocytometer.
8. Amber vials or polypropylene tubes for storage at –20°C.
9. Cell culture plates 96-well opaque-walled plates compatible
with fluorometer, with clear.
10. Multichannel pipettor.
11. MitoPT™ JC-1 Assay Kit (Catalogue #: 924, ImmunoChemistry
Technologies LLC, Bloomington, MN).
Mitochondria-Specific Nano-Emulsified Therapy for Myocardial… 105

12. Fluorescence plate reader with excitation 530–570 nm and


emission 580–620 nm filter pair, Synergy 2 multi-mode
microplate reader (BioTek Instruments, Winooski, VT).

3 Methods

3.1 Preparations Prepare all nanoemulsions using only clean glassware. Thoroughly
of Mitochondria- clean the glassware and spatulas with concentrated nitric acid fol-
Specific CSA-Loaded lowed by ethanol. Make sure no residue of white phospholipids or
Nanoemulsions drug remains in the glassware. Furthermore, use double-distilled
water (DDW) during the entire formulation processes to guaran-
tee purified grade final product.
Perform all the procedure at 60°C unless specified as follows:
1. Turn on the hot plate and adjust to 60°C. Warm the clean
25 mL beaker on the hot plate for 5 min, filled with DDI
water.
2. In a 25 mL pear-shaped glass flask, add 0.75 g of organic phase,
composed of 65% wt/wt argan oil or flax seed oil, and 35%
wt/wt a-tocopherol Vitamin E (VE). Mix oil component thor-
oughly, using vortex mixer and glass bead bath set at 60°C. In
case of mitochondria-targeted formulation, the mitochondrio-
tropic STTP ligand, dissolved in chloroform (1 mg/mL), will
be mixed with the warm oil phase mixture, as 5% wt ratio.
3. To the organic phase, add 2 mL of CSA dissolved in 100% etha-
nol (10 mg/mL), and mix using vortexer, until clear solution is
obtained.
4. Connect the pear-shaped glass to the rotary evaporator, and
slowly evaporate solvent under 25 PSI vacuum, set at 30 rpm
rotation, and 35°C water bath temperature, for approx.
20–30 min (see Note 1).
5. In a 15 mL glass tube, add 0.25 g total of surfactant mixture
(composed of 0.075 g of vitamin E PGS and 0.175 g of Solutol
HS-15, as weight ratio of 3:7, respectively). Using a heat gun,
mix surfactant components using vortex mixture, while moni-
toring the mixture temperature not to exceed 65°C.
6. While warm, quickly transfer the CSA-oily mixture to the 15 mL
glass tube containing the warm surfactant mixture, and vortex
under heat gun for 15–20 s.
7. Using 1 mL pipette, gradually add drop wise 4 mL of warm
DDI water onto the warm mixture inside the 15 mL glass tube,
mixing thoroughly; this is to make sure the drug-oily platform
will not cool down dramatically. Add a new drop promptly after
the previous drop blends well into platform.
106 Amy Faulk et al.

8. Keep stirring the platform gently using vortex mixer for 5 min,
under distant air heat gun exposure, while monitoring the mix-
ture temperature not to exceed 65°C.
9. The resulting milky macro-emulsion will them be homogenized
for 10 min, at 20,000 rpm setting.
10. Following, the resulting microemulsion will be sonicated for
using probe sonicator (8 W energy input, for three periods of
5 min each, resting for 1 min in between).
11. Finally, transfer the resulting nanoemulsion to the LIPEX™
extruder, and pass once, under 100 PSI nitrogen gas pressure
using first 0.2 mm filter disk; then, run another pass using the
100 nm filter (see Note 2).
12. Store the NE formulations at 4°C for later use.

3.2 Physical The nanoemulsion formulations will be characterized for particle


Characterization size and size distribution using the dynamic light scattering tech-
of STPP-Coupled nique with a Malvern nanosizer analyzer (Malvern instruments,
and Control Holtsville, NY) at 273° fixed angle and at 25°C temperature:
Nanoemulsions 1. Dilute NE formulation, for particle-size analysis, using DDI
water deionized at about 1,000-fold vol/vol, in plastic cuvette.
The numbered average oil droplet hydrodynamic diameter and
the polydispersity index will be determined (Fig. 3, panel a).
2. For the zeta potential, dilute NE samples in DDI water as
10,000-fold; then, employ a 1 ml syringe to inset solution inside
the electrophoretic cell of the Malvern nanosizer to avoid insert-
ing any air bubbles. The average surface charge will be mea-
sured (Fig. 3, panel b) (14).

3.3 Cell Viability Thaw CellTiter-Blue® reagent and bring to ambient temperature,
Assay Protocol and protect the CellTiter-Blue® reagent from direct light.
1. Set up 96-well assay flat bottom plates, containing approxi-
mately 2 × 104 cells in 100 mL of complete DMEM cell culture
media per well (15).
2. Allow H9C2 cells to attach to the bottom of the plate for 24 h.
Cell would be ready to the next step when they are about
60–70% confluent (see Notes 3 and 4).
3. Add doxorubicin HCl challenge (50 mM, diluted in serum-free
medium, SFM) to both positive control wells, as well as all
appropriate test well, so the final volume is 100 mL in each well.
Then, co-incubate at 37°C, for 2 h, followed by removal of
media from all wells (see Note 5).
4. Add the various NE vehicle controls, as well as test NEs
containing the different CSA and VE treatment, all diluted
100× at minimum, in SFM, and applied to test wells in two-
fold serial dilution pattern. Make sure the final volume is 100 mL
in each well.
Mitochondria-Specific Nano-Emulsified Therapy for Myocardial… 107

Fig. 3 Physicochemical characterization of STTP-coupled nano-emulsified lipid carriers, showing average


particle-size distribution (a) and average zeta-potential measurement (b) of typical CSA-loaded nanoemulsion
formulation

5. Culture cells for 30 h test exposure period.


6. Remove assay plates from 37°C incubator, and remove all NE
media and SFM controls.
7. Apply cell wash to all wells containing cells, by carefully and
slowly adding 100 mL of Hank’s balanced buffer saline (HBS),
leaving in well for 5 s and then removing HBS solution; repeat
this step twice for the entire plate, taking extreme caution to
minimize dislodging attached cells inside each well.
8. Finally, add 100 mL of SFM to each well, followed by the addi-
tion of 20 mL/well of CellTiter-Blue® reagent.
9. Shake plate for 10 s, to mix reagent well.
10. Incubate at 37°C, using the same standard cell culture condi-
tions for 1–2 h (see Note 6).
11. Insert the developed plate in the plate reader, and set assay pro-
tocol/method to shake plate for 10 s; then, record end point
fluorescence using ex.560/em.590 nm filter set.
12. Calculate results of fluorescence data; then, plot percent cell
viability [(test well fluorescence—untreated cell control
fluorescence)/100] vs. concentration of NE treatment
(see Note 7) (16).
108 Amy Faulk et al.

3.4 Modified Mitochondrial membrane potential, Dym, is an important parame-


MitoPT™ JC-1 ter of mitochondrial function used as an indicator of cell health. The
Protocols loss of mitochondrial membrane potential (ΔΨ) is a hallmark for
apoptosis. The JC-1 Assay Kit measures the mitochondrial mem-
brane potential in cells. JC-1(5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraeth
ylbenzimidazolylcarbocyanineiodide) is a lipophilic, cationic dye
that can selectively enter into mitochondria and reversibly change
color from green to red as the membrane potential increases.
In healthy cells with high mitochondrial Dym, JC-1 spontane-
ously forms complexes known as J-aggregates with intense red
fluorescence. On the other hand, in apoptotic or unhealthy cells
with low Dym, JC-1 remains in the monomeric form, which shows
only green fluorescence.

3.4.1 Reconstitution The MitoPT™ JC-1 dye reagent is supplied as a highly concen-
of the 100× or 200× trated lyophilized powder. It must first be reconstituted, using cell
MitoPT™ JC-1 Stock culture grade (sterile) DMSO solvent:
1. For the 100 test kit, reconstitute the 100-test vial with 500 mL
DMSO at room temperature (RT), forming a 100× stock.
2. Recap the vial and invert it several times to fully dissolve the
MitoPT™ JC-1 dye reagent.
3. Immediately use the 100×, or aliquot and store it at −20°C (see
Note 8).

3.4.2 Modified MitoPT™ 1. Prepare the 1× working strength MitoPT™ JC-1 solution by
JC-1 Protocol for 96-Well diluting the stock 1:100 with cell culture media, warmed to
Fluorescence Plate Reader 37°C (see Note 9).
2. Using clear 6-well flat bottom plates, seed 0.4 × 106 cells/well,
in approx 2.0 mL volume of complete growth DMEM medium,
and incubate at 37°C for 24–28 h, to allow A10 model cells to
attach and become approx. 60–70% confluent (see Note 10).
3. When cells are ready, remove media, and then, assign triplicate
wells as non-treated cell negative control that receive.
4. Add to cells in the rest of the wells doxorubicin HCl challenge
(50 mM, diluted in serum-free medium, SFM) to both positive
control wells, as well as all appropriate test wells; then, co-incu-
bate plates for 1 h, at 37°C, using the same standard cell culture
conditions.
5. Add the various NE vehicle controls, as well as test NEs con-
taining the different CSA and VE treatment, all diluted equivo-
cally, in SFM, and applied to test wells in twofold serial dilution
pattern. Make sure the final volume does not exceed 2 mL in
each well.
6. Culture cells for 20–24 h test exposure period (see Note 11).
7. Remove assay plates from 37°C incubator, and remove all NE
media and SFM controls.
Mitochondria-Specific Nano-Emulsified Therapy for Myocardial… 109

8. Apply cell wash to all wells containing cells, by carefully and


slowly adding 100 mL of HBS, leaving in well for 5 s, and then
removing HBS solution; repeat this step thrice, for all plates,
taking extreme caution to minimize dislodging attached cells
inside each well.
9. Remove cell wash solution, and then detach cells in each well
using 2 mL of trypsin LE/well.
10. Pellet cells by centrifugation at approx. 200 × g for 5 min at RT
(see Note 12).
11. Carefully remove and discard the supernatants.
12. Resuspend at between 0.5 and 1 × 106 cells in 1 mL of freshly
prepared 1× working strength MitoPT™ JC-1 solution pre-
pared fresh.
13. Gently vortex or pipette the cell pellets to disrupt any cell-
to-cell clumping.
14. Incubate the H9C2 cells (staining with the MitoPT™ JC-1 dye
reagent) at 37°C for 10–15 min in a CO2 incubator.
15. Warm the working strength 1× assay buffer to 37°C .
16. Add 2 mL of 1× assay buffer to each tube, and mix well using
vortex mixer.
17. Centrifuge the stained cells at <200 × g for 5 min at RT; then,
carefully remove and discard the supernatants; and then, gently
vortex the pellets to disrupt any cell-to-cell clumping.
18. Resuspend the cells in 1 mL of 1× assay buffer.
19. Take out a small (50 mL) aliquot of each cell population aliquot
to determine the concentration of both the induced and non-
induced cell populations. Then, add to 250 mL PBS (forming a
1:5 dilution of each), to count the cells using a hemocytometer
(see Note 13).
20. Centrifuge the remaining stained cells at 400 × g for 5 min at
RT; then, carefully remove and discard supernatants.
21. Gently vortex the pellets to disrupt any cell-to-cell clumping.
22. Adjust the volume of the induced cell suspension to match that
of the non-induced suspension. A minimum of 1 × 105 cells/
well is recommended to generate an adequate fluorescence sig-
nal using most 96-well plate readers (see Note 14).
23. For each sample to be tested, dispense 100 mL into each of 2–4
wells in a black round or flat bottom 96-well microtiter plate.
24. After inserting the plate, set the plate reader to perform an end-
point read, where the excitation wavelength is at 488–490 nm
and then the emission wavelengths to 527 nm for green
fluorescence and 590–600 nm for red fluorescence.
110 Amy Faulk et al.

3.4.3 Modified MitoPT™ Following the fluorescence microscope protocol, each sample to
Fluorescence Microscopy be stained requires only 0.5 mL of 1× MitoPT™ JC-1 solution
JC-1 Staining Protocol for (equal to 5 mL of 100× MitoPT™ JC-1 stocks):
Adherent Cells
1. Using 6-well plates, culture cells at about seed 0.2 × 106 cells/
well, in approx 2.0 mL volume of complete growth DMEM
medium, and incubate at 37°C for 24–28 h, to allow H9C2
model cells to attach and become approx. 60–70% confluent
(see Note 10). Cell density should not exceed the threshold
where cell sloughing occurs.
2. Follow steps 3–8 from the previous method of MitoPT™ JC-1
protocol for 96-well fluorescence plate reader (see 3.4.2) to
induce apoptosis.
3. Remove the cell wash solutions from both induced and non-
induced monolayer cultures.
4. Add sufficient fresh 1× MitoPT™ JC-1 solution (approx 0.1 mL)
to cover the cells on the cover slip, inside each well.
5. Incubate the cells, stained with the MitoPT™ JC-1 dye reagent,
at 37°C for 15 min in a CO2 incubator.
6. Warm the 1× assay buffer to 37°C; then, carefully remove and
discard staining media.
7. Wash the monolayer culture on the cover slips with 1 mL of 1×
assay buffer, twice, and then discard wash solution.
8. Add a drop of 1× assay buffer to cell culture slides; then, invert
each cover slip on a glass slide, cell surface down.
9. Examine using fluorescence microscope.

4 Notes

1. Complete removal of ethanolic solvent is confirmed when a


clear translucent running yellow color gel-like residue remains
in the flask that gets somewhat thicker as the flask temperature
cools down. The residue must be clear from any suspending
white CSA drug precipitates.
2. Pre-warm the thermobarrel LIPEX™ extruder to about 40°C
(measured externally), before running the NE sample, to guar-
antee smooth flow-pass through the filter. Make sure to run
sample through the larger pore-size filter disk first before the
smaller pore-size one, to avoid clogging of the filter disk.
3. Set up triplicate wells without cells to serve as the negative con-
trol to determine background fluorescence that may be
present.
4. Set up quadruplicate wells with untreated cells to serve as a
vehicle control. Add the same solvent used to deliver the test
Mitochondria-Specific Nano-Emulsified Therapy for Myocardial… 111

compounds to the vehicle control wells (17). Since, test cells are
subjected to both doxorubicin HCl and NE treatments diluted
in serum-free cell culture medium, hence, use the same serum-
free cell culture medium for untreated cell control wells.
5. DOX only treated cell wells will serve as the positive control for
cytotoxicity, using quadruplicate wells containing cells treated
with a DOX (50 mM, diluted in SFM) known to be toxic to the
A10 cell model system. Only SFM media (no NE treatments)
will be added to these wells, in the steps to follow in the assay.
6. Fluorescence generated can be stopped and stabilized by the
addition of 3% SDS. Typically, add 40 mL per 100 mL in each
well. The plate can then be stored at ambient temperature for
up to 24 h before recording data, provided that the contents are
protected from light and covered to prevent evaporation.
7. Optional: Subtract the average of fluorescence values of the cul-
ture medium background from all fluorescence values of experi-
mental wells (17).
8. The 1X working solution must be prepared immediately prior
to use; however, the reconstituted 100× or 200× stock can be
stored at –20°C for 6 months and used twice during that time.
As the 1× MitoPT™ JC-1 solution must be used immediately,
prepare the MitoPT™ reagents at the end of your metabolic
stress or apoptosis induction process.
9. Each sample to be stained requires only 0.5 mL of 1× MitoPT™
JC-1 solution (i.e., equivalent to 5 mL of 100× MitoPT™ JC-1
stock).
10. Cell density in the cell culture flasks should not exceed 106 cells
per mL (i.e., about 1–1.5 × 106 cells/well of the 6-well plate).
Cells cultivated in excess of this concentration may begin to
naturally enter apoptosis. Optimal cell concentration will vary
depending on the cell line used. Density can be determined by
counting cell populations on a hemocytometer.
11. Generation of experimental apoptosis or Dym-disrupted cells
oxidative stress may take few hours up to 48 h, depending on
model cell line, cell culture conditions, and the inducer test
concentration.
12. When concentrated, cells should have been grown to yield a
0.5 mL concentrated pool between 1 and 2 × 106 cells/mL.
13. After counting, compare the density of each. The non-induced
population may have more cells than the induced population, as
some induced cells may be lost during the apoptotic process. If
necessary, adjust the volume of the induced cell suspension to
match that of the non-induced suspension.
14. Resuspend the non-induced cell pellets in 500 mL to 1 mL of 1×
assay buffer to produce a cell suspension about 1 × 106 cells/mL,
112 Amy Faulk et al.

which is generally sufficient to generate enough signal. The vol-


ume may vary depending upon cell density. It is possible to get by
using a lower cell number/well, depending on the particular cell
model system and fluorescence detection plate reader, but this
should be predetermined for each individual case.

References
1. Lefrak EA, Pitha J, Rosenheim S, Gottlieb JA improve the apoptotic and cytotoxic action of
(1973) A clinicopathologic analysis of adri- sclareol. J Liposome Res 20:244–249
amycin cardiotoxicity. Cancer 32:302–314 11. Boddapati SV, D’Souza GG, Erdogan S,
2. Allen A (1992) The cardiotoxicity of chemo- Torchilin VP, Weissig V (2008) Organelle-
therapeutic drugs. Semin Oncol 19:529–542 targeted nanocarriers: specific delivery of lipo-
3. Doroshow JH (1983) Effect of anthracycline somal ceramide to mitochondria enhances its
antibiotics on oxygen radical formation in rat cytotoxicity in vitro and in vivo. Nano Lett
heart. Cancer Res 43:460–472 8:2559–2563
4. DiPiro JT, Talbert RL (2002) Pharmacotherapy: 12. Weissig V, Boddapati SV, Cheng SM, D’Souza
a pathophysiologic approach, 5th edn. GG (2006) Liposomes and liposome-like vesi-
McGraw-Hill, London cles for drug and DNA delivery to mitochon-
5. Gabizon AA, Lyass O, Berry GJ, Wildgust M dria. J Liposome Res 16:249–264
(2004) Cardiac safety of pegylated liposomal 13. Boddapati SV, Tongcharoensirikul P, Hanson
doxorubicin (Doxil/Caelyx) demonstrated by RN, D’Souza GG, Torchilin VP, Weissig V (2005)
endomyocardial biopsy in patients with Mitochondriotropic liposomes. J Liposome Res
advanced malignancies. Cancer Invest 15:49–58
22:663–669 14. Ganta S, Amiji M (2009) Coadministration of
6. Doroshow JH (1983) Anthracycline antibi- Paclitaxel and curcumin in nanoemulsion for-
otic-stimulated superoxide, hydrogen perox- mulations to overcome multidrug resistance in
ide, and hydroxyl radical production by NADH tumor cells. Mol Pharm 6:928–939
dehydrogenase. Cancer Res 43:4543–4551 15. Mu L, Elbayoumi TA, Torchilin VP (2005)
7. Tokarska-Schlattner M, Zaugg M, Zuppinger Mixed micelles made of poly(ethylene glycol)-
C, Wallimann T, Schlattner U (2006) New phosphatidylethanolamine conjugate and
insights into doxorubicin-induced cardiotoxic- d-alpha-tocopheryl polyethylene glycol 1000
ity: the critical role of cellular energetics. J Mol succinate as pharmaceutical nanocarriers for
Cell Cardiol 41:389–405 camptothecin. Int J Pharm 306:142–149
8. Wallace KB (2007) Adriamycin-induced inter- 16. Elbayoumi TA, Pabba S, Roby A, Torchilin VP
ference with cardiac mitochondrial calcium (2007) Antinucleosome antibody-modified
homeostasis. Cardiovasc Toxicol 7:101–107 liposomes and lipid-core micelles for tumor-
9. Zhou S, Starkov A, Froberg MK, Leino RL, targeted delivery of therapeutic and diagnostic
Wallace KB (2001) Cumulative and irrevers- agents. J Liposome Res 17:1–14
ible cardiac mitochondrial dysfunction induced 17. Lukyanov AN, Elbayoumi TA, Chakilam AR,
by doxorubicin. Cancer Res 61:771–777 Torchilin VP (2004) Tumor-targeted lipo-
10. Patel NR, Hatziantoniou S, Georgopoulos A, somes: doxorubicin-loaded long-circulating
Demetzos C, Torchilin VP, Weissig V, D’Souza liposomes modified with anti-cancer antibody.
GG (2010) Mitochondria-targeted liposomes J Control Release 100:135–144
Chapter 12

Formation of Pit-Spanning Phospholipid Bilayers


on Nanostructured Silicon Dioxide Surfaces for Studying
Biological Membrane Events
Indriati Pfeiffer and Michael Zäch

Abstract
Zwitterionic phospholipid vesicles are known to adsorb and ultimately rupture on flat silicon dioxide
(SiO2) surfaces to form supported lipid bilayers. Surface topography, however, alters the kinetics and
mechanistic details of vesicles adsorption, which under certain conditions may be exploited to form a sus-
pended bilayer. Here we describe the use of nanostructured SiO2 surfaces prepared by the colloidal lithog-
raphy technique to scrutinize the formation of suspended 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) lipid bilayers from a solution of small unilamellar lipid vesicles (SUVs). Atomic force microscopy
(AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) were employed to charac-
terize nanostructure fabrication and lipid bilayer assembly on the surface.

Key words Nanostructured surfaces, Phospholipid vesicles, POPC, BLM, QCM-D, AFM, Colloidal
lithography, Biosensor

1 Introduction

Phospholipid bilayers have chemical and physical properties that


closely resemble the biological cell membrane (1–3) and have there-
fore been frequently used in biosensor applications to tether mem-
brane proteins to the sensor surface (4–6). The bilayer provides a
natural environment for the protein and ideally functions as a cush-
ion, which prevents direct interaction of the protein with the sur-
face, two important prerequisites to preserve the protein’s
biorecognition sites and function (7, 8). On flat SiO2 surfaces, small
unilamellar phospholipid vesicles (SUVs) are known to adsorb and
rupture to form a supported phospholipid bilayer (9, 10). The kinet-
ics and mechanistic details of this process on flat surfaces have been
investigated using a number of surface analytical techniques, e.g.,
quartz crystal microbalance with dissipation monitoring (QCM-D)

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_12, © Springer Science+Business Media New York 2013

113
114 Indriati Pfeiffer and Michael Zäch

(11, 12) combined with atomic force microscopy (AFM) (13, 14),
which together with computer simulations (15) have provided a
thorough understanding of the process and thus ensure reproduc-
ible bilayer formation (14, 16). There has recently been a growing
interest for preparation methods, which are able to form continuous
lipid bilayers on substrates with nanoholes (17–19). Such pore-span-
ning membranes are attractive because they offer access to the liquid
reservoir on both sides of the lipid bilayer (20). Also, they are advan-
tageous for proteins with large extramembrane domains, which
might lose their functionality if reconstituted into a bilayer sup-
ported directly on a flat substrate (21). The nanometer size of the
pores grants a high stability of the suspended lipid membrane and
opens up possibilities to use this system for certain biotechnological
applications such as membrane protein arrays (22, 23) and ion chan-
nel protein biosensors (24, 25). Several different ways to form sus-
pended lipid bilayers on various types of surfaces and methods to
characterize their stability have been reported, including lipid paint-
ing methods or giant unilamellar vesicles spreading on micro-/
nanofabricated silicon nitride (Si3N4) surfaces (23, 26) and the use
of lipopolymer bilayers on porous aluminum oxide (Al2O3) surfaces
(27, 28). The formation of suspended bilayers on top of these sur-
faces was characterized electrochemically by measuring the electrical
resistance across the pores (21, 29). A drawback of this technique is
that no kinetic or mechanistic information of suspended bilayer for-
mation can be obtained, with poorly reproducible bilayer formation
as a possible consequence. In addition, the production of suitable
Si3N4 surfaces requires rather advanced micro-/nanofabrication pro-
cedures (30), and the anodization of aluminum to Al2O3 produces
less controllable size and dimension of the porous surfaces (21, 31).
Here, we describe a simple method termed colloidal lithography
(32, 33), which allows one to create surfaces with homogenous
nanoscale pits or through holes (34) and multiple surface chemis-
tries. As an example, we describe below the fabrication of pitted
surfaces with SiO2 forming the walls of the pits and Au constituting
the bottom of the pits (Fig. 1). In order to form a pit-spanning
bilayer, biotin-amidocaproyl bovine serum album in (BBSA) was
added to the system in a first step in order to passivate exposed Au
areas at the bottom of the pits against lipid vesicles adsorption (35).
Subsequently, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) lipid vesicles with 160 ± 40 nm average diameter were intro-
duced to the surface. Using this strategy, phospholipid vesicles that
adsorb on the SiO2 top surface rupture and form a continuous bilayer
that spans over the pits (Fig. 1f). Since we would like to follow the
bilayer formation process in situ using the QCM-D technique, we
produced the SiO2-pitted surface on top of Au-coated QCM-D sen-
sor crystals. Compared to supported bilayer formation on flat SiO2,
QCM-D revealed that the kinetics and the mechanism of vesicle
adsorption and bilayer formation are altered on these nanostruc-
tured surfaces (Fig. 2). The formation of a suspended phospholipid
Formation of Pit-Spanning Phospholipid Bilayers on Nanostructured Silicon… 115

a b - - - - c - - - -
+++++++++++++++ +++++++++++++++ +++++++++++++++

d e f

Strip-off

- - - -
+++++++++++++++

Fig. 1 Schematic illustration of the fabrication of nanostructured SiO2 surface using colloidal lithography
and the QCM-D measurement setup used to monitor the formation of a suspended phospholipid bilayer:
(a) Deposition of polyelectrolyte triple layer on gold-coated sensor crystal. The final PDDA polyelectrolyte
layer creates a positively charged surface. (b) Deposition of negatively charged polystyrene particle sus-
pension on top of polyelectrolyte triple layer. (c) Deposition of thin films of Ti and SiO2 using electron beam
evaporation. (d) Polystyrene particle stripping using an adhesive tape. (e) The end product; a homogenous,
pitted SiO2 surface. (f) Adsorption of BBSA and the formation of a suspended phospholipid bilayer on the
nanostructured silicon dioxide surface, as measured by the QCM-D technique

10 7

0 6

DD 5
-10
DD(10e-6)

4
-20 f eq
Df(Hz)

3
-30 2
f min
-40 1
Df
0
-50
t min
-1
2 3 4 5 6 7 8
Time (min)

Fig. 2 Example of QCM-D graphs showing changes in frequency and dissipation (Df, DD) as a function of time
and monitoring the kinetics of supported phospholipid bilayer formation on a flat SiO2 surface (dashed lines)
and suspended phospholipid bilayer formation on a BBSA-modified nanostructured SiO2 surface (solid lines).
Both graphs show similar feq, indicating that a continuous phospholipid bilayer was formed on both surfaces.
However, as shown by the values of fmin, the addition of nanotopography on the surface alters the critical
vesicular coverage (the surface concentration of vesicles requires for triggering the rupture of vesicles) (flat
surface, fmin, dash arrow; nanostructured surface, fmin, solid arrow), and accelerates lipid bilayer
formation
116 Indriati Pfeiffer and Michael Zäch

Fig. 3 3D topography images of pit-spanning bilayer as acquired at three different imaging forces using AFM.
The height scale is 25 nm for all three images, and the lateral dimensions are ca. 100 nm × 175 nm. The
apparent pit depth increases with increasing imaging force due to bilayer compliance, as illustrated by the
cross sections in the lower right panel. For the largest imaging force shown here, the bilayer is pushed all
the way down to the underlying BBSA layer. The theoretically expected pit diameter (as given by the colloid
size) and pit depth (as given by the total evaporated film thickness minus the thickness of a BBSA layer) are
indicated by the dotted line

bilayer was verified using AFM, which revealed a characteristic


dependence of the apparent pit depth on the applied imaging force
due to deformation of the pit-spanning membrane (Fig. 3). Results
were highly reproducible, owing to the homogeneity of surface nan-
otopographies produced by colloidal lithography.

2 Materials

Purified water with an electrical resistance of 18.2 MW at 25ºC was


used to prepare all solutions. All buffers and solutions used in the
experiments were prepared and stored at room temperature unless
otherwise specified. Waste solutions were handled as instructed by
local waste disposal regulations.

2.1 Colloidal 1. A gold-coated QCM-D sensor crystal with 5 MHz resonance


Lithography frequency and 13 mm diameter.
Components 2. A pair of tweezers (see Note 1).
Formation of Pit-Spanning Phospholipid Bilayers on Nanostructured Silicon… 117

3. Hydrogen peroxide, 30%.


4. Aqueous ammonia solution, 28%.
5. Water.
6. A flat beaker with a diameter of around 15 cm.
7. A 100 mL glass beaker.
8. A custom-made Teflon holder used to carry the QCM-D sen-
sor crystal (see Note 2).
9. Poly-(Diallyl-Dimethyl-Ammonium) chloride (PDDA) poly-
electrolyte: 2% w/w solution in water. Weigh 1 mg of PDDA
(20% w/w solution, MW = 200,000–350,000) and mix it with
49 mL of water in a 50 mL falcon tube (see Note 3).
10. Poly (sodium 4-Styrene-Sulfonate) (PSS) polyelectrolyte: 2%
w/w solution in water. Weigh 1 g of PSS powder
(MW = 70,000) in a 50 mL falcon tube. Add 49 mL of water
to dissolve (see Note 3).
11. Polystyrene particle suspension: 0.2% v/v solution in water.
Mix 200 mL of polystyrene particle suspension with average
diameter of 107 nm with 9.8 mL of water in a 15 mL falcon
tube to make a stock solution of 2% v/v. Store the stock solu-
tion at 4ºC. Prior to use, take 1 mL of stock solution and mix
it with 9 mL of water in a 15 mL falcon tube. Use immediately
(see Note 4).
12. N2 gun (see Note 5).
13. Clean room wipes.
14. Titanium (Ti) evaporation source.
15. Silicon dioxide (SiO2) evaporation source.
16. Adhesive tape (see Note 6).
17. A 20 mL flat beaker filled with isopropyl alcohol.
18. A 20 mL flat beaker filled with water.

2.2 Phospholipid 1. Vesicle preparation buffer: 100 mM NaCl, 10 mM Tris, pH 8.


Vesicle Solution Weigh 5.84 g NaCl and 1.21 g Tris, and transfer to a 1 L glass
Components beaker. Add water to a volume of 900 mL. Mix and adjust the
pH using HCl. Add water up to 1 L and filter through a
0.45 mM pore-size Nalgene filter. Store at room temperature
and degas prior to use.
2. Phospholipid: 1-palmitolyl-2-oleoyl-sn-glycero-3-phosphocho-
line (POPC) lysophilized powder. Store at −20ºC (see Note 7).
3. Chloroform.
4. A 1 L tank of liquid N2.
5. A 1 L beaker filled with warm water around 50ºC.
6. Polycarbonate membranes with 100 nm pore size.
118 Indriati Pfeiffer and Michael Zäch

2.3 QCM-D and AFM 1. Hellmanex cleaning solution: 1% v/v solution in water. Pipette
Measurement 1 mL of Hellmanex solution to a 100 mL glass bottle. Add
Components water up to 100 mL. Store at room temperature.
2. Measurement buffer: same buffer as for vesicle preparation (see
item 1 in Subheading 2.2).
3. Biotin-amidocaproyl bovine serum albumin (BBSA) stock solu-
tion: 1 mg/mL concentration in water. Add 2 mL of water to a
bottle containing 10 mg BBSA lysophilized powder. Gently tap
the bottle to help the protein to dissolve. Transfer this 2 mL
BBSA solution to a 15 mL falcon tube. Rinse the bottle with
1 mL of water, and then transfer the water to the same falcon
tube. Repeat the rinsing procedure one more time before adding
6 mL of water to the falcon tube to make a total of 10 mL BBSA
solution. Distribute this solution into Eppendorf tubes, each
containing 50 mL protein aliquots, and store at −20ºC.
4. A pair of tweezers: same as used for colloidal lithography
(see item 2 in Subheading 2.1).
5. AFM tip: commercially available MSCT-AUNM Microlever-
sharpened silicon nitride tip supported by a cantilever with a
spring constant below 30 pN/nm (or equivalent product).

2.4 Surface 1. Sodium dodecyl sulfate (SDS) solution: 10 mM. Weigh 0.72 g
Regeneration SDS powder and mix it with 250 mL of water in a glass beaker.
Component Store at 25ºC (see Note 8).
2. A small glass Petri dish.

3 Methods

3.1 Fabrication It is preferable to carry out all nanofabrication steps in a relatively


of Nanostructured dust-free environment. The use of a clean room is however not
Surface Using compulsory; a good laminar flow cabinet can be used as an
Colloidal Lithography alternative:
1. Cleaning the sensor crystal: fill a flat beaker (15 cm in diameter)
around one third with water, and prepare a hot bath with a
temperature of 80ºC (see Note 9). Arrange the sensor crystal
in the Teflon holder and put both inside a 100 mL glass beaker.
Fill this glass beaker with 50 mL of water, 10 mL of hydrogen
peroxide 30%, and 10 mL of ammonia solution 28%. Put this
beaker in the hot bath for 10 min, take it out, and remove the
sensor crystal. Rinse the sensor crystal thoroughly with water
and blow it dry immediately with N2. Let the mixture inside the
beaker cool down before disposing it into a base waste
container.
Formation of Pit-Spanning Phospholipid Bilayers on Nanostructured Silicon… 119

2. Deposition of polyelectrolyte triple layer on sensor crystal:


pipette PDDA solution onto the sensor crystal and let it stay
for 90 s. Make sure that the polyelectrolyte solution covers the
entire surface area, and prevent the surface from drying. Rinse
the sensor crystal thoroughly with water, and put the wet crys-
tal onto a triple layer of clean room wipes with the Au surface
facing upwards. Position the N2 gun perpendicular around
2 cm above the center of the crystal, and blow the crystal dry
in one shot (see Note 10). Repeat the procedure sequentially
using PSS, and another layer of PDDA.
3. Deposition of polystyrene particle suspension on top of poly-
electrolyte triple layer: pipette the 0.2% v/v polystyrene parti-
cle suspension onto the polyelectrolyte triple layer, which has
previously been deposited, and let it stay for 60 s. Rinse with
water and blow-dry the crystal in the same manner as after
polyelectrolyte deposition.
4. Deposition of metal layer: load the modified sensor crystal, Ti
source, and SiO2 source into a thin film deposition system.
Pump down the system to a pressure of 10−6 mbar (see Note 11).
Set the evaporation angle to normal incidence and electron
beam evaporate 1 nm of Ti, followed by 24 nm of SiO2 at a
deposition rate of 2 Å/s (see Note 12). Let the sources cool
down before venting the chamber and unloading the sample.
5. Stripping-off the polystyrene particles: lay the sensor crystal on
clean room wipes with the sensor surface facing upwards. Cut
the adhesive tape into square-shaped pieces with a size of
2 cm × 2 cm. Apply the tape on the sensor surface and press
gently. Avoid any air bubbles being trapped in between the
tape and the surface. By using a pair of tweezers, strip the tape
off the sensor surface at once (see Note 13).
6. Final cleaning: lay the nanostructured sensor crystal at the bot-
tom of a small, flat beaker containing 20 mL of isopropanol,
and sonicate it for 2 min. Take care so the modified surface will
not touch any surface of the glass beaker or other object such
as tweezers. Repeat the same procedure using water, and blow-
dry the sample immediately using N2 in the same manner as
described previously (see step 2 in Subheading 3.1).

3.2 Preparation 1. Weigh 5 mg of POPC lysophilized powder in the round-bot-


of Lipid Vesicles tom flask. Using a glass pipette, add 1 mL of chloroform to
dissolve the lipid powder. Evaporate the chloroform using a
constant stream of N2 for 1–2 h (see Note 14) to form a thin
lipid film on the round-bottom flask.
2. Dissolve the dried lipid film by adding 1 mL of vesicle prepara-
tion buffer and vortex it gently. Freeze the lipid solution by
dipping the round-bottom flask into liquid N2, and thaw it
120 Indriati Pfeiffer and Michael Zäch

immediately in the 50ºC warm water bath. Repeat this procedure


4–5 times.
3. Assemble the extruder (see Note 15) by following the distribu-
tor’s instructions. During extrusion, pass the lipid solution
back and forth 21 times through a polycarbonate membrane
with 100 nm pore diameter. Store the lipid solution under N2
gas in a 2 mL flask at 4ºC. Make sure to close the flask tightly
and additionally seal it using Parafilm.

3.3 QCM-D 1. General cleaning prior to the measurement (see Note 16): put
Measurements a blank sensor crystal in the measurement chamber, and pass
1 mL of 1% Hellmanex solution through all liquid handling
parts of the instrument (i.e., both the temperature and mea-
surement chamber loops if using a D300 system from Q-Sense).
Stop the flow and let the solution stay inside the measurement
chamber for 30 min. Resume the flow and rinse all liquid han-
dling parts with around 100 mL of water. Take out the blank
sensor crystal, and dry the chamber and tubing thoroughly.
2. Measurement: mount the clean, nanostructured sensor crystal
inside the measurement chamber. Close valves that connected
to all liquid handling parts. Connect a new 5 mL syringe to an
inlet of QCM-D system (if using D300 system from Q-Sense),
and fill it with measurement buffer. Open the valves and fill all
liquid handling parts with measurement buffer (see Note 17).
Set the temperature to 22ºC, and start the measurement by
stabilizing the frequency and dissipation baselines. Fill the inlet
syringe with 1.98 mL of measurement buffer, and pipette 20 mL
of 1 mg/mL BBSA stock solution into this buffer to make a
10 mg/mL BBSA solution. Introduce this solution into the
measurement chamber for 20 min, and rinse immediately with
measurement buffer until the frequency and dissipation shifts
reach stable values. Thereafter, in the same manner as BBSA
(pipette 80 mL of 5 mg/mL vesicle stock solution into 1.92 mL
buffer in the syringe), introduce a vesicle solution with a con-
centration of 200 mg/mL to the measurement chamber. The
adsorption of vesicles and the formation of a bilayer on the sur-
face can be directly monitored as a function of time by follow-
ing the changes in frequency and dissipation values until they
stabilize. The measurement chamber can then be rinsed with
measurement buffer in order to remove excess vesicles.

3.4 AFM 1. Clean the AFM fluid cell and tip holder (see Note 18) by
Measurements immersing them in isopropyl alcohol for 20 min. Rinse all parts
with water, measurement buffer, and water again before blow-
drying them.
2. Clean the cantilever chip by immersing it in measurement buf-
fer for 20 min, followed by careful rinsing with water and
Formation of Pit-Spanning Phospholipid Bilayers on Nanostructured Silicon… 121

blow-drying. Mount the cantilever chip in the tip holder and


coarse-align the laser deflection measurement system accord-
ing to your AFM supplier’s instructions.
3. Transfer the sensor crystal with the freshly formed bilayer from
the QCM-D instrument into the AFM fluid cell, making sure
that the sensor surface is covered with measurement buffer at
all times. Mount the fluid cell onto the AFM, fine-adjust the
laser deflection system, and let the instrument equilibrate until
temperature-induced drifts have settled to an acceptable level
(see Note 19).
4. Establish contact between your tip and the sensor surface, and
minimize the imaging force (see Note 20). Adjust scan size,
scan speed, and feedback gains.

3.5 Surface 1. After each measurement, remove the nanostructured sensor


Regeneration crystal from the AFM liquid cell and immerse it into the glass
Petri dish filled with 10 mM SDS solution. Incubate the crystal
for a minimum of 2 days before the next use; it may be stored
in SDS for longer periods of time.
2. Prior to the next measurement, sonicate the sensor crystal for
5 min in fresh SDS solution. Remove the crystal from the SDS
solution, rinse it thoroughly with water, and blow it dry with
N2 gas. Treat the crystal in a UV ozone chamber (e.g., FHR
UVOH 150 LAB) two times for 10 min, followed by rinsing
with water and blow-drying with N2. Use the cleaned crystal
immediately.

4 Notes

1. Stainless steel round-tip tweezers with 2 mm flat gripping point


are the best option for this purpose. The sensor crystal needs
to be held firmly during surface modification to prevent slid-
ing. On the other hand one cannot apply too much force when
holding the crystal in order for it not to crack or break.
Therefore, it is very important that the gripping point is thin
and flat.
2. Prior to surface modification, the sensor crystal has to be
cleaned. The Teflon holder will be used to hold the sensor
crystal during the cleaning procedure so that it will not move
around and avoiding the risk that the surface of the crystal will
touch the glass beaker.
3. A vortexer can be used to ensure better mixing.
4. Prior to each dilution, mix the stock solution of polystyrene
particle suspension thoroughly. Such a low concentration of
122 Indriati Pfeiffer and Michael Zäch

solution is necessary to avoid polystyrene particle aggregation


on the surface.
5. A N2 gun is required for a very critical, quick blow-dry step.
6. “Blue tape” is used because it has an adhesion level that is
strong enough to pull off the polystyrene particles but low
enough to be cleanly removed from the surface of the sensor
crystal without damaging it and without leaving any adhesive
residue behind. So far, we did not get good results with any
other type of adhesive tape.
7. Normally it is more cost-effective to order the lysophilized
lipid powder in 1 g quantity and aliquot it in smaller amounts
(50 mg) to avoid lipid hydration. Keep the lipid powder at
−20°C.
8. Based on our experience, a 10 mM concentration of SDS solu-
tion will start to crystallize when stored below 21ºC.
9. This cleaning procedure should be done inside a fume hood.
To make a hot bath, use a heating plate equipped with thermo-
couple and magnetic stirring function. Adjust the temperature
set point to 80ºC, and use a magnetic stirrer inside the hot
bath to homogenize the temperature.
10. Organize the working space in the laminar flow hood in such a
way that the N2 gun, the water bottle, and the falcon tubes
containing 2% w/w PDDA, 2% w/w PSS, and 0.2% v/v poly-
styrene particle suspension are ready and easily accessible.
Prepare one plastic pipette for each solution by blowing its
interior with N2. One may use the sink inside the laminar flow
hood if available, or find a beaker for waste disposal. Hold the
clean sensor crystal steadily using the tweezers with one hand
on top of the sink, and use the other hand to pipette the poly-
electrolyte solution onto the sensor crystal and to rinse the
crystal with water. Make sure that the tweezers are dry before
the next polyelectrolyte layer deposition step.
11. A good vacuum is required during metal evaporation in order
to reduce the possibility of contamination by residual gases in
the vacuum chamber.
12. A thin layer of titanium is used as adhesive layer between Au
and SiO2. It is important to sweep the electron beam across the
source during SiO2 evaporation so that the beam will not pen-
etrate the SiO2 source. Otherwise the evaporation rate may
jump suddenly in an uncontrolled way.
13. It is very important to be able to remove the tape completely
in one quick stripping. Otherwise the polystyrene particles will
slightly roll and as a result the pitted surface will not be
homogenous.
Formation of Pit-Spanning Phospholipid Bilayers on Nanostructured Silicon… 123

14. If a vacuum chamber is available, the best is to completely


evaporate the chloroform using a N2 stream for around 1 h and
then to place the round-bottom flask inside the vacuum cham-
ber for a few hours or overnight.
15. The most convenient way to make vesicles in this size range is
by using a lipid extruder. The lipid extruder system can be
obtained from several distributors. So far the best results were
obtained when using the extruder system from Avanti Polar
Lipids. The size distribution of the resulting vesicles in solu-
tion was 160 nm ± 40 nm as determined using an ALV dynamic
light scattering system equipped with a krypton-ion laser. The
size distribution of vesicles can be varied depending on
the number of times the vesicle solution is passed through the
polycarbonate membrane and the pressure used to push the
vesicle solution through the membrane (in our case, 21 times
and 1 kgf/cm2). Please follow carefully the distributor’s advice
for maintenance and cleaning of the lipid extruder.
16. We used a Q-Sense D-300 QCM-D system for our measure-
ments. However, the cleaning procedure can be adapted to all
types of QCM-D systems. The flow operation on the D300
system is based on gravitation, while in newer models (E1 and
E4) a peristaltic pump is used to control the flow rate. Please
refer to www.q-sense.com for more information regarding the
theoretical foundation of the QCM-D technique and modes of
operation.
17. Before starting each measurement, rinse all liquid handling
parts with buffer for couple of times to remove air bubbles that
may trap inside the liquid handling parts.
18. We used a PicoSPM microscope with a large-area scanner from
Agilent (formerly Molecular Imaging) in constant-force con-
tact mode for our experiments, but in principle any other AFM
system able to operate in liquid should work. A custom-made,
O-ring-sealed fluid cell able to accommodate standard QCM
sensor crystals was manufactured.
19. Variations of the imaging force due to cantilever bending
induced by thermal drift must strictly be avoided due to the
fragile nature of the pit-spanning membrane. If the imaging
force exceeds a critical value (typically around 1–2 nN, depend-
ing on lipid composition and tip radius), the AFM tip will pen-
etrate the lipid bilayer and faithful imaging of the pit-spanning
membrane will be impossible. It is recommended to use soft
cantilevers with spring constants below »0.05 nN/nm in order
to avoid large variations of the imaging force.
20. If there is a remaining thermal drift, the imaging force needs to
be readjusted during scanning for the reasons mentioned in
Note 18 above.
124 Indriati Pfeiffer and Michael Zäch

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Chapter 13

Characterization of Nanoparticle–Lipid Membrane


Interactions Using QCM-D
Rickard Frost and Sofia Svedhem

Abstract
In vitro characterization of nanoparticles is becoming increasingly important due to the rapid development
of novel nanoparticle formulations for applications in the field of nanomedicine and related areas.
Commonly, nanoparticles are simply characterized with respect to their size and zeta potential, and addi-
tional in vitro characterization of nanoparticles is needed to develop useful nanoparticle structure–activity
relationships. In this context it is highly interesting to characterize the interactions between nanoparticles
and model interfaces, such as lipid membranes. Here, we describe a methodology to study such interac-
tions using the quartz crystal microbalance with dissipation monitoring technique (QCM-D). In order to
mimic some aspects of the native cell membrane, a supported lipid membrane is formed on the QCM-D
sensor surface. Subsequently the membrane is exposed to nanoparticles, and the nanoparticle–lipid mem-
brane interactions are monitored in real time. The outcome of such analysis provides information on the
adsorption process (importantly kinetics and adsorbed amounts) as well as on the integrity of both the
nanoparticles and the lipid membrane upon interaction. QCM-D analyses are suitable for screening of
nanoparticle–lipid membrane interactions due to the fair throughput of the technique, which can be
complemented, when needed, by additional analyses by other surface-sensitive analytical techniques.

Key words Nanoparticles, Lipid membrane, Extrusion, Liposomes, QCM-D

1 Introduction
Due to the great complexity of native cell membranes, model sys-
tems are commonly used to learn more about the membrane prop-
erties and interactions. The most prominent and widely used model
systems are based on supported lipid membranes (1). Other com-
mon types of model systems include liposomes in suspension (2)
and Langmuir–Blodgett films at the liquid–air interface (3). All of
these systems have been used for nanoparticle–lipid membrane
interaction studies.
The main advantage of supported lipid membranes is the
confinement of the lipid membrane to a solid surface, which is useful
for the application of various surface-sensitive analytical techniques.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_13, © Springer Science+Business Media New York 2013

127
128 Rickard Frost and Sofia Svedhem

Common substrate materials for such studies include silica and


titania, and the lipid membrane formation onto these substrates
has been characterized in great detail by, e.g., the quartz crystal
microbalance with dissipation monitoring (QCM-D) technique.
The key feature of QCM-D, which is sensitive to small mass changes
on the sensor surface and to the viscoelastic (nanomechanical)
properties of the adsorbed material (4), is the ability of the tech-
nique to distinguish between supported membrane and adsorbed,
intact liposomes (5, 6). The QCM-D analysis is based on the piezo-
electric properties of thin, single crystalline quartz discs (sensors)
and gives two different responses: the shift in resonance frequency
(Df) of the quartz crystal, which is related to the amount of mass
adsorbed to the sensor surface, and the shift in dissipation (DD),
which originates from energy losses during the measurement.
A soft material on the sensor surface, as, e.g., adsorbed intact lipo-
somes, efficiently damps the oscillatory motion of the quartz crys-
tal and gives rise to a large dissipation response. This is opposite to
the small dissipation shift that is a characteristic for a supported
lipid membrane. In particular, the combination of (QCM-D) and
atomic force microscopy (AFM) has proven valuable to understand
the mechanisms of the process by which the lipid membrane forms
on the solid support (5, 7, 8), a process which depends on, e.g.,
the substrate material (8), the ion content, and the ionic strength
of the buffer used (9), as well as lipid composition, size, and charge
of the liposomes (8, 10). QCM-D is an acoustic technique, sensi-
tive to all mass that is acoustically coupled to the oscillatory motion
of the sensor and thereby complementary to, e.g., the more well-
established optical sensor techniques based on surface plasmon
resonance. Importantly, optical techniques are generally not able
to distinguish different mechanisms for the formation of supported
lipid membranes from each other (7, 11).
Here, we describe a methodology developed in our laboratory
to study the interactions between nanoparticles and supported
lipid membranes using QCM-D (Fig. 1). The described approach
is general and can be applied to all water-soluble nanoparticles.
More specifically, nanoparticles developed for drug delivery
(nanodrugs) can be characterized with respect to their membrane
interactions and also with respect to other properties such as pH
responsiveness, structural stability, or effect of surface modifications
(10, 12). We have also demonstrated lipid exchange between lipo-
somes and supported lipid membranes (13). Studies of inorganic
nanoparticles are in progress. The ability of this screening platform
to predict, e.g., cytotoxicity remains to be proven. It is however
tempting to suggest that conditions for the experiments may be
tuned such that disruption of the model membrane becomes indic-
ative for cytotoxic effects. In such cases, the QCM-D analysis can
be combined with AFM to visualize nanoparticle-induced mem-
brane damages.
Characterization of Nanoparticle–Lipid Membrane Interactions Using QCM-D 129

Fig. 1 Schematic illustration of a nanoparticle approaching a supported lipid


membrane

The procedures below describe the protocol that we commonly


follow for the preparation of liposomes (by extrusion), the sponta-
neous formation of supported lipid membranes on SiO2-coated
QCM-D sensors, and the subsequent real-time interaction analy-
sis. In addition to the use of a standard QCM-D flow module, a
protocol using an open QCM-D module suitable for the verification
of manual sample preparation procedures (commonly required in,
e.g., AFM analysis) is described. For modeling of the data, we refer
to other sources.

2 Materials
Chemicals and solvents should be of analytical grade. Water should
be deionized (typically to a resistivity above 18 MW cm) and
filtered, using, e.g., a MilliQ unit, Millipore, France.

2.1 Extrusion 1. Buffer for extrusion of liposomes. Here, phosphate buffered


Components saline (PBS): 0.0015 M KH2PO4, 0.0081 M Na2HPO4,
0.0027 M KCl, 0.137 M NaCl, pH 7.4 (see Note 1), which can
be conveniently prepared from tablets available from Sigma.
2. Extruder. Here, mini extruder, including syringes and stand, from
Avanti Polar Lipids, USA (Fig. 2a). Polycarbonate membranes
with suitable pore sizes (here, 100 and 30 nm) (see Note 2) and
membrane supports (Whatman, USA).
3. Lipid molecules (see Note 3). Here, e.g., 1-palmitoyl-2-oleyl-
sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleyl-
sn-glycero-3-phospho-L-serine (POPS), and 1-palmitoyl-2-
oleyl-sn-glycero-3-ethylphosphocholine (POEPC) (Avanti
Polar Lipids, USA).
130 Rickard Frost and Sofia Svedhem

Fig. 2 The main equipment needed in the protocol. (a) Mini extruder for production
of small unilamellar liposomes. (b) QCM-D E4 instrument with associated elec-
tronic unit, pump, and computer. An open QCM-D module is shown in the upper
inset, and the two faces of a QCM-D sensor are shown in the inset at the bottom

4. Chloroform (or other suitable solvents for the lipid stock


solution).
5. Round-bottomed flask.
6. Nitrogen gas.
7. Glass syringes for transfer of lipid stock solutions.
8. Ethanol for cleaning.
9. Optional: Equipment for determination of liposome size and
zeta potential.

2.2 QCM-D 1. QCM equipment that measures both frequency and dissipa-
Components tion responses. Here, QCM-D E4 instrument (Q-Sense,
Sweden) (Fig. 2b), including standard flow modules. Open
QCM-D module (Q-Sense, Sweden) (Fig. 2b).
2. SiO2-coated QCM-D sensors (Q-Sense, Sweden) (Fig. 2b).
Characterization of Nanoparticle–Lipid Membrane Interactions Using QCM-D 131

3. UV–ozone cleaner (or oxygen plasma cleaner).


4. Degassed and filtered PBS buffer (see Subheading 2.1).
5. Equipment for degassing of buffers (e.g., sonication under
reduced pressure).
6. Equipment for filtering buffer (e.g., plastic syringes and 0.2 mm
syringe filters).
7. Cleaning solutions. Here, 10 mM SDS in water, 2% Hellmanex
(Hellma, Germany), and Cobas cleaner (Roche, Germany).
8. Nanoparticles to be studied.
9. Buffer for dilution of nanoparticles.
10. Optional: Equipment for determination of nanoparticle size
and zeta potential.

3 Methods
3.1 Preparation There are several methods for the preparation of liposomes.
of Liposomes Extrusion was first suggested by MacDonald et al. in 1991 (14),
by Extrusion and a similar protocol is available from Avanti Polar Lipids:
1. If the supplier, provides the lipids in powder form, prepare
stock solutions of the lipids in chloroform (or another suitable
solvent, e.g., methanol). For example, weigh 40 mg of lipid in
a glass vial with a cap resistant to chloroform, and add 4.0 mL
of chloroform, to reach a final lipid concentration of 10 mg/
mL (see Note 4).
2. Add the desired amount of lipid solution(s) to a round-bot-
tomed flask (see Note 4). Keep the total amount of lipid to
5–10 mg per extrusion.
3. Evaporate the solvent in a fume hood using a low flow of nitro-
gen gas. Especially towards the end of the procedure, rotate
the flask while evaporating the last solvent such that a thin lipid
film forms on the inner walls of the flask.
4. Connect the flask to a vacuum pump to remove any residual
chloroform. Keep the low pressure for >1 h.
5. Rehydrate the lipids by adding buffer (see Note 1) to reach a
final lipid concentration of 5 mg/mL. Swirl the flask to wet the
lipid film or vortex to speed up the process. Upon dissolution
of the lipid film, the solution becomes turbid.
6. Extract the lipid solution into one of the two syringes belong-
ing to the mini extruder.
7. Mount a polycarbonate membrane with a pore size of 100 nm
in the extruder. Use two membrane supports on each side of
the membrane to avoid rupture of the membrane during
extrusion.
132 Rickard Frost and Sofia Svedhem

8. Press the lipid solution through the membrane 11 times


(see Note 5). In this process the lipid solution becomes less
turbid as small vesicles are formed (see Note 6).
9. Optionally, the liposomes can be reextruded 11 times through
a membrane with 30 nm pore size, to further improve on the
liposome size distribution.
10. Place the liposome solution in a glass vial, purge with nitrogen
gas, and store at 4°C.
11. Clean the mini extruder by rinsing the different parts in ethanol
and water.

3.2 Formation 1. Before conducting QCM-D experiments investigating nano-


of Supported Lipid particle–lipid membrane interactions, it is preferred to ensure
Membranes on QCM-D that the nanoparticle suspension is well defined. The nanopar-
Sensor Surfaces ticle size distribution, determined by, e.g., dynamic light scat-
and Exposure to tering, should be homogenous (i.e., be represented by a single
Nanoparticles peak, PDI < 0.2).
2. Clean the QCM-D instrument prior to use. Depending on
previous usage of the instrument, thorough cleaning (disas-
sembly of the flow modules) may be required. It is often
sufficient to rinse the system with 2% Hellmanex (>10 min)
and water (>5 mL/module). See also Note 12. Blow-dry the
flow modules with nitrogen gas.
3. Clean SiO2-coated QCM-D sensors in UV–ozone for
>30 min.
4. Mount the sensors, using a clean pair of tweezers, in the flow
module for the QCM-D E4 instrument and connect the tub-
ings to the pump (see Note 7).
5. Fill the module with buffer (here, PBS) at 100 mL/min and
turn off the flow (see Note 8).
6. Activate the temperature control, find the resonance frequen-
cies of the sensors (use several harmonics, e.g., the 3rd, 5th,
7th, 9th, 11th, and 13th overtone), and start the data acquisi-
tion. Wait until the baselines (both f and D) are stable.
7. Restart the data acquisition, in order to reset both QCM-D
responses (Df and DD ) to zero, and restart the flow
(100 mL/min).
8. After 10 min of stable baselines, stop the flow (to avoid air
bubbles in the system) and change solution from the buffer to
a solution of liposomes (0.1 mg/mL) (see Note 9). Restart the
flow (100 mL/min). The supported lipid membrane will form
spontaneously within approximately 10 min (Fig. 3).
9. Evaluate the Df and DD responses. For a supported lipid
membrane of good quality, characteristic QCM-D responses,
Characterization of Nanoparticle–Lipid Membrane Interactions Using QCM-D 133

Fig. 3 QCM-D responses (7th overtone) during the formation of a POPC membrane.
At t = 0 min the SiO2 surface is exposed to POPC liposomes in PBS. The liposomes
first adsorb intact, which generates a large decrease in frequency (corresponding
to mass uptake) and a large increase in dissipation (corresponding to the formation
of a soft layer) and later rupture and fuse into a supported lipid membrane. When
the liposomes rupture, the enclosed liquid is released from the liposomes, a
process that is accompanied by an increase in frequency and a decrease in dissi-
pation, and the result is a supported lipid membrane

Df » –26 Hz and DD < 0.5 × 10−6, should be obtained (see


Note 10).
10. If necessary, exchange the buffer in the QCM-D system to a
buffer suitable for the nanoparticles to be studied (see Note 11).
Do not forget to stop the flow while changing liquids.
11. Dilute the nanoparticle suspension to a suitable concentration,
e.g. ,10–100 mg/mL, shortly before use.
12. Pass the nanoparticle suspension over the formed lipid mem-
branes until the QCM-D responses have reached a steady
state.
13. If applicable, the ambient medium (buffer) can be exchanged
at this point to evaluate the responsiveness of the adsorbed
nanoparticles, e.g., by changing the pH or the ionic strength.
14. When the experiment is finished, rinse the system with an SDS
solution and water before emptying the tubing by pumping air.
Dismount the flow modules and replace the sensors with ones
that no longer are in use (maintenance sensors). Dry the sen-
sors with nitrogen gas, and store until next use. Alternatively, to
reduce surface contamination when reusing sensors frequently,
the sensors can be stored in 10 mM SDS until next use.
15. Mount the maintenance sensors and clean the system with
appropriate cleaning solutions, e.g., 2% Hellmanex and Cobas
cleaner (see Note 12). Rinse extensively with water. Depending
on the system under study, more thorough cleaning may be
needed.
16. Dry the flow modules and the maintenance sensors with
nitrogen gas
134 Rickard Frost and Sofia Svedhem

3.3 Validation 1. Clean the Teflon part of the open module and the o-ring by
of Manual Preparation sonication in ethanol (5 min) and water (5 min).
Procedures Using an 2. Clean a SiO2-coated QCM-D sensor in UV–ozone for
Open QCM-D Module >30 min.
3. Mount the sensor, using a clean pair of tweezers, in the open
module, and place the module on the E4 (or E1) instrument.
4. Add 300 mL of buffer (e.g., PBS), put on the lid, start the tem-
perature control, find the resonances of the quartz crystal, and
start the data acquisition (see Note 13).
5. When the baselines are stable, form the supported lipid mem-
brane by adding the liposome solution (0.1 mg/mL). When
adding a new solution, partially exchange the liquid by adding
300 mL of solution, mixing with the pipette, and withdrawing
300 mL of the solution (see Note 14). Repeat this step 3–5
times (see Note 15).
6. Proceed with the same steps as when performing the experi-
ment in a flow module (see Subheading 3.2), i.e., evaluate the
quality of the supported lipid membrane, exchange buffer (if
necessary), add nanoparticles, and rinse with buffer. The
pipetting may induce some small spikes in the QCM-D
response.
7. When the experiment is finished, empty and dismount the
module. Clean the Teflon part of the module and the o-ring by
sonication in ethanol (5 min) and water (5 min). Dry the clean
parts.

4 Notes
1. It is also common to prepare liposomes in buffers based on,
e.g., Tris or Hepes. If possible, it is preferred to use the same
buffer as the nanoparticles to be studied are dispersed in.
2. The membrane pore size that is used affects the final size of the
extruded liposomes (15). However, the hydrodynamic diameter
(typically measured with DLS) does not necessarily correspond
to the membrane pore size. Liposomes extruded through a
100 nm membrane followed by a 30 nm membrane typically
measure 80–90 nm in diameter. The applied pressure is another
parameter affecting the final size of the liposomes (15, 16).
3. Liposomes can be prepared with a wide variety of different
lipid compositions, yielding, e.g., liposomes with different net
charge. However, it should be noted that the lipid composi-
tion of the liposomes affects both their stability in suspension
and their ability to spontaneously rupture and fuse into a sup-
ported lipid membrane on a SiO2 surface.
Characterization of Nanoparticle–Lipid Membrane Interactions Using QCM-D 135

4. Use glassware, e.g., glass syringes, when handling chloroform.


Ordinary plastic pipette tips may release contaminants that
alter the properties of the lipid solution.
5. The more passages through the membrane that are performed,
the more homogeneous the liposome suspension becomes.
Often, 11 passages have been used to produce liposomes with
a narrow size distribution (low polydispersity). Use an odd
number of passages through the membrane to clear the lipo-
some solution from larger size contaminations.
6. Depending on the phase transition temperature of the lipids
that are used, the extrusion may need to be performed at ele-
vated temperatures. The extrusion must be performed at a
temperature exceeding the phase transition temperature of the
lipids. If necessary, the equipment can be preheated in an oven
and placed on a hot plate during the extrusion.
7. Before filling the modules with liquid, it is suggested to check
that it is possible to find the resonance frequencies of all sen-
sors that are to be used.
8. When starting a new measurement, it normally takes some
time (<20 min) before the baselines have stabilized (drift
<1 Hz during 10 min). During this time it is preferred not to
have any flow over the sensor surface to avoid contamination.
9. Dilute the liposome stock solution (5 mg/mL) in PBS to reach
a final concentration of 0.1 mg/mL. If negatively charged lip-
ids are used, e.g., POPS or POPG, it is preferable to add 5 mM
of MgCl2 to the buffer. Divalent cations, e.g., Mg2+ or Ca2+, aid
the formation of negatively charged lipid membranes. Note
that Ca2+ should not be added to phosphate buffers (to avoid
precipitation).
10. If the expected shifts in frequency and dissipation are not met,
i.e., if Df < –26 Hz and DD > 0.5 × 10−6, a lipid membrane of
good quality was not formed. Ideally DD should be close to
zero. Any increase in dissipation suggests that there are still
intact liposomes adsorbed to the surface. The most common
reason for not reaching the desired criteria is that the sensor
surface is contaminated before it is exposed to liposomes, or
simply needs to be replaced. A second reason for why the char-
acteristic QCM-D responses not always are obtained regards
the lipid composition. Liposomes of some lipid compositions
do not spontaneously fuse and form a supported lipid mem-
brane on a SiO2 support (8).
11. When changing buffer solution in the system, there will
often be a shift in Df and DD due to the change in bulk
composition.
136 Rickard Frost and Sofia Svedhem

12. The procedure necessary to clean the instrument after use


depends on what samples that have been used in the experi-
ment. One general cleaning protocol is as follows. Fill the sys-
tem with 2% Hellmanex (basic), turn off the flow, and leave for
30 min. Rinse briefly with water and fill the system with Cobas
cleaner (acidic), turn off the flow, and leave for 10 min. Rinse
extensively with water before drying the modules.
13. The lid to the open module prevents contamination of the
liquid from the surroundings and slows down the evapora-
tion process. However, if the lid is used, it will generate
reversible shifts in the QCM-D responses when it is removed.
When the lid is put back, the responses returns to their origi-
nal values.
14. Partially exchanging the liquid in the open module with a
pipette is performed to avoid exposing the surface to air.
However, this is a rather uncontrolled process. The rate of
which the new solution is added potentially influences the out-
come of the analysis. If the addition rate is critical for a specific
sample is easily evaluated in the open module. This informa-
tion is important when manually preparing samples for other
types of analyses, without real-time data acquisition. When
adding the liposome solution to form a supported lipid mem-
brane, the pipetting procedure is not crucial.
15. The number of liquid exchanges that is needed depends on the
two liquid compositions. Since the new solution is diluted
twofold in every liquid exchange, the liquid will never be com-
pletely exchanged. The dilution can be calculated according to
2n, where n is the number of liquid exchanges. This fact is
particularly important when removing one component from
the system. In such case, perhaps 15–20 liquid exchanges are
needed to sufficiently lower the concentration of that compo-
nent. When adding a component (e.g., the nanoparticles), the
dilution factor is not as crucial since the sample concentration
often can be adjusted beforehand. However, another factor
may be important in these cases. Since the analysis is performed
in batch mode, the sample could be depleted close to the sen-
sor surface if adsorption occurs. In such cases additional liquid
exchanges may be needed after some time.

Acknowledgment

The Swedish Environmental Research Council FORMAS is


acknowledged for financial support.
Characterization of Nanoparticle–Lipid Membrane Interactions Using QCM-D 137

References
1. Sackmann E (1996) Supported membranes: in supported phospholipid bilayers on titanium
scientific and practical applications. Science dioxide. Langmuir 22:3467–3473
271:43–48 10. Frost R, Grandfils C, Cerda B, Kasemo B,
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spherules (liposomes) as a model for biological of polymeric insulin-loaded nanoparticles
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Garnaes J, Schwartz DK (1994) Langmuir- 180–192
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(1997) Simultaneous frequency and dissipa- ported lipid structures and their biomolecular
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kinetics of lipid vesicle adsorption measured tion with model lipid membranes. J Colloid
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Asymmetric distribution of phosphatidyl serine Biophys J 85:996–1004
Chapter 14

Single-Cell Nanosurgery
Maxwell B. Zeigler and Daniel T. Chiu

Abstract
This chapter explains the steps necessary to perform laser surgery upon single adherent mammalian cells,
where individual organelles are extracted from the cells by optical tweezers and the cells are monitored
post-surgery to check their viability. Single-cell laser nanosurgery is used in an increasing range of meth-
odologies because it offers great flexibility. Its main advantages are (a) there is not any physical contact with
the cells so they remain in a sterile environment, (b) high spatial selectivity so that single organelles can be
extracted from specific areas of individual cells, (c) the method can be conducted in the cell’s native media,
and (d) in comparison to other techniques that target single cells, such as micromanipulators, laser nano-
surgery has a comparatively high throughput.

Key words Laser nanosurgery, Optical tweezers, Apoptosis, Opticution, Necrosis

1 Introduction

The effects of focused laser light on living bodies, ranging from single
cells to entire organisms, have been thoroughly researched (1–3). At
the scale of entire organisms, lasers have been used in neuroscience
applications, like the ablation of individual synapses in Caenorhabditis
elegans (4, 5), or in developmental biology with model organisms
such as Drosophila melanogaster (6) or zebra fish (7). Laser surgery is
also particularly advantageous for single-cell or subcellular studies
because mammalian cells are highly heterogeneous and segregated
into biologically relevant units (8), such as organelles, which are com-
parable in size to the diffraction-limited focal spot of a laser.
High numerical aperture (NA) objective lenses can focus laser
light down to spots on the order of hundreds of nanometers, which
provide excellent spatial resolution without the need to physically
manipulate the sample. When these diffraction-limited spots for
spatially localized ablation are paired with a single-beam gradient
optical trap, also known as optical tweezers, cell surgery can add
exogenous material (9) or remove organelles from individual cells (1).

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_14, © Springer Science+Business Media New York 2013

139
140 Maxwell B. Zeigler and Daniel T. Chiu

Optical tweezers are not limited to biological samples and can be


used to trap any object with a higher refractive index than its sur-
roundings, assuming it is of an appropriate size and the trapping
laser has sufficient power. A setup with optical tweezers and an
ablation laser is amenable to a wide array of complementary bio-
physical laboratory techniques, such as fluorescence microscopy,
and they have generated great interest for some time (10, 11).
Post-surgery, the cell may do one of three things: It may recover
and remain a viable cell; it may undergo laser-induced necrosis,
known as “opticution”; or it may undergo apoptosis over a period
of many hours. Our area of interest has been in the viability of dif-
ferent cell lines following cell surgery (1) and the effect of different
membrane disruption techniques on cell survival (3). The method
described here explains how to monitor the long-term viability of a
single cell after the removal of an organelle by laser nanosurgery.

2 Materials

2.1 Polydimethyl- 1. Sylgard elastomer kit (Dow Corning, Midland, MI).


siloxane Wells Used 2. Photoetched glass coverslips (Bellco, Vineland, NJ).
for Cell Surgery and
3. Scientific oven.
Staining
4. Plasma cleaner (Harrick Plasma, Ithaca, NY).
5. Vacuum pump.
6. Vacuum chamber.

2.2 Cell Culture 1. All cell types were grown in 75-cm2 culture flasks and stored in
Equipment and an incubator with 5% CO2 atmosphere at 37°C.
Reagents (a) NG108-15 cells (ATCC, Manassas, VA) grown in
Dulbecco’s modified Eagle’s medium (DMEM), supple-
mented with 8% fetal bovine serum (FBS) and 1% penicil-
lin/streptomycin.
(b) CHO-M1 cells (ATCC) grown in DMEM with 10% FBS
and 1% penicillin/streptomycin.
(c) ES-D3 (ATCC) grown in DMEM with 10% FBS and leu-
kocyte inhibitory factor to prevent differentiation.
2. 0.25% trypsin-EDTA solution.
3. Polydimethylsiloxane (PDMS) wells for cell surgery.
4. Milli-Q water.

2.3 Optical Setup 1. Microscope: TE2000 microscope (Nikon, Melville, NY) with
epi-illumination port, camera port, turret for optical filter
cubes, bright-field light source, and condenser.
Single-Cell Nanosurgery 141

2. Objective: 100× magnification 1.3 NA S Fluor oil immersion


objective (Nikon, Melville, NY).
3. Differential Interference Contrast (DIC) with de Sénarmont
optics for bright-field imaging to improve contrast.
4. Video capture: CCD camera (Cohu, San Diego, CA).
5. Ablation laser: Nanosecond pulse 337-nm N2 Ultraviolet laser
(Laser Sciences, Inc., Franklin, MA).
6. Laser for optical tweezers: continuous wave (CW) Nd:YAG
laser with a TEM00 Gaussian beam profile (Spectra-Physics,
Mountain View, CA).
7. Laser power meter (Ophir Optronics, Jerusalem, Israel).
8. Assorted neutral density (ND) filters, dichroics, lenses, optical
filters, mirrors, and optomechanics (Omega Optical,
Burlington, VT; Thorlabs, Newton, NJ; and Chroma Tech
Corp, Rockingham, VT).

2.4 Cell Viability 1. 2-μM ethidium homodimer.


Assay 2. 200-nM MitoTracker Red (Invitrogen, Grand Island, NY).
3. Annexin V Alexa Fluor 488 (Invitrogen, Grand Island, NY).
4. 488-nm CW sapphire laser (Coherent, Santa Clara, CA).

3 Methods

3.1 Making PDMS 1. Mix the Sylgard elastomer kit base and catalyst thoroughly in a
Wells 10:1 ratio in a disposable cup; 100 g of base and 10 g of cata-
lyst are enough to make ~12 wells.
2. Place the uncured silicone mixture in the vacuum chamber and
pump air out of the chamber to remove air bubbles.
3. Pour the PDMS into a large petri dish. We have found that plac-
ing evenly spaced glass vials with a diameter slightly smaller than
the “1” photoetched coverslips makes suitable molds to shape the
PDMS as it cures. To ensure that the vials do not move through
the PDMS before it hardens, keep the vials in place using a petri
dish lid with holes drilled such that the vials go through the holes
and thus are held in place by the holes in the lid.
4. Bake at 60°C for at least 1 h to cure.
5. Pull out the glass vials from the PDMS, remove the PDMS
from the petri dish, and cut out the individual wells.
6. Expose PDMS and photoetched glass coverslips to oxygen
plasma for 60 s.
7. Seal the PDMS to the coverslip by bringing both together;
apply light pressure to expunge any air pockets between the
glass and PDMS.
142 Maxwell B. Zeigler and Daniel T. Chiu

3.2 Membrane 1. Split cells into a PDMS well 24 h before surgery using the
Disruption cell’s native media. The cells should be split at a low density to
aid in identification during surgery and subsequent observa-
tion (see Note 1). Just prior to surgery, supplement the media
in the PDMS well with ~25% Milli-Q water to induce cell
swelling and help organelle extraction.
2. For the pulsed N2 laser to disrupt the membrane, use a beam
expander or lens pair to collimate and expand the beam to fill
the back aperture of the microscope objective. This will pro-
duce a submicron focal spot size. We would recommend
mounting at least one lens base on a rail to facilitate alignment
of the focal spot in the axial direction.
3. Each laser pulse needs ~800 nJ of energy per pulse when mea-
sured before the objective. Our particular laser has a 10% pulse-
to-pulse variability in pulse energy at 2 Hz at a 3-ns duration,
and the objective has a roughly 30% transmission efficiency. This
power is sufficient to disrupt the plasma membrane of an
NG-108 cell with a single pulse to allow organelle extraction
50% of the time. Greater laser powers could lead to excessive cell
damage and large cavitation bubbles; lower powers would have
no visible effect. We modulate the pulsed laser’s power using a
neutral density filter and select the laser pulses using a shutter
controlled by a transistor–transistor logic (TTL) switch.
4. Laser pulses should target the cell’s plasma membrane to
maximize membrane disruption and minimize cell damage
(see Note 2). Focusing on the coverslip leads to ablation of the
coverslip and immediate cell lysis; focusing on the cell cyto-
plasm creates large cavitation bubbles and excessive cell damage.
Regardless of where the laser focus is directed, morphological
changes will be evident for all cells following surgery.
5. If post-surgery cell viability is important and the cell line is par-
ticularly susceptible to death after surgery, a femtosecond near-IR
pulse laser, instead of a nanosecond UV pulse laser, may improve
cell viability. The mechanism of membrane disruption greatly
affects cell viability after surgery (3). Membrane disruption by
multiphoton absorption from a femtosecond laser produces a
smaller focal volume, smaller cavitation bubbles, and other effects
that help cell viability (2). The drawbacks of using a femtosecond
laser for surgery are added cost and complexity.

3.3 Optical Trap This section provides brief guidance on the construction of a simple,
Construction robust, single-beam, gradient-force, three-dimensional optical trap
with commercially available components. If more detail or explanation
of optical alignment is needed, more comprehensive guides are avail-
able (12). With additional modification, trap position sensing (13),
Single-Cell Nanosurgery 143

vortex-shaped optical traps (14), multiple traps (15, 16), force mea-
surements (17, 18), and a myriad of other techniques are possible (19)
but are beyond the scope of this chapter.
1. An inverted microscope is a stable and flexible platform for
optical trapping. The back epifluorescence port is convenient
for directing the trapping beam into the objective. Most
research-grade inverted microscopes also come with a turret
where multiple beam splitter cubes may be installed, which
makes it convenient to switch between cell surgery and
epifluorescence imaging modes. It is possible to build a plat-
form for optical trapping without a microscope, but this
requires a high degree of expertise in microscopy.
2. The objective lens is probably the most critical element of the
optical trapping setup. For stable trapping, a high NA objec-
tive lens is necessary to generate the tight focus (20), such as
the Nikon S Fluor 1.3 NA 100× oil immersion objective lens.
3. CW lasers emitting in the NIR, like the Nd:YAG laser, are used
for optical trapping because they produce sufficient power at a
wavelength that is relatively transparent to cells. Wavelengths
shorter than ~700 nm are more readily absorbed by cells and
should be avoided if possible because the high intensity in the
focal volume results in excessive photodamage. Also, the laser
should have an M2 < 1.1 and emit the TEM00 mode to produce
a light gradient steep enough to form a stable optical trap. For
the sake of safety, a shutter should be installed at the point
immediately after the beam exits the laser (see Note 3).
4. Place a dichroic mirror in the microscope’s laser filter cube so
that it will reflect IR and UV light but allow visible light to pass
through. After alignment of the IR laser, an emission filter used
should also block back-reflected IR and UV light from the
optical tweezers and the ablation laser (see Note 4).
5. The power of the trapping beam should be minimized, but still be
great enough to stably trap the organelle of interest. Most Nd:YAG
lasers emit significantly more power than is necessary. The power
is best modulated by using a λ/2 wave plate installed in a rotating
mount followed by a polarizing beam splitter and a beam block
that absorbs the excess laser power. With this configuration, the
laser’s power can be attenuated by rotating the wave plate.
Controlling the laser power by external means instead of adjusting
the drive current of the laser improves the laser power stability.
6. After the power attenuator, insert a pair of lenses into the beam
path that expand the beam to fill or slightly overfill the back
aperture of the objective. Beam expanders are commercially
available for this purpose.
7. The most convenient way to steer the optical trap is by using a
combination lens pair and a mirror to create a 4f system. Mount
144 Maxwell B. Zeigler and Daniel T. Chiu

Fig. 1 Schematic of optical setup. The outputs of a pulsed N2 laser (337 nm; 3-ns pulse width) and a CW
Nd:YAG laser (1,064 nm) were aligned collinearly and sent into the microscope. The two beams were directed
into the objective (NA = 1.3) by a dichroic mirror and focused onto the cell by the objective lens. The micro-
scope was equipped with Nomarski optics, but the components are omitted in the schematic for clarity. The
pulsed N2 laser (~0.7 μJ) was used to ablate a small hole (~1–3 μm) in the cell membrane, and the YAG-
trapping laser (200 mW) was employed to extract a subcellular organelle (arrow pointing away from focal
volume). To track individual cells post-surgery, we cultured cells in PDMS wells formed by sealing PDMS to
photoetched coverslips (upper left inset). HWP half-wave plate, BS polarizing beam splitter, BB beam blocker,
ND neutral density filter, M mirror, L lens. Reprinted with permission from (1)

the lens nearest the microscope on an x, y, z movable lens


mount and arrange the lens pair so that each lens is the same
distance from the central focal spot as its focal length. The
lenses closest to the objective can be used to fine-tune the
alignment of the optical tweezers. The movement of the final
lens along the optical axis can be used to adjust the height of
the optical trap above the objective. This adjustment will
ensure that the plane of the optical trap and the image are par-
focalized (see Note 5). The mirror is installed in a kinematic
mirror mount at the conjugate focal plane of the objective and
can be used to steer the optical trap (Fig. 1).

3.4 Organelle 1. Organelle trapping is performed using the optical tweezers


Trapping described in the previous section. For our setup, a power of
~200 mW at the nosepiece of the microscope generates a
sufficient gradient force to trap individual organelles. The
power transmitted through the microscope’s objective will
vary depending on the % transmission through the objective
and whether or not the trapping beam is larger than the back
aperture of the objective (13).
Single-Cell Nanosurgery 145

2. Organelles near the spot of membrane disruption can be


extracted from the cell after steering of the trapping beam
(see Note 6). Adding Milli-Q water to the media improves the
ease of organelle extraction without significantly reducing cell
viability, but a small amount of cytoplasm is still often extracted
with the targeted organelle (Fig. 2). The extracted organelles
are often lysosomes or mitochondria. These organelles can be
differentiated with the appropriate fluorescent stain.
3. We have observed that the high fluence of the IR laser leads to
fast photobleaching of organelle stains and possible photodam-
age to the organelle. If the extracted organelle is intended for
further analysis, such as single mitochondria capillary electro-
phoresis (21), photobleaching and photodamage to the organ-
elle can be reduced by using an optical vortex trap (14) instead
of the Gaussian optical trap described above.

3.5 Cell Viability 1. After surgery, incubate the cells at regular intervals in pre-warmed
Monitoring media for 10 min containing the viability reagents (see
Subheading 2.4). The particular dyes are chosen to determine cell
viability because (a) they can all be excited with a single 488-nm
laser, (b) their fluorescence is spatially and spectrally distinct so
they are easily resolved, and (c) in addition to cell necrosis, they
also suggest apoptosis prior to the morphological signs of apopto-
sis, which may take many hours to become apparent. Ethidium
homodimer is a membrane-impermeable dye that will brightly
stain the cell nucleus if the membrane loses integrity. MitoTracker
Red is a mitochondrial stain that will have a significant decrease in
brightness if the mitochondria lose the ability to maintain a pro-
ton gradient across their membrane. Annexin V Alexa Fluor 488
is a membrane-impermeable antibody that binds to phosphatidyl-
serine, a phospholipid that can be found on the external leaflet of
a cell’s lipid bilayer only during apoptosis.
2. After 10 min, remove the cells from the incubator and wash
with fresh pre-warmed media. Cell viability can be determined
by observing the fluorescent stains and cell morphology. Some
morphological indicators that a cell is dying, such as mem-
brane blebs, a granular appearance in their cytoplasm, more
spherical shape, and loss of adherence to the coverslip, can be
viewed under bright-field microscopy.
3. Cell apoptosis can be differentiated with bright-field DIC and
epifluorescence illumination (Fig. 3). The viable cell (a1, a2)
has a normal morphology under bright field and displays
bright-orange fluorescence under epifluorescent illumination
from the MitoTracker stain. The cell undergoing apoptosis
(b1, b2) has a more granular appearance under bright field.
Under epifluorescence, it has decreased emission intensity from
MitoTracker but a brightly fluorescent nucleus from the ethid-
ium homodimer and a green fluorescent plasma membrane as
Annexin V Alexa 488 binds to the lipid bilayer.
146 Maxwell B. Zeigler and Daniel T. Chiu

Fig. 2 Organelle extraction in successful surgeries. An NG108-15 cell (a) before, (b) during, and (c) after surgery;
the arrows point to the organelle that was extracted. A similar sequence showing a CHO cell (d) before, (e) during,
and (f) after surgery. A small cavitation bubble is visible in (e); (f) shows the organelle (arrow) that was completely
removed from the cell. An ES-D3 stem cell (g) before, (h) during, and (i) after surgery. Scale bars = 10 μm. Reprinted
with permission from ref. 1

4 Notes

1. Cells are known to migrate over time, a property dependent on


the cell line. If long-term observation is necessary, be sure to
either split the cells at a lower density or take images regularly
to track cell movement. Photoetched glass coverslips also help
to track individual cells.
2. When performing cell surgery, watch the surgery with a CCD
camera. Do not use the microscope’s oculars. IR light can leak
through the microscope’s oculars so place a light blocker over
them when appropriate to prevent accidental exposure.
3. Proper laser safety is crucial and should include the appropriate
laser goggles, beam blocks, shut-off keys, IR laser display cards,
and so on. Safety measures are especially important in cell laser
surgery because both the trapping and ablative lasers are out-
Single-Cell Nanosurgery 147

Fig. 3 Apoptosis assay. A healthy cell is observed using (a1) DIC microscopy and
then with (a2) epifluorescence microscopy. Another cell undergoes apoptosis, as
observed using (b1) DIC microscopy and (b2) epifluorescence microscopy. In
panel (a2), the intense fluorescence from MitoTracker Red indicates that the
mitochondria are able to maintain a potential gradient. In panel (b2), the cell is
undergoing apoptosis so the MitoTracker Red fluorescence is decreased. In addi-
tion, ethidium has penetrated the cell’s membrane and intercalated with the
cell’s DNA and Annexin V Alexa 488 has bound to the cell’s membrane. Reprinted
with permission from ref. 3

side the visible range and scattered light from either laser may
be intense enough to cause eye damage.
4. Back reflection from optical tweezers can be intense enough
to damage the camera so make sure its power is minimized
during alignment and that the proper IR emission filter is in
place.
5. The distance from the focal spot where an object is trapped by
the optical tweezers can change with the object’s size and com-
position. Some tweaking of the trap’s focal spot may be neces-
sary when trapping different samples to keep the objects in the
same plane as the imaging plane.
6. If the emission filter blocks 100% of the back-reflected IR and
UV light, then the trapping beam and ablation laser are invis-
ible to the camera. Organelle extraction is easier if you find a
method for tracking the position of the trapping beam and the
148 Maxwell B. Zeigler and Daniel T. Chiu

ablative pulsed laser so that they do not get lost between exper-
iments or are steered outside the field of view of the camera.
There are many ways to track the beams depending on the
application: It can be as complex as building a trapping posi-
tioning system or as simple as marking the laser beam focal
points on the monitor with a dry-erase marker.

Acknowledgments

We are grateful to NIH (NS062725, NS052637, GM085485) and


NSF (CHE0844688 and CHE0924320) for support of this work.

References

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Science 13(4507):505–513
Chapter 15

Single Quantum Dot Imaging in Living Cells


Jerry C. Chang and Sandra J. Rosenthal

Abstract
Direct visualization of biological processes at single-molecule level provides a detailed perspective which
conventional bulk measurements are hard to achieve. Among various classes of fluorescent tags used in
single-molecule tracking, quantum dots are particularly useful due to their unique photophysical proper-
ties. In this chapter, we describe the principles, methodologies, and experimental protocols for qdot-based
single-molecule imaging. The first half provides an overview of fluorescent microscopy and advances in
single-molecule tracking using quantum dots. The remainder of this chapter describes methods to carry
out qdot-based single-molecule experiments. Detailed protocols including qdot labeling, microscopy
setup, and single-molecule analysis using appropriate computational programs are given.

Key words Quantum dot, Biological labeling, Protein trafficking, Single-molecule, Fluorescence
microscopy

1 Introduction
In recent years, microscopy techniques in cell biology have seen
remarkable progress. Various new methods, such as near-field scan-
ning optical microscopy (NSOM) (1), single-molecule high-reso-
lution imaging with photobleaching (SHRIMP) (2), stimulated
emission depletion (STED) microscopy (3), and stochastic optical
reconstruction microscopy (STORM) (4), were developed to
achieve a significantly higher spatial resolution, which permits dis-
covery of subcellular structures in great detail. Despite the fact that
these imaging methods are designed to break the optical diffrac-
tion barrier, they are not very accessible to cell biology researchers.
The major shortcoming of these methods is that most of them are
still under development in experimental physics laboratories and
are not readily transferable to a standard laboratory microscope.
Also, the data analyses are usually extremely complicated, requiring
well-trained experts in the field.
Single-molecule fluorescent microscopy, derived from high-
speed fluorescence microscopy, is probably the most accessible

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_15, © Springer Science+Business Media New York 2013

149
150 Jerry C. Chang and Sandra J. Rosenthal

Fig. 1 Schematic representation of the diffraction pattern from a point emitter passed through an optical
device. Owing to diffraction, the smallest distance to which an imaging system can optically resolve separate
light sources at is limited by the size of the Airy disk

method for cell biologists to investigate single-molecule dynamics


in living cells. The basis of this method is to follow the real-time
movement of individual molecules by using fluorescent micro-
scope, resulting in a map of the dynamics upon observing many
individual events. Based on the above definition, an ultimate sensi-
tivity is required, which allows individual single molecules to be
monitored in a picoliter- to femtoliter-sized microscope sampling
volume. However, even if performed on an optimal designed
microscope equipped with a camera offering single photon sensi-
tivity per frame, optical detection of a single molecule is still diffrac-
tion-limited. To achieve a sub-pixel localization in single molecule
imaging, a 2D Gaussian approximation of point-spread function
(PSF) approach is typically performed (5). Single emitters viewed
through a microscope produce an Airy disc diffraction pattern in
the image plane (Fig. 1). In the 2D Gaussian approximation
approach, the diffraction pattern of a single emitter is analyzed in
terms of PSF where the localized centroid is determined by fitting
the PSF to a 2D Gaussian profile. The theoretical 2D paraxial PSF
of the wide-field fluorescence microscope can be calculated as (5)
2
⎡ J (k ·NA·r ) ⎤⎥
PSF = ⎢ 2 × 1 em , (1)
⎢ kem ·NA·r ⎥⎦

where r is the radial distance to the optical axis, NA is the numerical

aperture, J1 is the first-order Bessel function, kem = defines the
λ
emission wave number, and λ is the wavelength of light. The smallest
resolvable distance between two points of the 2D plane corre-
sponds to the first root of the PSF and is given by the Rayleigh
distances (6),

λ
d xyR = 0.61 . (2)
NA
Single Quantum Dot Imaging in Living Cells 151

As can be seen from the Eqs. 1 and 2, under the important


prerequisite in which single emitters are placed in extremely low
concentration to be spatially separated greater than the diffraction-
limited region, it is possible to identify the localization (x, y) of a
single molecule from an optical microscope image by fitting the
PSF. Indeed, the fundamental idea of the modern single-molecule
microscopy is reliant on PSF fitting to localize the centroid posi-
tion of single point emitters. Typically, fluorescent intensity distri-
bution from a single emitter can be fit with a 2D Gaussian (7, 8).
To calculate a subpixel estimate of the position from a single emit-
ter, the general method is to fit the intensity distribution with a 2D
Gaussian function and then calculate the local maximum
intensity:
( x− x0 )2 + ( y − y0 )2 (3)

w2
I xy = A0 + A·e ,

where Ixy is the intensity of the pixel, x0 and y0 is the designated


local maximum coordinates of the Gaussian, A is the amplitude of
the signal with local background A0, and w is the width of the
Gaussian curve. The smallest distance at which two emitters can be
recognized and separated is roughly equal to the full width at half
maximum (FWHM) of the w:

wFWHM = w· ln(4) = 1.177 w. (4)

The coordinate (x0, y0) acquired by fitting function Eq. 3 using


chi-squared minimization is not a true position, but only an estimate.
The accuracy is strongly dependent upon the respective signal-to-
noise ratio (SNR), which is defined as (8)
I0
SNR = , (5)
σ + σ I20
2
bg

2
where I0 is the maximum signal intensity above background, σbg is
2
the variance of the background intensity values, and σ I0 is the true
variance of the maximum signal intensity above the background.
Since w width is approximately equal to the wide-field diffraction
limit (for visible light is about 250 nm), the uncertainty of the
fitted coordinate (Δσ) is approximately given by

250
Δσ ≈ (nm). (6)
SNR

However, common organic fluorophores used in single-


molecule imaging have very limited photon yield which makes
them difficult to produce a sufficient SNR for single-molecule
imaging. It is also important to note that organic fluorescent dyes
also suffer from narrow Stokes shift (difference in excitation and
152 Jerry C. Chang and Sandra J. Rosenthal

emission wavelength). This drawback can increase background sig-


nals and further reduce SNR. In addition, photobleaching associ-
ated with organic fluorophores prevents long-term monitoring in
single-molecule microscopy. Hence, the shortcomings associated
with organic fluorophores place a high demand for new fluorescent
materials for single-molecule tracking.
Over the past decade, quantum dots (qdots) have shown tre-
mendous potential for in vitro and in vivo biological imaging (9–14).
Among the fluorophores developed for single-molecule tracking, a
fluorescent qdot is, perhaps, the ideal material for its enhanced
brightness, exceptionally high quantum yield, narrow emission
profile, large Stokes shift, and, most importantly, excellent photo-
stability. Another interesting property associated with qdots is the
fluorescent intermittency, or blinking, phenomenon (15), whereby
a time trace of fluorescent intensity from a single qdot can switch
between two distinct on/off states. This blinking phenomenon is
sometimes considered a minor drawback since it might cause tem-
porary trajectory data loss in single-molecule tracking (16). On the
other hand, blinking is often used to identify single molecules, pro-
viding a practical benefit (17, 18).
Using qdot-based single-molecule tracking, Dahan and coworkers
first reported the diffusion dynamics of individual glycine receptors
in living neurons (19). By conjugating Fab fragments of antibody
to single qdots, they were able to track the movement of individual
glycine receptors for more than 20 min. Similar approaches were
then employed for various protein targets including nerve growth
factor (NGF) (20), gamma-aminobutyric acid A receptor (GABAA
R) (21), glycosyl-phosphatidylinositol (GPI)-anchored protein
(22), serotonin transporter (23), and GM1 ganglioside (24). In
2008, Murcia et al. pushed the temporal resolution of single qdot
tracking up to an amazing 1,000 frames/s by using an extremely
sensitive double micro-channel plate (MCP) image intensifiers,
cooled intensified CCD camera (dual MCP ICCD) (25). Through
demonstration of single qdot tracking in living mice, Tyda et al.
further suggested to use single qdots as tumor-targeting nanocarri-
ers for in vivo drug delivery study (26). Recently, Zhang et al. uti-
lized single qdots as artificial cargo of synaptic vesicles to validate
the “kiss-and-run” model of neurotransmitter secretion (17).
Overall, qdot-based single-molecule imaging holds the promise to
reveal molecular biological mechanisms well beyond ensemble bio-
chemical techniques.
The process of qdot-based single-molecule microscopy is typi-
cally divided into three steps (Fig. 2). The first step is to acquire
time-lapse imaging after single target-specific labeling has been
made. Single fluorescent molecules should be able to produce dif-
fraction-limited blurred spots in each flame. The second step
involves imaging data processing and single-molecule localization
Single Quantum Dot Imaging in Living Cells 153

Fig. 2 Approach to single-molecule microscopy. (a) Time-lapse images of single fluorophore-tagged biomol-
ecules in living cells acquired from an optical fluorescent microscope system (epifluorescence, confocal, or
total internal reflection fluorescence (TIRF) microscope). (b) Estimation of the positions of single quantum dots
with subpixel accuracy is accomplished by fitting the individual spot intensity values with a two-dimensional
Gaussian distribution. After the positions of single quantum dots are identified, a trajectory of the target protein
(gray line) can be subsequently derived from the time-series imaging data. (c) The final step is to analyze the
diffusional properties (displacement, velocity, and diffusion coefficient) from single-molecule trajectories

from time-lapse images and is therefore normally anticipated as a


computationally demanding step. In the third step, the diffusion
dynamics can be analyzed from the trajectories of individual mol-
ecules, e.g., Brownian motion.
154 Jerry C. Chang and Sandra J. Rosenthal

2 Materials
2.1 Reagents 1. Biotinylated small molecule or antibody against an extracellular
epitope.
2. Qdot streptavidin conjugate (1 μM, Invitrogen Corporation,
Carlsbad, CA).
3. 35-mm cell culture dishes with cover glass bottom (MatTek
Corporation, Ashland, MA).
4. HeLa cells.
5. Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO,
Invitrogen Corporation, Carlsbad, CA).
6. Phenol red-free Dulbecco’s Modified Eagle Medium (DMEM)
(GIBCO, Invitrogen Corporation, Carlsbad, CA).
7. Penicillin-streptomycin antibiotic mixture (100×) (GIBCO,
Invitrogen Corporation, Carlsbad, CA).
8. Fetal bovine serum (FBS) (GIBCO, Invitrogen Corporation,
Carlsbad, CA).
9. L-glutamine (GIBCO, Invitrogen Corporation, Carlsbad, CA).

2.2 Equipment, 1. Fluorescent microscope (see Subheading 3.1).


Software, and 2. Microscope mounted heating chamber.
Accessories
3. Microscope image acquisition and analysis software (MetaMorph
7.6, Molecular Devices, Sunnyvale, CA).
4. Technical computing software for numerical analysis (Matlab
R2008b, MathWorks, Natick, MA).
5. Spin coater.

3 Methods

3.1 Imaging System A general strategy to identify whether the optical system is able to
Calibration Using detect individual qdots is to carry out time-lapse imaging of a very
Spin-Coated Single dilute qdot solution to avoid multiple qdots overlapping within dif-
Quantum Dots fraction-limited distance. Individual qdots are characterized by their
blinking properties (8, 18). The emission intensity of a single qdot is
shown in Fig. 3, where a single qdot blinks completely on and off
during a time-lapse sequence of 80 s at a 10-Hz frame rate. When
the fluorescence is produced by an aggregate structure consisting of
several qdots, such blinking effects are completely canceled out. The
protocol described below is based on a custom-built Zeiss Axiovert
200-M inverted fluorescence microscope coupled with a charge-
coupled device (CCD) camera (Cool-SnapHQ2, Roper Scientific,
Trenton, NJ). To track single qdots in real time, the acquisition rate
Single Quantum Dot Imaging in Living Cells 155

Fig. 3 A typical intensity over time plot from a single blinking qdot. As displayed
in the left panel, the intensity trajectory of a single qdot displays two dominant
states: an “on” state and an “off” state, termed blinking. A predefined intensity
threshold is shown by the dashed line. Right panel displays the probability den-
sity distribution

should be set at 10 Hz or higher. However, the imaging rate is usu-


ally limited by the frame readout time of the camera. This particular
CCD is chosen due to its adequate 60% quantum efficiency (QE)
throughout the entire visible spectrum range (450–650 mm) with a
frame rate >20 at 512 × 512 pixels (see Note 1). And again, as we
introduced in the background section, a more advanced back-illumi-
nated Electron Multiplying CCD (EMCCD) with sub-msec tempo-
ral resolution will be a much better choice. Imaging should be
performed with a high-resolution (63× or 100×) oil-immersion
objective lens with numerical aperture of 1.30 or greater.
The sample preparation steps are:
1. Prepare a clean microscope glass slide coverslip (or 35-mm cul-
ture dish with a coverslip at the bottom).
2. Add one drop (20 mL) of 100-pM Qdot® ITK™ carboxyl quan-
tum dots solution onto the coverslip.
3. Spin cast the qdot solution on the coverslip for 30 s at 500 rpm
(~30 × g for common compact spin coater) sufficient to disperse
qdot particles uniformly across the coverslip (see Note 2).
4. Mount the coverslip on the microscope stage.
5. Qdots are excited using a xenon arc lamp (excitation filter
480/40 BP) and detected with CCD camera through appropri-
ate emission filter (600/40 BP for Qdot 605). Acquire time-
lapse images (100 ms per frame, 60 s).

3.2 Single Quantum Single quantum dot labeling can be prepared through either a
Dot Labeling in Living direct labeling (one-step) procedure or an indirect (two-step) pro-
Cells tocol. In the direct labeling procedure, the target-specific probe
(small molecule organic ligand, peptide, or antibody) is directly
conjugated to the qdots surface to make ligand-qdot nanoconjugates.
Therefore, the cellular labeling strategy could be performed in one
156 Jerry C. Chang and Sandra J. Rosenthal

Fig. 4 Example of live-cell imaging of membrane proteins labeled with single qdots (a: bright-field image, b:
fluorescence image, and c: surface intensity plot of b)

step in which the live-cell sample is incubated with a target-specific


nanoconjugate prior to fluorescent imaging. In the two-step pro-
cedure, the cell sample is first incubated with biotinylated ligand to
yield the desired specific ligand-protein binding. After an appropri-
ate washing step, strep-qdots are added as the fluorescent tag of
the biotinylated ligand-protein complex for the single-molecule
imaging.
We provide a general protocol for single qdot labeling of adherent
cells which is applicable to most mammalian cell lines. The standard
protocol given below should be followed:
1. Prepare a 35-mm coverslip-buttoned culture dish with cells that
have reached about 50% confluence.
2. Wash the cells gently three times with phenol red-free culture
medium by repeatedly pipetting out.
3. Incubate cells with a biotinylated small molecule probe (0.5 nM
to 0.5 mM dependent upon the biological affinity) or antibody
(1–10 mg/mL) in red-free DMEM for 20 min at 37°C. (For
one-step labeling protocol, incubate cells with 10–50 pM ligand-
qdot nanoconjugates and skip steps 4 and 5) (see Note 3).
4. Wash cells gently three times with phenol red-free culture
medium.
5. Incubate the cells with Qdot streptavidin conjugate (0.1–
0.5 nM) in phenol red-free culture medium for 5 min at 37°C.
6. Wash the cells at least three times with phenol red-free culture
medium.
7. Place the culture dish on the microscope stage with mounted
heating chamber and heat to 37°C.
8. The labeling quality can be observed under fluorescent micro-
scope. Punctate qdot staining should be visible through the
eyepiece or CCD detector (Fig. 4). Single qdots can be identified
by their blinking property.
9. Acquire time-lapse images at 37°C. In our experiments, acquisi-
tion procedure typically lasts for 60 s at 10 Hz rate.
Single Quantum Dot Imaging in Living Cells 157

3.3 Tracking Tracking and trajectory construction is a computationally demanding


Programs for Single- step of following single Qdot-labeled biological targets through
Molecule Analysis successive images. One of the most important determinants of
modern single-molecule tracking techniques is the nanometer accu-
racy, which is heavily weighted by the PSF fitting to localize the
centroid position for subpixel resolution, normally demonstrated as
a fitting of a 2D Gaussian function to a PSF. For practical applica-
tion, estimated background-corrected intensities of an image are
normally filtered out, a necessary step for the calculation of centroid
position (x0, y0). After locations of single molecules are identified in
each frame, the next step is to link the detected single-molecule
positions. However, single qdot blinking brings additional difficulties
for the trajectory generation, as the spots can temporarily disappear.
A practical and most frequently used approach is to define a toler-
ance limit of blinking frames (usually 10 frames) and process an
additional association step in the trajectory generation algorithm to
merge multiple trajectories into one (27). This procedure allows
tracking to continue and thus compensates for the transient data
loss caused by qdot blinking. In addition, qdot blinking frequency
is dependent on the excitation power; hence, it is generally recom-
mended to perform single qdot tracking experiments with low-
power excitation if signal intensity is sufficiently high.
An ImageJ plug-in for single-molecule/single-particle track-
ing offers several user-friendly features including an easily under-
standable interface, free on-line tutorial, and computationally
efficient process. The program is free to download at ImageJ web-
site: http://rsbweb.nih.gov/ij/plugins/index.html (see Note 4).
In addition, particle tracking using IDL, developed by Crocker
and Grier, provides a total solution including 2D Gaussian fit for
spot localization, trajectory generation, as well as MSD calculation.
The algorithms with detailed tutorial are freely available at http://
www.physics.emory.edu/~weeks/idl/index.html. Matlab version
of these routines can be found at http://physics.georgetown.edu/
matlab/ (Fig. 5). With recent advances in computing power and
numerical software, the development of tracking algorithms has
evolved rapidly during the past few years in supporting better cor-
respondence for motion detection (28), high computational
efficiency (29), or 3D motion segmentation and localization (30).
All the tracking algorithms mentioned above may, however, require
technical training to operate since they all established under tech-
nical computing environments such as Matlab, IDL, or C++.
Below is a general tracking procedure using ParticleTracker:
1. Use the File/Open commend in the ImageJ to import the pre-
recorded TIF stack or uncompressed avi file (Fig. 6a). If the avi
file contains multichannel imaging data, use the Image/Color/
RGB Split to qdot data channel extraction.
2. Next, click the Plugins/Particle Detector & Tracker commend. If
RGB images or images with greater than 8 bits are loaded for
158 Jerry C. Chang and Sandra J. Rosenthal

Fig. 5 Snapshot of the interface of Matlab-based particle tracking program originally developed from particle
tracking using IDL algorithm. Data obtained from 600 frames of single Qdot imaging. Left panel indicates the
2D trajectory, and right panel shows the MSD over time

tracking, a checkable menu item will show up to ask whether the


images are converted to 8 bits. If running on a computer with
fewer than 2 GB of memory installed, it is strongly recommended
to convert to 8 bits to reduce the memory consumption.
3. As indicated in Fig. 6b, three basic parameters for particle detec-
tion are given. Radius: Approximate radius of the particles in
the images in units of pixels. Cutoff: The score cutoff for the
non-particle discrimination. Percentile: The percentile (r) that
determines which bright pixels are accepted as particles. Click
on preview detected and then the successfully detected spots
will be circulated. Here, we recommend to use Radius = 3,
Cutoff = 0, and Percentile = 0.1 as initial guess, but these values
might vary based on the images. Start with our recommended
parameter and change these values until most of the visible par-
ticles are detected after clicking the preview button.
4. After setting the parameters for the detection, set up the parti-
cle-linking parameters (Displacement & Link Range) in the bot-
tom of the dialog window (Fig. 6b). Here, the Displacement
parameter means the maximum number of pixels a particle is
allowed to move between two succeeding frames. The Link
Range parameter is used to specify the number of subsequent
frames that is taken into account to determine the optimal cor-
respondence matching. We recommend to use Displacement = 2
and Link Range = 10 as initial guess, and again, these parameters
can also be modified after viewing the initial results.
Single Quantum Dot Imaging in Living Cells 159

Fig. 6 Steps of single qdot tracking using ParticleTracker—an ImageJ plug-in. (a) A typical raw frame from a
time-lapse single Qdot labeling movie. White arrows indicate the Qdot-labeled target proteins. (b) Image con-
version and preview detection from the raw frame. Image conversion to Gray 8 is preferred to increase com-
putational efficiency. Red circle masks the successfully targeted spots for tracking after executing the Preview
Detected function. (c) Visualization of all trajectories after executing the Show Detected function. Particular
area of interest can be selected and zoomed in as indicated in yellow box. (d) Time-lapse trajectory from the
selected area of interest. Red line drawn indicates the “Gaps” in the trajectory

5. Next, push the OK button and the result window will then be
displayed (Fig. 6c) after seconds to minutes computational cal-
culation (for 600 frames of 128 × 128 pixel images take less than
1 min on a regular dual core PC). With the Filter Options but-
ton given on the dialog window, you can filter out trajectories
under a given length. Particular trajectory of interest can be
selected by clicking it once with the mouse left button (see yel-
low box of Fig. 6c).
6. The selected trajectory can be displayed in a separate window
by clicking on the Focus on Selected Trajectory button. The
160 Jerry C. Chang and Sandra J. Rosenthal

visualization of the selected trajectory can then be saved


individually in .gif format (Fig. 6d). Detected time-series trajec-
tory coordinates can also be exported in a single .txt file for
further analyses.
7. After exporting trajectory coordinates, MSD of a specific
trajectory can be obtained according to the formula below:
N −n
MSD(nΔt ) = ( N − n)−1 ∑ [(xi +n − xi ) + ( yi +n − yi ) 2 ],
2
(7)
i =1

where xi and yi are the position of particle on the frame i, Δt is the


time resolution, N is total number of frames, nΔt is the time interval
over which the MSD is calculated.

4 Notes
1. Detailed information regarding Photometrics CCD specification
can be found at http://www.photomet.com.
2. The spinning force is not critical in this step. In most cases, a
rotational speed as low as 500 rpm (~30 × g for common com-
pact spin coater) is sufficient to achieve a uniform spread when
using common compact spin coater.
3. The labeling concentration/cell type relationship should be
adjusted for the surface protein expression level. In our experi-
ments, we choose low concentrations for transfected cells. For
labeling endogenously expressing membrane proteins in living
cells, higher concentrations may be needed.
4. The algorithm used in the ParticleTracker program can easily
cause false linking of different molecules/particles between
frames. This could lead to incorrect trajectory construction. It
may be improved by manual relinking with visual inspection.
However, potential problems and limitations can still be associ-
ated with such manual relinking. Due to its respectable efficiency,
ParticleTracker program is suitable for preliminary screening
tests. For serious and in-depth analysis, we recommend to use
particle tracking using IDL or the Matlab-based tracking
routines.

Acknowledgments

The authors thank Drs. David Piston and Sam Wells for helpful
advice with single quantum dot tracking experimental setup. We
thank colleagues in the group, especially to Dr. James McBride and
Oleg Kovtun, for helpful discussions and suggestions. This work
was supported by grants from National Institutes of Health
(R01EB003778).
Single Quantum Dot Imaging in Living Cells 161

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Chapter 16

Fabrication of Fluorescent Silica Nanoparticles with


Aggregation-Induced Emission Luminogens for Cell Imaging
Sijie Chen, Jacky W.Y. Lam, and Ben Zhong Tang

Abstract
Fluorescence-based techniques have found wide applications in life science. Among various luminogenic
materials, fluorescent nanoparticles have attracted much attention due to their fabulous emission properties
and potential applications as sensors. Here, we describe the fabrication of fluorescent silica nanoparticles
(FSNPs) containing aggregation-induced emission (AIE) luminogens. By employing surfactant-free sol–gel
reaction, FSNPs with uniform size and high surface charge and colloidal stability are generated. The FSNPs
emit strong light upon photoexcitation, due to the AIE characteristic of the silole aggregates in the hybrid
nanoparticles. The FSNPs are cytocompatible and can be utilized as fluorescent visualizer for intracellular
imaging for HeLa cells.

Key words Fluorescent silica nanoparticles, Fluorescent probes, Aggregation-induced emission,


Sol–gel reaction, Cell imaging

1 Introduction

Fluorescent nanoparticles have been used widely in biology as


sensing, imaging or tracking probes (1, 2). Among them, semicon-
ductor quantum dots (QDs) have received particular interest because
of their high fluorescence and resistance to photobleaching.
Unfortunately, they are made of heavy metals and are thus inher-
ently toxic. Although scientists have tried to solve the problem by
various techniques, their cytotoxicity is still a considerable issue (3).
Silica nanoparticles (SNPs), on the contrary, are cytophilic,
transparent but nonfluorescent and hence are ideal host materials
for the fabrication of fluorescent silica nanoparticles (FSNPs) for
imaging application (4). FSNPs can be prepared by incorporating
fluorophores into silica networks via physical processes or chemical
reactions. The silica matrix acts as a protective shield, reducing the
possibilities of penetrations of oxygen and other harmful species
that may cause photobleaching of the embedded fluorophores.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_16, © Springer Science+Business Media New York 2013

163
164 Sijie Chen et al.

Fig. 1 (Left) TEM image of the FSNPs. (Right): photographs of solutions and suspensions of 1, SNPs, and
FSNPs in ethanol taken under 365 nm UV irradiation. Reproduced from ref. 11 with the permission of Wiley

However, most dye molecules emit intensely in the solution state


but become weakly fluorescent when aggregated in poor solvent or
in the solid state. Such phenomenon is known as aggregation
caused quenching (ACQ) effect (5) and has been an obstacle for
the development of highly emissive FSNPs.
In 2001, Tang discovered a phenomenon of aggregation-
induced emission (AIE) that is exactly opposite to the ACQ effect:
nonemissive, propeller-like luminogens such as hexaphenylsilole
are induced to emit efficiently by aggregate formation (6). The
AIE effect dramatically boosts the fluorescence quantum yields of
the luminogens, turning them from faint fluorophores to strong
emitters. Mechanistic investigations reveal that the AIE effect is
caused by the restriction of the intramolecular rotation in the
aggregate state, which blocks the nonradiative relaxation channel
and populates the radiative decay (7–10).
In this chapter, we describe a methodology of hybridizing AIE
luminogens with silica nanoparticles to generate highly emissive
FSNPs. The luminogens are chemically bound to the silica net-
works, which can prevent their leakage under harsh conditions.
The FSNPs are uniformly sized, surface-charged, and colloidally
stable. Whereas the solution of the AIE luminogens and the sus-
pension of the SNPs are invisible under the UV irradiation, intense
green light is emitted from the FSNPs (see Fig. 1). The FSNPs can
be function as a fluorescent visualizer for selectively imaging the
cytoplasm of HeLa cells (see Fig. 2) (11, 12).

2 Materials

2.1 Chemicals for 1. THF was purchased and distilled from sodium benzophenone
Synthesis ketyl under nitrogen immediately prior to use.
2. Tetraethoxysilane (TEOS).
Fabrication of Fluorescent Silica Nanoparticles… 165

Fig. 2 Fluorescent images of HeLa cells stained by FSNPs with different luminogen loadings. Concentration of
1 (μM): (a) 2, (b) 4, (c) 8. Reproduced from (11) with the permission of Wiley

3. Dimethylsulfoxide (DMSO).
4. (3-Aminopropyl)triethoxysilane (APS).
5. Phenylacetylene.
6. n-Butyllithium (n-BuLi).
7. Naphthalene.
8. Decylmethyldichlorosilane.
9. Lithium.
10. Dichloro(N,N,N ¢,N ¢-tetramethylethylenediamine)zinc.
11. 4-Iodophenol.
12. 1,2-Dibromoethane.
13. Dichlorobis(triphenylphosphine)palladium(II).
14. Deionized water was used in the experiment.

2.2 Cell Culture The following materials are used for culturing the HeLa cells.
1. Minimum Essential Medium, Fetal Bovine Serum, penicillin,
and streptomycin were purchased from Gibco, Invitrogen.
2. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
was purchased from Sigma.
3. 35 mm culture dishes were purchased from Corning.
4. Phosphate buffered saline (PBS).
5. Cover slides.
6. Cell culture CO2 incubator.

2.3 Instrument 1. NMR analysis was conducted on a Bruker ARX 400 spectrom-
eter with tetramethylsilane (TMS; d = 0) as internal standard.
2. Mass spectroscopy was carried out on a Finnigan TSQ 7000 triple
quadrupole spectrometer operating in a MALDI–TOF mode.
3. The morphologies of the FSNPs were investigated using JOEL
2010 TEM and JOEL 6700F SEM at an accelerating voltage
of 200 kV.
166 Sijie Chen et al.

4. Copper 400-mesh carrier grids covered with carbon-coated


formvar films were used for the TEM and SEM measurements.
5. Fluorescence spectra were taken on a Perkin–Elmer LS 50B
spectrofluorometer with a Xenon discharge lamp excitation.
6. Zeta potentials and particle sizes of the FSNPs were deter-
mined at room temperature by a ZetaPlus Potential Analyzer
(Brookhaven Instruments Corporation, USA).
7. The HeLa cells were imaged under an inverted fluorescence
microscope (Nikon Eclipse TE2000-U; λex = 330–380 nm, dia-
chronic mirror = 400 nm).

3 Methods

3.1 Synthesis 1. To a mixture of 4-iodophenol (2, 5.0 g, 22.7 mmol) and potas-
(See Scheme 1) sium carbonate (4.7 g, 34.1 mmol) in acetone was added
12.8 g (68.2 mmol) of 1,2-dibromoethane (3). The mixture
3.1.1 Synthesis of
was stirred and heated to reflux for 24 h.
1-(2-Bromoethoxy)-4-
Iodobenzene (4) 2. After filtration and solvent evaporation, the crude product was
purified by silica gel chromatography using chloroform/hexane
(1:4 v/v) as eluent.

3.1.2 Synthesis 1. To a THF solution of phenylacetylene (5, 4.0 mL, 36.4 mmol)
of Bis(phenylethynyl) was added 25.0 mL (40.1 mmol) of 1.6 M n-butyllithium
decylmethylsilane (7) solution in hexane at −78°C. After stirring at the same tem-
perature for 2 h, decylmethyldichlorosilane (6, 4.8 mL,
18.2 mmol) was added at −78°C. The mixture was warmed to
room temperature and stirred overnight.
2. The solvent was removed under reduced pressure. The mix-
ture was dissolved in dichloromethane and washed with water.
The organic layer was dried over magnesium sulfate and
filtered. The filtrate was evaporated and the crude product was
purified by a silica gel column using hexane as eluent.

3.1.3 Synthesis of 1. A mixture of lithium (0.056 g, 8 mmol) and naphthalene


1-Decyl-1-methyl-2,5-bis[4- (1.04 g, 8 mmol) in 8 mL of THF was stirred at room tem-
(2-bromoethoxy)phenyl]- perature under nitrogen for 3 h to form a deep dark green
3,4-diphenylsilole (10) solution of lithium 1-naphthalenide.
2. The viscous solution was added dropwise to a solution of 7
(0.77 g, 2 mmol) in 5 mL of THF over 2 min at room
temperature.
3. After stirring for 1 h, the mixture containing 8 was cooled to
0°C with an ice bath and diluted with 10 mL THF. ZnCl2–
TMEDA (2 g, 8 mmol) was then added to the mixture to give
a black suspension of 9.
Fabrication of Fluorescent Silica Nanoparticles… 167

Scheme 1 Synthesis of silole derivative 1 and fabrication of fluorescent silica nanoparticles (FSNPs).
n-BuLi n-butyllithium, THF tetrahydrofuran, Naph 1-naphthalenide, TMEDA N,N,N ′,N′-
tetramethylethylenediamine, DMSO dimethysulfoxide. Reproduced from ref. 11 with the permission
of Wiley

4. After stirring for an additional hour at room temperature, a


solution of 4 (1.63 g, 4.9 mmol) and Pd(PPh3)2Cl2 (0.08 g,
0.1 mmol) in 10 mL of THF was added. The mixture was
refluxed overnight.
5. After cooling to room temperature, 10 mL of 3 M HCl solution
was added and the mixture was extracted with dichloromethane.
The combined organic layer was washed with brine and dried
over magnesium sulfate.
168 Sijie Chen et al.

6. After solvent evaporation under reduced pressure, the residue


was purified by a silica gel column using ethyl acetate/hexane
(1:9 v/v) as eluent.

3.2 Fabrication 1. Silole–APS (1) was prepared by stirring a mixture of 4 μM of


of FSNPs 10 and 10 μM of APS in 100 μL of DMSO overnight.
2. The reaction mixture was concentrated under vacuum.
3. 1 was added into a mixture of 64 mL of ethanol, 1.28 mL of
ammonium hydroxide, and 7.8 mL of distilled water and
stirred at room temperature for 3 h to prepare the silole–silica
nanocores (see Note 1).
4. A mixture of 2 mL of TEOS in 8 mL of ethanol was then
added dropwise (see Note 2) into the mixture of the nanocores
and the reaction was stirred at room temperature for an addi-
tional 24 h (see Note 3) to coat the luminogenic nanocores
with silica shells.
5. After incubation, the mixture was centrifuged and the FSNPs
were redispersed in ethanol under sonication for 5 min. Such
process was repeated three times and the FSNPs were finally
dispersed in water for the cell imaging experiments.

3.3 Cell Imaging The HeLa cells were cultured in minimum essential medium con-
by FSNPs taining 10% fetal bovine serum and antibiotics (100 units/mL
penicillin and 100 μg/mL streptomycin) in a 5% carbon dioxide
3.3.1 Cell Culture
humidity incubator at 37°C. These cells were grown overnight on
a plasma-treated 25 mm round cover slip mounted onto a 35 mm
culture dish with an observation window (see Note 4).

3.3.2 Preparation of 1. Prepare desired concentration of FSNPs in PBS.


FSNPs for Imaging 2. Autoclave the nanoparticles suspension at 121°C for 20 min.
3. Sonicate the suspension for 5 min.
4. Filter the suspension using a 450 nm filter in biosafety cabinet
and add 250 μL of filtrate into 2 mL medium (see Note 5).
5. Remove the cell culture medium and add the medium pre-
pared in step 4.
6. Incubate the cell in a 5% carbon dioxide humidity incubator at
37°C for 24 h.

3.3.3 Cell Imaging 1. Wash the cell with PBS for three times (see Note 6).
2. Add MEM with HEPES to the dish.
3. Observe the cell under the fluorescent microscope through the
observation window.
Fabrication of Fluorescent Silica Nanoparticles… 169

4 Notes

1. During the fabrication of the FSNPs (Subheading 3.2, step 3),


the ethanol/ammonium hydroxide/distilled water mixture
should be stirred for more than 5 min before addition of 1.
2. To coat the fluorescent nanocores with silica shells
(Subheading 3.2, step 4), the TEOS/ethanol mixture should
be added dropwise. The rate should be well controlled and
uniform. Nanoparticles with sizes of ~200 nm are generated at
a rate of 3 s/drop.
3. The stirring speed is important for the particle fabrication. The rate
should be high and stable to furnish FSNPs with uniform sizes.
4. The following are treated to the dishes before cell culture.
(a) Drill a hole of around 10 mm diameter in the middle of
the dish.
(b) Stick a piece of cover slide to the dish by paraffin.
5. The FSNPs are sonicated and filtrated before use as autoclave
will destabilize the nanoparticles. The FSNPS will be more
uniform and well dispersed after these treatments.
6. The cells should be washed thoroughly before imaging as excess
FSNPs will emit outside the cell and disturb the observation.

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intramolecular rotation of 1,1-substituted 4266–4272
2,3,4,5-tetraphenylsiloles. Chem Mater 15: 12. Faisal M, Yu Y, Tang BZ (2011) Fabrication of
1535–1546 silica nanoparticles with both efficient
7. Yu G, Yin S, Luo Y (2005) Structures, elec- fluorescence and strong magnetization and
tronic states, photoluminescence, and carrier exploration of their biological applications.
transport properties of 1,1-disubstituted Adv Funct Mater 21:1733–1740
Chapter 17

Monitoring the Degradation of Reduction-Sensitive Gene


Carriers with Fluorescence Spectroscopy
and Flow Cytometry
Constantin Hozsa, Miriam Breunig, and Achim Göpferich

Abstract
Polycations like poly(ethylene imine) (PEI) or poly(L-lysine) (pLL) form nanometer-sized complexes with
nucleic acids (polyplexes) which can be used for gene delivery. It is known that the properties of these
carriers can be greatly improved by introducing disulfide bridges on the polymers, thus making them
reduction sensitive. However, little is known about how such modified carriers behave intracellularly.
Here, we describe a method that uses the reduction-sensitive fluorescent dye BODIPY FL L-cystine
to label PEI and pLL. Our probe is activated under reductive conditions leading to strongly increased
fluorescence intensity. Subsequently, we show how the intracellular route of polyplexes made from these
labeled polymers can be monitored by flow cytometry.

Key words Poly(ethylene imine), Poly(L-lysine), Redox-sensitive gene carrier, Disulfides, Flow cytometry,
BODIPY FL L-cystine, Polyplexes

1 Introduction

Most strategies in gene therapy aim at altering the cellular gene


expression by either inserting therapeutic genes or inhibiting the
expression of undesirable gene products (1). In both cases, nucleic
acids are exogenously introduced into living cells with gene deliv-
ery vehicles. Currently, there is a growing interest in nonviral vec-
tors because of their higher safety compared to viral vectors and
their great flexibility for modification (2, 3). The two most com-
monly used classes of nonviral vectors are cationic lipids like
DOTAP and polycations such as poly(ethylene imine) (PEI) or
poly(L-lysine) (pLL) (4). Materials from both groups interact with
negatively charged nucleic acids to form nanometer-sized com-
plexes termed lipoplexes or polyplexes, respectively (2).
Such complexes must remain highly stable against ion exchange
reactions (i.e., displacement of the cargo) in the bloodstream but

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_17, © Springer Science+Business Media New York 2013

171
172 Constantin Hozsa et al.

have to release their cargo quickly after their uptake in target cells.
An inefficient release of nucleic acids leads to a low gene expression
level (DNA) or a low silencing effect with small interfering RNA
(siRNA) (5). Therefore, it is essential to create carriers that satisfy
both conflicting demands on the complex stability (6). One possi-
bility is to create environment-responsive polyplexes by exploiting
the large redox-potential gradient between extra- and intracellular
compartment. In this case, disulfide bonds serve as a trigger: They
remain stable outside cells but are readily cleaved at the high cyto-
solic glutathione concentration (7). There are numerous examples
in the literature employing this strategy. For instance, both short-
chained pLL and PEI were cross-linked to high molecular weight
branched products. Polyplexes made from those products show an
increased nucleic acid condensation capability and plasma stability as
well as high transfection efficiency (6, 8, 9). Disulfide cross-linked
PEI (S2-lPEI) is also known to be less cytotoxic than its non-cleavably
branched counterpart. Disulfides are also used to reversibly attach
cell-targeting ligands (small molecules, peptides, or antibodies) or
hydrophilic polymers as polyethylene glycol (PEG) to prevent
unspecific binding on the polyplex surface (2, 7).
As beneficial as the use of disulfides might be, there are a num-
ber of facts to be considered. Among others, the choice of the
cross-linking agent is important as well as the cross-linking ratio
(9). Polyplexes can easily be “overstabilized”, so it is indispensable
to adjust the exact spatiotemporal point of the linker cleavage to
the type of nucleic acid to be delivered: RNA has to be released
earlier than DNA as its target is in the cytosol, not the nucleus (8).
“Naked” DNA (i.e., without carrier), however, does not efficiently
pass nuclear pores (3, 10).
Unfortunately, the cellular trafficking of reduction-sensitive
gene carriers has hardly been investigated in detail so far. Where,
when, and how the carrier’s disulfides are cleaved is a matter of
dispute (7). The most likely location is the cytosol, but the cell
membrane, the endolysosomes, and the nucleus are also discussed
(10, 11).
To gain a better understanding of those processes, we devel-
oped a fluorescent probe based on the reduction-sensitive dye
BODIPY FL L-cystine (BP-Cys2, Fig. 1). This dye consists of a
cystine residue that carries two BODIPY molecules linked to its
amines. In this dimeric form, it is virtually nonfluorescent due to
the strong self-quenching of both fluorophores (12, 13). When
the bridging cystine disulfide is cleaved, a significant fluorescence
intensity increase indicates the presence of reductive conditions.
Polyplexes made with our probe can address several questions
such as the following: (1) Does the delivery system actually reach
reductive cellular compartments? (2) At which point does the
disulfide cleavage begin and how fast is it? (3) How are S2-cross-
linked polycations degraded?
Degradation of Reduction Sensitive Carriers 173

N N N
B N
B
F F F
NH HN F
O O
HO2C S S CO2H

Fig. 1 Structure of the self-quenching and reduction-sensitive dye BODIPY FL L-cystine: Cleavage of the bridging
disulfide bond results in an increased fluorescence intensity

In the following sections, we describe a quick and simple


method to label lPEI, S2-lPEI, and pLL with BP-Cys2. We also
explain the removal of unbound dye and the quality control of the
end product. Moreover, we shortly describe the cross-linking of
lPEI with cystine. Finally, we demonstrate the use of these labeled
polymers in flow cytometry.

2 Materials

Ultrapure water (18 MΩ cm or 0.055 µΩ/cm) and analytical grade


chemicals should be used in all experiments. Please note that
BODIPY FL Cys2, NEM, and all labeled products must be kept
frozen (£ −18°C) and protected from light (wrapped in aluminum
foil). DMTMM and pLL are to be stored under argon atmosphere.
Where indicated, solutions should be prepared fresh immediately
before use.

2.1 BODIPY FL 1. Solvents for labeling and purification: Water, methanol,


L-Cystine Labeling dimethyl-sulfoxide (DMSO), 0.1 M NaOH, and 10 mM HCl.
Components 2. 50 mM borate buffer, pH = 9, for purification of labeled PEI.
For preparation, add 4.77 g of Na2B4O7⋅10 H2O (sodium
tetraborate) to about 150 ml of water in a glass beaker (see
Note 1). Stir the solution with a magnetic stir bar until the
borate is completely dissolved. Remove the stir bar and adjust
the pH to 9 with HCl. Add water to a volume of 250 ml. Keep
refrigerated.
3. Phosphate-buffered saline (PBS buffer, see Note 2) for stock
solutions of the labeled products.
4. Linear polycations: lPEI is available in different molecular
weights from various manufacturers or can be synthesized by the
hydrolysis of poly(2-ethyl-2-oxazoline) (see Note 3). Linear
pLL in different sizes is also commercially available (see Note 4).
For this work, we used 6.3 kDa lPEI and 5.2 kDa pLL·HBr.
174 Constantin Hozsa et al.

5. Dye stock solution: 1 mg BODIPY FL L-cystine (M = 788.44 g/


mol, see Note 5) dissolved in 100 μl DMSO (γ = 10 g/l). Store
at −20°C and protected from light.
6. Coupling reagent stock solution: DMTMM (4-(4,6-Dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, M = 276.72 g/
mol) in methanol (γ = 10 g/l). Must be prepared fresh prior to each
use (see Note 6 and (14)).
7. Glassware: Small scale syntheses (approx. 200 mg) can easily be
done in 4 ml glass vials with snap-caps. Product handling and
recovery is improved with silanized glassware (see Note 7).
8. Ultrafiltration tubes with a molecular weight cutoff (MWCO)
suitable for the retention of the polymer (see Note 8).
9. Smaller items: magnetic stirrer and small magnetic stir bar
(ca. 5 mm), aluminum foil, pH indicator paper, syringe
(1–2 ml) and corresponding hypodermic needle, syringe
filter (0.22 μm pore size), TLC plates (silica gel) and separa-
tion chamber, UV lamp (254 or 366 nm), and argon.
10. Larger equipment: freeze dryer, alternatively a vacuum pump
with desiccator or a SpeedVac (see Note 9).

2.2 lPEI Cross- The cross-linking of lPEI is quite similar to the labeling procedure.
Linking Components Additional components needed: t-Butyl carbamate protected cys-
tine (Boc-Cys-OH)2, 1 M HCl, and NaOH pellets. The molecular
weight of the cross-linked product can be determined by size-
exclusion chromatography (see Note 23).

2.3 Cell Culture We typically use Chinese hamster ovary cells (CHO-K1) in our
and Flow Cytometry transfection experiments but this method was successfully tested
on other cell lines as well (13, 15). The following items are
needed:
1. CHO-K1 cells grown overnight (37°C, 5% CO2) in Ham’s
F12 medium (+10% fetal calf serum) in 24-well tissue-culture-
treated polystyrene plates (80,000 cells per well).
2. 1.5 ml microcentrifuge tubes and 1 ml, 200 μl and 20 μl
pipettes.
3. Stopwatch.
4. Trypsin and a laboratory centrifuge.
5. PBS buffer.
6. Ice for storing stock solutions and cells.
7. Cellular thiol-blocking solution: N-Ethylmaleimide (NEM) in
PBS (γ = 78.12 mg/l, c = 625 μM) (see Note 10).
8. Nucleic acid stock solutions: Plasmid DNA and/or siRNA in
water (γ » 1 g/l, see Note 11).
Degradation of Reduction Sensitive Carriers 175

2.4 Spectroscopy 1. UV/Vis spectrophotometer (400–600 nm), fluorometer


Components (lex = 488 nm, lem = 500–650 nm), and suitable microtiter
plates or cuvettes.
2. PBS buffer.
3. 2-Mercaptoethanol cleavage solution for a final concentration
of 100 mM (see Note 12).

3 Methods

All procedures (except cell culture experiments) are performed at


room temperature. The polymer amounts used here can be adapted
to your needs (see Note 13).

3.1 Labeling of This section describes the labeling of lPEI, Cys2-lPEI


Polycations with (see Subheading 1), and pLL (see Note 14). Differences in the
BODIPY FL L-Cystine work up procedures are indicated.
1. Dissolve about 30 mg (see Note 15) of the dried polymer in
400 μl H2O and 200 μl DMSO. Depending on the polymer,
the dissolution process might take several minutes. Add more
water if the polymer does not fully dissolve (200–400 μl), i.e.,
the solution is not clear.
2. Add 20–30 μl (200–300 μg/0.25–0.38 μmol) of the
BODIPY FL Cys 2 stock solution (see Note 16 ). The solu-
tion should now be orange and show an intense fl uorescence
under UV.
3. Dissolve 10 mg of DMTMM·H2O in 1 ml of methanol and
then add 63–96 μl (n(DMTMM) = 2–3 μmol) to the polymer
solution. This amount corresponds to four equivalents of
DMTMM for each BODIPY FL Cys2 carboxylic group.
4. Protect the reaction setup from light and stir it for at least 4 h.
5. Transfer the solution into an ultrafiltration tube and rinse the
reaction vessel with 10% DMSO (in water). Fill the tube with
the same solvent to its maximum volume and start the cen-
trifugation (see Note 17).
6. Discard the permeate, add more DMSO solution, and repeat
the centrifugation until the permeate is free from unbound dye
(see Note 18).
7. Repeat step 6 with borate buffer (pLL: 0.1 M NaOH). Then
use water until the permeate is neutral and switch to 10 mM
HCl (see Note 19).
8. Filter the retentate through a syringe filter (see Note 20) and
freeze-dry it in a silanized glass vial (see Notes 7 and 9). The
final product is a green fluorescent, brittle foam.
176 Constantin Hozsa et al.

3.2 Cross-Linking 1. Dissolve 200 mg lPEI (32 μmol) and 40 mg of (Boc-Cys-OH)2


of Linear PEI with (91 μmol; 2.8 equivalents per PEI molecule) in 800 μl and
(Boc-Cys-OH)2 850 μl, respectively, in methanol (see Note 21). Combine both
solutions then add 115 mg of DMTMM·H2O (361 μmol; two
equivalents per carboxylic group) in 500 μl methanol and stir
the reaction for at least 4 h.
2. Evaporate the methanol by carefully heating it up (50–60°C)
while stirring.
3. To remove the BOC-protecting group, add 1 M HCl (2 ml)
and stir for 1 h at room temperature. Do not close the reaction
vessel as CO2 is forming.
4. Transfer the solution into a 50 ml centrifuge tube and add
about 10–15 ml of water. Place the tube in an ice bath and
carefully add concentrated NaOH solution (15 ml or 4–5
NaOH pellets) to form a white PEI precipitate. Fill the rest of
the tube with water (do not overfill).
5. Centrifuge (4°C/12–15,000 g) until the precipitate is com-
pletely sedimented (see Note 22). Discard the supernatant.
6. Fill the tube with water and stir up the precipitate thoroughly.
Repeat steps 5 and 6 until the supernatant is neutral.
7. Dissolve the crude product in 1 M HCl (see also Note 21) and
perform an ultrafiltration with the same solvent (repeat four to
five times).
8. Filter and dry your final product as described under
Subheading 3.1 (see Note 23).

3.3 Quality Control Quality control with UV/Vis spectroscopy is a very important step
with UV/Vis because the spectroscopic properties of BODIPY FL Cys2 tend to
Spectroscopy undergo unforeseeable changes during the labeling process result-
ing in nonfluorescent products. We observed this behavior also
with succinimidyl ester activated BODIPY and 5(6)-Carboxytet-
ramethylrhodamine (5(6)-CO2H-TAMRA).
1. Dissolve the labeled polymer hydrochloride in PBS. A concen-
tration between 2 and 10 mg/ml is usually sufficient.
2. Measure the absorbance between 400 and 600 nm with pure
PBS as blank.
3. Check for the characteristic BODIPY FL absorbance maxi-
mum (Fig. 2a) at about 504 nm. A typical product spectrum
is shown in Fig. 2b. A hypsochromic shift (Fig. 2c, dotted
line) or a strong sideband next to the main absorbance band
(Fig. 2c, black line) indicates the formation of dye aggre-
gates. In that case, you can try lowering the labeling density
or use another polymer type (see Note 14).
Degradation of Reduction Sensitive Carriers 177

a 480 nm b 504 nm c 465 nm 502 nm


504 nm
absorbance

549 nm

400 450 500 550 600 400 450 500 550 600 400 450 500 550 600
λ/nm λ/nm λ/nm

Fig. 2 Absorbance spectra of BP-Cys2 (a) and BP-Cys2-labeled polymers: disulfide cross-linked lPEI
(b), branched PEI 25 kDa (c, black line), and disulfide cross-linked poly(L-lysine) (c, dotted line) measured in
PBS. Note that the second absorbance band (480 nm) of BP-Cys2 is only present when measuring in PBS.
There is no difference between the absorbance of the cleaved and uncleaved probe

3.4 Quality Control Fluorescence spectroscopy of the final product allows estimating
with Fluorescence the overall fluorescence intensity and the increase of fluorescence
Spectroscopy intensity after disulfide cleavage (see Note 24). Most importantly,
it shows potential changes in the emission spectra that might indi-
cate dye degradation or the formation of dye aggregates.
1. Prepare two 800 μl polymer samples (PBS, γ(polymer) » 0.25–
1.25 mg/ml) and add 200 μl PBS or 2-mercaptoethanol
(500 mM), respectively (see Notes 12). The samples’ absor-
bance should be below 0.1 at 504 nm to avoid self-absorbance
which lowers the fluorescence intensity.
2. Measure both samples (lex = 488 nm, lem = 500–650 nm) using
PBS as blank (see Note 25).
3. Check the emission spectrum of BODIPY FL. It must not
change during the labeling process (Fig. 3a). Look for any shift
in the emission maximum, signal broadening (Fig. 3b, black
line) or a band at around 610 nm (data not shown). In those
cases, the fluorescence intensity will be too low for reasonable
measurements.

3.5 Flow Cytometry In this section, we explain how nucleic acid complexes based on
the labeled polymers can be used in vitro (see Subheading 2.3).
We describe the basic setup, the polyplex formation, and the data
analysis in an experiment that follows the BODIPY FL fluorescence
intensity during the cellular uptake of pLL-DNA nanoparticles.
The use of N-Ethylmaleimide as a thiol-blocking agent proves the
involvement of intracellular SH-groups in the probe’s cleavage.
NEM untreated cells show a significantly higher increase of
fluorescence intensity.
1. Prepare two 24-well polystyrene tissue-culture plates (for 16
samples in triplicate) with CHO-K1 cells for the addition of
pLL-DNA polyplexes (incubation times: 4 h, 2 h, 1 h, 45 min,
178 Constantin Hozsa et al.

a b
511 nm 522 nm
fluorescence intensity

500 550 600 650 500 550 600 650


λ(emission)/nm λ(emission)/nm

Fig. 3 Fluorescence spectra of labeled polymers (lex = 488 nm): (a) disulfide cross-linked lPEI in absence
(black line) and in presence (dotted line) of 100 mM 2-mercaptoethanol. The intensity increase is about
twofold. (b) Comparison between S2-lPEI (dotted line) and branched PEI 25 kDa (black line). Note the
redshifted, broader emission band (not drawn to scale. bPEI 25 kDa has a significantly lower fluorescence
intensity than S2-lPEI)

30 min, 15 min; see Note 26) in the presence and absence of


NEM (twelve samples). Cells without polyplex treatment serve
as reference (four samples: 4 h, 15 min; ±NEM).
2. Before each polyplex addition, the cells are preincubated with
25 μM NEM (see Note 27) for 1 h: Replace the medium with
a mixture of 240 μl Ham’s F12/10% FCS and 10 μl freshly
prepared NEM stock solution or PBS, respectively.
3. After 50 min, start the polyplex formation: Add 20 μl PEI stock
solution to 1 μg DNA dissolved in 20 μl PBS (see Note 28). Keep
it in the dark for 10 min. Wash the cells once with PBS, then
add 200 μl Ham’s F12 (w/o FCS) and the polyplex solution
(references: 40 μl PBS). Shake the well plate gently and place
it back in the incubator.
4. Repeat steps 2 and 3 with all samples at the given time points.
5. Wash all samples with PBS and detach the cells with 250 μl
trypsin (see Note 29). Stop the reaction by adding 300 μl
Ham’s F12/10% FCS.
6. Wash all samples at least once before resuspending them in
300 μl PBS.
7. Adjust your flow cytometer as if using FITC stained samples
(lex = 488 nm; detection: “FITC-channel” (usually FL1)
530/30 nm). For finding the right PMT voltage settings, first
measure the samples with the highest (4 h polyplex incubation
w/o NEM) and the lowest (untreated cells) expected
fluorescence intensity.
Degradation of Reduction Sensitive Carriers 179

a b

normalized fluorescence intensity (FL1)


absolute fluorescence intensity (FL1)

1
0 60 120 180 240 0 60 120 180 240
t(polyplex incubation)/min t(polyplex incubation)/min

Fig. 4 Flow cytometry—fluorescence intensity of BP-Cys2-pLL/DNA polyplexes in the presence (gray symbols)
or absence (white symbols) of 25 μM NEM as a function of polyplex incubation time. Polyplex treated (open
circles) and untreated/reference (open squares) cells: (a) absolute values, (b) normalized values

8. Data analysis: Plot the sideward scattering (SSC) intensity


against the forward scattering (FSC) intensity. Gate all living,
intact cells and plot their BODIPY FL fluorescence intensity
(FL1) against FSC.
9. Finally, plot the mean FL1 intensity against the polyplex incu-
bation time (Fig. 4a). Negligible changes in the intensity
(15 min → 4 h) of the references indicate a reliable experiment.
The presence of NEM tends to shift the measured values
slightly upwards so a normalization against the first time point
is advisable (Fig. 4b; see Note 30).

4 Notes

1. It is not necessary to use a volumetric flask. The accuracy of the


glass beaker is sufficient for this application.
2. Ready to use PBS buffer is inexpensive and can be found in any
cell culture lab.
3. Selected lPEI manufacturers: Polysciences Inc., Warrington,
PA, USA (http://www.polysciences.com); Polymer
Chemistry Innovations Inc., Tucson, AZ, USA (http://www.
polychemistry.com). PEI hydrochloride and PEI as a freebase
can be labeled. There is no influence on reactivity, but
PEI·HCl is insoluble in organic solvents. Please also note that
commercially available PEI might contain large amounts of
water. For an accurate gravimetric determination of its
amount, it has to be dried in vacuum at » 95°C until all water
is evaporated (no bubbles in the molten polymer). For a
review of lPEI syntheses, see (16) and (17).
180 Constantin Hozsa et al.

4. Highly monodisperse linear pLL can be obtained from


Alamanda Polymers, Inc., Huntsville, AL, USA (http://www.
alpolymers.com). The choice of the counterion is irrelevant.
Keep pLL under argon.
5. To our knowledge, BODIPY FL L-cystine is only available
from Invitrogen Corporation, Carlsbad, CA, USA (http://
www.invitrogen.com). Catalog Number: B-20340.
6. Solid DMTMM should be kept frozen and under an argon
atmosphere. It is highly hygroscopic. Please be aware that it
contains about 12% (w/w%) water as an impurity. Hence, 1 mg
solid DMTMM·H2O consists of 3.18 μmol DMTMM and
6.66 μmol H2O.
7. Silanization can be done with Sigmacote (Sigma-Aldrich) or
any comparable product. Please refer to the manufacturer’s
instructions.
8. We prefer ultrafiltration (centrifugal tubes) for the removal of
unbound dye as it is much faster and has a higher product
recovery than dialysis. As a rule of thumb from the manufac-
turers, select a membrane with a cutoff between 0.2 and 0.3
times the value of the molecular weight of the polymer.
However, we were able to recover 50% of 3.2 kDa pLL with a
3 kDa membrane. If the MWCO is too small compared to the
polymer mass, the product purification might take a very long
time. Ultrafiltration tubes are available in many sizes (usually
0.5, 4, and 15 ml). For small samples (< 20 mg), use a tube
volume of 4 ml. To our experience, the filtration process is
faster in swing bucket than in fixed angle centrifuges.
9. If no freeze dryer is available, put the frozen product in a desic-
cator and use a standard laboratory vacuum pump.
10. Caution: NEM is highly toxic! Use proper safety equipment.
Always prepare a fresh solution before use: Dissolve 2 mg of
NEM in 639 μl PBS (c (NEM) = 25 mM) then dilute 25 μl of
this solution with 975 μl PBS (c (NEM) = 625 μM). Keep this
solution on ice and protected from light.
11. TE buffer can be used as well (10 mM Tris–HCl, pH = 7.0,
1 mM EDTA). RNA is highly sensitive to degradation by
RNase and base hydrolysis under basic conditions. Make sure
to use RNase free chemicals.
Be careful in the choice of your gene product. The emission
spectra of proteins like (E)GFP are very similar to BODIPY FL
(lem = 509 nm) and will interfere in any measurement.
12. Caution: 2-Mercaptoethanol (BME) is toxic, malodorous
(fume hood!), and absorbed through skin. It decomposes
under the influence of water to hydrogen sulfide, so stock
Degradation of Reduction Sensitive Carriers 181

solutions must always be prepared fresh. For a 500 mM stock


solution, add 965 μl PBS to 35 μl of pure BME (c = 14.3 M).
13. We tested our method with the following amounts of polycat-
ions: labeling 9–50 mg, cross-linking 40–220 mg.
14. To our experience, the labeling of branched PEI (bPEI) and
Cys2-pLL leads to the formation of BODIPY aggregates with
altered absorbance and emission characteristics. The resulting
products show little or no fluorescence.
15. 30 mg of dried polymer correspond to about 5 and 6 μmol of
6.3 kDa lPEI and 5.2 kDa pLL·HBr, respectively.
16. A labeling ratio from 10 to 20 (n (polymer)/n (BODIPY)) is
usually sufficient. Lower ratios increase the risk of forming
nonfluorescent BODIPY aggregates, while higher ratios yield a
low fluorescence signal of the end product.
17. At maximum speed, one centrifugation step usually takes
between 20 and 30 min. The filtration devices have a dead-
stop volume which prevents them from running dry.
18. Use TLC (eluant: methanol, detection: UV, Rf(BODIPY) ≈ 0.8,
labeled polymer remains on baseline) to check for unbound
dye. The product should be stirred up with a pipette (do not
touch the filtration membrane!) before each consecutive cen-
trifugation step. If you are unable to the finish the ultrafiltration,
the product can be stored in the tube overnight.
19. PEI precipitates at higher pH values, while BODIPY FL
L-cystine has an increased solubility. Do not use higher concen-
trated HCl (above 10 mM, pH = 2) as the dye degrades over
time under more acidic conditions.
20. This filtration is necessary because the polymer hydrochloride
electrostatically attracts dust.
21. The molecular weight of the cross-linked product is influenced
by factors like the size of the lPEI, the amount of cross-linker,
and the reaction volume. If the product turns out to be insol-
uble (gel-like, i.e., overly cross-linked) under acidic conditions,
use less cross-linker but keep the reaction volume constant.
22. The precipitate might swim on top in the first two centrifuga-
tion steps due to the higher density of the concentrated NaOH
solution. In that case, you can use a syringe with a long needle
to remove the solution under the crude product.
23. The molecular weight can be determined by gel filtration chro-
matography: g (S2−lPEI) = 20 mg/ml, 150 mM NaCl + 0.1%
TFA at 1 ml/min, Novema 300 Å SEC column (PSS Polymer
Standards Service GmbH, Mainz, Germany) at 40°C, refrac-
tive index, and UV detector (247 nm).
182 Constantin Hozsa et al.

Table 1
An example of how to plan a flow cytometry experiment The following
parameters were used: polyplex incubation: 4 h and 15 min;
NEM incubation: 1 h; polyplex formation: 10 min

t (polyplex incubation) 4h 15 min


NEM addition 8:00 11:45
Polyplex formation 8:50 12:35
Polyplex addition 9:00 12:45
Trypsinization 13:00 13:00

24. The increase in fluorescence depends on factors like labeling


density and polymer type. Therefore, no general rule can be
given. It usually varies between 30 and 100% but a 30-fold
increase is possible under certain conditions. You should always
check if a particular polymer batch can be used for an experi-
ment. Each instrument (e.g., spectrometer vs. cytometer) has
a unique detector linearity. We even found samples with an
increase as low as 30% to be very useful in flow cytometry.
25. An excitation at 504 nm would result in higher fluorescence
intensity but most microscopes and flow cytometers are
equipped with 488 nm light sources. In addition, due to the
small Stokes shift of BODIPY FL, the excitation light might
interfere with detection of the emitted light at 511 nm. It is
advisable to measure the cleaved sample first as a higher emis-
sion intensity is to be expected. Adjust the measurement
parameters (PMT voltage, band-passes) accordingly, and then
measure the uncleaved sample and the blank under the same
conditions.
26. This experiment requires thorough time planning. Frequently,
different solutions have to be added at the same time. Combined
with the large number of samples, this might quickly lead to
confusion. It is strongly advised to create a spreadsheet that
automatically calculates the time point for each work step (for
an example, see Table 1).
27. NEM is cytotoxic in higher concentrations. Although in many
publications concentrations around 100 μM are used, we
found that this is killing virtually all CHO-K1 cells after 1 h of
incubation. According to our data, 25 μM is sufficient to
efficiently block cellular thiols while leaving most cells intact.
28. The ratio of positively charged polymer amine nitrogen atoms
to negatively charged nucleic acid phosphorus atoms is specified
by the N/P value. The higher it is the stronger polyplexes are
formed; it typically lies between 8 and 24. The N/P value is
calculated as follows:
Degradation of Reduction Sensitive Carriers 183

n(P) estimation:
1 μg double-stranded DNA contains approximately 3.237 nmol P
(RNA: 3.150 nmol)
n(N) estimation (pLL·HCl):
m (pLL.HCl )
n(N)pLL × HCl = × [N (lysine residues) + 1]
M (pLL.HCl )
with M (pLL.HCl) = 18.05 g/mol + N (lysine residues) × 164.62 g/mol,

e.g., 1 μg H2N-(Lys)25-OH·HCl corresponds to


M (pLL.HCl ) = 4133.55 g/mol, n(pLL.HCl) = 241.9 pmol,
and n(N)pLL.HCl = 6.290 nmol

n(N) estimation (PEI·HCl): PEI’s nitrogen content (mass frac-


tion w(N)) has to be determined with CHN analysis or is pro-
vided by the manufacturer,
e.g., 1 μg PEI with a nitrogen content of 17.62% corresponds to
m(PEI.HCl) × ω(N) 1 μg × 0.1762
n(N)PEI.HCl = = = 12.58 nmol.
M (N) 14.0067 g/mol

29. PEI or pLL can lead to an increased cell adhesion making com-
plete trypsinization impossible. In that case, press the well plate
(lid!) on a flat, smooth surface and shake it vigorously in a cir-
cular motion.
It
30. Calculation of normalized fluorescence intensities: I t =
n

I t0
I tn : normalized intensity, It: absolute intensity. I t 0 : absolute
intensity at first time point t0.
Corresponding propagation of uncertainty for the standard
deviation s:
2 2
⎛ 1⎞ ⎛ I ⎞
σ(I ) = ⎜ ⎟ σ 2 (I t )+ ⎜ − t 2 ⎟ σ 2 I t 0
n
c
⎝ I t0 ⎠ ⎝ I t0 ⎠
( )

References

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using polymer therapeutics. In: Satchi-Fainaro, delivery systems for siRNA. Oligonucleotides
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Springer, Berlin plexes with regulated densities of charge and
3. Won YY et al (2009) Missing pieces in under- disulfide cross-linking directed to enhance
standing the intracellular trafficking of polycation/ gene expression. J Am Chem Soc 126:
DNA complexes. J Control Release 139:88–93 2355–2361
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mers for efficient gene and siRNA delivery. 14. Kunishima M et al (1999) 4-(4,6-dimethoxy-
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269:2550–2561 130:57–63
11. Lin C, Engbersen JF (2009) The role of the 16. Lungwitz U et al (2005) Polyethylenimine-
disulfide group in disulfide-based polymeric gene based non-viral gene delivery systems. Eur
carriers. Expert Opin Drug Deliv 6:421–439 J Pharm Biopharm 60:247–266
12. Da Poian AT et al (1998) Kinetics of intracel- 17. Brissault B et al (2003) Synthesis of linear
lular viral disassembly and processing probed polyethylenimine derivatives for DNA trans-
by Bodipy fluorescence dequenching. J Virol fection. Bioconjug Chem 14:581–587
Methods 70:45–58
Chapter 18

Quantification of Intracellular Mitochondrial


Displacements in Response to Nanomechanical Forces
Yaron R. Silberberg and Andrew E. Pelling

Abstract
Mechanical stress affects various aspects of cell behavior, including cell growth, morphology, differentiation,
and genetic expression. Here, we describe a method to quantify the intracellular mechanical response to an
extracellular mechanical perturbation, specifically the displacement of mitochondria. A combined
fluorescent-atomic force microscope (AFM) was used to simultaneously produce well-defined nanome-
chanical stimulation to a living cell while optically recording the real-time displacement of fluorescently
labeled mitochondria. A single-particle tracking (SPT) approach was then applied in order to quantify the
two-dimensional displacement of mitochondria in response to local forces.

Key words Atomic force microscopy, Mitochondria, Single-particle tracking, Nanomechanics,


Mechanotransduction, Force transmission

1 Introduction

The atomic force microscope (AFM) (1) has become an invaluable


tool for investigating biological systems. The ability to study living
cells in fluid under physiological conditions has facilitated both
nanoscale imaging (2) and the measurement of various mechanical
and material properties of living cells, such as viscoelasticity (3, 4)
and mechanical dynamics (5, 6). Recent technical developments
have integrated traditional microscopy methods, such as
fluorescence and laser scanning confocal microscopies, into AFM
systems (7–9). This has enabled the simultaneous measurement of
material properties of living cells and their biological responses and
signaling pathways to be made.
Mitochondria are semiautonomous and highly dynamic organ-
elles, which have the ability to change their shape and their loca-
tion inside the living cell (10). Localization and rearrangement of
mitochondria in higher eukaryotes is known to be dependent on
the microtubule cytoskeleton (11, 12). More recent research sug-
gests that actin filaments have an important role as well, such as

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_18, © Springer Science+Business Media New York 2013

185
186 Yaron R. Silberberg and Andrew E. Pelling

facilitating mitochondrial organization in yeast and vertebrate


neurons (13, 14) and controlling mitochondrial movement and
morphology (15). Given the strong association of mitochondria
with the cytoskeleton, it is predicted that forces locally applied via
the AFM tip would affect their arrangement if the cytoskeleton is
capable of mechanical transduction (16–18).
Feature-point tracking is a single-particle tracking (SPT) algo-
rithm that enables an efficient, automated, two-dimensional detec-
tion and tracking analysis of particles trajectories (19). The method
is suited to digital video and time-lapse fluorescence imaging,
which typically generates low-intensity data. Feature-point track-
ing detects particle positions in a digital video or image sequence
and generates particle trajectories over time. One of its main advan-
tages is that it does not make any assumptions regarding the
smoothness of the trajectories. Thus, it is extremely useful for many
biological applications where the type of motion is not explicitly
known in advance or when the motion is random and can change
rapidly. Furthermore, by not assuming a motion model, the algo-
rithm integrity is not biased when several modes of motion are
incorporated by a single trajectory. Therefore, this method is useful
for tracking both natural and force-induced motion (post-pertur-
bation) of an organelle in the same time-lapse sequence, such as in
the case of tracking mitochondrial displacements.
This protocol describes a straightforward approach for com-
bining time-lapse fluorescent imaging while performing extracel-
lular mechanical indentation using the AFM. The quantification of
the mechanical response of mitochondria, by applying feature-
point tracking approach using ImageJ’s plug-in ParticleTracker,
allows to correlate the effect nanomechanical forces have on the
living cell and to conclude on the mechanical force transmission in
the live cell. Examples for the application of this system include the
comparison between basal and force-induced mitochondrial dis-
placements (20) and the analysis of the effect of cytoskeleton dis-
ruption on force induction in the live cell (21).

2 Materials

1. Cell culture: Any standard cell type that can be stained with a
live-cell mitochondria dye, such as MitoTracker Red (Life
Technologies, Grand Island, NY, USA), can be used. In our
case, NIH-3T3 fibroblasts were used.
2. Growth medium: GlutaMAX I media (Life Technologies, Grand
Island, NY, USA) supplemented with 10% fetal bovine serum
and 100 IU/ml penicillin and 100 μg/ml streptomycin.
3. Culture dishes: For epi-fluorescence imaging, either plastic or
glass-bottom dishes are suitable. Note: If the AFM apparatus con-
tain a specialized stage, then specific dish type might be needed.
Quantification of Intracellular Mitochondrial… 187

4. Fluorescent dye: MitoTracker Green (Life Technologies,


Grand Island, NY, USA) and MitoTracker Red stock solutions
should be kept at −20°C in DMSO at concentration of 1 mM.
Cells are incubated with either MitoTracker Green at 1 μM for
30 min or MitoTracker Red at 100 nM for 10 min, before
replacing with fresh media.
5. AFM: Should be suitable for working in liquid and integrated
onto an epi-flourescence inverted optical microscope to allow
for simultaneous operation. In our case, we employed a
NanoWizard I AFM (JPK Instruments); however, several man-
ufacturers now offer similar AFMs for combined AFM and
optical microscopy. Contact-mode cantilever for liquid should
be used and calibrated prior to experiment. In our case, MSCT-
AUWH AFM cantilevers (Veeco) with pyramidal-shaped tips
were calibrated, and the spring constant was experimentally
determined to be 0.05 ± 0.01 N/m (22).

3 Methods

3.1 Image 1. Plate the cells on the day prior to experiment into suitable
Acquisition dishes (must be compatible with the fluorescence microscopy
layout and the AFM apparatus) (see Note 1).
2. On the day of experiment, incubate the cells with MitoTracker
Red at 100 nM for 10 min, before replacing with fresh media
(see Note 2).
3. Leave cells to equilibrate in the incubator (37°C) for a further
30 min prior to the indentation experiment (see Note 3).
4. Calibrate the AFM (cantilever sensitivity and spring constant)
by using a suitable method such as the “thermal fluctuation”
method (22).
5. Put the dish containing the cells on the microscope stage (pref-
erably temperature-controlled) (see Notes 2–6), and carefully
position the AFM head on top of the stage and lower the can-
tilever holder into the media, paying attention not to crash the
cantilever’s tip into the bottom of the dish. Try to avoid having
bubbles caught on the cantilever (see Note 7).
6. Optically choose live, interphase cells and position the AFM
cantilever and tip above the center of the nucleus (see Note 8).
7. Set time-lapse fluorescent image capturing at the desired inter-
val (such a one image per second).
8. After at least two images had been captured, perform indenta-
tion at the desired force (see Fig. 1 of the experimental layout).
9. Retract the AFM cantilever after the next image had been cap-
tured and stop time-lapse imaging.
188 Yaron R. Silberberg and Andrew E. Pelling

Fig. 1 Experimental layout of image acquisition. A sequence of images was taken at 1 s intervals. Three images
were picked for analysis: two images taken prior to AFM indentation (images 1 and 2) and the one image that
followed the indentation (image 3). Changes between image 1 and 2 reflect basal mitochondrial movement,
while changes between image 2 and image 3 reflect the basal movement together with force-induced move-
ment that resulted from the AFM indentation. The pyramidal AFM tip can be seen in the middle of the cell in
image 3. Scale bars are 10 μm

3.2 Image Analysis Mitochondria displacement analysis is carried out by the method of
feature-point tracking (see Introduction). A sequence of three
images is analyzed, and the comparison is made between two pairs
of images: changes from image 1 to image 2, which were captured
prior to AFM indentation, reflect the basal movement of mito-
chondria in the cell (control), and changes from image 2 to image
3, between which AFM indentation takes place, reflect the basal
movement in addition to the force-induced movement (Fig. 1).
Thus, this experimental layout has a built-in control that allows the
normal, basal movement to be distinguished from movement that
results from force application and indentation.
For analysis of mitochondria displacements, any particle-track-
ing software can be used; however, ImageJ, a public-domain image
processing and analysis program developed at the National Institute
of Health (ImageJ, http://rsbweb.nih.gov/ij/), together with the
ParticleTracker plug-in (ParticleTracker, http://courses.washington.
edu/me333afe/ImageJ_tutorial.html), proved to be a suitable
tool for that purpose (20, 21). This plug-in is used to detect par-
ticles and calculate trajectories in an image sequence using the
feature-point tracking algorithm (19). The tracking algorithm
consists of two main steps: detection of feature points in every
frame and linking of these points into trajectories:
1. Load the three sequential fluorescent images into ImageJ, and
convert them to a stack (Image > Stacks > Images to Stack) (see
Note 8).
2. Run the ParticleTracker plug-in. Since image conditions, such
as the intensity and noise levels, vary between each image set,
several parameters need to be adjusted manually in order to
facilitate correct particle detection and avoid false detections
Quantification of Intracellular Mitochondrial… 189

Fig. 2 Tracking mitochondrial displacements using ParticleTracker. After the three images are combined into a
stack, the ParticleTracker plug-in identifies feature points according to manually entered parameters (a) a
region for analysis is then chosen, (b) and the trajectories in that region are shown together with coordinates
of each determined feature point at each of the three images (c). Before processing the results, all trajectories
are manually verified and false links (c,white arrow ) are removed. Scale bars are 10 μm

due to background noise. These include the approximate


radius of the particle (in pixels), which should be bigger than a
single-point radius but smaller than the distance between two
separate points (usually set to 3), and a percentile (%) that
determines the sensitivity of the algorithm to background noise
when deciding the local maxima of featured points (i.e., how
bright the particle needs to be in order to be accepted as a
feature point). The percentile is the percentage of the upper
end intensity range that will be considered as feature points,
and is usually set between 0.2 and 1.5%.
3. ImageJ marks with red circles all the detected feature points
(Fig. 2a). If the image is too noisy, adjustments to the above
parameters should be made to make sure no “noise” was mis-
taken as feature points.
190 Yaron R. Silberberg and Andrew E. Pelling

Fig. 3 Mean mitochondrial displacement calculated with the feature-point tracking


algorithm. The average mitochondrial displacement increased in ~40% following
AFM indentation, from 114 ± 6 nm for the natural displacement to 160 ± 10 nm
post-indentation (mean ± s.e.m; * < 0.001)

4. Click on “visualize all trajectories.” Then, select an appropriate


region for the analysis (e.g., where single mitochondrial struc-
tures can clearly be distinguished from each other) (Fig. 2b),
and click “Trajectories in area info.” Data on all the trajectories
in that region is extracted, including the x and y coordinates of
each particle in each of the images in the stack (Fig. 2c). You
can then copy and paste the list of coordinates to a separate
ASCII file.
5. Manual inspection of the trajectory list is recommended, espe-
cially for more noisy images, in order to remove invalid trajec-
tories (see Note 9).
6. The final step is the calculation of absolute displacements of
each particle from the extracted trajectories, which can be done
either manually or using an appropriate data processing tool,
such as MatLab. Note that the second column of the output
data contains the x coordinate and the third column contains
the y coordinate and that every line represents a different image
in the stack.
As images 1 and 2 are taken prior to indentation and image 3
taken following indentation, the absolute basal (natural) displace-
ment (in pixels) is given by (x 2 − x 1)2 + (y 2 − y 1)2 ,and the force-
induced movement is given by (x 3 − x 2)2 + (y 3 − y 2)2 (the
numbers following x and y represent the respected images).
Figure 3 shows the results for natural and post-indentation dis-
placement of mitochondria, calculated using the feature-point
tracking algorithm (21). A total of 323 individual mitochondria
Quantification of Intracellular Mitochondrial… 191

were analyzed, in 42 regions for a total of 21 cells. The average


natural displacement calculated to be 114 ± 6 nm, while the aver-
age post-indentation displacement was 160 ± 10 nm (mean ± s.e.m;
*P < 0.001). That is, mitochondrial displacement increased by
~40% in response to indentation.

4 Notes

1. When investigating force transmission on a single-cell level, it


is important to make sure the experiment is conducted on sin-
gle cells not on a confluent layer, where intracellular mechanics
are interlinked and affected by neighboring cells. However,
experiments can also be devised to investigate the movement
of internal cellular structures in cell monolayers.
2. It important to take into consideration medium evaporation
from the dish. The AFM cantilever should be fully immersed in
the fluid. In our case, 50 mm glass-bottom FluoroDish™ cul-
ture dishes (World Precision Instruments, Inc., UK) were used,
containing 3 ml of media.
3. Use of HEPES buffer is recommended for maintaining appro-
priate pH levels in the medium. Typically, cells in a 50 mm dish
are stable for about 1 h in ambient conditions. We do not rec-
ommend working beyond 2 h without medium replacement or
devising a scheme to control the pH of the medium (e.g., cre-
ating a 5% CO2 atmosphere).
4. The experiment length should be minimized, as temperature
and pH are not stable under these conditions and also cell are
susceptible to contamination. Ideally, the experiment should
not extend longer than 1 h.
5. Following the experiment, cells should be discarded and not
put back into the incubator, as contamination from the envi-
ronment and/or AFM tip are prone to happen.
6. Owing to their viscoelastic properties, the cells’ response to
force will vary not only according to the magnitude of the
force but will also be affected by loading rate; thus, tip velocity
should be taken into consideration and kept constant through-
out the experiments.
7. Applying a drop of ethanol or DMSO on the cantilever tip
helps avoiding air bubbles being trapped underneath the tip
when lowering the AFM cantilever into the media.
8. When choosing a region for tracking (in ImageJ or other soft-
ware), take into consideration that when tracking a 2D move-
ment only, it is better to choose mitochondria that are near or
at the edges of the cell, where the cell is very flat and 2D motion
can be assumed.
192 Yaron R. Silberberg and Andrew E. Pelling

9. The output trajectories data will normally include a few invalid


trajectories (sometimes up to ~10% of the total calculated tra-
jectories, depending on image quality, particle density, and
analysis parameters), which results from false linking of particles
between two frames (Fig. 2c, white arrow). These false trajec-
tories are often of high magnitude and will greatly affect the
overall calculated average; thus, it is important to filter them
out. However, they can be easily noticed as their magnitude of
displacement (the change in x or y coordinates) will be consid-
erably higher than the usual displacement of true trajectories.
10. As mitochondrial motion is both random Brownian motion
and directed filament-based displacement, a convenient way to
validate the displacement analysis is to compare between mito-
chondrial movement in control cells and that in cells treated
with cytoskeleton-disrupting drugs such as cytochalasin and
nocodazole (21).

Acknowledgments

Y.R.S. would like to thank the Japanese Society for the Promotion
of Science (JSPS) for a post-doctoral fellowship grant. A.E.P.
acknowledges generous support from the Canada Research Chairs
program, the Province of Ontario Early Researcher Award, and the
Natural Sciences and Engineering Research Council. The authors
would like to gratefully acknowledge the tremendous support and
mentorship of Professor Michael Horton (1948–2010) and his
inspiration for this work.

References

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van Hulst NF, Greve J (1994) Viscoelasticity visiae. Science 305:1147–1150
of living cells allows high resolution imaging 7. Haupt BJ, Pelling AE, Horton MA (2006)
by tapping mode atomic force microscopy. Integrated confocal and scanning probe
Biophys J 67:1749–1753 microscopy for biomedical research.
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Chapter 19

Imaging Select Mammalian Organelles Using Fluorescent


Microscopy: Application to Drug Delivery
Paul D.R. Dyer, Arun K. Kotha, Marie W. Pettit,
and Simon C.W. Richardson

Abstract
The microscopic imaging of specific organelles has become a staple of the single-cell assay and has helped
define the molecular regulation of many physiological processes. This definition has been made possible by
utilizing different criteria to identify specific subpopulations of organelles. These criteria can be biochemi-
cal, immunological, or physiological, and in many cases, markers regulate fusion to the organelle they
define (e.g., Rab-GTPase proteins). Single-cell imaging technology allows, within the context of drug
delivery, an evaluation of the intracellular trafficking of both biological and synthetic macromolecules.
However, it should be remembered that there are many limitations associated with this type of study and
quantitation is not easy. The temporal dissection of novel and default trafficking of both macromolecular
“drugs” and macromolecular drug delivery systems is possible. These methodologies are detailed herein.

Key words Endocytosis, Drug delivery, Organelle, Fluorescent microscopy, Live-cell imaging,
Polymers

1 Introduction

Why do we care about cell imaging? Despite being subject to many


limitations, including the misinterpretation of fixation artifacts, the
interpretation of static depictions of dynamic systems, a high degree
of difficulty interpreting quantitative information, microscopy is
still a very useful tool to both the cell biologist and the drug deliv-
ery scientist. However, false assumptions based upon propter hoc
arguments, i.e., “because a protein is visible on a specific compart-
ment, it functions there,” must be identified and rejected. Further,
cognizance of the limitations of the systems is also critical, as essen-
tially ex vivo systems, i.e., cells grown in culture, may or may not
behave the same way as cells in a functioning organism. Fluorescent
quenching (as an effect of fluorophore concentration) and changes
in fluorescent yield due to vesicular acidification make qualitative

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_19, © Springer Science+Business Media New York 2013

195
196 Paul D.R. Dyer et al.

Fig. 1 Shows a cartoon representing the trafficking of material between the organelles of the secretory and
endocytic system of mammalian cells

and temporal data easier to interpret reliably than quantitative data


defining drug uptake. However, that said, quantitation is not
impossible but beyond the scope of this chapter (1). The useful
application of microscopy to drug delivery is defined by experi-
mental design, and can vary from affirming the cellular uptake and
internalization of fluorescently labeled material, to examining the
kinetics associated with the movement of material between intrac-
ellular compartments (see Fig. 1) (1–4). Mitigating the caveats
outlined above is the philosophical view that some data is infinitely
more than no data and that in an imperfect world, we need to start
somewhere.
In this chapter, we will document protocols that have been
used to examine the trafficking of macromolecular material to a
select panel of intracellular compartments in a qualitative or tem-
poral manner. This is explored in both live and fixed cells in order
to try to control for fixation artifacts. Particular attention has been
paid to sample preparation as, given the current technology base,
imaging is viewed as routine and not rate limiting. Discussion per-
taining to specific markers delimiting intracellular compartments
(within the context of drug delivery) can be found in an earlier
review (4, 5).
Imaging Select Mammalian Organelles Using Fluorescent Microscopy 197

This chapter has three objectives and these are to describe


methodologies that:
(a) Establish subcellular markers in fixed cells (see Fig. 1).
(b) Localize “hard to fix” materials, i.e., biocompatible polymers.
(c) Facilitate the visualization of material in live cells.
Examples of select markers are shown in both non-transfected
(see Fig. 2) and transfected (see Fig. 3) squamous epithelial (Vero)
cells.

2 Materials

2.1 Materials, Transient transfections were performed using Lipofectamine®


Equipment, and Cells (Invitrogen, Paisley, UK). Vero cells (designated CCL-81) were
from the American Type Culture Collection (ATCC) (Middlesex,
UK). PD10 columns (GE Healthcare, Fairfield, CT, USA) were
used for conjugate-fluorophore separation. Cells were imaged
using either a Nikon 90i overhead epifluorescent microscope
attached to a Nikon digital camera (DS-Qi1Nc) and a computer
running Nikon NIS-Elements Advanced Research software. The
principal objective used for fluorescent imaging was an oil immer-
sion CFI Plan Apochromat VC 60X N2 (NA 1.4, WD 0.13 mm).
Live-cell imaging was performed using a Nikon Eclipse Ti
inverted microscope with a heated stage (set to 37°C) attached to
a Nikon (DS-Fi1) digital camera also connected to the Nikon NIS-
Elements Advanced Research software. The principal objective
used for inverted fluorescent imaging was a CFI Super Plan Fluor
60× (dry) (NA 0.70, WD 2.61–1.79 correction collar
0.1–1.3 mm).

2.2 Solutions and 1. Phosphate-buffered saline (PBS): This was prepared as a 10×
Culture Media stock by adding sodium chloride (80 g), potassium chloride
(2 g), disodium hydrogen phosphate (14.4 g), and potassium
dihydrogen phosphate (2.4 g) to 800 ml of double distilled
deionized (dddi) H2O. The final volume was adjusted to
1,000 ml with dddi H2O. This was diluted appropriately using
dddi H2O (see Note 1).
2. Richardson Piper (RP) media: Magnesium acetate (1 mM),
calcium chloride (1 mM), glucose (5 mM), glutamate (5 mM),
fetal bovine serum (10% v/v), were made up to their stated
concentration in 1× PBS. This was then filter sterilized through
a 0.2 μm filter (see Note 2).
3. Buffered formalin: First 10× PBS (5 ml) was added to a glass
beaker. Dddi H2O was added to bring the volume to 30 ml.
The preparation was heated to approximately 80°C.
Paraformaldehyde (PAF) (1 g) was added as well as 5 N sodium
198 Paul D.R. Dyer et al.

Fig. 2 Panels (a–c) cells fixed in aldehyde, having been incubated for 1 h with 25 μg/ml WGA-TxR (with
200 μM leupeptin), were washed with PBS and incubated for a further 4 h in complete media supplemented
with 200 μM leupeptin. Little co-localization between the early endocytic structures labeled by the anti-early
endosomal antigen 1 (EEA1) antibody and the WGA-TxR was evident. This was not surprising as the majority
Imaging Select Mammalian Organelles Using Fluorescent Microscopy 199

hydroxide (1 ml). The reagent was subject to aspiration to dis-


solve the PAF. Hydrochloric acid (5 N) was then added
(900 μl). More hydrochloric acid was added drop-wise to bring
the pH to 7.0 before dddi H2O was added to bring the final
volume to 50 ml (see Note 3).
4. Saponin extraction buffer: PIPES buffer (10 ml of 400 mM pH
6.8 in water, stored at 4°C) was added to a 50 ml sterile plastic
conical tube. EGTA (5 ml of a 50 mM stock) was also added.
Saponin (500 μl of a 5% w/v solution in water, store at −20°C)
and 5 μl magnesium chloride solution (1 mM in H20) were
also added. Dddi H2O was added to a final volume of 50 ml
(see Note 4).
5. Triton permeabilization buffer: PBS (5 ml of a 10× stock solu-
tion) was added to a 50 ml sterile plastic tube. Glycine was
added to bring its final concentration to 50 mM. Triton-X-100
was added to a final concentration of 0.2% v/v (see Note 5).
6. Blocking buffer: PBS (5 ml of a 10× stock) was added to a
50 ml sterile plastic tube. Serum (1 ml) was added and the final
volume adjusted to 50 ml using dddi H2O (see Note 6).
7. Mounting media (see Note 7): N-propyl gallate (10 mg) was
added to a sterile 1.5 ml Eppendorf tube. PBS (100 μl of a 10×
stock) was added as well as dddi H2O (400 μl) (see Note 8).
Glycerol (500 μl) was then added and the preparation gently
heated to (60–70°C) to facilitate the dissolution of the
N-propyl gallate. The preparation was then allowed to cool to
room temperature.

3 Methods

3.1 Establishing As reported (4–6), robust markers for subcellular endocytic and
Subcellular Markers in secretory organelles are often proteins that are integral to main-
Fixed Cells taining organelle identity (“gate keepers”). Examples include teth-
ering proteins, receptors, or overexpressed GTPases fused in frame

Fig. 2 (continued) of the WGR-TxR was documented (d–f) as occupying late endocytic structures (Fig. 1).
Panels (d–f) shows cells fixed in methanol having been treated with WGA-TxR as above. Here a high degree of
co-localization was evident between WGA-TxR and antibodies specific for lysosomal-associated membrane
glycoprotein 1 (LAMP1), LAMP 1 being a marker for later endocytic structures (Fig. 1). Panels (g–i), cells sub-
ject to aldehyde fixation were immunostained with a monoclonal antibody specific for Golgi matrix (GM) protein
of 130 kDa (GM130) and a polyclonal (sheep) anti-Trans-Golgi network protein of 46 kDa (TGN46) antibodies.
Here a high degree of co-localization was evident, though TGN46 signal may also appear upon puncta sur-
rounding the medial-Golgi. The anti-GM130 antibody may be seen decorating a reticular structure that corre-
sponded to the Golgi ribbon (medial-Golgi). GM130 is known to facilitate tethering to the cis-Golgi. This reticular
structure is visible below (k). Panels (j–l) show aldehyde fixed cells that have been treated with WGA (as above)
and immunostained using an anti-GM130 antibody. Here little co-localization of signal from the late endocytic
structures labeled with WGA-TxR and the Golgi labeled with the anti-GM130 antibody was evident
200 Paul D.R. Dyer et al.

Fig. 3 Transfected cells transiently expressing either enhanced green fluorescent protein (eGFP)-rat sarcoma
((Ras)-related in brain) (Rab) 5a (b, h) or eGFP-Rab7a (e, k), 48 h after transfection. Panels (a, d) show cells
that have been treated with WGA as described in Fig. 1 after the cells were transfected. A very small degree
of co-localization was evident between eGFP-Rab5 and WGA-TxR (a–c) and eGFP-Rab7a and the anti-EEA1
Imaging Select Mammalian Organelles Using Fluorescent Microscopy 201

to eGFP. In order to image these proteins, either immunostaining


or transfection may be employed. The trafficking of fluorescent
physiological probes subject to endocytic capture may also be
characterized (temporally) relative to these immunological or
eGFP-tagged markers (see Note 9). To this end:
1. A glass coverslip (22 mm × 22mm × 0.5mm) was placed in a
small glass beaker and the coverslip completely submerged in
absolute ethanol. The beaker was then placed in a class II cell
culture hood containing a lit spirit burner.
2. Using a set of metal micro-forceps, the glass coverslip was
removed from the alcohol and the excess liquid removed.
Taking care not to ignite the reservoir of ethanol in the beaker,
the ethanol still on the coverslip was ignited. The coverslip was
then sterile (see Note 10).
3. Within the class II hood, a sterile glass coverslip was placed in
each well of either a sterile 6-well TC coated plate or a 35-mm-
diameter sterile TC-coated dish.
4. Squamous epithelial cells suspended in complete media were
then added to each well at a density of 5 × 105 cells/well for
transfection experiments or 1 × 105 for non-transfection
immunofluorescence (IF) experiments (see Notes 11 and 12).
5. The final volume of media in each well was then adjusted to
2 ml, and the cells left in a cell culture incubator overnight,
i.e., at 37°C (5% v/v CO2).
6. If the transient expression (via transfection) of marker proteins
was required, plasmid DNA (1 μg/well) was placed in 0.5 ml/
well of serum- and antibiotic-free cell culture media. This
preparation was sterile (see Note 13).
7. The DNA preparation (step 6 above) was then mixed with an
equal volume of serum-free and antibiotic-free media contain-
ing a 5× excess (relative to DNA by weight) of Lipofectamine®.
This preparation was sterile.
8. The transfection reagent (from the previous step) was left at
room temperature for 30 min to come to equilibrium.
9. After 30 min., 1 ml of the lipofection reagent was applied per
well, to cells that had been washed 3× with sterile PBS pH 7.4.
The cells were then left under standard incubation conditions
for 4–6 h.
10. After 4–6 h, the lipofection reagent was removed, and the cells
were washed three times with sterile PBS before being left in
complete media under standard incubation conditions (see
Note 14) prior to examination.

Fig. 3 (continued) antibody (j–l). A high degree of co-localization between cells expressing eGFP-Rab7a and
WGA-TxR (d–f) was evident. Separately, cells immunostained with the anti-EEA1 antibody, and expressing the
eGFP-Rab5a transgene (g–i) also showed a high degree of co-localization. All cells were fixed with aldehyde
202 Paul D.R. Dyer et al.

3.2 Immunostaining The following steps were performed in rapid succession, having made
the necessary reagents immediately beforehand (see Note 15).
1. Cells were set up as described above (Subheading 3.1, steps
1–5).
2. Following an appropriate incubation time, the cell monolayer
was washed three times with PBS (see Notes 16 and 17).
Fixation may be undertaken with formalin (aldehyde) or cold
solvent (4).
3. Fixation with formalin (see Note 18): Buffered formalin (2%
w/v) (Subheading 2.2, item 3) was added to the cell mono-
layer immediately after the final PBS wash had been removed.
The cells were left in the buffered formalin for 20 min at room
temperature.
4. Fixation by solvent extraction: Immediately after the final PBS
wash was removed, prechilled absolute methanol (−20°C) was
added (2 ml/well). The dish was then left at −20°C for 5 min
(see Note 19).
5. Following fixation, the cells were washed three times with PBS,
and the final wash removed.
6. The Triton permeabilization buffer (Subheading 2.2, item 5)
was added to the cell monolayer after fixation in aldehyde. The
cells were incubated in this buffer for 5–20 min at room tem-
perature (see Note 20).
7. Blocking nonspecific antibody binding: After a further 3× PBS
washes, blocking buffer (1–2 ml) (Subheading 2.2, item 6) was
added to each well. The cells were left in blocking buffer at
room temperature for 60 min.
8. Primary antibody hybridization: Antibody was diluted to an
appropriate concentration using 1% v/v serum solution made
up in PBS. The diluted antibody (40 μl) was placed on a strip of
Parafilm™ within a humidified sealable container (see Note 21).
Following this incubation, the cells were washed three times
with PBS.
9. Secondary antibody hybridization: A dilution of 1:100 to
1:300 of fluorophore-labeled secondary antibody diluted in
1% v/v serum in PBS was made. Hybridizations were per-
formed as before (step 8 above) with the exception that the
coverslips were left in the dark for 30–45 min. After this incu-
bation, the coverslip(s) were washed three times with PBS.
10. Mounting media (~30 μl) (Subheading 2.2, item 7) was placed
on a glass slide, and the edge of one coverslip was lowered into
it, cells facing the glass slide. Mounting media was then smeared
across the microscope slide, ensuring that the area that the
coverslip would eventually occupy was covered. The angle
between the coverslip and the glass slide was then reduced
Imaging Select Mammalian Organelles Using Fluorescent Microscopy 203

until the two were in contact ensuring there were no trapped


air bubbles.
11. The coverslip was then positioned and excess mounting media
removed. A folded tissue was then used to absorb any excess
liquid while gently pushing the coverslip into the slide (see
Note 22).
12. The edges of the coverslip were sealed to the microscope slide
with nail varnish and the slide stored at −20°C (see Note 23).

3.3 Localizing “Hard This can be achieved by localizing material relative to a well-characterized
to Fix” Materials, i.e., endocytic probe (see Note 24) such as Texas red-conjugated BSA
Biocompatible (see Note 25) or Texas red-conjugated WGA. Having established
Polymers the temporal kinetics of the probe relative to immunological mark-
ers, it was possible to use an aforementioned physiological probe in
conjunction with an unknown (i.e., a fluorophore-labeled poly-
mer) to delineate the intracellular trafficking of material that will
not fix well but is subject to compartmentalization. That said, the
test material to be evaluated must have been conjugated to a
fluorophore. This approach allows the omission of a permeabiliza-
tion step, which would allow the postfixation movement of mate-
rial. Although Texas red-conjugated WGA can be obtained
commercially, Texas red-conjugated BSA must be made and charac-
terized by the researcher.
1. Bovine serum albumin (BSA) (100mg) was dissolved in 9.5 ml
of PBS and placed in a glass, lightproof container.
2. Texas red®-X, succinimidyl ester (TxR-NHS) was dissolved in
1 ml of dimethyl sulfoxide (DMSO).
3. TxR-NHS (0.5 ml) was then added to the BSA solution and
left at room temperature for 30 min. Residual TxR-NHS was
frozen @ −20°C for use at a later date.
4. PD10 columns (GE Healthcare, Fairfield, CT, USA) (x4) were
equilibrated with 30 ml of PBS.
5. Free fluorophore was separated from the conjugate by loading
2.5 ml of the reaction onto each (of four) PD10 columns and
eluted in a volume of 3.5 ml/column. Characterization of the
conjugates was performed as per the manufacturer’s instruc-
tions (TxR-NHS) (2) (see Note 26).
6. Vero cells were plated onto either 35 mm diameter or 6-well
sterile TC coated places containing a sterile coverslip and left
under optimal culture conditions for 24 h (Subheading 3.1,
steps 1–5).
7. The cell culture media was removed, and cells were washed
three times with sterile PBS.
8. Wheat germ agglutinin (WGA)-Texas red (TxR) (5–50 μg/
ml) or BSA-conjugated Texas red (BSA-TxR) (5–10 mg/ml)
204 Paul D.R. Dyer et al.

was then added to the cells, co-incubated with leupeptin


(200 μM). These reagents were typically administered in a final
volume of 0.5 ml cell culture media.
9. Media containing WGA-TxR was left on the cells at 37°C in
5% v/v CO2 for 1 h and media containing BSA-TxR for 4 h at
37°C in an atmosphere of 5% v/v CO2.
10. Both physiological probes were then “chased” (temporally)
into late endocytic structures, typically over a 4–48 h time
span. The chase phase was performed after removing the media
containing the fluorescent marker, washing the cells 3× with
sterile PBS (pH 7.4), and replacing the PBS with complete
media also containing 200 μM leupeptin.
11. The cells were then fixed and subject to immunological char-
acterization (Subheading 3.2) (2, 6).
12. Using fresh cells, the unknown material was incubated with
the marker (WGA-TxR or BSA-TxR) under experimental con-
ditions that result in a known distribution of the WGA-TxR/
BSA-TxR.
13. Following the pulse and chase phases, the cells are fixed with
aldehyde, and the permeabilization step is omitted completely.
The cells are then mounted as previously described and imaged
(2, 4, 6).

3.4 Live-Cell Sterile 6-well TC treated plates (or individual sterile 35 mm TC


Imaging treated dishes) were ideal for this application. Live-cell imaging
was performed using an inverted microscope with a heated stage,
preheated to 37°C. This temperature was maintained throughout
the experiment. To minimize photobleaching and oxidative cell
damage, the exposure of the cells to all light sources was kept to a
minimum. The lid was removed from the dish containing the cells
in order to remove condensation. If necessary, the media was
replaced when evaporation was deemed to be a problem (2, 4).
1. At an appropriate time, complete media was removed and the
cells washed three times with sterile 1× PBS (pH 7.2).
2. Prewarmed (37°C) RP media (~2 ml) containing 200 μM leu-
peptin (where appropriate) was then applied to the cells.
3. Once the region of interest was selected using phase contrast
microscopy (and the appropriate fluorescent channels), images
were taken every 10 min over 4 h.
4. After completing the imaging process, the pictures from both
channels were merged using the software provided by the
microscope manufacturer (i.e., NIS-Elements Advanced
Research software by Nikon). The data was exported as an avi
file and edited in Final Cut Studio (Apple Computing Ltd.
Cupertino, USA).
Imaging Select Mammalian Organelles Using Fluorescent Microscopy 205

3.5 Pre-Fixation This methodology was deployed in order to remove a cytosolic


Extraction pool of immunoreactive protein. This may impact upon the signal
to noise ratio of protein pools that may be hard to image (6) (see
Note 27).
1. Cells were prepared as described (Subheading 3.1, steps 1–5)
2. Following the first set of three PBS washes, saponin extraction
buffer (~1 ml) was added to the cells prior to fixation.
3. The saponin extraction buffer was left on the cells at room tem-
perature for 60 s before the cells were then washed three times
with PBS.
4. Cells were then fixed either using solvent or aldehyde.
5. If the cells have been fixed using aldehyde, do not incubate
in Triton permeabilization buffer for more than 5 min
(see Note 20).
6. Cells can be subject to immunolabeling or mounted
(Subheading 3.2) as previously described.

4 Notes

1. This is an isosmotic reagent that is buffered to pH 7.4. It is


very useful for washing cells and removing serum components.
It is also occasionally used (when its pH is dropped below its
buffering capacity) to remove non-covalently bound material
from the outside of cells. It can be autoclaved or used non-
sterile depending upon the application.
2. This is a proprietary cell culture media for live-cell imaging
and, as it uses a phosphate buffering system as opposed to a
carbonate buffering system, does not require an atmosphere of
5% v/v CO2 to maintain its pH (2). RP media also contains no
phenol red indicator. This media has been used for 2–4 h at a
time successfully, though much longer time frames are proba-
bly ill-advised. This is because once the cells are on the micro-
scope stage, the lid of the dish is often removed to prevent
condensation building up, or to allow reagents to be added.
Consequently, after the lid is removed, the media is no longer
sterile. Also, if possible it is a good idea to change the media
regularly to allow for evaporation, which will raise the salt con-
centration. Osmotic stress is known to modulate endocytosis
(7) potentially introducing artifacts into an experimental
system.
3. Formaldehyde, under normal atmospheric pressure and at
ambient temperature, is a gas, and when dissolved in water, the
resultant solution is formalin. One solid precursor of formalde-
hyde is paraformaldehyde (PAF). Fresh isosmotic, buffered
206 Paul D.R. Dyer et al.

formalin can be prepared from PAF. For best results, make this
solution fresh, less than an hour before using. When preparing
buffered formalin, do not add the PAF to the liquid before
heating, as this will cause the release of a considerable volume
of formaldehyde gas. Occasionally it may be desirable to
decrease the fixation time, and if this is the case, then a 4% w/v
PAF solution may be prepared. This will reduce the fixation
time from 20 min at room temperature to 4 min at room
temperature.
4. This buffer is applied to the cells prior to fixation and may be used
to deplete an immunoreactive cytosolic pool of material prior to
fixation and immunolabeling. Make fresh every time (6).
5. This buffer extracts lipid from cellular membranes, postfixation,
and allows antibodies to assimilate their intracellular targets
without the impediment of biological barriers. The removal of
intracellular barriers will also allow the free movement of non-
fixed material. It is necessary to make this solution fresh every
time. Do not store Triton-X-100 as dilute stock solution. If
this buffer is being used in conjunction with saponin extraction
buffer (Subheading 3.5), do not incubate the cells for more
than 5 min with this buffer at room temperature.
6. Store serum as multiple 1ml aliquots at −20°C in sterile 1.5 ml
Eppendorf tubes. Blocking buffer can be kept at 4°C if sterile.
Serum from any species that will not react with the primary or
secondary antibody will provide an adequate block, though
serum from the same species, the secondary antibody was
raised in is ideal. Fetal bovine serum or goat serum offers ade-
quate noise suppression when used in conjunction with goat
anti-mouse or goat anti-rabbit secondary antibodies.
7. Using products such as Vectashield® (Vector Laboratories,
Peterborough, UK) for mounting works well if a high volume
of fluorescence microscopy is being undertaken. However, the
short shelf life of this relatively expensive product can be pro-
hibitive. After a couple of months at 4°C, there is a tendency
to see unacceptably high levels of autofluorescence in the red
channel. Consequently, an inexpensive and perfectly accept-
able alternative can be made (mounting media).
8. Warm the preparation gently (do not boil) in a water bath or
microwave oven. Typically multiple bursts of energy (<10 s at
full power) will be sufficient when using a commercial 800 W
microwave oven. Store this at room temperature in the dark
for up to 1 week.
9. These methodologies relate to the use of squamous epithelial
cells, which lend themselves to microscopic examination, espe-
cially by epifluorescence microscopy by virtue of being intrinsi-
cally “flat” (looking a little like a small “fried egg” (see Notes
Imaging Select Mammalian Organelles Using Fluorescent Microscopy 207

2 and 3)). Consequently the cell densities quoted here have


been optimized for Vero and other squamous epithelial cells.
More cuboidal cell morphologies may be better suited to imag-
ing using a confocal aperture.
10. Momentarily release your grip to allow the alcohol to cover the
entire coverslip. A gentle but firm grip was maintained upon
the coverslip during manipulations, as if the coverslip was
gripped too firmly, or if there was excess alcohol upon ignition,
the coverslip was prone to cracking or shattering. This was not
dangerous, though having a sharp disposal container to hand
was convenient.
11. For live imaging experiments, the coverslip may be omitted.
12. Cell density and the timing of experiments in relation to seed-
ing cell density are particularly important as it is much harder
to image cells at confluence than it is at 80% confluence (on
account of an increased volume in the Z-axis at confluence). If
noise is a problem associated with signal above and below the
focal plane, a confocal aperture may be used.
13. For an eGFP fusion protein-encoding plasmid, utilizing either
an SV40 or CMV promoter.
14. Transgene expression is strongly evident after 24 h and optimal
after 48 h. It is still normally evident 72 h after transfection.
Expression levels varied greatly from transfected cell to trans-
fected cell, with up to 20% of the living cells expressing the
transgene. At 24 h post lipofection, many LAMP positive
structures appeared vacuolated as a consequence of the trans-
fection process. Some toxicity was also seen.
15. It was vitally important to know which side of the coverslip the
majority of the cells were attached (i.e., the side facing up dur-
ing culture) and to keep track of this during the experiment
(i.e., when manipulating the coverslips).
16. With practice, this can be poured directly into a sink rather
than aspirated without losing the coverslip.
17. The PBS, unless otherwise stated, was at room temperature. If
required, PBS may be prechilled to 0°C or acidified to pH 5. If
acidified PBS has been used, the acid wash was followed (rap-
idly) with 3× washes using PBS at pH 7.4. Using a 500 ml
wash bottle filled with PBS, run down the wall of the plate, was
least likely to disrupt the monolayer of cells.
18. Either aldehyde or solvent fixation was used depending upon
the antibodies being deployed. Solvent fixation will remove
lipids, which spatially constrain macromolecular material
within the intracellular vesicle. Consequently solvent fixation
should not be used with “hard to fix” material (Subheading
3.2, step 4).
208 Paul D.R. Dyer et al.

19. In the instance of solvent extraction, the first PBS wash was
added directly to the methanol. The cells were then washed
another two times with PBS. In order to prevent the cells dry-
ing out, (methanol is volatile) the next PBS wash was added
immediately after removing the previous one. Each time, the
cells were left in wash for 5 min before the PBS was removed.
After the final wash, the cells were incubated with blocking buf-
fer (Subheading 3.2, step 2). Do not extract the cells with
Triton permeabilization buffer as the cells have already had
their membrane lipids extracted by the solvent (4).
20. If the cells have not been subject to transfection or saponin
pre-fixation extraction, an incubation time of 20 min has
worked well. If the cells were transfected or subject to saponin
pre-fixation extraction, then a maximum incubation time of
5 min should be used. Longer incubation times resulted in
very few intact cells remaining on the coverslip. Following
incubation with the Triton permeabilization buffer, the cells
were washed 3× with PBS as before (4).
21. A strip of Parafilm “M”TM was placed on top of two paper tow-
els moistened with PBS, in a sealable container. The primary
antibody was diluted using 1:1 blocking buffer (Subheading
2.2, item 6). The coverslip was placed face (cells side) down
onto the antibody, and the preparation was left at room tem-
perature for 60 min. Antibody final dilution should be about
10× less than is used for immunoblotting. After the incubation
period, the coverslip was transferred back into the 6-well plate
(cells side up) and washed 3× in PBS pH 7.4.
22. It was critical to avoid any sheer force coming from lateral
movement or rotation of the coverslip once all the excess
mounting media was removed.
23. The glycerol in the mounting media prevented sample damage
from crystal formation at low temperature. Once the excess
mounting media was removed, repositioning the coverslip was
avoided as this also could sheer cells. Trapping air bubbles
between the coverslip and the microscope slide was avoided
through the use of excess mounting media.
24. Such as albumin (i.e., BSA) (8), conjugated to Texas red®
N-hydroxy-succinimidyl-ester (TxR-NHS) (2, 4).
25. This can be done the day before.
26. This preparation should contain minimal free (unconjugated)
TxR. The exact quantity of unconjugated TxR can be deter-
mined by further fractionation of the purified sample over a
PD10 column, and the Mol% loading can be determined by
following the manufacturer’s instructions. Unused TxR-NHS
can be stored at −20°C. This reagent is best used at a final con-
centration of BSA of 5–10 mg/ml. The high concentration of
Imaging Select Mammalian Organelles Using Fluorescent Microscopy 209

BSA-TxR (relative to WGA-TxR) was necessary as endocytic


capture was inefficient (i.e., fluid phase capture).
27. This extraction resulted in vesicular structures adopting a more
expanded “donut” like appearance. This procedure can be
used to remove an immunoreactive cytosolic pool of a protein
that may mask the decoration of the target protein on other
intracellular structures (4). Other labs have successfully used
streptolysin O (9) in place of saponin.

References
1. Galush W, Nye J, Groves T (2008) Quantitative biologically active polymers. Expert Opin Drug
fluorescence microscopy using supported lipid Deliv 8(4):403–407
bilayer standards. Biophys J 95:2512–2519 6. Richardson SC, Winistorfer SC, Poupon V,
2. Richardson SC, Wallom KL, Ferguson EL, Luzio JP, Piper RC (2004) Mammalian late
Deacon SP, Davies MW, Powell AJ, Piper RC, Vps orthologues participate in early endosomal
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microscopy to define polymer conjugate locali- Biol Cell 15:1197–1210
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and live target cells. J Control Release Parker PJ, Michell RH (1997) Osmotic stress
127:1–11 activates phosphatidylinositol-3, 5-bipgosphate
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of bioresponsive poly(amidoamine)s in vitro JP (1998) Fusion of lysosomes with late endo-
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specific pharmaceutical nanotechnology. Wiley, Goud B, Johannes L (1998) Direct pathway
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Chapter 20

Real-Time Particle Tracking for Studying Intracellular


Trafficking of Pharmaceutical Nanocarriers
Feiran Huang, Erin Watson, Christopher Dempsey, and Junghae Suh

Abstract
Real-time particle tracking is a technique that combines fluorescence microscopy with object tracking and
computing and can be used to extract quantitative transport parameters for small particles inside cells.
Since the success of a nanocarrier can often be determined by how effectively it delivers cargo to the target
organelle, understanding the complex intracellular transport of pharmaceutical nanocarriers is critical.
Real-time particle tracking provides insight into the dynamics of the intracellular behavior of nanoparticles,
which may lead to significant improvements in the design and development of novel delivery systems.
Unfortunately, this technique is not often fully understood, limiting its implementation by researchers in
the field of nanomedicine. In this chapter, one of the most complicated aspects of particle tracking, the
mean square displacement (MSD) calculation, is explained in a simple manner designed for the novice
particle tracker. Pseudo code for performing the MSD calculation in MATLAB is also provided. This chap-
ter contains clear and comprehensive instructions for a series of basic procedures in the technique of par-
ticle tracking. Instructions for performing confocal microscopy of nanoparticle samples are provided, and
two methods of determining particle trajectories that do not require commercial particle-tracking software
are provided. Trajectory analysis and determination of the tracking resolution are also explained. By pro-
viding comprehensive instructions needed to perform particle-tracking experiments, this chapter will
enable researchers to gain new insight into the intracellular dynamics of nanocarriers, potentially leading
to the development of more effective and intelligent therapeutic delivery vectors.

Key words Real-time, Particle tracking, Nanocarrier, Confocal microscopy, Mean square displacement,
ImageJ, MATLAB

1 Introduction

1.1 Real-Time Nanocarriers for drug or gene delivery have a number of potential
Particle Tracking advantages over systemic drug delivery, including more specific
targeting and higher efficiency. However, the success of these ther-
apeutics can often be limited by how effectively they reach target
organelles—a process potentially made difficult by the number of
intracellular biological barriers to nanocarrier delivery. For exam-
ple, nanocarriers can be hindered by the highly crowded cytoplasm,
which includes a dense cytoskeletal network. Even if gene delivery

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_20, © Springer Science+Business Media New York 2013

211
212 Feiran Huang et al.

vectors or other nucleus-targeted nanocarriers are able to navigate


the cytoplasm and reach the perinuclear region, they still may be
blocked from entering the nucleus. Understanding the dynamics
and transport mechanisms of nanoparticles inside cells will allow
for the design of more intelligent and efficient nanocarriers. Many
fluorescence techniques, such as fluorescence recovery after photo-
bleaching (FRAP), only reveal ensemble information about
nanocarrier transport dynamics. Real-time particle tracking, in
contrast, can reveal both ensemble and individual particle trans-
port information. This technique can be used to extract detailed
quantitative nanoparticle transport information, such as diffusivity,
velocity, transport mode, and directionality—all of which can be
used to characterize the dynamics of intracellular nanocarrier trans-
port (1, 2). Such insight can be harnessed to improve the design
and development of novel delivery systems for pharmaceuticals,
such as drug and genes.
Despite these potential advantages, real-time particle tracking
has not been widely used in the field of nanotherapeutics, partially
due to the difficulty in understanding the calculations behind the
technique. In a typical particle-tracking experiment (Fig. 1),
fluorescently labeled nanocarriers are added to live cells and imaged
using a fluorescence confocal microscope. Captured high-resolu-
tion time-series images are processed to extract x, y coordinate data
over time, which are then used to reconstruct the trajectories of
the particles. The data are subsequently used to calculate mean
square displacement (MSD) and other transport parameters, such
as diffusion coefficient, or velocity. This chapter begins by describ-
ing the MSD calculation, the core of the data analysis and often a
confusing concept, in an approachable way designed for research-
ers inexperienced with this technique. Additionally, programmatic
language is provided in Subheading 3 that explains how to per-
form the MSD calculation using MATLAB.
Comprehensive instructions are given that detail how to suc-
cessfully perform the basic procedures in a typical particle-tracking
experiment. Specifically, real-time particle tracking of nanoparti-
cles in live cells and in glue samples (for determining tracking
resolution) will be described in parallel in Subheading 3. The fol-
lowing procedures will be explained: preparing the glue samples
to determine tracking resolution, preparing the live cell imaging
samples, imaging samples with a confocal microscope, tracking
particles (to determine trajectories) with ImageJ and MATLAB
programs, computing MSD, and determining tracking resolution
with glued nanoparticles.

1.2 MSD Calculation MSD is a measure of a particle’s motion which is typically calculated
in the analysis stage of a particle-tracking experiment. It is calcu-
lated after particle trajectories have been determined, and once cal-
culated, it can be used to derive other transport parameters, such as
Particle Tracking of Nanocarriers 213

Confocal imaging

MSD (μm2)

Time Lag (s)

Tracking Computing MSD

Fig. 1 Steps in a typical real-time particle-tracking experiment. Images are taken of fluorescently labeled
nanoparticles inside live cells using confocal microscopy. Next, image analysis is performed to find the parti-
cles’ positions in each frame and then to determine the particle trajectories. Finally, mean square displacement
(MSD) and other transport parameters are calculated using the trajectory data

diffusivity. Two-dimensional MSD is most commonly used for


intracellular tracking and therefore will be discussed in this chapter.
MSD, <Δr2(τ)>, is determined as a function of time lag, τn = nΔt,
where Δt is the time between frames and n is the number of time
intervals. MSD is calculated by finding the average particle square
displacement for all possible time lags for a particle trajectory, where
square displacements between frames can easily be calculated using
the x and y coordinate data from the particle trajectories,

Δr 2 = (xi +n − xi ) + (y i +n − y i )
2 2

For the smallest τ (n = 1), the particle square displacements are


simply the square displacements between each frame. For time lags
longer than one frame interval, the particle displacements are
calculated between all frames that are of that particular time lag
apart. For example, if the time lag is 3-frame intervals (n = 3), then
particle displacement would be calculated between frames 1 and 4,
214 Feiran Huang et al.

frames 2 and 5, frames 3 and 6, etc. Note that each τn for all integer
values of n from 1 to N − 1 should be used, where N is the total
number of frames for that trajectory. As an example, Fig. 2 demon-
strates the mean square displacement calculation for the case of a
five-frame trajectory. The number of displacements, m, that can be
computed for a particular time lag is m = N − n. For example, for
the smallest time lag τ = Δt, there are N − 1 displacement values; for
the largest time lag, between frame 1 and frame N, τ = (N − 1)Δt,
there is only 1 displacement value.
Once all of the displacements have been found for a particular
time lag, the MSD for that time lag can be calculated by finding
the arithmetic mean of the square of the displacements
<Δr2(τ)> = <Δr12,Δr22, …, ΔrN − n2>, where <…> denotes mean.
Mathematically, the following equation summarizes the calculation
of two-dimensional MSD of a given trajectory of x,y coordinates at
a particular τn:
1 N −n
〈Δr 2 (τ)〉 = 〈Δr 2 (n Δt )〉 = ∑
N −n i
[(xi +n − xi )2 + (y i +n − y i )2 ]

Next, ensemble MSD can be determined across all particles. To


calculate ensemble MSD, a simple arithmetic mean of the MSD’s of
all particle trajectories in an experiment is determined. Note that
longer trajectories and smaller time scales both result in more dis-
placement values, and thus more statistically meaningful MSD cal-
culations. Therefore, MSD values calculated using the upper range
of the time lag or using very short trajectories are considered unreli-
able since few displacement values were used in these calculations.
More discussions of MSD calculation and various transport param-
eters that can be derived from it are found in references (3–10).

2 Materials

2.1 Cell Culture 1. HeLa cells (ATCC).


2. Complete medium: Dulbecco’s Modified Eagle’s Medium (Life
Technologies, Grand Island, NY, USA) supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin.
3. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4.
4. 0.25% trypsin–EDTA for cell passing.

Fig. 2 (continued) calculated between particles in every second frame (P1 →P3, P3 →P5). Rather, displacements
are calculated between particles in all frames that are 2 time intervals apart (P1 → P3, P2 → P4, P3 → P5 ), as
shown by the arrow. Once all the square displacements have been found for a particular time lag, the mean of
those displacements is calculated, Δr2 = <Δr12, …, Δrm2 >. Finally, MSD is typically plotted as a function of time
lag, as shown in (c), to allow for further analysis of transport parameters
Particle Tracking of Nanocarriers 215

For a 5 frame trajectory, N = 5:

a
P5(5,5) P1: t = 0, (x1,y1)
P2: t = Dt, (x2,y2)
P1(1,3)
P3: t = 2Dt, (x3,y3)
P4(4,4)
P4: t = 3Dt, (x4,y4)
P2 (2,2) P3 (3,2) P5: t = 4Dt, (x5,y5)

P5 T1 = Dt, n= 1, m= 4
b1
Δr12 = (x2–x1)2 + (y2 –y1)2 (2 – 1)2 + (2 –3)2 = 2
Δr22 = (x3–x2)2 + (y3 – y2)2 (3 – 2)2 + (2 –2)2 = 1
P4
P1 Δr32 = (x4–x3)2 + (y4 – y3)2 (4 – 3)2 + (4 –2)2 = 5
P2 P3 Δr42 = (x5–x4)2 + (y5 – y4)2 (5 – 4)2 + (5 –4)2 = 2
<Δr2(t1)> = <Δr12, Δr22 , Δr32, Δr42> <2, 1, 5, 2> = 2.5

P5 T2 = 2Dt, n = 2, m = 3
b2
Δr12 = (x3 – x1)2 + (y3 – y1)2 (3 – 1)2 + (2 – 3)2 = 5
P4 Δr22 = (x4 – x2)2 + (y4 – y2)2 (4 – 2)2 + (4 – 2)2 = 8
P1 Δr32 = (x5 – x3)2 + (y5 – y3)2 (5 – 3)2 + (5 – 2)2 = 13
P2 P3 <Δr2(t1)> = <Δr12, Δr22, Δr32> <5, 8, 13> = 8.67

b3 P5 T3 = 3Dt, n = 3, m = 2
Δr12 = (x4 – x1)2 + (y4 – y1)2 (4 – 1)2 + (4 – 3)2 = 10
P4 Δr22 = (x5 – x2)2 + (y5 – y2)2 (5 – 2)2 + (5 – 2)2 = 18
P1
<Δr2(t1)> = <Δr12, Δr22 > <10, 18> = 14
P2 P3

b4 P5 T3 = 4Dt, n = 4, m = 1
Δr12 = (x5 – x1)2 + (y5 – y1)2 (5 – 1)2 + (5 – 3)2 = 20
P4 <Dr2(t1)> = Δr12 20
P1
P2 P3

c 30
20
MSD

10
0
1 2 3 4
τ (Δt)

Fig. 2 Example MSD calculation for a five-frame trajectory. (a) Particle positions in each frame, where Pi denotes
the and position for each frame, i. The displacement squared, Δri 2 = (xi+n – xi )2 + (yi+n – yi )2, is calculated between
the x,y particle positions in frames that are time intervals apart, with n increasing by 1 for each successive time
lag. (b1–b4) Displacements used for a time lag of τn = nΔt, where = 1, 2, 3, and 4, and the corresponding calcula-
tions to find MSD for each time lag. m = N – n is the number of displacements, . The displacements used are denoted
by the arrow. For example, (b2) depicts the displacements used for t = 2Δt, so the square displacements are calcu-
lated between particle positions in frames that are 2 time intervals apart. Note that displacements are not simply
216 Feiran Huang et al.

2.2 Imaging 1. Carboxylate-Modified FluoSpheres polystyrene nanoparticles


(Life Technologies, Grand Island, NY, USA), red
(580/605 nm), 100 nm, 2% in weight.
2. Hoechst 34580 if nucleus identification is desired.
3. Microscope slides and coverslips.
4. Loctite 495 superglue.
5. Lab-Tek™ 8-well chambered coverglass (culture area = 0.8 cm2/
well) (Thermo Fisher Scientific, Waltham, MA, USA).
6. LSM 5 LIVE confocal microscope (Zeiss, Thornwood, NY,
USA) with a 63×/1.4 oil objective, the appropriate filters for
the nanoparticles being studied, and an environmental cham-
ber maintained at 37°C and 5% CO2.

2.3 Image 1. For tracking particles with ImageJ, a “spot tracker” plug-in
Processing needs to be downloaded from http://bigwww.epfl.ch/sage/
and Data Analysis soft/spottracker/ and installed. Documentation of the plug-in
is in Ref. 11.
2. MATLAB 7.0 (MathWorks) or newer needs to be installed for
MSD calculation and particle tracking with MATLAB. For
tracking particles with MATLAB, download publicly available
subroutines and instruction document “Instructions_feature_
track_pretrack.pdf” from the Maria Kilfoil research group
Web site, http://people.umass.edu/kilfoil/downloads.html
in “Particle pretracking and tracking and 2D feature finding”
section. Store all M-files in the current directory. MATLAB’s
Image Processing Toolbox is also required.

3 Methods

3.1 Cell Culture 1. Maintain cells in complete medium at 37°C in 5% CO2.


2. Pass cells at 1:10 ratio every 3–4 days.

3.2 Preparing Glue 1. Dilute PSNP 1:100 in ultrapure water.


Sample to Determine 2. Spot 10 μL of the PSNP solution onto a microscope slide, and
Tracking Resolution spot a small amount of superglue (about 50–100 μL) on top of
the PSNP.
3. Press down a coverslip over the glue and wait until dry.

3.3 Preparing Live 1. Plate cells onto a Lab-Tek 8-well chambered coverglass at den-
Cell Imaging Sample sity of 2 × 104 cells/well (20% confluency), and incubate in
complete medium for 24 h.
2. Replace the medium with 0.4 mL fresh complete medium con-
taining 0.1 mg/mL PSNP (see Note 1).
Particle Tracking of Nanocarriers 217

Fig. 3 Example trajectories (insets) obtained with ImageJ spot tracker plug-in for
PSNPs

3. Incubate samples for desired time (usually 15 min to 24 h), at


37°C and 5% CO2.
4. If nucleus identification is desired, add medium containing
1 μg/mL Hoechst to cells and incubate at 37°C for 30 min
before imaging (see Note 2 for an alternative nuclear labeling
method).
5. Wash cells with PBS and replenish with medium.

3.4 Imaging Sample Using a high-magnification objective (63× oil), perform time-se-
with Confocal ries imaging for 200 frames at 50 ms per frame with a 2× zoom
Microscope (see Note 3). For imaging glued particles, find a population of
beads near the glue/particle interface where the particles are
immobilized by the glue. For imaging particles in living cells, find
the best focal plane with reference to the Hoechst-stained cell
nucleus. A single image with both Hoechst channel and nanopar-
ticle channel can be taken before and after the time-series images
to ensure that the images stay on the same optical plane.

3.5 Tracking The ImageJ plug-in explained below can only track one particle at a
Particles with Image J time in a time-series image stack that was obtained from the confocal
imaging. See Fig. 3 for PSNP trajectories obtained with ImageJ.
218 Feiran Huang et al.

1. Open the time-series file with ImageJ, and crop the stack of
images to the particle to be tracked by drawing a rectangle
around it and selecting image > crop, making sure that the par-
ticle stays within the field of view during the frames to be
tracked.
2. Select plug-ins > spot tracker > spot tracker 2D.
3. Draw a small rectangle around the particle and select Add to
specify the initial location of the particle.
4. Modify the parameters of the plug-in to fit the time-series
properties. Maximum displacement refers to the maximum
movement in pixels below which the particle can be success-
fully tracked. The other parameters (intensity factor, intensity
variation, movement constraint, and center constraint) specify
the different weights for the cost function of the algorithm.
More information can be obtained by clicking Help.
5. Click Track to obtain a table of the tracking data, which includes
x position, y position, confidence interval, pixel intensity at the
center, velocity from one frame to the next, and a mean inten-
sity over a 1 × 1 pixel window. At this point, Display Results can
be chosen to visualize the trajectory of the particle.
6. After performing the tracking, the plug-in will output the data in
a table that includes frame number, x position, y position, and
various other quantities that may be desired. This table can be
copied and further analyzed to calculate other useful parameters
such as MSD. Select File > Save As… to save table in .xls format.

3.6 Tracking with The MATLAB programs from Maria Kilfoil research group can
MATLAB Programs track multiple particles simultaneously. The following procedure is
based on a complete tutorial written by Vincent Pelletier and Maria
Kilfoil (Instructions_feature_track_pretrack.pdf). Small changes
must be made to the program files to run on Macintosh computers
(see Note 4).
1. The following explains the default arrangement of the files (for
custom arrangement, see Note 5). Organize images from each
experiment into one root folder containing subfolders named
“fov#” with a different # for each field of view. Each subfolder
should contain time-series images named “fov#_####.tif,”
with the first # referring to the subfolder and the second ####
referring to the index of the frame in four-digit format. The
root folder should contain time information vectors called
“time” and saved as “fov#times.mat” with the # referring to
the corresponding subfolder. All downloaded M-files should
also be stored in the root folder. In MATLAB, make the root
folder the current directory.
2. Run mpretrack_intit.m to determine the correct parameters for
accepting real particles and rejecting false features. This func-
tion requires inputting basepath, featsize (size of the feature),
Particle Tracking of Nanocarriers 219

barint (minimum intensity), barrg (maximum Rg squared),


barcc (maximum eccentricity), IdivRg (minimum ratio of
Intensity/pixel), fovn (ID# for the series of images), frame
(frame number of a representative image), and three more
optional parameters. The inputs for all functions are detailed in
the comments found in the function files and the tutorial.
3. The output of mpretrack_init.m will be a matrix containing
accepted features (MT) and a matrix containing rejected fea-
tures (M2). These matrices contain the following columns
from left to right (all units in pixels or pixels^2): x positions, y
positions, integrated intensity, square radius of gyration, and
eccentricity. A figure with accepted features surrounded by
green circles and rejected features in red will be displayed. It
takes several runs to optimize the input parameters until the
correct features are accepted.
4. Run mpretrack.m using as inputs basepath, fovn, numframes
(number of frames in the series), the parameters optimized in
step 3, and three other optional parameters. This function creates
a matrix named MT containing accepted features for all frames in
that field of view. MT has the same first five columns as the out-
put of mpretrack.init.m, as well as a sixth column for frame num-
ber and a seventh column for the image time (taken from
fov#_times.mat). This matrix is stored as the following file:
(basepath ‘Feature_finding MT_#_Feat_Size_#.mat’).
5. Run fancytrack.m to determine trajectories from particle data
determined by mpretrack.m. The following are inputs for
fancytrack.m: basepath, FOVnum (field of view number), featsize
(feature size for accessing the right MT file), and optional trajec-
tory parameters, maxdisp (maximum displacement the particle
may make between successive frames), goodenough (minimum
length requirement, in number of frames, for a trajectory to be
retained), and memory (how many consecutive frames a feature
is allowed to skip). If unsatisfactory trajectories are determined,
an empty matrix is output, or errors occur, try optimizing these
optional trajectory parameters. For example, the default of
optional parameter goodenough is 100 and may cause errors if
the total number of frames is near or less than 100.
6. The output of fancytrack.m is a file named res_fov#.mat
(# refers to the field of view number) in the folder (basepath
‘Bead_tracking\res_files\’). This file contains a matrix, res, with
the same first seven columns as the output from step 4 (MT )
and an additional eighth column containing the trajectory ID
number. The matrix is automatically saved in .mat form. If
desired, MATLAB’s built-in function xlswrite can be used to
save the matrix as an excel spreadsheet; however, this is not
necessary if MSD is calculated using MATLAB as described in
Subheading 3.7.
220 Feiran Huang et al.

Begin

Input X, Y, N

n = 1 to N-1
step 1

sum = 0
m = N-n

i = 1 to m
step 1

sum = sum+(X(i+n)-X(i))^2+(Y(i+n)-Y(i))^2

msd(n) = sum/m

Output msd

End

Fig. 4 Flowchart of a subroutine for computing MSD of a single trajectory. X and


Y are the particle position vectors, N is the total number of frames, n is the num-
ber of time intervals for a particular time lag, m is the number of displacements
for a particular n, and i is the index of m

3.7 Computing MSD 1. Format data outputted from ImageJ or other tracking software
with MATLAB to X, Y vectors. If the trajectories are different in length, then
the data need to be trimmed to the same length. Usually a
trajectory that has more than 200 frames is sufficient for statis-
tically significant calculations (see Note 6).
2. Create a subroutine for computing MSD of one trajectory.
A main program needs to be created to calculate multiple tra-
jectories by calling this subroutine in a loop. The following
pseudo-code of the subroutine can be translated easily to
MATLAB code (see Fig. 4 for flowchart). X, Y vectors are the
position data, N is number of frames, and msd is the calculated
MSD vector. Note that the frame interval and the time lag vec-
tors are not needed in the computation.
MSD computing subroutine:
Begin
input X, Y, N
for n = 1 to N-1
sum = 0
m = N-n
for i = 1 to m
Particle Tracking of Nanocarriers 221

sum = sum + (X(i + n)-X(i))^2 + (Y(i + n)-Y(i))^2


msd(n) = sum/m;
output msd
End
3. Ensemble MSD can be computed by averaging MSD of all
particles. For an MSD with N time lags, the first N − 100 points
are considered significant (see Note 6).

3.8 Determining The glued particles are immobile and will reflect the vibrational
Tracking Resolution noise of the experimental setup. Thus, by calculating the MSD of
from Glued glued PSNP, the tracking resolution of the system is determined.
Nanoparticles Calculate the arithmetic mean (M) of the first 50 points of the
ensemble MSD. Then, use σ = (Μ/2)1/2 to obtain the tracking res-
olution σ (see Note 7 and Fig. 5).

a 0.6

0.5

0.4
μm2)
MSD (μ

0.3

0.2

0.1

0
0 10 20 30 40
Time Lag (s)

b x 10-4
5
σ = 0.012 μm
4
μm2)

3
<MSD> (μ

0
0 0.2 0.4 0.6 0.8 1
Time Lag (s)

Fig. 5 Example MSD plots of PSNPs. (a) Five PSNPs were tracked in live HeLa
cells. (b) PSNPs immobilized with superglue were tracked to determine tracking
resolution. Tracking resolution was calculated by taking the mean of the ensem-
ble MSD of the first 20 time lags
222 Feiran Huang et al.

4 Notes
1. Too high of a concentration of nanoparticles may complicate
particle-tracking image analysis.
2. Labeling the nucleus helps to locate the plane crossing the cell
cytoplasm and nucleus and to assure the nanoparticles are inside
the cell. An alternative way to outline the cell is by transfecting
it with green fluorescent protein (GFP) plasmid DNA. 24–48 h
after transfection, the cytoplasm and nucleus will be labeled
with expressed GFP and ready for nanoparticle addition.
3. Optimal time interval depends on fluorescence properties of
particles and how fast the particles are moving. For PSNP
endocytosed by mammalian cells, a time interval of 50–400 ms
works well. The duration of the movie also depends on whether
the cell moves during the course of the movie. To maximize
the temporal resolution, excitation laser power needs to be
optimized so that the fluorescence signal is strong enough for
particle tracking but will not cause sample photobleaching
within the recording time.
4. Mac users need to make minor changes to the program files to
account for differences in file path naming. Backward slashes
(\) should be changed to forward slashes (/) in the following
locations: line 72 of mpretrack_init.m, lines 70 and 77 of
mpretrack.m, and lines 51 and 59 of fancytrack.m.
5. The default arrangement of the files and folders, basepath can
be set to null. Readers can customize the arrangement and
name format of the folders and image files by changing lines 71
and 72 in mpretrack_init.m and lines 74 and 77 in mpretrack.m,
accordingly.
6. The error of an MSD data point is m−1/2, where m is the num-
ber of displacements for a particular time lag. If 10% is an
acceptable error, then m = 100 and n = N − 100. Only the MSD
of first n time lags are acceptable. If N = 200, then n = 100, i.e.,
the first 100 points of the MSD are significant.
7. In practice, <Δr2(0)> = 2σ2 ¹ 0, due to the errors introduced by
experimental conditions and particle-tracking algorithm. Glued
PSNPs are assumed to be fixed on the slide during the track-
ing, thus any displacements are considered to be errors.
Therefore, the system tracking resolution is determined by the
<Δr2> of the glued PSNPs.

Acknowledgment

This work was supported by the National Institutes of Health


(RC2GM092599 and T32EB009379).
Particle Tracking of Nanocarriers 223

References

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3. Qian H et al (1991) Single particle tracking. ery system in the interstitium of a human tumor
Analysis of diffusion and flow in two-dimen- xenograft. Breast Cancer Res 11:R43
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4. Kusumi A et al (1993) Confined lateral diffu- ing for studying intracellular transport of nano-
sion of membrane receptors as studied by single therapeutics. In: Weissig V, D’Souza GGM
particle tracking (nanovid microscopy). Effects (eds) Organelle-specific pharmaceutical nano-
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5. Saxton MJ, Jacobson K (1997) Single-particle ogy of living cells: principles and applications.
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Annu Rev Biophys Biomol Struct 26:373–399 11. Sage D et al (2005) Automatic tracking of indi-
6. Suh J et al (2005) Real-time multiple-particle vidual fluorescence particles: application to the
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Chapter 21

Interactions of Nanoparticles with Proteins:


Determination of Equilibrium Constants
Lennart Treuel and Marcelina Malissek

Abstract
The behavior of nanoparticles towards proteins is an important aspect across wide areas of nanotoxicology
and nanomedicine. In this chapter, we describe a procedure to study the adsorption of proteins onto nano-
particle surfaces. Circular dichroism (CD) spectroscopy is utilized to quantify the amount of free protein
in a solution, and the experimental information is evaluated to derive equilibrium constants for the protein
adsorption/desorption equilibrium. These equilibrium constants are comparable parameters in describing
the interactions between proteins and nanoparticles.

Key words Nanoparticles, Protein structure, Protein adsorption, Protein corona, Equilibrium constant,
Dissociation constant, Circular dichroism spectroscopy

1 Introduction
The behavior of nanoparticles (NPs) towards proteins is a key
aspect in understanding the fate of NPs in biological systems.
The efficiency of this interaction can be a decisive factor for the
effect of a nanoparticle within a biological system (1–5). Most
interactions of proteins with nanoparticles lead to structural
changes in the protein (6–10), and an effect of ligand chemistry
on the quantity of adsorbed protein and the extent of denaturation
has also been reported (11). These structural changes upon adsorp-
tion to a nanoparticle surface can result in the loss of biological
activity and also in an activation of immune response (12, 13).
In this chapter, we describe how circular dichroism (CD)
spectroscopy can be used for quantitative studies of the protein
corona formation around colloidal nanoparticles. This technique
is utilized to monitor the loss of protein structure as a function of
nanoparticle surface area present in an observed volume. From the
spectral information, the amount of free protein can be determined
and this experimental information can be evaluated to derive

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_21, © Springer Science+Business Media New York 2013

225
226 Lennart Treuel and Marcelina Malissek

Fig. 1 CD structure shows typical spectra for pure secondary structures occur-
ring in proteins

equilibrium constants for the protein adsorption / desorption


equilibrium. These equilibrium constants are comparable parameters
in describing the interactions between proteins and nanoparticles,
and we describe how they can be determined from CD spectro-
scopic measurements largely independent of the nanoparticle
properties (5).

1.1 Principle of Circular dichroism (CD) spectroscopy essentially measures differ-


Circular Dichroism ences in the absorption of left-handed and right-handed polarized
Spectroscopy light. These differences arise from structural asymmetries. In the UV
spectral region (260–180 nm), characteristic signals arising from sec-
ondary protein structures can be detected. These signals are related
to peptide bonds located in differently folded environments. For a
more detailed discussion of the physical basics of circular dichroism
spectroscopy, the reader is referred to the many excellent textbooks
that have been published on this topic among which those by G.D.
Fasman (14) and by N. Berova, K. Nakanishi, and R.W. Woody
(15) can be especially recommended by the authors. Figure 1 shows
typical spectra for pure secondary structures occurring in proteins.
Spectral deconvolution methods (16–20) can be used to extract
the relative contributions of the individual structural elements from
protein spectra.

1.2 Derivation of The following descriptions regarding the derivation of equilibrium


Equilibrium Constants constants, the determination of free protein content, and the deter-
mination of surface sites have been published by L. Treuel et al.
(5), and they are in parts reproduced from this source with kind
permission from the copyright holder, Wiley-VCH Verlag GmbH
& Co. KGaA.
Interactions of Nanoparticles with Proteins: Determination of Equilibrium Constants 227

For the derivation of the necessary equation we assume that


free proteins [P] and surface adsorption sites [S] are in equilibrium
with proteins adsorbed on the surface sites [PS]:
P + S  PS. (1)
Thus, the following equilibrium constant KD can be derived
for the adsorption/desorption process:

KD =
[P ]⋅ [S]. (2)
[PS]
The total surface available for protein adsorption is quantita-
tively expressed by the number of free surface sites per unit vol-
ume. Provided, a monodispersed distribution of particles is
assumed, the number of surface sites is determined by the sum
over all particles ni times the maximum number of protein mole-
cules that can fit on the surface one nanoparticle Nmax divided by
the Avogadro constant NA.

∑ n ·Ni max

[S] = i

NA
. (3)

This number can be calculated using a spherical geometry and


literature size values for the protein. The amount of protein
adsorbed [PS] onto the surface at equilibrium is given by the initial
amount of protein present in the mixture [P0] minus the amount
of free protein in solution [P].

[P ]− [P ] = [PS]
0 (4)
After substitution of this expression into Eq. 2 and simple rear-
rangements the following expression is obtained:
[P ] − 1 = [S]
0 1 (5)
[P ] KD
To determine KD, (P0/P) − 1 is plotted against the surface site
concentration in mol/L, yielding a linear relation with a gradient
of 1/KD.

2 Materials
Pipettes and pipette tips in the range of 10–1,000 mL are needed
for the preparation of the samples. For handling proteins at these
low concentrations, we advise to use specially functionalized test
tubes that have a low affinity to bind proteins (e.g., LoBind® tubes,
Eppendorf). All solutions should be prepared using ultrapure
deionized water (18 MW ´ cm at 25°C). Glassware must be carefully
cleaned (aqua regia, ultrapure deionized water).
228 Lennart Treuel and Marcelina Malissek

2.1 Circular The sensitivity and spectral range (for these studies wavelengths
Dichroism between 260 and 180 nm should be accessible) might require
Spectrometer modifications of the spectrometer (see Note 1).

2.2 Preparation In the UV range used here (from 260 to 180 nm) special quartz
of the Cuvettes cuvettes are necessary to ensure a sufficient UV transparency (e.g.,
1 mm Suprasil® quartz cuvette, Helma). Moreover, these cuvettes are
temperature resistant and resistant to most chemicals, for example,
acids which can therefore be used as cleaning agents. The cuvettes
must be protein free and free of any nanoparticle residues and must
be cleaned between two measurements. In the following, we
describe a general cleaning procedure, and the reader is referred to
Note 2 for further considerations.
“To remove protein residues and ions, clean your cuvettes
carefully between the individual measurements with a cuvette
cleaning agent (e.g., Helmanex®, Helma).
1. Preparing cleaning agent: Mix the stock solution of the cuvette
cleaning agent (e.g., Helmanex® Fa. Helma) with ultrapure
water as indicated by the supplier.
2. Place the cuvette into a beaker containing the cleaning solu-
tion. Heat the solution to between 50 and 60°C and keep it at
this temperature for about 20 min. Thereafter, wash the cuvette
several times with ultrapure water.
3. Preparation of aqua regia: To remove metallic nanoparticles
from the cuvette, clean with aqua regia (see Notes 2 and 3). To
produce aqua regia 3 aliquots of concentrated hydrochloric
acid (HClaq) are mixed with 1 aliquot concentrated nitric acid
(HNO3 (aq)). These are all very strong acids and also gaseous
Cl2 is produced in the reaction (see Note 3). Pay attention to
the relevant safety instructions for all components! Mixing the
two acids produces aqua regia, which can be identified by a
golden yellow color.
4. Boil your cuvette with a little (as little as possible to fully sub-
merge your cuvette) aqua regia for about 5 min.
5. After cleaning with aqua regia, the cuvettes have to be carefully
rinsed with ultrapure water 3–5 times. To check if all aqua
regia is rinsed off, test the pH of the rinsing water and wash
until a neutral pH is reached. Avoid traces of fingerprints.

2.3 Preparation Prepare a protein stock solution in a suitable buffer depending on


of Protein Solution the type of protein and the type of nanoparticle used in the study
(see Note 4). Typical protein concentrations for protein–nano-
particles interaction studies are 0.05–0.5 mg/mL depending on
the affinity. The stock solution should have a concentration
between 10 and 100 mg/mL. Test spectra should be acquired at
the diluted concentrations with protein contents between 0.5
Interactions of Nanoparticles with Proteins: Determination of Equilibrium Constants 229

and 0.05 mg/mL. The final concentrations need to yield a


sufficient CD signal. Attention must also be paid to the depen-
dence on the nanoparticle concentration and size as described in
Subheading 2.4.
1. Weigh in your protein into a low bind test tube and add buffer
solution to prepare the stock solution with the desired
concentration.

2.4 Preparation 1. Calculating the concentration range of the sample: To choose


of NP Solution the concentration of the nanoparticles relative to the protein
concentration, calculate the number of proteins needed to
form a monolayer on the nanoparticles surface. Select your
nanoparticle concentration assuring that the total nanoparticle
surface in the mixture will be sufficient to theoretically accom-
modate all proteins present.
2. Test the stability of the nanoparticle suspension in the buffer
used for the preparation of the protein solution (see Note 4).

3 Methods
3.1 Preparation of 1. Prepare low binding tubes.
Protein–Nanoparticles 2. Pipette the pre-calculated amount of protein stock solution
Samples into each of the tubes
3. Fill the tubes with the calculated amount of water (see Note 5).
4. Shake your protein solutions and incubate them for about
5 min at room temperature (20°C).
5. Add the different pre-calculated volumes of your nanoparticle
suspension to the protein solution. This procedure produces
samples with a range of nanoparticle concentrations and a con-
stant protein concentration.
6. Incubate your prepared samples for 3–5 h at room tempera-
ture (20°C) until an adsorption equilibrium is established (see
Note 6).
7. Perform a final test of NP stability after the equilibration. The
sample should be stable for at least 12 h.

3.2 Measuring of the 1. Record a CD spectrum of the buffer used for the sample prep-
Protein–Nanoparticle aration. The measuring parameters (slid width, scanning step,
Samples integration time/scanning speed) have to be the same as those
that will be used for measuring the samples. This background
spectrum is necessary, because some buffers can also produce a
circular dichroism signal in this wavelength region.
2. Record CD spectra of the residual chemicals that may be present
in your nanoparticle solution from the synthesis procedure or
230 Lennart Treuel and Marcelina Malissek

Fig. 2 Circular dichroism spectra of pure bovine serum albumin (black line) and
bovine serum albumin at three different nanoparticle concentrations (citrate sta-
bilized gold nanoparticles, diameter = 20 nm)

as surface ligands. Some of these chemicals (e.g., TPPTS ) may


by themselves influence the protein structure (see Note 7).
3. Test the UV absorption of the nanoparticles used in the study
(see Note 8).
4. Carry out CD measurements with the incubated protein–
nanoparticle solutions. Test the reproducibility of the CD
measurements (see Note 9) (Fig. 2).
5. Subtract the background.

3.3 Determination of The following descriptions and the corresponding notes regarding
Adsorption Equilibrium the derivation of equilibrium constants, the determination of
Constant (KD) free protein content, and the determination of surface sites have
been published by L. Treuel et al. (5) and are in parts reproduced
from this source with kind permission from the copyright holder,
Wiley-VCH Verlag GmbH & Co. KGaA:
1. Conversion of the measured data from mdeg to the molar elliptic-
ity: First, the acquired CD signal has to be converted to the
mean residue ellipticity, MRE (symbol [Q]) using the following
equation:
Θ 208nm ·M
[Θ] = . (6)
n·l ·c ·10
With Q208 nm being the observed CD in mdeg, M the molar
mass of the protein, n the number of amino acid residues in the
protein, l the path length in cm, and c the protein concentra-
tion in g/L.
2. Calculation of structural content: Determine the relative struc-
tural contribution of a-helix structural elements in the protein
Interactions of Nanoparticles with Proteins: Determination of Equilibrium Constants 231

using spectral deconvolution (see Note 9). For alternative


procedures: (see Note 10). Commercial as well as open source
software exists for this procedure (e.g., CDNN (21, 22)).
3. Calculation of free protein content: This helical content is used
to determine the amount of free protein in solution assigning
the loss of CD spectroscopic signal intensity to the loss of pro-
tein structure in the interaction process. This allows the deter-
mination of the amount of free protein present in the solution
[P]. The a-helix content in the pure protein solution (without
nanoparticles present) is set to be 100% of intact protein. This
is referring to [P0] as described in the introduction of this
chapter. Any loss in the a-helix content is attributed to loss of
free protein and hence adsorption of proteins on NP surface
sites. Subsequently the a-helix content of the protein is deter-
mined from the CD signal at various nanoparticle concentra-
tions as described. The amount of free protein [P] can then be
determined in relation to [P0] according to Eq. 7.
Helix protein + NP
·[P0 ] = [P ]. (7)
Helix pure protein

4. Calculation of (P0/P) − 1: For the determination of KD values,


a plot of (P0/P) − 1 against the surface site concentration is
required. Having determined [P], and knowing [P0], the initial
protein concentration in mol/L, [(P0/P) − 1], can now be
calculated as described in the introduction.
5. For the determination of KD values, the surface site concentration
is required. This is calculated according to the following
procedure (the determination of surface sites on the nanoparticle
follows the assumption of a spherical particle geometry).
The total number of protein molecules that can adsorb onto
these surface sites is obtained by dividing the surface area of the
nanoparticle by the interacting area of the protein (informa-
tion about the size, shape, and structure of many proteins can
be found in the literature or in protein data bases, e.g., RCSB
protein data bank (23) or swissprot (24)). With this informa-
tion, it is possible to calculate the number of proteins fitting on
one nanoparticle assuming a monolayer coverage by dividing
the surface area of a nanoparticle by the surface area of the
protein according to the following equation:

NPsurface area ⎡⎣ nm 2 ⎤⎦
Number of surface sites / NP = . (8)
protein surface area ⎡⎣ nm 2 ⎤⎦

Now, the surface site concentration can be determined by


simply multiplying this value with the number of nanoparti-
cles in 1 L and dividing by the Avogadro constant
(NA = 6.022 × 1023 mol−1).
232 Lennart Treuel and Marcelina Malissek

Fig. 3 KD plot—a linear fit of this plot yields a gradient of 1/KD and thus KD

c surface sites ⎡⎣L−1 ⎤⎦


C surface site concentration [mol / L] = . (9)
N A ⎡⎣ mol −1 ⎤⎦

6. Determination of the KD value with a linear plot: To now determine


KD, (P0/P) − 1 is plotted against the surface site concentration.
A linear fit of this plot yields a gradient of 1/KD and thus KD.
An example of such a plot is shown in Fig. 3.

4 Notes
1. A UV lamp emitting between around 260 nm and 180 nm is
needed for the measurements described here. Typically, Xenon
lamps will be used for this purpose. At these wavelengths
oxygen will be photolyzed and subsequently ozone will be
formed. This is a health hazard and also the ozone will damage
optical parts in the CD spectrometer. Therefore, the spectrom-
eter has to be oxygen free which is usually achieved by flowing
nitrogen through the housing. If in doubt, please refer to the
literature (14, 25) or contact the manufacturer of your spec-
trometer for further advice.
2. Most of the metallic nanoparticle residues can be removed
with aqua regia. However, the procedure will not work for
some other nanoparticles. In this case, more suitable solvents
(depending on the individual type of nanoparticle used in the
experiment) are advised.
3. Aqua regia is produced by mixing hydrochloric acid and nitric
acid according to the following reaction:
Interactions of Nanoparticles with Proteins: Determination of Equilibrium Constants 233

HNO3(aq) + 3HCl (aq) → NOCl (aq) + 2Cl nasc (g) + 2H 2O(l) . (10)
This and the gaseous chlorine formed in the reaction are
very hazardous chemicals and close attention must be paid to the
relevant safety instructions. The synthesis must be carried out
in a fume hood and should only be carried out with a sufficient
chemical background and relevant practical experience.
4. The buffer usually has to be chosen according to the require-
ments of the protein under consideration. However, the
presence of the ionic components from this buffer might com-
promise the stability of the colloidal nanoparticle system. This
is an important point since agglomeration of the nanoparticles
will falsify the calculation of the surface sites and lead to com-
plex light scattering effects during the measurements (26–28).
It is therefore necessary to test the stability of the nanoparticle
suspension in the buffer at the relevant concentrations. This
can be done using dynamic light scattering (DLS) (29–31) or
Brownian motion nanoparticle sizing (BMNS) (5, 32). The
suspensions have to be stable in buffer for at least 12 h. Some
proteins might be stable at this pH without a buffer making it
possible to refrain from using a buffer system.
5. To keep the protein concentration constant during the mea-
surements, the total volume of the samples is kept constant
throughout the measurement series. This is achieved by always
using the same amount of protein stock solution. Different
relative quantities of water / buffer and nanoparticle suspension
are then added to vary the nanoparticle concentration. In this
procedure, the sum of the solvent and the nanoparticle suspen-
sion volume is kept constant (only the ratio is varied) and hence
a constant total volume is achieved (usually 1–2 mL).
6. The described values of 3–5 h are equilibration times for typi-
cal nanoparticle and protein concentrations. However, these
times will strongly depend on the nanoparticle and protein
concentrations (absolute and relative concentrations) used in
the measurements. Therefore, it is strongly advisable to test the
equilibrium time by performing time dependent CD studies at
a constant relative NP/protein ratio. Equilibrium is reached
when the acquired CD spectra show no further change.
7. It has to be tested, if any residual chemicals present in the
nanoparticle suspension affect the protein structure. These can
be residues from the nanoparticle synthesis but also some
ligands, e.g., TPPTS (3,3¢,3″-Phosphinidynetris(benzenesulph
onic acid) trisodium salt). To exclude the possibility of such an
influence, CD spectra of the protein under consideration
together with these chemicals have to be recorded and com-
pared to the spectra of the pure protein. The concentrations of the
234 Lennart Treuel and Marcelina Malissek

individual chemical substances should be chosen to represent


the maximum possible concentration in the final solution.
8. Test the UV absorption of the nanoparticles by recording a
UV/Vis spectrum of your nanoparticles. If the nanoparticles
absorb too strongly in the wavelength region between 260 and
180 nm, the particles might not be suitable for this type of
experiment. It has also to be tested if the particles themselves
produce a CD spectrum. In this case, they might also be unsuit-
able for the experiment.
9. To test the reproducibility of the CD measurements, repeat
the experiment for a sufficient number of times. If the data
shows a bad reproducibility, this can point to an insufficient
equilibration time or to a destabilized colloid.
10. The deconvolution of CD spectra can be carried out with dif-
ferent methods many of which are used by commercial soft-
ware solutions (e.g., CDNN (17, 21, 22), SELCON3 (33),
CONTIL/LL (34), CDSSTR (34)). Alternatively, simplified
procedures to determine structural content such as that
described by Lu et al. (18, 35–37) can be used and yield com-
parable results in many cases. In the procedure the a-helix
content of the protein is determined based on the different
spectral intensities at 220 and 208 nm, respectively. This
method is commonly used in the contemporary interpretation
of CD spectroscopic measurements (5, 35–38).

References

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Chapter 22

Tracing the Endocytic Pathways and Trafficking Kinetics


of Cell Signaling Receptors Using Single QD Nanoparticles
Katye M. Fichter and Tania Q. Vu

Abstract
Cellular signaling is the fundamental process through which cells communicate with each other and
respond to their environment. Regulation of this cellular signaling is crucial for healthy cellular function.
Malfunctions in signaling are the cause for many diseases and disorders and therefore are under heavy
investigation. The molecular mechanisms that underlie cellular signaling rely upon complex and dynamic
processes of receptor intracellular trafficking. The specific endosomal pathways and kinetics through which
receptors are intracellularly transported regulate the strength and duration of cellular signaling. In even
more subtle and complex aspects, the cell orchestrates the individual motions of many receptors, through
multiple different pathways, simultaneously. Despite the fundamental role of endosomal trafficking in
signal regulation, it has been technically challenging to study since intracellular trafficking is complex and
dynamic, with millions of individual receptors simultaneously undergoing trafficking in different endocytic
stages. Here, we describe the use of single nanoparticle quantum dot (QD) probes to quantitatively inves-
tigate the endocytic trafficking pathways that receptors undergo following ligand activation. This new
capability to directly visualize and quantitate cellular signaling at the level of individual receptors inside the
cell has broad and important value for understanding fundamental cell signaling processes and the action
and effect of therapeutics upon signaling.

Key words Quantum dots, Receptors, Cellular signaling, Intracellular trafficking, Endocytosis, Receptor
recycling, Serotonin, GPCRs

1 Introduction
Cellular signaling is the fundamental process through which cells
communicate with each other and their environment and as such
represents a large field of study with many biomedical applications
(1–8). Once thought to be distinct from each other, endocytosis
and cell signaling have found significant functional overlap (9).
In recent years a significant role for endocytosis has been implicated
in the regulation of cellular signaling strength (10). For example, a
delicate balance of internalization and intracellular traffic controls
the number of receptors that are available at the surface of the

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_22, © Springer Science+Business Media New York 2013

237
238 Katye M. Fichter and Tania Q. Vu

Fig. 1 Some of the typical intracellular pathways involved in the trafficking of


GPCRs. Vesicles bud from the plasma membrane and are directed toward early
endosomes (EE) where the cargo is sorted by the cell. The receptors can be
directed toward lysosomes (L) for degradation or recycled either quickly, through
Rab 4-associated vesicles, or slowly through recycling endosomes (RE) studded
with rab coupling proteins (asterisks). N denotes the nucleus

plasma membrane, and thus the number of receptors capable of


activation (11). It is the intracellular trafficking through mem-
brane-bound endosomes in which the cell may direct receptors
either back to the surface of the cell for subsequent rounds of acti-
vation, or toward lysosomes for degradation and signal downregu-
lation (11–14).
The pathways and kinetics of intracellular trafficking are very
responsive to subtle nuances such as the duration of agonist expo-
sure, concentration of agonist, presence of drugs, and other bio-
chemicals. (15–18). Investigating these processes is very difficult
since intracellular trafficking is a very complex process that employs
multiple different intracellular pathways that individual receptors may
use simultaneously with different trafficking kinetics (Fig. 1) (13).
Additionally, recent studies are uncovering the existence of signal-
ing endosomes from which activated receptors may continue to
initiate signaling cascades while traveling intracellularly (19, 20).
Unfortunately, classical techniques for investigating cellular inter-
nalization and intracellular trafficking lack the degree of resolution
necessary to observe how subpopulations of receptors are regu-
lated through this process. Surface biotinylation and radiological
assays must be performed on a large number of cells, which blinds
investigators from the contributions of individual receptors (21, 22).
While many organic fluorophores have been used to observe single
cells, it is very difficult to visualize single or small groups of receptors
with these labels because of the relative dimness of organic
fluorophores and their susceptibility to photobleaching.
Tracing the Endocytic Pathways and Trafficking Kinetics of Cell Signaling Receptors… 239

Quantum dots are extremely bright and photostable nanopar-


ticles that have proved valuable for studying the molecular tracking
of receptors on the surface of cells because of their ability to label
single or small groups of receptors (23–26). Here, we present new
straightforward and robust methods to identify the intracellular
trafficking pathways of receptors and the kinetics through which
they proceed through those pathways. Our lab has expertise in
using single QD nanoparticles for superior resolution in labeling
single or small groups of receptor (27–32). Here, our labeling
scheme involves genetically encoding three copies of the hemag-
glutinin (HA) epitope (33) onto the extracellular N terminus of
the human serotonin subtype 1A (5-HT1A) receptor. A single QD
probe, consisting of a biotinylated anti-HA antibody combined with
streptavidin-coated QDs at 1:1 stoichiometry, is then used to label
the HA-tagged receptors with high affinity (see Note 1). However,
this general scheme is widely applicable to just about any receptor
system with a corresponding high-affinity epitope tag. Recently, we
have shown that the presence of the QD, as bound to the receptor
through this method, does not appear to alter the trafficking behavior
(pathway or kinetics) of the 5-HT1A receptor in live cells (27).
Here, we describe methods to investigate the intracellular path-
ways and trafficking kinetics of 5-HT1A receptors with our QD
labeling system. Cellular internalization is initiated with exposure to
an agonist (i.e., serotonin). After activation, the receptors are allowed
to traffic for a set of predetermined time points (0–60 min). The
cells are then fixed and immunolabeled with markers for various
endocytic compartments. The cells are imaged and then image anal-
ysis is used to verify the presence of single QDs and to co-localize the
QD-labeled receptors with the various endocytic compartments.
This data is then plotted with time to give a picture of the dynamics
of the receptors through each endosomal compartment. While we
have used this method to investigate the trafficking of 5-HT1A (27),
it is generally applicable most other receptor systems. Furthermore,
this method presents an ideal assay platform to determine the effect
of therapeutics on the trafficking/signaling behavior of receptors,
such as the effect of selective serotonin reuptake inhibitors (SSRIs)
on the dynamics of serotonin receptor signaling.

2 Materials
2.1 General 1. N2a (neuroblastoma) cells, transfected with pDNA encoding
Reagents/Materials the human 5-HT1A gene tagged with the hemagglutinin
epitope (HA-5-HT1A) (see Note 1). Culture N2a cells on six
glass coverslips (one coverslip for each duration of 5-HT acti-
vation) in each of six separate, small Petrie dishes to facilitate
the receptor activation protocol (Subheading 3.2 below).
2. Dulbecco’s Modified Eagle Medium (D-MEM).
240 Katye M. Fichter and Tania Q. Vu

3. Serotonin HCl (chemically known as 5-hydroxytrytamine, or


5-HT).
4. Phosphate-buffered saline (PBS), pH 7.4.
5. 0.1 M borate buffer, pH 8.5.
6. Streptavidin-conjugated QD655 (Strep-QD655) (Life Technologies,
Grand Island, NY, USA).
7. NHS-PEO4-biotin.
8. 10,000 MWCO slide-a-lyzer dialysis unit (Thermo Scientific).
9. 4% paraformaldehyde (PFA) in PBS.
10. 0.2% Triton X-100 in PBS.
11. 10% normal goat serum (NGS) in PBS and/or 10% bovine
serum albumin (BSA) in PBS.

2.2 Immunoreagents 1. Monocolonal mouse anti-HA.


2. Polyclonal rabbit anti-Rab 4.
3. Polyclonal goat anti-Rab11-F1P1(N-15).
4. Monocolonal rat anti-LAMP1.
5. Alexa-conjugated secondary antibodies: anti-rabbit, anti-goat,
and anti-rat (Life Technologies, Grand Island, NY, USA).

2.3 Imaging 1. Inverted Zeiss Axiovert 200M (or other comparable


Equipment/Analysis epifluorescent or confocal microscope) equipped with 63× (or
Software higher), 1.4 NA oil objective, and digital CCD camera. A digi-
tal CCD camera capable of capturing time-lapse movies at a
frame rate of at least 30 frames per second is necessary to verify
the presence of single QDs (Subheading 3.4).
2. An imaging chamber, compatible with the coverslips of choice.
3. Appropriate filters for QDs and organic dyes (e.g., Chroma,
QD655 filter set # 32012).
4. Appropriate acquisition software for your digital camera.
5. ImageJ (34) with the JACoP plug-in (35) (both are available
for free download at http://rsbweb.nih.gov/ij).
6. Microsoft Excel or similar spreadsheet program.
7. Autoquant X or similar deconvolution software (if not using a
confocal microscope).

3 Methods
3.1 Biotinylation of Store all QD solutions at 4°C. Never freeze QD solutions. Because
Anti-HA Antibody and of potential QD aggregation, it is not recommended to store QD
Preparation of QD solutions at a concentration less than 100 nM.
Stock Solution 1. Biotinylate the anti-HA antibody by reacting 10 μg of antibody
with a threefold molar excess of NHS-PEO4-biotin in PBS.
Tracing the Endocytic Pathways and Trafficking Kinetics of Cell Signaling Receptors… 241

Allow the reaction to incubate 30 min at room temperature


(RT). Purify the biotinylated antibody from excess NHS-
PEO4-biotin via dialysis against 1 L PBS using a 10,000
MWCO slide-a-lyzer unit. Dialysis typically consists of 4–5
changes of dialysate over the course of 4–5 h. The biotinylated
antibody should then be aliquoted into 10-μL aliquots and
unused aliquots should be stored at −80°C.
2. Prepare separate, 50-μL dilutions of both the biotinylated anti-
HA and Strep-QD655 at a concentration of 100 nM in 10%
NGS. Store solutions at 4°C. Allowing the QDs to incubate in
10% NGS for 24–48 h before forming the QD probes has been
found to help decrease the nonspecific binding of QDs to
cells.

3.2 Agonist-Induced Ensure all reagents to be used with live cells are warmed to 37°C
Activation of 5-HT prior to cell incubation (including 4% PFA).
Receptors
1. Aspirate media from cells and replace with D-MEM (or media
containing dialyzed serum) (see Note 2). Allow cells to incu-
bate at 37°C and 5% CO2 for at least 4 h before beginning the
activation.
2. Set a timer to facilitate the time-dependent 5-HT activation
step. Create one alarm at 15 min to alert you to end 5-HT
activation. Create separate alarms to alert you to fix cells at 5,
10, 20, 30, and 60 min after 5-HT activation. Label each dish
with the appropriate incubation time (5, 10, 20 min, etc.).
Leave one dish to the side as a control (no activation).
3. Prepare QD-anti-HA conjugates. This step will prepare 6 mL
of QD-Ab conjugates (combined at a 1:1 stoichiometry) at a
final concentration of 250 pM. Add 15 μL 100 nM biotiny-
lated anti-HA to 5,970 μL 10% NGS and mix. To this mixture
add 15-μL Strep-QD655 and mix again. Allow the solution to
incubate for 2 min, and then use immediately.
4. Label cells with QD-anti-HA conjugates. Aspirate media from
each dish. Add 1 mL of the above QD/Ab conjugate solution
to each dish. Incubate for 5 min RT. Wash the cells six times
with warm PBS rapidly, with no incubation in between. Leave
the cells in PBS temporarily until completing the next step.
5. Prepare a solution of 10-μM 5-HT in warmed D-MEM (or media
containing dialyzed serum). Use this solution immediately
after preparing.
6. Aspirate PBS from cells, replace with 5-HT solution, and activate
the timers. Incubate cells for 5, 10, 20, 30, and 60 min at 37°C
and 5% CO2.
242 Katye M. Fichter and Tania Q. Vu

7. At the time of the alarm to fix cells, (5 and 10 min) remove the
appropriate coverslip from the incubator and fix in 4% PFA for
30 min. Store cells in 0.1 M borate buffer, pH 8.5 (see Note 3)
at 4°C until immunolabeling.
8. At the time of the alarm to end 5-HT activation (15 min), cells
activated for 5 and 10 min should already be fixed. Remove the
remaining cells from the incubator, aspirate the 5-HT solution
and replace with D-MEM (or media containing dialyzed
serum). Return cells to the incubator and continue to fix the
rest of the cells at the appropriate time (20, 30, and 60 min).
Remember to also fix the control dish (no activation). Store all
cells in 0.1 M borate buffer, pH 8.5 (see Note 3) at 4°C until
immunolabeling.

3.3 Immunolabeling For extended storing time (<12 h), store cells in 0.1 M borate buf-
Cells for Endocytic fer, instead of PBS, to maximize QD fluorescence.
Compartments
1. Permeabilize cells by incubating them with 0.2% Triton X-100
for 20 min and wash three times with PBS, with a 15 min incu-
bation in between each wash.
2. Block cells in 10% NGS for at least 1 h.
3. Incubate cells in the first primary antibody (e.g., polyclonal
rabbit anti-Rab 4 at a dilution of 1:500 in 10% NGS for 4 h at
room temperature). Afterwards, wash the cells three times with
PBS, with a 15-min incubation between each wash.
4. Block cells again in 10% NGS for at least an hour (if using an
anti-goat antibody, substitute with 10% BSA).
5. Incubate cells in the first secondary antibody (e.g., Alexa488
donkey anti-rabbit at a dilution of 5 μg/mL in 10% NGS or
BSA for 1 h at room temperature). Wash the cells three times
with PBS, with a 15-min incubation between each wash.
6. Repeat steps 2–5 for each additional primary and secondary
antibody you wish to use (see Note 4).

3.4 Verify the To ensure the data collected from these experiments reveal infor-
Presence of Single QDs mation on the level of single or small groups of receptors, it is
necessary to verify that the QDs in the cell sample are single QDs.
1. Load a coverslip containing QD-labeled cells into an imaging
chamber and fill the chamber with 0.1 M borate buffer pH 8.5
(see Note 5).
2. Observe the fluorescence from the QD channel. All QDs should
be blinking rapidly and should exhibit approximately the same
fluorescence intensity. Larger, brighter puncta that are blinking
slowly, or not at all, are evidence of QD aggregates.
3. Record a time-lapse fluorescence movie in the QD channel at
a rate of 30 frames per second for 500 frames.
Tracing the Endocytic Pathways and Trafficking Kinetics of Cell Signaling Receptors… 243

Fig. 2 Exemplary intensity trace of a single QD displaying multiple square on/off


wave pulses (some examples marked with an asterisk) and constant intensity of
“on” and “off” states

4. Use ImageJ to open the movie file. Zoom in on an individual


QD puncta as far as possible. Use the rectangle marquee to
select the brightest pixels of the QD puncta (i.e., 4–6 pixels for
an image with a resolution of ~0.13 μm/pixel).
5. Generate a plot of the fluorescence intensity rate of the QD
puncta by choosing Plugins → T-functions → Intensity vs. Time
Monitor. Copy the frame number and average pixel intensity
output to a worksheet in Excel. Repeat steps 4 and 5 several
times to obtain a representative population of QD puncta.
6. For each QD puncta, create a fluorescence intensity trace. Create
an xy scatter plot using the frame number (or convert to time) as
x input and the average pixel intensity as y input. Inspect the plot
carefully. The trace should demonstrate discrete square on/off
wave pulses. The intensity of the “on” and “off” states should
remain constant throughout the plot (Fig. 2).

3.5 Image Acquisition 1. Place a coverslip in an imaging chamber and fill the chamber
with 0.1 M borate buffer pH 8.5.
2. Find a cell expressing HA-5-HT1A by looking for high QD
binding.
3. Capture a z-stack of each fluorescent channel of the cell. Ensure
that the stack includes the entire cell from top to bottom. Use a
reasonable distance between slices (z-slice) to ensure sampling
of the entire vertical height of the cell (e.g., 0.275 μm between
slices for a 100× 1.4 NA objective).
4. Capture one bright-field (DIC or phase) image for each cell.
5. Repeat steps 2–4 to capture image data for at least 5–10 cells.
6. Repeat steps 2–5 for each coverslip at each time point.
244 Katye M. Fichter and Tania Q. Vu

Fig. 3 The intracellular trafficking kinetics of the 5-HT1A receptor. Receptor


trafficking through the fast recycling pathway (Rab 4) maximized at 10 min, while
recycling through the slow pathway (rab coupling protein, RCP) maximized
around 30 min. 5-HT1A receptors do not appear to significantly accumulate in
lysosomes (LAMP1) after short-term exposure to 5-HT. Bar depicts duration of
exposure to 5-HT

3.6 Image Analysis 1. If images were not acquired using a confocal microscope,
deconvolve each z-stack using Autoquant X or other similar
deconvolution software.
2. Open a deconvolved QD stack and corresponding deconvolved
endosome stack (e.g., Rab 4) from one cell in ImageJ. If neces-
sary, perform a background subtraction of each stack and adjust
the brightness and contrast.
3. Run the JACoP plug-in by selecting Plugins → JACoP. Ensure
the QD stack is listed as Image A and the Rab 4 stack is listed
as Image B. Click the threshold button and use the slider to
choose a threshold for each image. Slide though the entire
stack to ensure choosing an appropriate threshold. Push the
analyze button.
4. Copy the M1 value to an excel spreadsheet under the duration
of agonist exposure for that particular coverslip. Repeat steps
2–3 for each cell images. Calculate the average M1 and the
standard deviation of M1 for each coverslip.
5. Plot the average M1 against time for each coverslip. This plot will
allow you to visualize the time course through with receptors
traffic through Rab 4 endosomes (Fig. 3).

4 Notes
1. The hemagglutinin (HA) epitope is a peptide sequence
(YPYDVPDYA) (33) for which very high-affinity antibodies
have been created. We prefer to use HA-tagged receptors for
targets since antibodies targeting native epitopes of many
Tracing the Endocytic Pathways and Trafficking Kinetics of Cell Signaling Receptors… 245

GPCRs have been found to have less-than-optimal affinity.


This HA peptide can be fused to your receptor sequence via
basic molecular cloning techniques. In this protocol, we have
fused three copies of the HA epitope to the N-terminal (extra-
cellular) end of the receptor. By fusing the HA tag to the extra-
cellular end of the receptor, we aim to avoid interferences with
the signaling and trafficking machinery that interacts with the
C-terminal (intracellular) end of the receptor.
2. Because serum typically contains serotonin, cells cultured in full
serum may downregulate the activation of serotonin receptors
to mitigate this exposure. We recommend removing the serum
from cells before ligand activation to minimize the impact of
this downregulation on receptor trafficking. Alternatively, cells
can be cultured in dialyzed serum for a few days before activation
to alleviate cellular downregulation before receptor activation.
3. QDs may quench when stored in PBS for longer durations.
When necessary to store cells labeled with QDs for longer than
12 h, 0.1 M borate buffer, pH 8.5 is recommended as the storage
buffer to maintain QD fluorescence.
4. To avoid complications due to cross-species reactivity with sec-
ondary antibodies, separate experiments may be designed to
investigate receptor co-localization with multiple different
endocytic compartments.
5. Many mounting mediums may cause QDs to quench or stop
blinking. At the time of this writing, most mounting mediums
compatible with QDs require dehydration in ethanol and
washing with toluene, which may destroy the integrity of cel-
lular structures. For this reason, we recommend imaging cov-
erslips in an imaging chamber.

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Chapter 23

Cellular Internalization of Quantum Dots


Yue-Wern Huang, Han-Jung Lee, Betty Revon Liu, Huey-Jenn Chiang,
and Chi-Heng Wu

Abstract
Cell-penetrating peptides (CPPs) can facilitate uptake of quantum dots (QDs) for a variety of basic and
applied sciences. Here we describe a method that utilizes simple noncovalent interactions to complex QDs
and CPPs. We further describe methods to study uptake mechanisms of the QD/CPP complex. The
inhibitor study coupled with the RNA interference (RNAi) technique provides a comprehensive approach
to elucidate cellular entry of the QD/CPP complex.

Key words Quantum dots, Cell-penetrating peptides, Inhibitors, siRNA, Clathrin, Caveolin, Lipid
raft, Macropinocytosis

1 Introduction
Nanomaterials have found numerous biomedical applications in
recent years. The applications include, but not limited to, drug
delivery, disease staging and therapeutic planning, sentinel lymph
node mapping and removal, cellular receptor trafficking monitor-
ing, and nanoelectronic biosensing. Among nanomaterials,
fluorescent semiconductor quantum dots (QDs) have been widely
used to deliver and monitor biologically active molecules into cells
(1–3). QDs have unique physical and chemical properties such as
photostability, high quantum yield, narrow emission peak, resis-
tance to degradation, and broad size-dependent photolumines-
cence (4). These properties enable QDs for long-term multiplexing
imaging. QDs are extremely slow in entering the cell. One solution
to overcome the slow uptake is to conjugate with cell-penetrating
peptides (CPPs) (5–7). We have developed protocols to (1) enhance
efficiency of cellular uptake of QDs mediated by cell-penetrating
peptides (8, 9) and (2) study molecular mechanisms of action of
QD internalization.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_23, © Springer Science+Business Media New York 2013

249
250 Yue-Wern Huang et al.

2 Materials
2.1 General 1. Quantum dots (QDs): Carboxyl-functionalized QDs with a
Components CdSe/ZnS core-shell have emission and excitation peak wave-
lengths at 520 and 505 nm, respectively. Store at 4°C.
2. Synthetic nona-arginines (SR9): SR9 are synthesized by solid-
phase peptide synthesis and then purified by high performance
liquid chromatography (HPLC) using a reverse phase column.
The desired purity of SR9 should be ³90%. Store at −20°C.
3. Cell culture medium: Ham’s F-12 modified medium (Cellgro,
VA, USA) supplemented with 10% fetal bovine serum (FBS)
(Hyclone, Utah, USA) and 1% penicillin–streptomycin. Add
50 mL FBS and 5 mL penicillin–streptomycin into 445 mL
Ham’s F-12 modified medium. Store at 4°C.
4. Phosphate buffered saline (PBS): Dissolve 8 g NaCl, 0.2 g
KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 800 mL dis-
tilled H2O. Adjust pH to 7.4 and make up to 1 L with distilled
H2O. Sterilize by autoclaving. Store at 4°C.
5. Trypsin–EDTA: 0.25% (w/v) trypsin supplemented with
0.53 mM EDTA in PBS. Mix 5 mL trypsin (5×) and 50 mL of
53 mM EDTA together. Make up to 50 mL with PBS. Store
at −20°C.
6. Tris-acetate–EDTA (50× TAE) buffer: Weigh 242 g Tris and
then transfer into a graduated cylinder containing 500 mL
double distilled water (ddH2O). Add 57.1 mL glacial acetic
acid and 100 mL of 0.5 M EDTA. Make up to 1 L with ddH2O.
Store at 4°C.

2.2 Components 1. 0.6% agarose gel: Dissolve 3 g agarose in a 100-mL graduated


of Gel Retardation cylinder containing 50 mL TAE buffer (1×). Heat agarose
solution in a microwave until agarose is completely dissolved.
Cool down the agarose solution to 60°C. Insert the comb
onto the glass plate and then pour the agarose liquid into glass
plate. Remove the air bubbles under or between the teeth of
the comb. Wait the agarose gel to become solid before use.
2. UV transilluminator.

2.3 Components 1. Medium for fluorescence imaging: Ham’s F-12 modified


of Fluorescence medium supplemented with 1% FBS. Add 5 mL FBS into
Image Studies 495 mL Ham’s F-12 modified medium. Store at 4°C.
2. Dish for fluorescence imaging: 35-mm glass-bottom tissue culture
plates (MatTek, Massachusetts, USA).
3. Phenol red-free medium (Invitrogen, California, USA).
4. Epifluorescent microscopy or confocal microscopy.
Cellular Internalization of Quantum Dots 251

2.4 Components 1. Temperature treatment: Cells are treated at either 37°C or 4°C.
of Energy-Dependent 2. A combination of metabolic inhibitors: 0.15% sodium azide,
Cellular Uptake 15 mM sodium fluoride, and 2 mg/mL antimycin A.

2.5 Components 1. Inhibitors of clathrin-dependent pathway: Chlorpromazine


of Pathway-Specific stabilizes the nascent clathrin-coated vesicles while monodan-
Inhibitors sylcadaverine (MDC) inhibits relocation of clathrin and adap-
tor protein complex-2 (AP-2) from the plasma membrane to
the endosomal membrane. The mechanism of hypertonic
sucrose (0.45 M) involves the dispersion of clathrin lattices on
the plasma membrane. Mix chlorpromazine or MDC stock solu-
tion in Ham’s F-12 modified medium supplemented with 10%
FBS and 1% penicillin–streptomycin at final concentrations of
10 mM and 25 mg/mL, respectively. For hypertonic studies,
dissolve 0.154 mg sucrose in 1 mL Ham’s F-12 modified medium
supplied with 10% FBS and 1% penicillin–streptomycin.
2. Inhibitors of caveolin-dependent pathway: Filipin and nystatin
are used to deplete cholesterol. Mix the filipin or nystatin stock
solution in Ham’s F-12 modified medium supplemented with
10% FBS and 1% penicillin–streptomycin at final concentra-
tions of 3 and 20 mg/mL, respectively.
3. Inhibitors of macropinocytosis: 5-(N-ethyl-N-isopropyl)
amiloride (EIPA) and cytochalasin D (CytD) are used to inhibit
Na+/H + exchange or cap the barbed, fast-growing ends of
actin filaments, respectively. Mix the EIPA or CytD stock solu-
tion in Ham’s F-12 modified medium supplemented with 10%
FBS and 1% penicillin–streptomycin at final concentrations of
30 mM or 1 mg/mL, respectively.

2.6 Components 1. Small interfering RNA (siRNA): The sequences of siRNAs for
of siRNA Experiments clathrin heavy chain and caveolin-1 are as follows:
Clathrin heavy chain:
5¢-CCCUAAACACCUCAACGAU[dT][dT]-3¢ (sense)
5¢-AUCGUUGAGGUGUUUAGGG[dT][dT]-3¢
(antisense)
Caveolin-1:
5¢-CAUUAUGACCGGGCUCAUA[dT][dT]-3¢ (sense)
5¢-UAUGAGCCCGGUCAUAAUG[dT][dT]-3¢ (antisense)
2. Lipofectamine 2000: Dilute lipofectamine stock solution in
the OPTI-MEM I-reduced serum medium according to the
manufacturer’s instructions (Invitrogen).
3. Treatment medium: Desired siRNA is premixed with OPTI-
MEM I-reduced serum medium (Invitrogen) before complexing
with lipofectamine.
252 Yue-Wern Huang et al.

4. Lysis buffer: 150 mM NaCl, 1% NP-40, and 50 mM Tris


(pH 8.0) supplemented freshly with 1% protease inhibitor
cocktail. Weigh 0.876 g NaCl and then transfer to a 100-mL
cylinder containing 50 mL ddH2O. Add 1 mL NP-40 and
5 mL of 1 M Tris solution (pH 8.0). Make up to 100 mL with
ddH2O. Store at −20°C. Add 1% protease inhibitor cocktail
before use.
5. Bio-Rad protein assay (1×): Dilute 1 mL of Bio-Rad protein
assay (5×) (Bio-Rad, California, USA) with 4 mL ddH2O.
6. Standard solutions: Prepare bovine serum albumin in a series of
five dilutions. The range depends on predicted concentrations
in samples.
7. Resolving buffer: 1.5 M Tris–HCl (pH 8.8). Add 100 mL
ddH2O to a graduated cylinder. Weigh 181.7 g Tris and 4 g
sodium dodecyl sulfate (SDS), and then transfer them into the
cylinder. Make up to 900 mL with ddH2O. Mix and adjust pH
to 8.8 with HCl. Make up to 1 L with ddH2O. Store at 4°C.
8. Stacking gel buffer: 0.5 M Tris–HCl (pH 6.8). Weigh 60.6 g
Tris and 4 g SDS. Prepare a 1-L solution as in the previous
step. Store at 4°C.
9. 30% acrylamide/bisacrylamide solution: Weigh 29.2 g acryl-
amide monomer and 0.8 g N,N¢-methylenebisacrylamide.
Transfer them to a graduated cylinder containing 80 mL
ddH2O. Make up to 100 mL ddH2O and filter through a
0.45-mm filter (Corning, Massachusetts, USA). Store at 4°C in
the darkness.
10. 10% ammonium persulfate solution: Dissolve 0.1 g ammonium
persulfate in 1 mL ddH2O. Store at 4°C in the darkness.
11. N,N,N¢,N¢-tetramethyl-ethylenediamine (TEMED): Store at
4°C.
12. SDS-PAGE buffer (5×): 0.125 M Tris–HCl (pH 8.3), 0.96 M
glycine, 0.5% SDS. Weigh 15.1 g Tris, 72 g glycine, and 5 g
SDS. Transfer all of them into a 1-L cylinder containing
700 mL ddH2O. Adjust pH to 8.3 with HCl. Make up to 1 L
with ddH2O. Store at 4°C.
13. SDS loading buffer (5×): 300 mM Tris–HCl (pH 6.8), 10%
SDS, 0.05% bromophenol blue, and 40% glycerol. Prepare
1 mL of 3 M Tris–HCl (pH 6.8), 1 g SDS, and 0.5 g bro-
mophenol blue. Transfer them to 5 mL ddH2O. Make up to
9 mL with ddH2O. Store at 4°C. Mix 100 mL mercaptoethanol
and 900 mL SDS loading buffer before use.
14. Nitrocellulose membranes (Bio-Rad).
15. Western blot transfer buffer: 0.025 M Tris, 0.192 M glycine,
and 20% methanol. Weigh 14.4 g glycine and 3 g Tris, and
then transfer them to 200 mL methanol. Make up to 1 L
with ddH2O.
Cellular Internalization of Quantum Dots 253

16. Phosphate buffered saline-Tween 20 (PBS-T): Dissolve 8 g


NaCl, 0.2 g KCl , 1.44 g Na2HPO4, 0.24 g KH2PO4, and 1 mL
Tween 20 in 800 mL distilled H2O. Adjust pH to 7.4 and
make up to 1 L with distilled H2O. Sterilize by autoclaving.
Store at 4°C.
17. Blocking solution: 5% milk in PBS. Dissolve 2.5 mg nonfat dry
milk in PBS and then make up to 50 mL with PBS. Store at 4°C.
18. Diluent solution: 5% milk in PBS-T. Dissolve 2.5 mg nonfat
dry milk in PBS and then make up to 50 mL with PBS-T.
Store at 4°C.
19. Mini PROTEAN® 3 System glass plates: Bio-Rad (catalogue
#1653311).
20. Pierce ECL western blotting substrate (catalogue #32106).

3 Methods
3.1 Formation of QDs To test whether SR9 peptides complex with QDs, QDs are mixed
and SR9 Noncovalent with SR9 at various molar ratios (1:10, 1:20, 1:30, and 1:60) fol-
Binding lowed by separation with a 0.6% agarose gel. That the complexes
mobility decrease as the amount of SR9 increases indicates the for-
mation of noncovalent QD/SR9 complexes.

3.1.1 QDs/SR9 1. The concentrations of QDs and SR9 stock solutions are 10 and
Noncovalent Binding 625 mM, respectively.
2. Mix 5 mL of QDs stock solution with SR9 stock solution to
reach various molecular ratios; make up to a final volume of
40 mL with PBS.
3. Incubate the QDs/SR9 mixture at room temperature for
20 min before use.

3.1.2 Gel Retardation 1. Prepare the QDs/SR9 mixture of various molecular ratios.
Assay 2. Prepare 0.6% agarose gel. After gel becomes solid, fill the elec-
trophoresis tank with TAE buffer (1×).
3. Load QDs/SR9 mixture into wells.
4. Turn on the power supply. Set the voltage at 130 V and then
perform the electrophoresis for 60 min.
5. After 60 min, stop the electrophoresis and then capture the
image by a UV transilluminator.

3.2 Dependent We select the final concentration of QDs at 150 nM as this concen-
Uptake of Qds/SR9 tration is below the cytotoxic level. To determine the optimal molar
ratio of cellular uptake, cells are incubated with QDs and SR9 at
different ratios. The ratio is determined by two criteria: the kinetics
of uptake and the imaging quality. For instance, QD uptake
increases as the molar ratio of SR9 increases from 1:10 to 1:30; the
254 Yue-Wern Huang et al.

uptake reaches the highest level at 1:30. However, good imaging


quality can be obtained at 1:20. Thus this ratio is chosen. The fol-
lowing procedure describes time-dependent uptake:

1. Seed 1.2 × 105 cells into 35-mm glass-bottom dishes and then
allow attaching for 48 h.
2. To achieve a molar ratio of 1:20 (QD:SR9). Prepare stock
solutions of QD (10 mM) and SR9 (625 mM). Mix 15 mL QD
and 4.8 mL SR9 in 980.2 mL Ham’s F-12 modified medium
supplemented with 1% FBS.
3. Incubate the QDs/SR9 mixture at room temperature for
20 min before use.
4. Discard the old medium.
5. Wash cells with 1 mL PBS three times.
6. Add 1 mL phenol red-free medium into the dish and then
detect fluorescence intensity using fluorescent microscopy.

3.3 Energy-Dependent To determine whether uptake of QDs/SR9 is energy dependent, cells


Endocystosis are incubated with QDs/SR9 under varying metabolic conditions.
In temperature studies, cells are treated at either 37°C or 4°C. In
metabolic inhibition experiments, cells are incubated in the absence
or presence of a mixture of metabolic inhibitors.

3.3.1 Temperature 1. Seed 1.2 × 105 cells into 35-mm glass-bottom dishes and then
Treatment allow cells to attach for 48 h.
2. Preincubate cells at 4°C or 37°C for 1 h.
3. To achieve a molar ratio of 1:20 (QD:SR9). Prepare stock
solutions of QD (10 mM) and SR9 (625 mM). Mix 15 mL QD
and 4.8 mL SR9 in 980.2 mL Ham’s F-12 modified medium
supplemented with 1% FBS.
4. Incubate the QDs/SR9 mixture at room temperature for
20 min before use.
5. Discard the old medium.
6. Add 1 mL of the QDs/SR9 mixture into the dish and place the
dish at either 4°C or 37°C for another hour.
7. Wash cells with 1 mL PBS three times.
8. Add 1 mL phenol red-free medium into the dish and then
detect fluorescence intensity using fluorescent microscopy.

3.3.2 Metabolic 1. Seed 1.2 × 105 cells into 35-mm glass-bottom dishes and then
Inhibition Studies allow cells to attach for 48 h.
2. Preincubate cells with a combination of metabolic inhibitors
(0.15% sodium azide, 15 mM sodium fluoride, and 2 mg/mL
antimycin A.) for 1 h at 37°C.
Cellular Internalization of Quantum Dots 255

3. Discard the old medium.


4. To achieve a molar ratio of 1:20 (QD:SR9). Prepare stock
solutions of QD (10 mM) and SR9 (625 mM). Mix 15 mL QD
and 4.8 mL SR9 in 980.2 mL Ham’s F-12 modified medium
supplemented with 1% FBS.
5. Incubate the QDs/SR9 mixture at room temperature for
20 min before use.
6. Add 1 mL QDs/SR9 mixture into the 35-mm glass-bottom
dish and place the dish at 37°C for another hour.
7. Wash cells with 1 mL PBS three times.
8. Add 1 mL phenol red-free medium into the dish and then
detect fluorescence intensity using fluorescent microscopy.

3.4 Pathway- To investigate the uptake mechanism of QDs/SR9, inhibitors are


Specific Inhibitor used to disrupt three major pathways of endocytosis: clathrin-
Experiments dependent, caveolin-dependent, and macropinocytosis.
1. Seed 1.2 × 105 cells into 35-mm glass-bottom dishes and then
allow cells to attach for 48 h.
2. To achieve a molar ratio of 1:20 (QD:SR9). Prepare stock
solutions of QD (10 mM) and SR9 (625 mM). Mix 15 mL QD
and 4.8 mL SR9 in 980.2 mL Ham’s F-12 modified medium
supplemented with 1% FBS.
3. Incubate the QDs/SR9 mixture at room temperature for
20 min before use.
4. Mix a desired inhibitor in Ham’s F-12 medium supplemented
with 10% FBS.
5. Discard old culture medium. Incubate cells with a specific
inhibitor for 30 min at 37°C. See Subheading 2.5 for final con-
centrations of inhibitors. Add QDs/SR9 mixture into the cells
to incubate for another hour.
6. Wash cells with 1 mL PBS three times.
7. Add 1 mL phenol red-free medium into the dish and then
detect fluorescence intensity using fluorescent microscopy.

3.5 siRNA RNAi technique is applied to complement the use of pharmaco-


Experiments logical inhibitors. Clathrin heavy chain and caveolin-1 siRNAs
depress expression of critical components of the clathrin and caveolar
pathways.

3.5.1 siRNA Treatment 1. Seed 1.0 × 105 cells into 35-mm glass-bottom dishes and then
and Imaging Experiment allow cells to attach for 48 h.
2. Desired siRNA is complexed with Lipofectamine 2000 reagent
in OPTI-MEM-reduced serum medium for 30 min according
to the manufacturer’s instruction.
256 Yue-Wern Huang et al.

3. Discard old culture medium.


4. Add 1 mL siRNA-lipofectamine mixture into the dish, and
then incubate for 4 h at 37°C.
5. After 4 h, add FBS containing Ham’s F-12 modified medium
to cells to achieve a final concentration of 5%.
6. After another 68 h, mix 15 mL QDs stock solution (10 mM)
and 4.8 mL SR9 stock solution (625 mM) in 980.2 mL Ham’s
F-12 modified medium supplemented with 1% FBS.
7. Incubate the QDs/SR9 mixture at room temperature for
20 min before use.
8. Add 1 mL QDs/SR9 mixture into the dish for 1 h.
9. Wash cells with 1 mL PBS three times.
10. Add 1 mL phenol red-free medium into the dish and then
detect fluorescence intensity using fluorescent microscopy.

3.5.2 siRNA-Mediated 1. Seed the 3.0 × 105 cells into 60-mm dishes and then allow cells
Protein Downregulation to attach for 48 h.
Experiment 2. Desired siRNA is complexed with Lipofectamine 2000 reagent
in OPTI-MEM I-reduced serum medium for 30 min accord-
ing to the manufacturer’s instruction. A nonspecific-targeting
siRNA is employed as a negative control.
3. Discard old culture medium.
4. Add 5 mL siRNA-lipofectamine mixture into the 60-mm dish
for 4 h at 37°C.
5. After 4 h, add FBS containing Ham’s F-12 modified medium
to cells to achieve a final concentration of 5%.
6. After another 68 h, follow the instruction in Subheading 3.6.3.

3.5.3 Western Blot 1. The analysis is conducted 3 days after transfection with siRNA.
Analysis 2. Discard old medium.
3. Wash the cells with 2 mL PBS three times and then place the
dish on ice.
4. Add ice-cold 200 mL lysis buffer into the dish.
5. Scrape cells off the dish using an ice-cold plastic cell scraper,
and then transfer the cell lysate into 1.5 mL Eppendorf tubes.
6. Set the 1.5 mL Eppendorf tubes on ice for 30 min.
7. After 30 min, centrifuge 1.5 mL Eppendorf tubes at 4°C,
12,000 rpm for 30 min.
8. Transfer the supernatant to another 1.5 mL Eppendorf tubes.
Store at 4°C for fresh use. Store the rest of samples at −20°C
for future use.
9. Measure the protein concentration of the sample according to
the Bio-Rad protein assay.
Cellular Internalization of Quantum Dots 257

10. Mix the 10 mL SDS loading buffer (5×) with 40 mL samples.


Boil the mixture at 95°C for 5 min. Then set the samples on
ice for at least 10 min.
11. Make 10% ammonium persulfate in ddH2O.
12. Assemble the gel casting apparatus, making sure that the sand-
wich of glass plates and spacers seal properly.
13. Prepare the separating gel solution according to the acrylam-
ide concentration needed. Vortex before use.
14. Various percentages of separating gels

Final acrylamide
conc 5% 6% 7% 8% 9% 10% 12% 13% 15%
30% acryl/0.8% 2.5 3.0 3.5 4.0 4.5 5.0 6.0 6.5 7.5
bisacryl (mL)
H2O (mL) 8.8 8.3 7.8 7.3 6.8 6.3 5.3 4.8 3.8
4× Tris–HCl/SDS 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7
pH 8.8 (mL)
10% ammonium 200 200 200 200 200 200 200 200 200
persulfate (mL)
TEMED (mL) 10 10 10 10 10 10 10 10 10

15. Load the apparatus with 4.5 mL of the separating gel


solution.
16. Top with 100 mL of isoamyl alcohol.
17. After polymerization, pour off the isoamyl alcohol, and rinse
with distilled water.
18. Remove any water droplets from the inside of the casting appa-
ratus with a Whatman paper or a paper towel. Insert the comb
for the stacking gel.
19. Prepare the stacking gel solution. Add 3 mL ddH2O, 1.3 mL
Tris–HCl/SDS (pH 6.8, 4×), 0.9 mL of 30% acrylamide/0.8%
bisacrylamide, 80 mL of 10% ammonium persulfate, and 5 mL
TEMED. Vortex mixtures.
20. Load the stacking gel solution; do not introduce air bubbles
underneath or between the teeth of comb. Remove air bubbles
by pipetting up and down. Allow the stacking gel to polymer-
ize completely (~45 min) before removing comb.
21. Remove the glass and gel sandwich from the casting apparatus.
22. Clip the sandwich to the electrophoresis apparatus. Carefully
remove the comb from the gel and fill the top of the apparatus
with SDS-PAGE electrophoresis buffer (1×).
23. Flush the wells with buffer. Carefully load samples and 5 mL
pre-stained protein molecular weight markers into the bottom
258 Yue-Wern Huang et al.

of the wells using a flat-tipped pipette tip. The sample protein


loaded into each wells is 30 mg.
24. Fill the bottom of the electrophoresis apparatus with SDS-
PAGE electrophoresis buffer (1×) and connect the apparatus
to the power supply.
25. Perform the gel at 80 V. After the protein markers pass through
the stacking gel, increase the voltage to 120 V.
26. When the dye reaches the bottom of the separating gel, turn
off the power supply. Remove the gel sandwich.
27. Remove the stacking gel.
28. Use the Bio-Rad gel blotting sandwiches kit to transfer protein
from the gel to nitrocellulose membrane.
29. Set the transfer system and filled with transfer buffer. The ampere
is 200 mA and transferring time is 2 h.
30. After 2 h, block the nitrocellulose with 5 mL blocking solution
for 1 h.
31. Wash with 5 mL PBS-T for 5 min three times.
32. Prepare 1st antibody with diluent solution. Clathrin heavy
chain is detected using a mouse monoclonal antibody at a dilu-
tion of 1:200. Mix 10 mL antibody with 2 mL diluent solution
and keep on ice. Caveolin-1 protein is detected using a rabbit
monoclonal antibody at a dilution of 1:1,000. Mix 2 mL anti-
body with 2 mL diluent solution and keep on ice.
33. Incubate the nitrocellulose in 1st antibody-containing diluent
solution. Keep shaking for 12 h at 4°C.
34. After 12 h, wash with 5 mL PBS-T for 5 min three times.
35. Prepare 2nd antibody with diluent solution. Goat anti-mouse
IgG-HRP secondary antibody is used for clathrin 1st antibody
at dilution of 1:1,000. Goat anti-rabbit IgG-HRP secondary
antibody is used for caveolin 1st antibody at dilution of 1:1,000.
Mix 2 mL antibody with 2 mL diluent solution and keep on ice.
Incubate the nitrocellulose in 2nd antibody-containing diluent
solution. Keep shaking for 1 h at room temperature.
36. After 1 h, wash with 5 mL PBS-T for 5 min three times.
37. The blots are probed with the ECL Western blot detection
system.

4 Notes
1. It is important that both inhibitors and siRNAs are used, as
they are complementary methods to investigate uptake mecha-
nisms. In general, inhibitors are relatively nonspecific. Thereby,
they can inhibit multiple pathways of cellular uptake, which
mislead false results.
Cellular Internalization of Quantum Dots 259

2. Imaging of cellular uptake can be tricky. An image from a single


plane sometimes can be difficult to determine whether
QD/CPPs are on the membrane surface or inside the cell. A 3D
image would be the most appropriate venue to resolve this issue.
3. The uptake efficiency and kinetics are QDs-, CPPs-, and cell
type specific.

Acknowledgments

The authors thank Robert S. Aronstam for technical editing. This


work was supported by Award No. R15EB009530 from the
National Institute of Biomedical Imaging and Bioengineering
(to Y.-W. Huang) and the National Science Council (NSC
97-2621-B-259-003-MY3 to H.-J. Lee), Taiwan.

References

1. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, 5. Dietz GP, Bahr M (2004) Delivery of bioactive
Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir molecules into the cell: the Trojan horse
SS, Weiss S (2005) Quantum dots for live cells, approach. Mol Cell Neurosci 27:85–131
in vivo imaging, and diagnostics. Science 6. Wang YH, Hou YW, Lee HJ (2007) An intracel-
307:538–544 lular delivery method for siRNA by an arginine-
2. Bharali DJ, Lucey DW, Jayakumar H, Pudavar rich peptide. J Biochem Biophys Methods
HE, Prasad PN (2005) Folate-receptor- 70:579–586
mediated delivery of InP quantum dots for bio- 7. Tunnemann G, Ter-Avetisyan G, Martin RM,
imaging using confocal and two-photon Stockl M, Herrmann A, Cardoso MC (2008)
microscopy. J Am Chem Soc 127: Live-cell analysis of cell penetration ability and tox-
11364–11371 icity of oligo-arginines. J Pept Sci 14:469–476
3. Hoshino A, Fujioka K, Oku T, Nakamura S, 8. Xu Y, Liu BR, Lee HJ, Shannon KB, Winiarz JG,
Suga M, Yamaguchi Y, Suzuki K, Yasuhara M, Wang TC, Chiang HJ, Huang YW (2010) Nona-
Yamamoto K (2004) Quantum dots targeted to arginine facilitates delivery of quantum dots into
the assigned organelle in living cells. Microbiol cells via multiple pathways. J Biomed Biotechnol
Immunol 48:985–994 2010:1–11
4. Gerion D, Cheng F (2004) Fluorescent 9. Liu BR, Li JF, Lu SW, Leel HJ, Huang YW,
CdSe/ZnS nanocrystal − peptide conjugates Shannon KB, Aronstam RS (2010) Cellular inter-
for long-term, nontoxic imaging and nuclear nalization of quantum dots noncovalently conju-
targeting in living cells. Nano Lett 4: gated with arginine-rich cell-penetrating peptides.
1827–1832 J Nanosci Nanotechnol 10:6534–6543
Chapter 24

Electrochemical Scanning Tunneling Microscopy


and Spectroscopy for Single-Molecule Investigation
Andrea Alessandrini and Paolo Facci

Abstract
The technique of electrochemical scanning tunneling microscopy (ECSTM) and spectroscopy (ECSTS)
for studying electron transport through single redox molecules is here described. Redox molecules of both
biological and organic nature have been studied by this technique with the aim of understanding the trans-
port mechanisms ruling the flow of electrons via a single molecule placed in a nanometer-sized gap between
two electrodes while elucidating the role of the redox density of states brought about by the molecule.
The obtained results provide unique clues to single-molecule transport behavior and support the concept
of single-molecule electrochemical gating.

Key words ECSTM, ECSTS, Redox molecules, Redox metalloproteins, Blue copper proteins, Cyclic
voltammetry

1 Introduction
Scanning tunneling microscopy and spectroscopy with full control
of substrate and tip potential in an electrochemical cell represent
powerful tools for investigating the phenomenology and mecha-
nisms of interfacial electron transport in redox molecules down to
single-molecule level (1). These techniques have been exploited for
studying transition metal complexes (2–4), molecular wires con-
taining viologen moieties (5, 6), redox metalloproteins (e.g., the
blue copper protein azurin (7–9), cytochrome c (10)), and deriva-
tives of redox cofactors (e.g., quinones with thio-alkyl chains) (11)
chemisorbed at noble metal surfaces (e.g., Pt and Au). Both ECSTM
and ECSTS allow the retrieval of information on the role of the
molecular redox moieties in mediating the electron transport pro-
cess in the tunneling gap between two electrodes. In comparison to
UHV-performed measurements, these techniques allow operation
in buffered aqueous media, especially important for metallopro-
teins, while enabling control of the potential (i.e., Fermi level) of

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_24, © Springer Science+Business Media New York 2013

261
262 Andrea Alessandrini and Paolo Facci

both substrate and tip with respect to a reference electrode. Such a


goal is reached by assembling a four-electrode electrochemical cell
where substrate and tip play the role of two working electrodes
(12); the tip has to be insulated in order to minimize capacitance
and Faradaic contributions to the overall current, and a bipoten-
tiostat is needed to drive the potential of the two working elec-
trodes (13). The result is an imaging/electron tunneling
spectroscopy in physiologic-like conditions and the possibility of
scanning the energy scale in search for electronic levels brought
about by the molecular adsorbate, possibly involved in electronic
transport. These possibilities make the ECSTM and ECSTS tech-
niques very promising for the characterization of single molecules
in the context of molecular electronics. The exploitation of the
potential of the electrolyte solution allows assembling a transistor-
like device in which the positioning of a third electrode near to the
tip and the substrate (drain and source) is not required. In fact, the
role of the gate electrode is assured by the electrolyte solution
potential which is able to affect the levels of the involved redox
molecule. Many experimental studies have demonstrated the pos-
sibility of obtaining tunneling current enhancement at constant bias
voltage by means of electrolyte gating for a redox molecule sand-
wiched between tip and substrate. The enhancement is obtained by
an alignment of the redox energy levels of the molecule with the
Fermi levels of both tip and substrate. Typically, in an ECSTM
investigation the focus of the measurements is the apparent height
of the molecules with respect to an internal reference feature in the
image. The same area of the sample is imaged at constant bias volt-
age between the tip and the substrate for different values of the
substrate potential. The images are acquired in the constant-current
mode of the STM technique. As a consequence, an enhancement of
the tunneling current is reflected in an increased height of the imaged
structure. The apparent height of the redox molecules is measured
with respect to that of a structure whose apparent height is not
affected by changes in the substrate potential. The substrate poten-
tial is chosen in a range which includes the redox equilibrium poten-
tial of the molecule investigated as determined by electrochemical
methods such as cyclic voltammetry. The above technique is usually
named “spectroscopy-like imaging.” In ECSTS experiments the tip
is brought into close proximity to the sample until a specified tun-
neling current set point is reached. At that point the z-feedback is
switched off and the potential of the working electrode on which the
molecule is adsorbed is swept while tunneling current is measured.
By this approach the correlation of tunneling current and potential
is directly obtained and the results are easier to compare even to
theoretical models (14). The results of the investigation are reported
in a plot of the tunneling current as a function of the potential of the
electrode on which the redox molecule is immobilized.
Here, the methods used to study the interfacial electron transport
properties of the metalloprotein azurin will be discussed in detail.
Electrochemical Scanning Tunneling Microscopy and Spectroscopy for Single-Molecule… 263

Both the strategies for ECSTM spectroscopy-like imaging and for


the ECSTS technique with the molecule immobilized on the tip
will be discussed in the case of asymmetric junction conditions.
The methods can be easily transferred to the study of other metal-
loproteins once a suitable technique to immobilize them on a con-
ductive substrate has been developed. Similarly, the methods to
study the electron transport features of a molecule carrying the
hydroquinone/benzoquinone redox couple will be discussed.

2 Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water to attain a resistivity of 18.2 MΩ cm at 25°C) and
analytical grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Follow waste disposal
regulation when disposing waste materials. Follow strictly regula-
tions on personal lab safety when dealing with hazardous reagents,
wearing personal protection equipment, and always operating
under aspiration hood.

2.1 Solutions 1. Imaging buffers: 50 mM NH4Ac pH 4.6 or 7.6. Store at 4°C.


and Reagents 2. Azurin: commercial azurin has been used throughout without
any further purification at typical concentration of 1 mg/ml.
Add 910 μL of pure water to the lyophilized protein directly in
the commercial vial to reconstitute solution. Store at 4°C.
3. 2-(6-Mercaptoalkyl)hydroquinone: 10−5 M in EtOH (99.8%).
4. 6-mercapto-1-hexanol (MCH) 10−5 M in EtOH (99.8%).
5. Gold for evaporation, purity 99.99+.
6. Freshly cleaved muscovite mica substrates (see Subheading 3).
7. Imaging substrates: thermally evaporated and recrystallized
200-nm-thick gold on mica (see Note 1).
8. Tips: electrochemically etched Pt/Ir (80:20) and Au 0.25-mm-
thick wires (see Note 2) with UHV wax (see Subheading 3)
and/or electropolymerizable varnish (ClearClad Co.) insulation
(see Note 3).
9. Electrodes: 0.5-mm-thick Pt wire as counter electrode;
0.5-mm-thick Ag wire as quasi-reference electrode (see Note 4).
10. Substrate electrical-contact clip: 1-mm-thick stainless steel wire.
11. “Piranha” solution: H2SO4/H2O2 (70/30 V/V) freshly pre-
pared in a 50-ml beaker (see Note 5).
12. Imaging cell: The bottom of the cell is formed by the imaging
substrate; walls consist of a 10-mm-high and 2-mm-thick
PTFE ring (Molecular Imaging. Co.).
13. Plastic pipettes.
264 Andrea Alessandrini and Paolo Facci

3 Methods
Here the methods to assemble the sample, to prepare the tip,
and to perform the measurements both for azurin and
2-(6-Mercaptoalkyl)hydroquinone will be presented.
Carry out all the procedures at room temperature unless oth-
erwise specified.

3.1 Substrate 1. Cut a piece of muscovite mica in shape of a square, 20 mm in


Preparation side. Cleave it with a sharp blade by inserting the blade laterally on
a corner of the slide and lifting some sheets in order to expose
a freshly cleaved surface. Pay attention handling the blade.
2. Evaporate 200-nm-thick Au film on the prepared mica sheet
(see Note 1).
3. Flame anneal the evaporated substrate and check surface qual-
ity by STM in air (see Note 6). Repeat the procedure until flat
triangular terraces, some hundreds of nanometers in size with
surface reconstruction of Au(111), are evident (Fig. 1).

3.2 Tip Preparation 1. Cut a 30-mm-long, 0.25-mm-thick Pt/Ir wire and install it in
the etching cell (see Note 2).
3.2.1 Pt/Ir Tip
Preparation 2. Insulate the tip by mounting it in a device similar to that
reported in Fig. 2. Put a small piece of Apiezon wax (about
1 mm3) across the slit cut in a piece of bulk copper (cubic
shape, 8 mm in side) mounted at the end of a soldering iron
(Fig. 2) and melt it. Then, with the help of a micrometer screw,
pass the tip in vertical direction from below through the melted
wax film spanning the slit. The very tip dewets (phenomenon

Fig. 1 STM images of Au (111) surfaces used as substrates for ECSTM experiments. (a) Image showing the
typical triangular features of Au(111) surfaces. (b) Au(111) surface showing the characteristic herringbone
23 × 3 reconstruction of Au(111)
Electrochemical Scanning Tunneling Microscopy and Spectroscopy for Single-Molecule… 265

Fig. 2 Set-up for the coating of ECSTM tips. The electrochemically etched tip is
passed through a film of melted wax. The tip will be coated by the wax but in the
very apex, due to dewetting. The inset shows the soldering iron details

neither visible by eyes nor under optical microscope) leaving a


small (some tens of square microns) metal area uncoated.
3. Test the tip for proper insulation by:
(a) Installing the electrochemical cell (see Subheading 3.3
below).
(b) Installing the tip in the microscope measuring chamber
filled with the imaging buffer (the tip should be far from
the substrate).
(c) Applying a series of tip potentials and checking that the
leakage current be below 5 pA at each potential value. The
potential range in which the tip current response will
remain under the aforementioned threshold will define the
operation window for the tip.
(d) Save the tip for the experiment.

3.2.2 Au Tip Preparation 1. Cut a 30-mm-long, 0.25-mm-thick Au wire and install it in the
etching cell (see Note 2).
2. Etch it in HCl 37% applying a bias of 5 V tip positive with
respect to a Pt counter electrode.
3. Go through points b and c in step 3 of Subheading 3.2.1 for
insulation and tip leakage test.

3.3 Installing 1. At the beginning and at the end of each experimental session,
the Electrochemical check the potential of the silver wire against a standard refer-
Cell (Both ECSTM ence electrode (here SCE) and refer all the data to the selected
and ECSTS) standard reference electrode.
266 Andrea Alessandrini and Paolo Facci

2. Install the substrate on the sample holder plate by pressing


over it the PTFE ring and fixing it with two clips. Connect the
stainless steel wire to act as a substrate electrical contact so that
it touches the substrate outside the cell. With the help of a
multimeter check for electrical continuity between the sub-
strate and the wire.
3. Connect counter electrode and quasi reference, paying atten-
tion they neither touch the metal substrate nor each other.
4. Fill the cell with the imaging buffer (typically 500 μL) while
controlling the substrate potential by the potentiostat. Check
carefully for buffer leaks; if any, disassemble the cell, dry care-
fully the substrate and the ring with N2 flow, and assemble it
again adjusting the clip pressure to fix the problem.

3.4 Sample 1. Proceed as for installing the electrochemical cell (step 1 of


Preparation Subheading 3.3).
3.4.1 Azurin for ECSTM 2. Fill the cell with 200 μL protein solution (1 mg/ml).
3. Keep it incubating for about 30 min at room T.
4. Remove protein solution with a micropipette paying attention
to leave enough residual solution on the surface to avoid sur-
face dewetting (see Note 7).
5. Refill the measuring chamber with the imaging buffer to rinse
the chamber.
6. Remove the rinsing buffer almost completely, avoiding to
dewet completely the sample surface.
7. Repeat the last two steps three times.
8. Fill the measuring chamber with 500 μL imaging buffer.

3.4.2 Azurin for ECSTS 1. Incubate insulated gold tips with azurin solution by fixing tips
on the bottom of a Petri dish and dropping 100 μL of azurin
solution over the tip apex. Place the Petri dish at 4°C and let
incubate overnight.
2. Rinse the tip in 50 mM NH4Ac, pH 4.6.
3. Install the tip into the tip holder.

3.4.3 2-(6-Mercaptoalkyl) 1. Flame anneal a Au(111) substrate and immediately immerse it in


hydroquinone for ECSTM a 10−5 M 6-mercapto-1-hexanol (MCH) solution in ethanol.
2. Incubate the substrate overnight in a sealed container to avoid
ethanol evaporation.
3. Rinse copiously the substrate with ethanol and immerse it in a
10−5 M 2-(6-mercaptoalkyl)hydroquinone solution in ethanol
(see Note 8).
4. Incubate the substrate for 30 min.
5. Rinse the substrate copiously with ethanol.
Electrochemical Scanning Tunneling Microscopy and Spectroscopy for Single-Molecule… 267

6. Mount the substrate in the ECSTM and assemble the electro-


chemical cell, filling it with 500 μl of 50 mM NH4Ac pH 4.6
or 7.6 aqueous solution while controlling the substrate poten-
tial by the potentiostat.
7. Install the tip into the tip holder.

3.4.4 2-(6-Mercaptoalkyl) 1. Flame anneal a Au(111) substrate and immediately immerse


hydroquinone for ECSTS it in a 10−5 M 2-(6-Mercaptoalkyl)hydroquinone solution in
ethanol.
2. Incubate the substrate overnight in a sealed container to avoid
ethanol evaporation.
3. Rinse the substrate copiously with ethanol.
4. Mount the substrate in the ECSTM and assemble the electro-
chemical cell, filling it with 500 μl of 50 mM NH4Ac pH 4.6
or 7.6 aqueous solution while controlling the substrate poten-
tial by the potentiostat.
5. Install the tip into the tip holder.

3.5 Performing 1. Install the insulated tip into the tip holder.
ECSTM Measurements 2. Using a dummy substrate approximately of the same thickness
on Azurin than the real one, regulate tip–substrate distance at less than
about 0.5 mm by optical inspection.
3. Assemble the electrochemical cell as described.
4. Set the substrate potential at a value within the ideal capaci-
tance window of the sample (see Note 9).
5. Set the Vbias at 400 mV (tip positive) and the current set point
at 1 nA.
6. Start approach.
7. Once set point is reached, start scanning at scanning frequency
not exceeding 4 Hz (number of lines per second. The exact
value depends upon the microscope used and on the scan size:
larger scan sizes require lower scan frequency).
8. Check for thermal drift by controlling that subsequent frames
are not “shifted” (see Note 10).
9. Change the substrate potential stepwise. Wait for capacitance
currents to be over by checking the substrate current on the
bipotentiostat. Once current is stabilized, start a new scan on
the same area (see Note 11).
An example of the above procedure is shown in Fig. 3. After
obtaining the sequence of images, the apparent height of azurin
molecules with respect to an internal reference for each substrate
potential should be measured (in the specific case of Fig. 3, the
reference is constituted by non-electroactive Zn-azurin molecules)
and reported in a plot as a function of the substrate potential (it is
268 Andrea Alessandrini and Paolo Facci

Fig. 3 Sequence of ECSTM images of a sample containing a mixture redox-active and redox-inactive azurin
molecules for different values of the substrate potential (reported in each image). Scan size: 130 nm. The
redox-active molecules change their apparent height with respect to the redox-inactive ones. The height of
one molecule with respect to the non-changing molecules is reported in the plot as a function of the substrate
potential. In the inset the average of the same measurement over six molecules is shown

also possible to refer the apparent height to the overpotential,


which represents the difference between the substrate potential
and the equilibrium redox potential of the azurin molecules).

3.6 Performing 1. Follow the same steps as in the case of ECSTM (steps 1–6,
ECSTS Measurements Subheading 3.5) but using the azurin-coated gold tip.
on Azurin 2. Position the tip potential in a potential range in which no tun-
neling current enhancement is expected, according to data
from ECSTM.
3. Approach the tip towards a Au(111) substrate.
4. Once the set point current is reached, wait for thermal drift to
be negligible, by checking the tip piezo voltage to be stable.
5. Disable the feedback on tunneling current by lowering the
servo gain parameters to about 0.1.
6. Start scanning the tip potential in the potential range
(−0.2 ÷ 0.4 V vs. SCE) while recording the tip current.
7. Check for the stability of the tunneling gap by controlling the
value of the tunneling current at the start and at the end of the
Electrochemical Scanning Tunneling Microscopy and Spectroscopy for Single-Molecule… 269

Fig. 4 ECSTS curves obtained with an azurin-coated tip. The continuous curves
(black Curves) represent curve obtained with the azurin-coated tip. The other
curves have been performed as a check: (triangles) represent data obtained with
a tip coated with non-electroactive azurin; (circles) represent data obtained with
a tip not modified with azurin (bare gold)

cyclic potential scan (the positions should be in regions of non-


enhancement in tunneling current).
8. Discard the curves if the initial and final values of the tunneling
current differ too much.
9. Repeat the measurement for different values of both the tun-
neling current initial set point and the bias voltage between the
tip and the substrate.
10. Compare the obtained results with the case in which there are
no proteins on the tip surface or with another molecule which
is not electroactive.
An example of the results of this kind of measurement is
reported in Fig. 4.

3.7 Performing 1. Perform the same steps (1–10) as in Subheading 3.5.


ECSTM Measurements 2. The presence of protruding bright spots is a signature of the
on 2-(6-Mercaptoalkyl) presence of 2-(6-mercaptoalkyl)hydroquinone molecules
hydroquinone/MCH inserted in the MCH layer.
Adlayer
3. Once a sequence of ECSTM images has been obtained, measure
the apparent height of the bright spots which are present in the
images and report them in a plot as a function of the substrate
potential. In this case the apparent height is measured with
respect to the non-electroactive MCH layer as shown in Fig. 5.

3.8 Performing 1. Mount the 2-(6-Mercaptoalkyl)hydroquinone-modified Au(111)


ECSTS Measurements substrate in the electrochemical cell while controlling the
on 2-(6-Mercaptoalkyl) substrate potential by the potentiostat.
hydroquinone Adlayer 2. Mount an insulated tip into the tip holder.
270 Andrea Alessandrini and Paolo Facci

Fig. 5 Scheme of the molecular layer in the case of a mixed self-assembled


monolayer and the apparent height Δh which has to be measured in each image

3. Select a substrate potential which does not induce a tunneling


current enhancement, according to ECSTM data on the same
molecule.
4. Select a tip/substrate bias voltage (typically 100 mV) and a set
point tunneling current (typically 0.1 nA).
5. Approach the tip to the sample until the set point tunneling
current is reached.
6. Wait for stabilization of the tunneling junction with the z-feed-
back on the current on.
7. Switch off the feedback system.
8. Start scanning the substrate potential measuring the corre-
sponding tunneling current.
9. Check for the stability of the set-up by comparing the initial
tunneling current with the final value (both initial and final
potential should be outside the region of tunneling current
enhancement).
10. Switch on the feedback system to reestablish control on the
tunneling gap.
11. Repeat the same measurement for different values of the bias
voltage and for different values of current set point.
Electrochemical Scanning Tunneling Microscopy and Spectroscopy for Single-Molecule… 271

4 Notes
1. Use a metal thermal evaporator. Insert freshly cleaved muscovite
mica sheet and fix it in the sample holder. Load a boot with
gold (pellets or wire). Start the evaporator and operate it to
10−7 mbar. Once the target pressure has been reached, heat the
substrate at 450°C for 4 h in order to degas it and to remove
all residual water and organics. After that, start evaporation at
a rate of 0.1 ÷ 0.2 nm/s up to a thickness of 200 nm. After
that, anneal the sample for 6 h at 450°C. Subsequently, switch
the pumps off along with the sample heater and wait for room
temperature thermalization (typically 4 h). Open the vacuum
chamber and extract the sample.
2. Pt/Ir (80:20) tips are made by electrochemical etching in satu-
rated CaCl2 using a two-electrode electrochemical cell. One
electrode is the Pt/Ir wire, whereas the counter electrode is a
piece of nuclear graphite. An ac voltage of 24 V (p–p) and a
current of 0.27 A are used. The wire has to be immersed 3 mm
below the solution meniscus. Full erosion of the immersed part
of the Pt/Ir wire takes typically 10 min, as it is revealed by the
zeroing of the current flowing between the two electrodes. Pay
attention to avoid the formation of waves at the air–solution
interface (e.g., due to mechanical vibration) since that will
result in a blurred etching plane and in blunt tips. As long as
the solution tends to age, etching time and performances will
worsen. If you notice that etching times increase significantly
above 10 min, change the saturated solution for a fresh one.
3. ClearClad™ electropolymerizable varnish (HSR 401 from
LVH Coatings Ltd., UK) can be used to insulate the tip before
or in place of wax coating. Insulation is accomplished by filling
a 50-mL beaker with the varnish diluted in water (1:3 V:V).
In the beaker insert your tip and around it a Pt-coiled wire
(diameter of the coil 3 mm) acting as a counter electrode.
Apply 8 V dc between the tip (negative) and the counter.
Monitor the current flow and continue till current is zeroed.
Afterwards, lift the tip, rinse it in pure H2O, and cure it in an
oven at 90°C for 30 min.
4. Silver wire is not exactly a reference electrode, but it is used
more conveniently in ECSTM and ECSTS experiments due to
its reduced size. Its potential in the working solution has to
be calibrated against that of a real reference (e.g., Ag/AgCl,
Hg/HgCl). It is strongly recommended to test Ag wire poten-
tial in the working buffer before and after any experiment in
order to check for potential shifts. If any, the average between
the two values (before and after the experiment) is usually
taken for quoting any further potential value. To measure the
272 Andrea Alessandrini and Paolo Facci

Ag wire potential against a reference, connect a voltmeter


between the Ag wire and a reference electrode and immerse both
of them in the working solution, recording the readout value.
5. Pay extreme attention to “piranha” solution. It is extremely
oxidizing and reacts violently with organics. Always wear antia-
cid gloves and protective goggles and never leave the solution
unattended. When you prepare it, it is extremely important
to add acid to H2O2 and not the other way around, since it
can result in splashes of acid. Dispose it by first neutralizing it
with KOH.
6. Flame annealing can be performed with different types of
flames. The best is hydrogen flame but is very dangerous and
not everywhere allowed. We use to work with an ethanol flame.
The aim is to heat up the gold film in order to get atomically
flat terraces suitable for high-resolution imaging in STM.
Switch on the flame; pick the substrate up with stainless steel
tweezers; facing the flame tip with the gold surface, touch the
flame tip with the substrate inclined at about 45° with respect
to the flame vertical axis. Do not leave the sample in the flame
still for more than 1–2 s; otherwise you run the risk of over-
heating it. Pay attention to avoid burning the underlying mica.
Check with STM imaging in air the formation of large, flat
Au(111) terraces (Fig. 1) and repeat the procedure till you get
the optimal quality of your sample. Pay attention that over-
heating will destroy the film, enhancing its roughness and
making it useless for STM/STS experiments.
7. It is of paramount importance to avoid that the protein sample
crosses the liquid meniscus. In fact such an action can easily
induce protein denaturation due to the action of water-based
solution surface tension. Therefore, once wet, protein sample
should always remain immersed in liquid.
8. The method described allows, by a substitution mechanism, to
assemble a mixed monolayer in which the molecules in the
second incubation step are able to replace some of the previ-
ously immobilized molecules or to insert themselves in defects
present in the previous self-assembled monolayer. The concen-
tration of the second incubation solution should be adjusted in
order to have a significant number of substituted molecules in
the monolayer.
9. Although there are no strict rules on this aspect, it is usually
desirable to avoid any redox reaction at surface during tip–
sample approach in order to avoid disturbance to the feedback
system. A good strategy is that of presetting the substrate
potential at a value where both the substrate and the molecular
adsorbates thereby present are known to be redox inactive.
One can check in advance for such a condition by performing
Electrochemical Scanning Tunneling Microscopy and Spectroscopy for Single-Molecule… 273

cyclic voltammety measurements in the imaging buffer and


selecting regions where no Faradaic contributions are present.
10. Small thermal drifts or failure in tip repositioning can be cor-
rected either relocating the tip starting point before each frame
scan or by post-processing image realignment if some internal
markers are present in the images (e.g., Au monoatomic steps,
a surface defect, some adsorbed features other than the mole-
cules under investigation).
11. This aspect is important, since the presence of residual capaci-
tance currents will superimpose to the tunneling one hamper-
ing molecular behavior identification.

Acknowledgment

The authors acknowledge partial financial support by Italian MIUR


FIRB “Italnanonet.”

References

1. Alessandrini A, Corni S, Facci P (2006) mediated by the active site Cu ion. Chem Phys
Unraveling single metalloprotein electron Lett 376:625–630
transfer by scanning probe techniques. Phys 9. Alessandrini A, Salerno M, Frabboni S et al
Chem Chem Phys 8:4383–4397 (2005) Single-metalloprotein wet biotransis-
2. Albrect T, Guckian A, Ulstrup J et al (2005) tor. Appl Phys Lett 86:133902
Transistor-like behavior of transition metal 10. Chi Q, Zhang J, Arslan T et al (2010) Approach
complexes. Nano Lett 5:451–1455 to interfacial and intramolecular electron trans-
3. Albrecht T, Moth-Poulsen K, Christensen JB fer of the diheme protein cytochrome c4
et al (2006) In situ scanning tunnelling spec- assembled on Au(111) surfaces. J Phys Chem
troscopy of inorganic transition metal com- B 114:5617–5624
plexes. Faraday Discuss 131:265–279 11. Petrangolini P, Alessandrini A, Berti L et al
4. Albrecht T, Guckian A, Ulstrup J et al (2005) (2010) An electrochemical scanning tunneling
Transistor effects and in situ STM of redox microscopy study of 2-(6-mercaptoalkyl)hyd-
molecules at room temperature. IEEE Trans roquinone molecules on Au (111). J Am Chem
Nanotechnol 4:430–434 Soc 132:7445–7453
5. Li Z, Han B, Meszaros G et al (2006) Two- 12. Alessandrini A, Facci P (2007)
dimensional assembly and local redox-activity of Electrochemically assisted scanning probe
molecular hybrid structures in an electrochemical microscopy: a powerful tool in nano(bio)sci-
environment. Faraday Discuss 131:121–143 ence. In: Erokhin V, Ram MK, Yavuz O (eds)
6. Pobelov IV, Li Z, Wandlowski T (2008) Biophysical aspects of nanotechnology.
Electrolyte gating in redox-active tunneling Elsevier, Amsterdam
junctions—an electrochemical STM approach. 13. Siegenthaler H (1995) STM in electrochemis-
J Am Chem Soc 130:16045–16054 try. In: Wiesendanger R, Güntherodt H-J
7. Facci P, Alliata D, Cannistraro S (2001) (eds) Scanning tunneling microscopy II, 2nd
Potential-induced resonant tunneling through edn. Springer, Berlin
a redox metalloprotein probed by electrochemi- 14. Albrecht T, Guckian A, Kuznetsov AM et al
cal scanning probe microscopy. Ultramicroscopy (2006) Mechanism of electrochemical
89:291–298 charge transport in individual transition
8. Alessandrini A, Gerunda M, Canters G et al metal complexes. J Am Chem Soc 128:
(2003) Electron tunneling through azurin is 17132–17138
Chapter 25

Intracellular Delivery of Biologically Active Proteins


with Peptide-Based Carriers
Seong Loong Lo and Shu Wang

Abstract
The medical applications of protein-based therapeutics are hampered by low bioavailability associated with
inefficient intracellular delivery. Various delivery materials have been developed and tested to interact with
protein cargos in a manner of stabilizing proteins extracellularly and facilitating cellular uptake of proteins,
thus enhancing delivery efficiency. Peptides that can form stable complexes with proteins through non-
covalent interaction appear to be a promising tool to improve intracellular delivery of proteins. Here we
describe the preparation of complexes formed between β-galactosidase and peptide-based carrier, protein
transfer of the complexes, and the methods to evaluate delivery efficiency qualitatively and quantitatively.

Key words Protein delivery, Protein transduction, Peptide, Self-assembling complex, Non-covalent

1 Introduction
Rapid advances in functional genomics and proteomics have
revealed numerous attractive diseased-related intracellular targets
and hence significantly accelerated the discovery and development
of protein-based therapeutics. However, delivery of biologically
active proteins into cells is always challenging due to various extra-
and intracellular barriers that affect protein stability and delivery
efficiency. Over past few decades, there has been significant effort
put into this field. Physical methods (1–9), covalent modification
of protein (10–12), and lipid-based carriers (13, 14) have been
applied for protein delivery. However, the use of these methods
usually suffers from different inherent limitations including high
rate of cell mortality (15, 16), loss of protein function (17, 18),
and allergic-type immune reactions (19, 20).
Peptide-based carriers have emerged as one of the protein
delivery vectors with great potential (21) because of lower immu-
nogenicity, large-scale synthesis with well-established chemical
methods, and incorporation of natural or synthetic sequences with

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_25, © Springer Science+Business Media New York 2013

275
276 Seong Loong Lo and Shu Wang

biological activities such as cell targeting domain and cell penetrating


domain. This chapter describes the basic methods for (1) non-cova-
lent complex formation between protein cargo and peptide-based
carrier, (2) protein delivery using these complexes, and (3) evaluation
of delivery efficiency by quantitative and qualitative methods.

2 Materials

2.1 Reagents 1. Mammalian cells (exponentially growing).


2. Complete cell culture medium: Dulbecco’s modified Eagle’s
medium, 10% fetal bovine serum (FBS), 1% penicillin–strepto-
mycin, 1% L-glutamine.
3. Opti-MEM® reduced serum media (Life Technologies, Grand
Island, NY, USA).
4. Ultrapure laboratory grade water (deionized water with sensi-
tivity of 18 MΩ cm at 25°C).
5. Phosphate-buffered saline (PBS): 0.1 M sodium phosphate
(pH 7.4), 0.15 M NaCl.
6. Lyophilized peptides.
7. β-galactosidase 119 kDa subunit (Active Motif, Carlsbad,
CA, USA).
8. β-galactosidase staining kit (Active Motif, Carlsbad, CA,
USA).
For each well in a 96-well plate.
1× fixing solution: 90 μl of PBS, 10 μl of 10× fixing
solution.
Complete staining solution: 138 μl of PBS, 7.5 μl of X-gal,
1.5 μl of staining solution 1, 2, and 3 respectively.
9. Galacto-Light Plus™ beta-Galactosidase Reporter Gene Assay
System (Life Technologies, Grand Island, NY, USA).
For each well in a 96-well plate.
Lysis solution: 30 μl.
Reaction buffer: 69.3 μl of reaction buffer diluent, 0.7 μl of
100× Galacto-Plus substrate.
Accelerator-II: 100 μl.
10. Bovine serum albumin (BSA).
11. Dc protein assay kit (Bio-rad, Hercules, CA, USA).
For each 5 μl of sample.
Reagent A’: 24.5 μl of reagent A, 0.5 μl of reagent S.
Reagent B: 200 μl.
Intracellular Delivery of Biologically Active Proteins with Peptide-Based Carriers 277

2.2 Equipment 1. Humidified CO2 incubator for cell culture


2. Tissue culture vessels
3. Cell culture well plate
4. Inverted microscope
5. Single-tube luminometer
6. Microplate spectrophotometer (capable of absorbance mea-
surement at wavelength of 280 nm and 650–750 nm).

3 Methods
Prepare all solutions using ultrapure laboratory grade water or PBS
at room temperature (unless indicated otherwise).

3.1 Preparation 1. Prepare peptide stock solution by reconstituting lyophilized


of Peptide/ b- peptide in appropriate volume of ultrapure water (see Note 1).
Galactosidase 2. Prepare the working solution by further diluting peptide
Complex stock solution to a concentration of 50 μM with ultrapure water
(see Note 2).
3. Prepare β-galactosidase solution of 0.25 mg/ml by reconsti-
tuting lyophilized protein in PBS (see Note 2).
4. Dilute 0.5 μg of β-galactosidase (for delivery into cells seeded
in a well of a 96-well plate) in 8 μl of PBS.
5. Based on the required peptide–β-galactosidase molar ratio
(typically ranging from 5 to 150), dilute appropriate quantity
of peptide working solution in PBS to 10 μl.
6. Add the peptide solution from step 5 into β-galactosidase solu-
tion from step 4. Mix immediately by vortexing for 10 s.
7. Incubate the mixture for 30 min at room temperature.

3.2 Protein Delivery 1. The day before protein delivery, harvest mammalian cells,
into Mammalian Cells grown in complete cell culture medium, by trypsinization and
replate them into 96-well plate at density of 10,000–12,000
cells/well. Incubate the cells for 24 h at 37°C in a humidified
incubator with 5% CO2.
2. Remove the medium from the wells and wash the cells with
100 μl of PBS (prewarmed to 37°C) (see Note 3).
3. Add 50 μl/well of Opti-MEM reduced serum media to the
cells, followed by 20 μl/well of the peptide/β-galactosidase
complex.
4. Incubate the cells for 4 h at 37°C in a humidified incubator
with 5% CO2.
5. Top up the wells with 80 μl of complete cell culture medium.
Incubate the cells for 20 h in the humidified CO2 incubator.
278 Seong Loong Lo and Shu Wang

3.3 Qualitative 1. Prepare fresh 1× fixing solution immediately before use.


Evaluation Using 2. Remove the medium from the wells and wash the cells with
b-Galactosidase 100 μl of PBS (see Note 3).
Staining Kit
3. Fix the cells with 100 μl of 1× fixing solution for 10 min at
room temperature (see Note 4).
4. While waiting for fixation, prepare complete staining solution.
5. Wash the cells twice with 100 μl of PBS.
6. Stain the cells with 150 μl of complete staining solution for 2 h
in humidified CO2 incubator.
7. Check the cells and take images under inverted microscope
(see Note 5).
8. For long-term storage of the plate, replace complete staining
solution with 70% glycerol in PBS, seal the plate with parafilm
and store at 4°C.

3.4 Quantification of 1. Prepare lysis solution and reaction buffer at room temperature
b-Galactosidase Using immediately before use.
Galacto-Light Plus™ 2. Remove the medium from the wells and wash the cells with
Beta-Galactosidase 100 μl of PBS (see Note 3).
Reporter Gene Assay 3. Lyse the cells at room temperature with 30–50 μl of lysis solu-
System tion (see Note 6).
4. Mix 5–20 μl of cell lysate with 70 μl of reaction buffer in a
luminometer tube (see Note 7). Vortex briefly and incubate
for 30 min.
5. Add 100 μl of accelerator-II and vortex briefly. Place tube in
luminometer and measure chemiluminiscent signal for 0.1–1 s/
tube (see Note 8).
6. To determine the actual amount of β-galactosidase, generate a
standard curve by serially diluting 5 ng/μl of β-galactosidase in
lysis solution, followed by measurement of chemiluminiscent
signal (follow steps 4 and 5).
7. For normalization against total protein, serially dilute 2.5 mg/ml
of BSA in lysis solution. Pipette the BSA standards and cell lysate
samples into a clean 96-well plate. Add reagents A’ and B from Dc
protein assay kit to the samples. Incubate for 15 min with gentle
agitation; measure absorbance at wavelength of 650–750 nm.
Generate a standard curve using BSA standards and calculate total
protein concentration of cell lysate (see Note 9).

4 Notes
1. Hydrophilic peptides have a tendency to bind more water.
Therefore, it is important to measure the concentration of
peptide solution after reconstitution of lyophilized peptide.
Intracellular Delivery of Biologically Active Proteins with Peptide-Based Carriers 279

For peptides containing amino acids with aromatic ring


(tryptophan and tyrosine), concentration can be determined
by estimating the molar extinction coefficient and measuring
absorbance at 280 nm with spectrophotometer. For other pep-
tides without such features, amino acid analysis is required to
determine the peptide concentration.
2. To prevent or minimize peptide or protein degradation, always
store the stock solution in individual aliquots at −20 or −80°C
after use to avoid freeze-thaw cycles.
3. When dealing with multiple wells of cells, use multichannel
pipette to avoid morphological changes or cell detachment due
to PBS washing.
4. Some mammalian cell lines might be very sensitive and might
undergo morphological changes during fixation. To avoid such
scenario, the fixation time could be reduced to 5 min.
5. Before imaging, replace complete staining solution with 100 μl
of PBS for better contrast.
6. Put on a shaker for 15 min to improve lysis rate. Check under
inverted microscope to confirm complete lysis before proceed
to next step.
7. The volume of cell lysate required for measurement depends
upon the β-galactosidase concentration. Use 15 μl as a starting
point for optimization.
8. Due to the light emission kinetics of the reaction, accelerator-
II should be added in the sequence of adding reaction buffer
in the previous step.
9. Bubbles will affect absorbance measurement. If bubbles form,
use a clean, dry tip to pop them.

Acknowledgments

This work was supported by Institute of Bioengineering and


Nanotechnology, Agency for Science, Technology and Research
(A*STAR), Singapore.

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Chapter 26

Lipo-oligoarginine-Based Intracellular Delivery


Jae Sam Lee and Ching-Hsuan Tung

Abstract
Efficient cellular delivery, including plasma membrane permeability and intracellular metabolic stability, is
a crucial factor determining the success of therapeutic agents. Cell-penetrating peptides (CPPs) have been
widely used for the intracellular delivery of various bioactive molecules into cells to modify cellular func-
tions. We have developed an improved CPP-based cellular delivery vector, named lipo-oligoarginine pep-
tide (LOAP), by conjugating an oligoarginine peptide with a fatty acid moiety. The prepared LOAPs were
further stabilized by introducing different combinations of D-Arg residues into the peptide backbone and
were systematically evaluated for their membrane-penetrating properties and metabolic stabilities in cells.

Key words Cell-penetrating peptide, Oligoarginine, Fatty acid, Cellular uptake, Drug delivery
system, Metabolic stability

Abbreviations

MBHA 4-Methylbenzhydrylamine
Fmoc 9-Fluorenylmethyloxycarbonyl
Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl
Mtt Methyltrityl
TA Thioanisole
EDT Ethandithiol

1 Introduction
Cell-penetrating peptides (CPPs) have been employed to deliver
various membrane impermeable bioactive molecules such as pro-
teins, peptides, nanoparticles, and nucleic acids into cells. Among
the CPPs studied, the human immunodeficiency virus 1 (HIV-1)
Tat protein has gained much attention due to its efficient internal-
ization capacity and relatively low cytotoxicity (1). The functional
domain on the Tat protein that controls translocation through the
cell membrane is a short 9-mer peptide, RKKRRQRRR (1–3). The
positively charged arginine residues in the Tat peptide are crucial

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_26, © Springer Science+Business Media New York 2013

281
282 Jae Sam Lee and Ching-Hsuan Tung

for membrane penetration; replacing these with other positively


charged amino acids such as lysine, histidine, or citrulline abolishes
the ability of the protein to penetrate the membrane, suggesting
that the cationic guanidine moiety on the arginine side chain plays
an important role in this unique membrane translocation property
(4). It has been reported that the cellular uptake efficiency of argi-
nine-rich CPPs depends on the number of arginine residues
(5 < R < 15) (4, 5). No internalization was observed in the case of
peptides with three or less arginine residues (6), and severe cell
toxicity was seen in CPPs having 15 or more arginine residues (5).
Interestingly, the internalization efficiency of CPPs is increased
when L-arginines are replaced by different combinations of a pro-
tease resistant D-arginine sequence (5, 7).
A major obstacle in developing potent therapeutic agents is
their poor bioavailability, which prevents them from entering the
lipid bilayer of the plasma membrane, resulting in a diminished
therapeutic efficacy. Hydrophobic enrichment of therapeutic agents
has been attempted to achieve improved delivery into cytoplasmic or
intracellular compartments (8). Similarly, fatty acid moieties that
could advance cellular association with hydrophobic cell membranes
have been incorporated into various CPPs, such as proline-rich,
arginine-rich, or cationic peptides (9–11). However, studies on a
myristoylated random neutral peptide revealed that a myristoyl
group alone was not enough to promote cellular uptake of the
reference peptide (12). It was also reported that a hydrophobic
peptide modified by a single farnesyl group did not associate
strongly with membrane (13). These data imply that conjugation
of a fatty acid to a peptide in itself is insufficient to promote cellular
uptake, and a second signal is required to achieve stable association
of CPPs with cell membrane. Neither a fatty acid (e.g., myristic acid)
nor a basic peptide (e.g., oligoarginine) alone is adequate for
plasma membrane binding; however, both these moieties when
combined within the same protein or peptide template might
exhibit enhanced association with the cell membrane. We have pre-
viously reported that oligoarginine peptides modified with fatty
acid moieties exhibit enhanced cellular uptake efficiency (5, 7, 12),
confirming that oligoarginine sequences tethered to fatty acid
moieties might form effective delivery vectors, because such
modifications could enhance membrane association and potentially
lead to improved delivery of cargo molecules into the intracellular
compartment.
Although the metabolic stability of CPPs is an important fac-
tor for their efficient cellular translocation and for sustained release
of cargo molecules into target cells, most CPPs are metabolically
unstable and are rapidly degraded before reaching their targets
(14–18). The degradation process usually begins with the com-
bined action of exoproteases, which cleave the C-terminal and/or
N-terminal residues, as well as endoproteases, which cleave internal
Lipo-oligoarginine-Based Intracellular Delivery 283

Table 1
Sequences of synthesized LOAPs

Name Sequencea
R7 B(R)7K(FITC)-NH2
C12R9 Lauroyl-B(R)9K(FITC)-NH2
C12dR9 Lauroyl-B(dR)9K(FITC)-NH2
C12dR9-1 Lauroyl-B(dR)5(R)4K(FITC)-NH2
C12dR9-2 Lauroyl-B(R)4(dR)5K(FITC)-NH2
C14R11 Myristoyl-B(R)11K(FITC)-NH2
C14dR11 Myristoyl-B(dR)11K(FITC)-NH2
B β-alanine, dR D-arginine, and FITC fluorescein isothiocyanate
a

amide bonds at specific sites. To minimize proteolytic degradation,


the structures of these peptides are often modified so that they are
no longer labile to proteases. We recently introduced a series of
stable lipo-oligoarginine peptide (LOAP) backbone, in which
L-Arg residues were replaced with D-Arg residues (Table 1).
Although there was no apparent difference in cellular uptake (15, 19),
a C14dR11 peptide (where all Arg residues are in D configuration)
expressed superior metabolic stability and enhanced intracellular
retention without adverse effects on cell viability compared with its
unmodified parent C14R11 peptide (5). This modified LOAP
might potentially be an excellent delivery vector to transport vari-
ous payloads.
Conjugation of fluorescence markers to CPPs has emerged as
a useful method for analyzing cellular uptake, intracellular distribu-
tion, and metabolic stability of CPPs by confocal laser scanning
microscopy (5, 7, 12). Furthermore, fluorescence probes enable
direct comparison of cellular uptake efficiencies by flow cytometry
(5, 7, 20, 21). Here we describe methods for the systematic evalu-
ation of various LOAPs generated by the introduction of different
fatty acid moieties at the N-terminus of oligoarginine peptides,
focusing on synthesis and purification, cellular uptake, intracellular
distribution, cell toxicity, and metabolic stability in Jurkat cells.

2 Materials
Prepare all solutions using ultrapurified water and analytical grade
chemicals and reagents.

2.1 Reagents for 1. Rink amide MBHA resin (0.72 mmol/g), and amino acids includ-
Lipo-oligoarginine ing Fmoc-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Lys
Peptide Synthesis (Mtt)-OH, and Fmoc-β-Ala-OH, 2-(1H-benzotriazole-1-yl)-1,
284 Jae Sam Lee and Ching-Hsuan Tung

1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N-


hydroxybenzotriazole (HOBt), piperidine, dimethylforma-
mide (DMF), and N-methyl-2-pyrrolidone (NMP). Peptide
synthesis is accomplished using the ABI 433A peptide synthe-
sizer (Life Technologies, Grand Island, NY, USA).
2. Fatty acyl chloride (lauroyl chloride, myristoyl chloride, palmitoyl
chloride), N,N-di-isopropylethylamine (DIEA), methanol,
and anhydrous dichloromethane (DCM), used in the conjuga-
tion of fatty acid with the oligoarginine peptide. Ninhydrin test
is performed using the Kaiser test kit (Sigma-Aldrich,
Milwaukee, WI, USA). A vacuum manifold reservoir and 15 ml
reservoir filter (Fisher Scientific, Waltham, MA, USA) are also
needed.
3. For FITC labeling of LOAPs, trifluoroacetic acid (TFA),
Wheaton glass 20 ml scintillation vials, and fluorescein isothio-
cyanate (FITC).
4. For cleavage and purification of LOAPs, cleavage cocktail
chemicals can be obtained (Fisher Scientific, Waltham, MA,
USA). The Vydac C18 column and reverse-phase high-perfor-
mance liquid chromatography (HPLC) are available (Varian,
Palo Alto, CA, USA).
5. The MW of LOAPs can be measured using an AB4800 matrix-
assisted laser desorption/ionization time-of-flight (MALDI-
TOF) Analyzer (Applied Biosystems, Norwalk, CT).

2.2 Cell Culture 1. Maintain human Jurkat cells from the American-Type Culture
Collection in RPMI-1640 supplemented with 10% heat-inacti-
vated fetal bovine serum and 1% penicillin/streptomycin. 75 cm2
culture flasks and 24-well culture plates are also needed.
2. Trypsin-ethylenediaminetetraacetic acid (EDTA) and propid-
ium iodide (PI) are used in flow cytometry analysis. Falcon cup
cell strainer 70 μm (Becton Dickinson, Franklin Lakes, NJ,
USA) is needed to prevent cell clumping. The FACS LSR II
instrument (Becton Dickinson, Franklin Lakes, NJ, USA)
is used for analysis with WinMDI software (The Scripps
Institute).
3. For cell cytotoxicity analysis, CytoTox 96® Non-radioactive
Cytotoxicity Assay (Promega, Madison, WI, USA) and Phenol
red-free RPMI 1640 are needed. We performed our analysis
on the SpectraMax M2 microplate reader (Molecular Devices,
Sunnyvale, CA, USA).
4. For microscopic observation, Olympus FluoView™ 1000 laser
scanning confocal microscope (Center Valley, PA) can be used.
Glass slide and cover slip are also needed.
Lipo-oligoarginine-Based Intracellular Delivery 285

3 Methods
LOAPs are synthesized by conventional Fmoc solid-phase chemis-
try with β-alanine and lysine at either end for conjugation of fatty
acid and FITC moieties, respectively. All amino acids and fatty
acids are pre-activated with HBTU. Starting with the polystyrene
resin, peptides are built “backwards,” from the C-terminus to the
N-terminus. After synthesis, the peptide is cleaved from the resin,
yielding the crude form of the peptide (Scheme 1). Generally,
yields of crude peptides are ~90% pure, but HPLC purification can
be performed if higher purity is desired.

3.1 Lipo-oligoarginine 1. Load an ABI Model 433A peptide synthesizer equipped with
Peptide Synthesis UV monitoring of Fmoc deprotection and conditional cycle
feedback, with 0.25 mmol Rink amide MBHA resin
(0.7 mmol/g) and HBTU/HOBt as the activating and cou-
pling reagents in NMP and DIEA.
2. The amino acids used in the synthesis of the LOAPs are Fmoc-
Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Lys(Mtt)-OH,
and Fmoc-β-Ala-OH. Position the nonnatural amino acid
β-Ala at the N-terminus of the oligoarginine peptide to reduce
proteolytic degradation.

Scheme 1 Synthesis of LOAPs. Reagents and conditions. (a ) Amino acids used are Fmoc-β-Ala-OH, Fmoc-
Arg(Pbf)-OH, and Fmoc-Lys(Mtt-OH), with HBTU/HOBt and 20% piperidine/DMF, 0.5 h. (b ) Conjugation of the
fatty acyl chloride in 5 ml of DIEA/NMP solution (2 M) at room temperature for 2 h. (c ) Removal of Mtt in 5 ml
TFA in DCM (1:99, v/v) for 2 min, 9–12 times, followed by labeling with FITC in 5 ml of DIEA/DMF (1:4) at room
temperature overnight. (d ) Cleavage of LOAPs using TFA:TA:EDT:anisole (90%:5%:3%:2%) at room temperature
for 3–4 h
286 Jae Sam Lee and Ching-Hsuan Tung

3. Deprotect the Fmoc moiety by 20% piperidine/DMF and


activate the resin with NMP/DMF (0.4 M) for each cycle
(see Note 1).

3.2 Fatty Acid 1. Suspend the peptide resin (0.08 mmol) in 5 ml of freshly
Conjugated prepared DIEA/NMP solution (2 M).
Oligoarginine Peptides 2. Add fatty acyl chloride (0.32 mmol) dropwise into the resin
slurry and stir the mixture gently at room temperature for 2 h.
3. Check the completion of the reaction via the Kaiser test kit. If the
coupling of the acyl group to the amino group is not complete,
the test will yield a blue color.
4. Collect the raw product by filtering through a vacuum mani-
fold reservoir and wash with DCM (5×), NMP (5×), and
methanol (5×).

3.3 FITC Labeling 1. Selectively remove the 4-methyltrityl (Mtt) protecting group
with Lipo-oligoarginine on the lysine side chain using 5 ml of TFA in DCM (1:99, v/v)
Peptide for 2 min with stirring. Repeat this step 9–12 times, followed
by washing with DCM (5×), NMP (5×), and methanol (5×)
(see Note 2).
2. React the free lysine-containing peptide with fourfold excess of
FITC (0.4 mmol, 155.8 mg) in 5 ml of DIEA/DMF (1:4), and
stir the slurry overnight at room temperature in the dark.
3. Wash the bright yellow LOAP resin with NMP and methanol.

3.4 Cleavage and 1. Cleave the fully dried LOAPs from the resin using the cleavage
Purification of LOAPs cocktail (90% TFA, 5% TA, 3% EDT, and 2% anisole) for 3–4 h.
Longer reaction times will be needed to completely remove
the Pbf protecting groups from the longer arginine sequences.
Precipitate the yellow LOAPs with diethyl ether or methyl-t-
butyl ether (see Note 3).
2. Purify the peptide solution by reversed-phase HPLC equipped
with a Vydac C18 column using 0.1% TFA and acetonitrile
(ACN) as elution buffers. Detect peptide peaks by UV absorp-
tion at 220 and 265 nm. Lyophilize the eluted peptide to
obtain a yellow cotton-like solid (see Note 4).
3. Characterize the molecular mass of each synthetic LOAP by
MALDI-TOF (M + H)+ (see Note 5).

3.5 Cellular 1. For cellular internalization and cytotoxicity assays, prepare


Internalization of Jurkat cells (3.5 × 105 cells/ml) in a 24-well plate in complete
LOAPs in Jurkat Cells culture medium and incubate overnight at 37°C in a humidified
5% CO2 incubator.
2. Determine the concentration of each LOAP solution by mea-
suring FITC absorption using a SpectraMax M2 Microplate
(Molecular Devices, Sunnyvale, CA) at 490 nm (ε = 67,000) in
phosphate buffered saline (PBS) buffer (pH = 7.2).
Lipo-oligoarginine-Based Intracellular Delivery 287

3. Wash the cells with serum-free media and PBS twice, and add
freshly prepared LOAP stock solutions (100 μM) to the culture
medium in each well, to a final LOAP concentration of 10 μM.
4. Incubate for 10 min at 37°C. Wash with PBS three times, and
incubate cells with 200 μl trypsin-EDTA for 3 min at 37°C to
remove cell surface-bound peptides (see Note 6).
5. Following trypsin treatment, add 200 μl of culture medium to
neutralize the protease and centrifuge at 250 × g for 3 min.
Wash with PBS and incubate with 200 μl ice-cold propidium
iodide (1 μg/ml) in PBS + 2% FBS to assess cell viability.
6. Cells staining with propidium iodide will be excluded from
the flow cytometry analysis. Count and characterize a total of
1–2 × 104 events using a FACS LSR II instrument and WinMDI
software (Fig. 1).
7. Determine the toxicity of LOAPs to Jurkat cells using the
CytoTox 96® Non-radioactive Cytotoxicity Assay (Fig. 2a).
8. Load the LOAP-treated cells (5 μl, 3.5 × 103 cells) onto a regular
microscopic glass slide and cover with a cover glass slip.

Fig. 1 FACS analysis of LOAP cellular uptake in Jurkat cells. Cells were incubated with 10 μM of LOAPs for
10 min at 37°C. The cells were washed, trypsin treated, PI stained, and analyzed by flow cytometry with
1–2 × 104 events. Each histogram indicates (a) control, (b) R7, and (c) C14R11, respectively. (d) Cellular uptake
of LOAPs. The median fluorescence intensity of R7 in Jurkat cells was set as 1
288 Jae Sam Lee and Ching-Hsuan Tung

Fig. 2 Cell toxicity and intracellular distribution of LOAPs in Jurkat cells.


(a) Effects of LOAPs on cell toxicity. Cells were incubated with 10 μM of LOAPs
for 60 min at 37°C. Data were normalized to the maximum amount of lactate
dehydrogenase (LDH) released from the cultures treated with lysis solution alone
and corrected for baseline LDH release from cultures exposed to buffer only.
Experiments were performed in triplicate, and data are represented as mean ± SD.
(b) Visualization of intracellular distribution. Cells were treated with 10 μM of
LOAPs for 10 min at 37°C. Cell images were obtained by confocal microscopy

9. Capture fluorescence images using an Olympus FluoView TM


1000 laser scanning confocal microscope equipped with 60×
oil immersion objective, with excitation by 488 nm laser line
(to excite the FITC tag on the LOAPs; Fig. 2b)

3.6 Metabolic 1. Monitor the metabolic degradation of LOAPs by analytical


Stability of LOAPs reversed-phase HPLC (920-LC, Varian) equipped with a
fluorescence detector, with excitation at 495 nm and emission
at 521 nm (Fig. 3a).
2. At each indicated time point, collect the whole culture media
containing the LOAPs for HPLC quantification and mass
analysis.
3. Prepare elution solvent A consisting of H2O (0.1% TFA) and
solvent B consisting of ACN (0.1% TFA). Set the linear gradient
as 0–60% solvent B over 30 min with 1 ml/min flow rate for a
10 μl sample volume.
Lipo-oligoarginine-Based Intracellular Delivery 289

Fig. 3 Release kinetics and cleavage site of LOAPs. (a) Jurkat cells were incubated
with 10 μM of LOAPs at 37°C for 10 min. Cells were then washed completely;
further incubated with a fresh peptide-free culture medium for 6, 24, 48, or 72 h;
and harvested for flow cytometry analysis. The fluorescence intensity of LOAPs
at 0 h was set as 100%. (b) Schematic illustration of the cleavage site in LOAPs.
It was found that most internalized LOAPs were hydrolyzed between the lauroyl
chain and beta-alanine residue, releasing the intact peptide into the culture medium

4. Collect aliquots of FITC-conjugated sequences (C-terminus)


and identify the molecular weight by MALDI-TOF mass
(Fig. 3b).

4 Notes
1. Swell Rink amide MBHA resin in 5 ml of DMF for 5 min, and
remove the Fmoc group of Rink amide MBHA resin using
20% piperidine/DMF. Dissolve HBTU in a solution of HOBt
in DMF and add the first amino acid into this solution along
with NMP and DIEA. Transfer the mixture solution directly to
the reaction vessel. HBTU activation is highly efficient, espe-
cially when difficult couplings are predicted, such as with Arg
and the branched side chains of Ile, Val, and Leu. Generally, a
coupling time of 2–4 h is sufficient. Remove the Fmoc group
of each amino acid using 20% piperidine/DMF. Verify Fmoc
290 Jae Sam Lee and Ching-Hsuan Tung

group removal using the ninhydrin test. Modify reaction


conditions to compensate by extending the deprotection and/or
coupling periods when aggregation occurs. After removing the
Fmoc group, wash the resin with NMP to remove the piperi-
dine. Before coupling, the carboxyl group of the amino acid
must be activated by adding HBTU, which can be dissolved in
a solution of HOBt in DMF.
2. The coupling of the fluorophore (e.g., FITC) to the CPP is
usually carried out by covalent linkage to the N-terminal
α-amino group or to a lysine ε-amino group in the peptide.
The side-chain Mtt group can be selectively removed with 1%
TFA in DCM (22). In order to remove the Mtt group com-
pletely from the LOAP, the peptide resin should be washed at
least 9–12 times using 1% TFA in DCM with gentle shaking
for 2 min each time. In washing steps 1–3, the color of the
solution will remain unchanged. During washing steps 3–6,
the color of the solution will change from pale to dark yellow.
In washing steps 6–9, almost all the color will disappear.
3. Wash the bright yellow resin from the previous step with NMP
(5 × 5 ml) and methanol (5 × 5 ml), and dry for 60 min in a
vacuum. Treat the peptide resin with 10 ml of the deprotection
cocktail, and stir gently for 3–4 h. Prepare and assemble col-
umns for the extraction of peptide from the resin. Fill a 50-ml
conical tube with 30 ml ether, and place the tube in ice to keep
the ether cold. Place the column via a clamp and stand in hood.
Slowly add the cocktail to column. Once the cocktail has stopped
“dripping” from the column, add slight pressure to the top of
the column using the air line to remove any remaining peptide
from the column. Remove tube from under column, cap,
shake, and release pressure. Incubate the ether-peptide mix-
ture for 10 min, then centrifuge the tube at 2,500 rpm (780 ´ g)
for 5 min at room temperature. Decant supernatant into a
waste carboy. Allow the ether to evaporate under low vacuum
drying. Add 15 ml of 10% acetic acid to the centrifuge tube.
Freeze and lyophilize. Characterize the molecular weight of
the peptide using MALDI mass spectrometry, and store at
−20°C.
4. To obtain highly pure peptide, approximately 200 mg/ml
crude peptide (the amount depends on the complexity of the
HPLC trace of the crude product) can be purified by prepara-
tive HPLC (Vydac 218TP1022 column) using a gradient of
60–100% B over 60 min (A = 0.1% TFA/water and B = 0.1%
TFA/ACN) at a flow rate of 8 ml/min. Collect all column
fractions and pool those containing the ideal product. The
amount of pure material recovered depends on the sequence
but is usually greater than 90% of that loaded.
Lipo-oligoarginine-Based Intracellular Delivery 291

5. Dissolve LOAPs in a mixture of 70% ACN and 30% H2O (0.1%


TFA) and desalt using a ZipTipC18 tip (Millipore, Billerica, MA).
Spot a 1 μl sample directly onto a MALDI target plate. Apply 1 μl
matrix solution (α-cyano-4-hydroxycinnamic acid, 5 mg/ml in
50:50 ACN/H2O) on the sample spot and allow to dry. Blow
the dried MALDI spot with compressed air before inserting
into the mass spectrometer (AB4800 MALDI-TOF) Proteomics
Analyzer. Operation conditions: The instrument must be oper-
ated in positive ion reflector mode, with the mass range as
required. Acquire 2,000 laser shots and calculate the average
for each sample. Perform an external calibration using a cali-
bration mixture including four different masses (904.468,
1296.685, 1570.677, and 2465.199). Applied Biosystems
software package 4000 Series Explorer (v. 3.6 RC1) can be
used to analyze the acquired data.
6. A very important challenge in quantifying CPP uptake is to
distinguish between membrane-associated and intracellular
CPPs. For this purpose, trypsin/EDTA is an effective method
to detach membrane-associated peptides from the cell surface.

Acknowledgment

This work was supported in part by NIH CA135312, CA114149,


and CA126752.

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Chem Lett 11:2315–2317 peptoid molecular transporters. Proc Natl
15. Elmquist A, Langel U (2003) In vitro uptake Acad Sci USA 97:13003–13008
and stability study of pVEC and its all-D analog. 20. Vives E, Richard JP, Rispal C, Lebleu B (2003)
Biol Chem 384:387–393 TAT peptide internalization: seeking the
16. Trehin R, Nielsen HM, Jahnke HG, Krauss U, mechanism of entry. Curr Protein Pept Sci
Beck-Sickinger AG, Merkle HP (2004) 4:125–132
Metabolic cleavage of cell-penetrating peptides 21. Duchardt F, Fotin-Mleczek M, Schwarz H,
in contact with epithelial models: human calci- Fischer R, Brock R (2007) A comprehensive
tonin (hCT)-derived peptides, Tat(47–57) and model for the cellular uptake of cationic cell-
penetratin(43–58). Biochem J 382:945–956 penetrating peptides. Traffic 8:848–866
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derived cell-penetrating peptides with J Pept Sci 6:264–270
Chapter 27

Fluorescent Spherical Monodisperse Silica Core–Shell


Nanoparticles with a Protein-Binding Biofunctional Shell
Achim Weber, Marion Herz, and Günter E.M. Tovar

Abstract
The production of uniform protein-binding biofunctional fluorescent spherical silica core–shell nanoparticles
by a modified Stöber method is described. Fluorescent particle cores with diameters of 100 nm are synthe-
sized in a two-step reaction. Functional shells for subsequent coupling reactions are provided by generat-
ing organic shells providing amine and carboxyl groups at the nanoparticles’ shell surface. Conjugation of
proteins to the nanoparticles is achieved using N-(3-dimethylaminopropyl)-N¢-ethylcarbodiimide hydro-
chloride (EDC) as coupling agent. The characterization of the nanoparticle systems and their surface
functionalization is done by microelectrophoresis, dynamic light scattering (DLS), and a colorimetric
detection of the amount of nanoparticle-attached protein via a bicinchoninic acid (BCA) assay. Fluorescently
spiked nanoparticle cores with biofunctional shells for molecular recognition reactions may be used as
imaging tools or reporter systems.

Key words Silica nanoparticles, Nanoparticles, Core–shell, Surface modification, Monodisperse,


Fluorescent dye

1 Introduction
The synthesis of submicron particles with narrow size distribution,
uniform shape, and surface modification is of interest in various
fields in life science such as molecular biological assays in cell and
tissue engineering, molecular biotechnology, biological imaging,
or microarrays (1–6). The production of nanoparticulate sol–gel
particles is simple, their size can be tailored by a systematic varia-
tion of reaction conditions, and the flexible silica chemistry pro-
vides versatile possibilities for surface modifications and preparation
of a nanoscopically thin organic shell based on the silanol groups
that are present on the nanoparticles’ surface (7, 8). These silanols
can be used to integrate amine groups, carboxyl groups, or other
functionalities into the particle shell using organo-functional
silanes.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_27, © Springer Science+Business Media New York 2013

293
294 Achim Weber et al.

Fig. 1 Typical scanning electron micrograph of silica nanoparticles prepared by


the hydrolysis of tetraethoxysilane in the presence of ethanol containing ammonia
(Subheading 3.1)

By incorporating a fluorescent dye into the core of these


chemically stable and biocompatible particles, you gain a system of
high brightness and photostability which can be used for identify-
ing or characterizing biological samples such as cells (4). One of
the major advantages of nanoparticles is their small size which leads
to a dramatically increased specific surface. A large number of dye
molecules can be incorporated into one single silica particle such
that much higher optical signals are generated than with isolated
dye molecules.
Here, the synthesis of spherical silica nanoparticles with a
hydrodynamic diameter of 100 nm is demonstrated via a modified
Stober method (see Fig. 1), which consists of the hydrolysis of tet-
raethoxysilane in a mixture of ethanol and aqueous ammonia (7).
After the formation of the nanoparticles, additional layers of reactive
functional groups, amine and carboxy, are attached (see reaction
schemes in Fig. 2). Fluorescent-dyed particles are synthesized in a
two-step reaction. In the first step, the fluorescent dye Alexa Fluor®
488 carboxylic acid succinimidyl ester is chemically bound to
(3-aminopropyl)triethoxysilane, an amine containing silane agent
(see fluorescence image in Fig. 3). In the second reaction step, the
silanized dye and tetraethoxysilane are hydrolyzing and co-condens-
ing to form dye-doped silica nanoparticles (9, 10). A protein is cova-
lently bound to carboxyl-functionalized silica nanoparticles by using
carbodiimides that form amide linkages between the carboxyl groups
at the particle surface and the amino groups of proteins (11).
Fluorescent Spherical Monodisperse Silica Core–Shell Nanoparticles… 295

Fig. 2 Reaction scheme of the surface modifications. (a) Functionalization of silica particles with (3-aminopro-
pyl)triethoxysilane, (b) reaction of silica surface covered by amino groups with succinic anhydride, and (c)
formation of an amide bond between the carboxylic group of the silica particles and the amine group of the
antibody in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)

Fig. 3 Fluorescence microscopy image of Alexa Fluor® 488-stained silica nanoparticles attached to a microscope
slide, 10× magnification
296 Achim Weber et al.

2 Materials
All solutions are prepared by the use of Milli-Q grade water
(<18.2 mW/cm) and stored at room temperature (unless described
otherwise).

2.1 Synthesis 1. Magnetic stirrer.


of Unmodified Silica 2. Stirring bar.
Nanoparticles
3. 500 mL closable bottle.
4. Graduated volumetric cylinder, 1 L.
5. Volumetric pipette, 20 mL.
6. Graduated pipette, 20 mL.
7. Piston stroke pipette, 1–10 mL with appertaining tips.
8. Suction bulb.
9. Centrifuge for high sample volume.
10. Centrifugal tubes, 500 mL.
11. Plastic beaker, 5 L.
12. Beaker, 600 mL.
13. Glass funnel.
14. Dialysis tube, MWCO 12–14 kDa, diameter 29 mm, flat width
45 mm, 6.4 mL/cm (volume/length).
15. Dialysis clips.
16. Syringe.
17. Syringe filter, pore size 0.45 mm with a membrane consisting
of mixed cellulose esters.
18. Conductivity electrode.
19. Ethanol (HPLC grade, >99.9%).
20. Ammonium hydroxide solution (puriss p.a. plus, ³25% in water).
21. Milli-Q grade water.
22. Tetraethoxysilane (TEOS, 99%).

2.2 Surface 1. Magnetic stirrer.


Modification with 2. Stirring bar.
Amino-Functional
3. 100 mL closable bottle.
Groups
4. Volumetric pipette, 100 mL.
5. Graduated pipette, 10 mL.
6. Suction bulb.
7. Piston stroke pipette, 100–1,000 mL with appertaining tips.
8. Centrifuge for high sample volume.
Fluorescent Spherical Monodisperse Silica Core–Shell Nanoparticles… 297

9. Centrifugal devices, 30 mL teflon.


10. Centrifugal devices, 30 mL PP.
11. Centrifugal tubes, 1.5 mL.
12. Syringe.
13. Syringe filter, pore size 0.45 mm with a membrane consisting
of mixed cellulose esters.
14. Ammonium hydroxide solution (puriss p.a. plus, ³25% in water).
15. (3-Aminopropyl)triethoxysilane (APTES, 99%).
16. Milli-Q grade water.
17. 100 mM acetate buffer solution, pH 4.7 (ACB47): add approx-
imately 800 mL Milli-Q grade water to a glass beaker. Pipette
11.44 mL of acetic acid to the beaker and then add 3.999 g of
sodium hydroxide. Mix and adjust the pH with 1 M caustic
soda. Transfer the solution to a 1 L graduated cylinder and
make up to 1 L with Milli-Q grade water. Store at room
temperature.

2.3 Surface 1. Magnetic stirrer.


Modification with 2. Stirring bar.
Carboxyl Functional
3. 50 mL closable bottle.
Groups
4. Graduated pipette, 10 mL.
5. Suction bulb.
6. Centrifuge for high sample volume.
7. Centrifugal devices, 30 mL PP.
8. Centrifugal devices, 30 mL teflon.
9. Balance.
10. Spatula.
11. Plastic beaker, 5 L.
12. Beaker, 600 mL.
13. Glass funnel.
14. Dialysis tube, MWCO 12–14 kDa, diameter 29 mm, flat width
45 mm, 6.4 mL/cm (volume/length).
15. Dialysis clips.
16. Syringe.
17. Syringe filter, pore size 0.45 mm with a membrane consisting
of mixed cellulose esters.
18. Conductivity electrode.
19. 100 mM phosphate buffer solution, pH 7.0 (PB70): add
approximately 800 mL Milli-Q grade water to a glass beaker
(see Note 1). Weigh 8.8995 g disodium hydrogen phosphate
298 Achim Weber et al.

dihydrate and 6.8995 g sodium dihydrogen phosphate mono-


hydrate and transfer to the beaker. Mix and adjust the pH with
1 M caustic soda. Transfer the solution to a 1 L graduated cyl-
inder and make up to 1 L with Milli-Q grade water.
20. Tetrahydrofuran (>99.9%).
21. Succinic anhydride (>99%).
22. Milli-Q grade water.

2.4 Fluorescent 1. Centrifuge for small sample volume.


Dye Labeling of 2. Centrifugal tubes 1.5 mL.
Unmodified Silica
3. Piston stroke pipette 1–10 mL with appertaining tips.
Nanoparticles
4. Piston stroke pipette 10–100 mL with appertaining tips.
5. Piston stroke pipette 100–1,000 mL with appertaining tips.
6. Shaker for centrifugal tubes.
7. Alexa Fluor® 488 carboxylic acid succinimidyl ester.
8. Ethanol (HPLC grade, >99.9%).
9. (3-Aminopropyl)triethoxysilane (APTES, 99%).
10. Milli-Q grade water.
11. Ammonium hydroxide solution (puriss p.a. plus, ³25% in water).
12. Tetraethoxysilane (TEOS, 99%).

2.5 Surface 1. Centrifuge for small sample volume.


Modification 2. Centrifugal tubes 1.5 mL.
of Fluorescent
3. Piston stroke pipette 1–10 mL with appertaining tips.
Dye-Labeled
Nanoparticles with 4. Piston stroke pipette 10–100 mL with appertaining tips.
Amino-Functional 5. Piston stroke pipette 100–1,000 mL with appertaining tips.
Groups 6. Shaker for centrifugal tubes.
7. Ammonium hydroxide solution (puriss p.a. plus, ³25% in water).
8. Tetraethoxysilane (TEOS, 99%).
9. (3-Aminopropyl)triethoxysilane (APTES, 99%).
10. 100 mM acetate buffer, pH 4.7 (ACB47)—see preparation
noted in Subheading 2.2.
11. Ethanol (HPLC grade, >99.9%).
12. Milli-Q grade water.

2.6 Surface 1. Centrifuge for small sample volume.


Modification of 2. Centrifugal tubes 1.5 mL.
Fluorescent Dye-
3. Piston stroke pipette 100–1,000 mL with appertaining tips.
Labeled Nanoparticles
with Carboxyl 4. Balance.
Functional Groups 5. Spatula.
Fluorescent Spherical Monodisperse Silica Core–Shell Nanoparticles… 299

6. Shaker for centrifugal tubes.


7. 100 mM phosphate buffer, pH 7.0 (PB70)—see preparation
noted in Subheading 2.3.
8. Tetrahydrofuran (>99.9%).
9. Succinic anhydride (>99%).
10. Milli-Q grade water.

2.7 Functionalization 1. Centrifuge for small sample volume.


of Carboxy-Modified 2. Centrifugal tubes 1.5 mL.
Nanoparticles with
3. Shaker for centrifugal tubes.
Protein
4. Piston stroke pipette 100–1,000 mL with appertaining tips.
5. Balance.
6. Spatula.
7. N-(3-Dimethylaminopropyl)-N¢-ethylcarbodiimide hydrochlo-
ride (EDC): 8 mM in Milli-Q grade water.
8. Trehalose solution: 5% trehalose in Milli-Q grade water.
9. 100 mM 2-(N-morpholino)ethanesulfonic acid sodium salt
(MES), pH 4.5 (MES45): Add approximately 800 mL Milli-Q
grade water to a glass beaker (see Note 1). Weigh 21.72 g MES
and transfer to the beaker. Mix and adjust the pH with HCl.
Transfer the solution to a 1 L graduated cylinder and make up to
1 L with Milli-Q grade water.
10. 100 mM phosphate buffer saline, pH 7.4 (PBS74): add approx-
imately 800 mL Milli-Q grade water to a glass beaker (see
Note 1). Weigh 8 g sodium chloride, 0.2 g potassium chloride,
0.2 g potassium dihydrogen orthophosphate, and 1.44 g diso-
dium hydrogen phosphate dihydrate and transfer to the bea-
ker. Mix and adjust the pH with 1 M caustic soda. Transfer the
solution to a 1 L graduated cylinder and make up to 1 L with
Milli-Q grade water.
11. Triton X-100 (PBS/Tr): 0.1% Triton X in PBS74.
12. Protein, e.g., gelatin, other proteins or antibodies are also usable.

3 Methods
All reactions are done at room temperature (unless described
differently).

3.1 Synthesis 1. Measure out 400 mL of ethanol in a 1 L volumetric cylinder


of Unmodified Silica and pour it into a 500 mL closable bottle.
Nanoparticle Cores 2. Put a stirring bar into the bottle and place it onto a magnetic
with a Hydrodynamic stirrer. Stir at 500 rpm.
Diameter of 100 nm
300 Achim Weber et al.

3. Add 20 mL of Milli-Q grade water, 10.3 mL of ammonium


hydroxide solution, and 5.35 mL (corresponding to 5 g) of
TEOS while stirring. Then close the bottle and let the reaction
run for at least 12 h (see Notes 2 and 3).
4. Transfer the resulting particle suspension into two 500 mL
centrifugal devices and tare. Centrifuge at 10,000 rpm for
60 min (Beckman Coulter Avanti J, Rotor JA-10, 17,700 × g).
Discard the supernatant. Resuspend the particle pellets thor-
oughly in 100 mL of Milli-Q grade water with the help of an
ultrasonic bath (see Note 4).
5. Preincubate a 30 cm piece of dialysis tube in Milli-Q grade
water for approximately 5 min.
6. Fill a 5 L plastic beaker with Milli-Q grade water, put a stirring
bar into it, and place the beaker onto a magnetic stirrer.
7. Seal the dialysis tube at one end using two clips and fill in the
particle suspension with the help of a glass funnel. Rinse the
centrifugal devices and the funnel with Milli-Q grade water
and close the dialysis tube at the top with again two clips. Place it
into the beaker filled with Milli-Q grade water (see Note 5).
8. Gently stir at about 200 rpm such that the water inside the
beaker will be moved while the dialysis tube floats motionless.
9. Exchange water daily. Dialysis may be stopped when the con-
ductivity of the water is below 1.3 mS/cm (see Note 6). This
usually takes 5–7 days.
10. Finally pass the particle suspension through syringe filters with
a pore size of 0.45 mm.
11. A SEM image of these particles is seen in Fig. 1.

3.2 Surface 1. Suspend 100 mg of silica particles in 100 mL Milli-Q grade


Modification with water and pour it into a closable bottle.
Amino-Functional 2. Put a stirring bar inside the bottle and place the bottle onto a
Groups magnetic stirrer. Stir at about 500 rpm.
3. Add 8 mL of ammonium hydroxide solution and 200 mg of
APTES (20% w/w of the used particle mass) while you keep
on stirring (see Notes 2 and 3).
4. Close the bottle and make sure that the reaction time is kept at
exactly 1 h (see Note 7).
5. Split the particle suspension into four centrifugation devices
made of teflon (see Notes 8 and 9). Centrifuge and resuspend
the particles in ACB47. Transfer suspension to centrifugal
devices made of PP and repeat washing several times (see Note 10).
Finally pass the particle suspension through syringe filters with
a pore size of 0.45 mm.
Fluorescent Spherical Monodisperse Silica Core–Shell Nanoparticles… 301

3.3 Surface 1. Centrifuge 500 mg of amino-modified silica nanoparticles at


Modification with 20,000 rpm for 20 min in 30 mL centrifugal devices made of
Carboxylic Groups PP (Beckman Coulter Avanti J, Rotor JA-25.50, 48,384 × g).
2. Throw away the supernatant and resuspend the particle pellets
in 25 mL of PB70 with the support of an ultrasonic bath. The
particle suspension should also be transferred into centrifugal
devices made of teflon because of the ongoing working step.
3. Centrifuge at 10,000 rpm for 30 min (Beckman Coulter Avanti
J, Rotor JA-25.50, 12,096 × g). Aspire the supernatant again
and resuspend the particle pellet in 25 mL of tetrahydrofuran
whereby the suspension is poured into a 50 mL closable bottle
(see Note 11).
4. Add 875 mg of succinic anhydride to the reaction suspension
and homogenize it by using the ultrasonic bath for 1 h while
you sonify for 5 min and let it rest for 10 min. Repeat that for
four times and make sure that the bath is ice cooled at all time
as tetrahydrofuran has a high vapor pressure and the reaction
suspension is getting warm very fast.
5. Shake the particle suspension for at least 16 h.
6. Pour the modified particle suspension into centrifugal devices
made of teflon and rinse carefully to make sure that all particles
are collected.
7. Wash the particles with centrifugation and resuspension into
50 mL of Milli-Q grade water as mentioned above.
8. Then fill the thoroughly resuspended particles inside a dialysis
tube as described in Subheading 3.1 and exchange again the
water until the conductivity is below 1.3 mS/cm. That will usu-
ally take 3–5 days.

3.4 Fluorescent 1. Add 79 mL of ethanol to 1 mg of Alexa Fluor® 488 fluorescent


Labeling of dye in a 1.5 mL centrifugal tube and homogenize the mixture.
Unmodified Silica 2. Add 3.47 mg of APTES to 1 mL of ethanol in a separate
Nanoparticles 1.5 mL centrifugal tube and mix it carefully.
3. For silanization of the dye, pipette 79 mL of the APTES solu-
tion to the Alexa dye solution and shake the mixture constantly
for 2 h (see Note 12).
4. Centrifuge 10 mg silica particles in a 1.5 mL centrifugal device
at 15,000 rpm for 20 min (Hermle Z216 MK, Rotor 220.87
V11, 21,380 × g).
5. Aspire the supernatant and resuspend the particle pellet in
354 mL of Milli-Q grade water.
6. Add 35 mL of ammonium hydroxide solution, 16.7 mL of the
silanized Alexa dye solution, and 3 mL of TEOS to the carefully
resuspended particles (see Notes 2 and 3).
302 Achim Weber et al.

7. Shake the suspension for 24 h at room temperature.


8. Centrifuge the particles at 15,000 rpm for 20 min.
9. Throw away the supernatant and wash the dye-doped particles
three times with ethanol and one time with Milli-Q grade
water as described above.

3.5 Surface 1. Pipette the corresponding volume of 10 mg dye-doped nano-


Modification of particle suspension into a 1.5 centrifugal tube and add 100 mL
Fluorescent Dye- of ammonium hydroxide solution.
Labeled Nanoparticles 2. Furthermore add 3.7 mL TEOS and 11 mL APTES and shake
with Amino-Functional the reaction mixture for 24 h at room temperature.
Groups 3. Wash the amino-modified particles with centrifugation and
resuspension two times with 1 mL ethanol, one time with
1 mL ACB47, and two times with 1 mL Milli-Q grade water as
mentioned in Subheading 3.4.
4. Characterize the particles with zeta potential measurements
explained in Subheading 3.8.

3.6 Surface 1. Centrifuge 10 mg of dye-doped and amino-modified silica


Modification of nanoparticles at 15,000 rpm for 20 min in a 1.5 mL centrifugal
Fluorescent Dye- device.
Labeled Nanoparticles 2. Aspire the supernatant and resuspend the particle pellet in
with Carboxy- 1 mL of PB70 with the support of an ultrasonic bath.
Functional Groups 3. Again centrifuge the particle suspension at 15,000 rpm for
20 min. Throw away the supernatant and resuspend the particle
pellet in 1 mL of tetrahydrofuran (see Note 11).
4. Add 17.5 mg of succinic anhydride to the reaction suspension
and homogenize it by using the ultrasonic bath for 1 h while
you sonify for 5 min and let it rest for 10 min. Repeat that reac-
tion cycle for four times; make sure that the bath is ice cooled
at all time as tetrahydrofuran has a high vapor pressure and the
reaction suspension is getting warm very fast.
5. Shake the particle suspension for at least 16 h.
6. Wash the particles three times with centrifugation and resus-
pension into 1 mL of Milli-Q grade water as described in
Subheading 3.4.

3.7 Functionalization 1. Add 45 mg of protein and 100 mL of EDC solution to 1 mg of


of Carboxy-Modified carboxy-modified silica nanoparticles (see Note 13).
Silica Nanoparticles 2. Fill up to 1 mL with MES45.
with Proteins
3. Shake the suspension for 16 h at 4°C.
4. Centrifuge the suspension at 15,000 rpm for 10 min and aspi-
rate the supernatant very carefully (Hermle Z216 MK, Rotor
220.87 V11, 21,380 × g).
Fluorescent Spherical Monodisperse Silica Core–Shell Nanoparticles… 303

5. Resuspend the particle pellet in 1 mL of PBS/Tr and centrifuge


the suspension again at 15,000 rpm for 10 min (see Note 14).
6. Repeat the washing procedure with PBS74 and at least resus-
pend the pellet in 1 mL of 5% trehalose solution.
7. Storage of the produced particles is possible at 4°C for several
weeks.

3.8 Particle 1. The particle size distribution is measured by dynamic light


Characterization scattering (DLS) using a Zetasizer 3000HSA (Malvern
Instruments, UK) as well as the measurement of the zeta
potential via microelectrophoresis. Only distilled/deionized
water (Milli-Q-grade water purification system, TKA, Germany)
is used for all analytical measurements. The values shown in
Table 1 are an average of five measurements. The hydrody-
namic particle diameter is determined at a fixed scattering angle
of 90°. Measurements are performed with 500 mg particles dis-
persed in 3 mL Milli-Q grade water for size distribution and
buffer solution for zeta potential distribution.
2. Electron microscopic investigation is done with a field emis-
sion scanning electron microscope Leo 1530 VP Gemini (Carl
Zeiss AG, Germany). The picture shown in Fig. 1 is recorded
with a 1,000× magnification. For gaining a particle monolayer,
the suspension is diluted to 100 mg/mL with Milli-Q grade
water.
3. The fluorescence image is obtained using an Olympus BX60
fluorescence microscope fitted with a 2.5× lens, an HQ-filter set
for Cy2 sel, Alexa 488, and a soft imaging system color view
camera. Illumination of the nanoparticles is done at 495 nm
with a mercury lamp; emission is collected at 519 nm. The con-
centration of the sample is 10 mg/mL in Milli-Q grade water.

Table 1
Hydrodynamic diameter determined by dynamic light scattering and zeta
potential measured via microelectrophoresis both of initial silica
nanoparticles, after surface modification with (3-aminopropyl)triethoxysi-
lane (APTES) and after ongoing functionalization of amino-modified
nanoparticles with succinic anhydride

Zeta potential (mV)


<dDLS > (nm) pH = 4.7 pH = 7.0
Silica 108 −14 −21
Amino-silica 94 +48 −16
Carboxyl-silica 123 −24 −34
304 Achim Weber et al.

4. The detection of protein concentration is done spectrophoto-


metrically by comparison with known protein standards via
bicinchoninic acid (BCA) assay. A set of protein standards has
to be prepared with concentrations between 2 and 0 mg/mL
(blank) preferably using the same diluent as the sample. 20 mL
of each standards and sample are pipetted into individual wells
of a 96-microwell plate to which 200 mL of “BCA” reagent is
added. Incubation time is 30 min at 37°C. After cooling down
to room temperature, absorbance is measured at 562 nm
(see Note 15).

4 Notes

1. Putting some water inside of the beaker helps to dissolve the


salts very quickly.
2. Holding ammonium hydroxide solution close to the reaction
bottle while you are pipetting helps avoiding spillage and makes
sure that the correct amount will be introduced into the reaction
mixture. Because of the high vapor pressure of the ammonium
hydroxide solution, it will drop out of a graduated pipette.
3. Work under a hood while pipetting the ammonium hydroxide
solution. It is harmful when inhaling it and will burn your ana-
tomical airway.
4. Join the two pellets to one by using only the amount of 100 mL
Milli-Q grade water. By doing that, you are able to keep the
working volume small. So put only about 50 mL of water into
the first centrifugal device and resuspend the pellet by using
ultrasonic sound. Make sure that there are no clusters left in
the suspension. Always check that by overturning the device
and observing the suspension by bright light. If there are no
clusters left, add the suspension to the second pellet and start
resuspending that as well.
5. Use the remaining 50 mL of Milli-Q grade water to rinse the
centrifugal devices. It is better to use smaller volumes for rins-
ing and repeat the procedure 2–3 times.
6. It is not necessary to change the water of the dialysis more than
two times a day. The system needs approximately 8–10 h to
equilibrate.
7. One hour reaction time is perfect for saturating the particles’
surface with amine groups. If the reaction time is exceeded, the
particles will start to agglomerate because of the excess amount
of silane inside the reaction mixture.
8. Centrifugation devices made of teflon are necessary because of
the high basic pH inside the suspension, which would destroy
other plastic material like polypropylene.
Fluorescent Spherical Monodisperse Silica Core–Shell Nanoparticles… 305

9. By working with less volume in many centrifugal devices, the


purification effect is better. You will use more buffering solu-
tion for the same amount of particles. Again after your last
centrifugation, join the four pellets with little amount of
ACB47 to gain one particle suspension.
10. Working with teflon centrifugal devices makes it harder to
resuspend particle pellets with ultrasonic sound. The material
is soft and the energy can hardly penetrate the devices.
Switching to harder centrifugal devices allows centrifugation at
higher speed velocity and makes resuspension much easier.
11. The particles are totally discharged inside of this media, so it is
normal that the suspension is not stable and the particles look
as they seem to agglomerate. Make sure that no particle sus-
pension will touch the inner surface of the bottle when pour-
ing the suspended particles inside. The solvent will dry
immediately, and there will be no way to bring the dried par-
ticles back into the reaction suspension after that. So they will
not be modified.
12. Storage of the prepared dye is possible at −18°C for several
months.
13. EDC should be dissolved in Milli-Q grade water not until it is
needed for the coupling trial. Otherwise hydrolysis of EDC
will take place, and the activation of the carboxy groups may be
prevented.
14. For destabilizing the particle pellet, knock the centrifugal
device at a table several times. That will reduce the time needed
for resuspension of the pellet in an ultrasonic bath and preserve
the coupled antibody at the particles’ surface.
15. For additional information about the BCA assay, please visit
www.thermo.com/pierce.

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10. Herz M et al. (2009) In vitro study of mouse 11. Hermanson GT (2008) Bioconjugate tech-
fibroblast tumor cells with TNF coated and niques, 2nd edn. Elsevier, Amsterdam
Chapter 28

Direct Quantification of PTD Transduction


Using Real-Time Monitoring
Mi-Sook Lee and Song Her

Abstract
Protein transduction domains (PTD or cell-permeable proteins) have attracted much attention as drug
carriers because of their ability to penetrate cellular membranes. Although numerous PTD have been
identified and their properties elucidated, their mechanism of action has not been fully understood due to
the absence of a reliable quantification method. This chapter provides a direct method for quantifying cel-
lular transduction of PTD in vitro and in vivo using bioluminescence imaging (BLI). This methodology
exploits noninvasive techniques to create an environment suitable for the real-time imaging of PTD transduc-
tion and is therefore a promising tool for studying the mechanism of PTD transduction and the in vivo
application of new therapeutic candidates.

Key words Drug carrier, Protein transduction domains, Cell-permeable proteins, Cellular transduc-
tion, Bioluminescence imaging, Real-time imaging

1 Introduction
Protein transduction domains (PTD) have attracted increasing
attention in intracellular therapeutic protein delivery (1), and
quantifying cellular transduction of PTD is important for compre-
hending both their mode of transduction and the efficacy of new
drugs (2). Current approaches for quantifying PTD transduction,
including flow cytometry and confocal fluorescence microscopy,
are based on the fluorescent labeling of peptides, but flow cytom-
etry can lead to false-positive results originating from cell surface-
bound peptides (3). Also, fluorescence imaging by confocal
fluorescence microscopy, which can monitor the subcellular local-
ization of peptides and discriminate between internalized and
extracellular fluorescent peptides, is limited by statistical problems
(4). Alternatively, direct peptide detection by MALDI-TOF using
isotope-labeled peptide was recently reported (5, 6). Although this
method allows for the direct quantification of cell-permeable

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_28, © Springer Science+Business Media New York 2013

307
308 Mi-Sook Lee and Song Her

Fig. 1 Schematic illustration of bioluminescence in living cells following PTD-Fluc transduction. Inset :
Structures of PTD-Fluc containing 11 amino acids (YARVRRRGPRR) and Fluc as a control. After PTD-Fluc trans-
duction, luminescence is emitted by an ATP-dependent luciferin–luciferase reaction (C cytosol, N nucleus)

proteins and the discrimination of extracellular membrane-bound


and intracellular peptides, it can only be used with cell lysates and
is not suitable for in vivo measurements.
Bioluminescence imaging (BLI) based on luciferase activity is
a rapid and sensitive method for in vitro and in vivo studies of
ongoing biological phenomena. The most commonly used
luciferase for BLI is firefly luciferase (Fluc), which requires
ATP-Mg2+ and oxygen in the presence of the substrate, D-luciferin,
to produce bioluminescence (7). Using firefly luciferase-tagged
PTD (PTD-Fluc), we quantified internalized PTD by the real-time
monitoring of ATP-dependent luciferase activity in vitro and
in vivo (Fig. 1). This chapter provides a basic protocol for the
real-time quantification of PTD transduction using BLI.

2 Materials
2.1 Luciferase Assay 1. Purified PTD-Fluc (8) (see Note 1).
of Purified Protein 2. Purified Fluc (8) (see Note 1).
3. Luciferin substrate solution: Combine 1 mM D-luciferin
(Xenogen, Alameda, CA, USA) (see Subheading 2.2), 3 mM
ATP (ATP disodium salt), and 15 mM MgSO4 in 30 mM
HEPES (pH 7.8) using fresh, deionized ATP-free water. Store
the solution at −20°C in polypropylene or glass tubes.
4. 96-well black microplate with a clear bottom.
Real-Time Quantification of PTD Transduction 309

2.2 PTD-Fluc 1. HeLa cells or other biologically relevant cell lines of interest.
Transduction In Vitro 2. Dulbecco’s modified Eagle’s medium (DMEM) complete
media: DMEM supplemented with 10% (v/v) fetal bovine
serum and 1× (v/v) pen/strep (100× antibiotic solution:
10,000 U of penicillin and 10,000 U of streptomycin).
3. 1× Phosphate-buffered saline without Mg2+ and Ca2+.
4. D-Luciferin firefly (potassium salt, MW = 318.42, #XR-1001;
Xenogen) stock solution: Reconstitute a 1.0 g of D-luciferin in
33.3 ml of sterile 1× PBS without Mg2+ and Ca2+ to make a
30 mg/ml (0.1 M) stock solution. Filter sterilize through a
0.2-μm syringe filter. Store in aliquots at −20°C and protect
from light (see Note 2).
5. Syringe filter, 0.2 μm.

2.3 PTD-Fluc 1. Male 4-week-old ICR mice weighing approximately 30 g each


Transduction In Vivo (SPF grade).
2. 0.5-cc, 27-gauge insulin syringes.

2.4 BLI Equipment 1. Isoflurane anesthesia chamber.


(Xenogen) 2. IVIS-200.
3. Caliper/Xenogen IVIS® Living Image software, version 3.0.

3 Methods
3.1 Cell-Free Prior to the in vitro or in vivo analysis of PTD transduction, the
Luciferase Assay following protocol using PTD-Fluc and Fluc should be carried
out to accurately determine the activity and concentration of each
protein.
1. Bring the luciferin substrate solution to room temperature
before starting.
2. Transfer 10 μl each of PTD-Fluc and Fluc in Ni-NTA elute
buffer to a 96-well black microplate. Commercially available
purified Fluc can be used as a standard for calibration (see
Note 3).
3. Add 90 μl of D-luciferin substrate solution and use the sub-
strate solution without purified protein as a blank.
4. Immediately acquire an image using the IVIS-200 imaging
system (see Subheading 3.2, step 7).
5. Generate a luciferase standard curve for light emission (photons/
second, p/s) vs. concentration (ng/ml).
6. Based on the standard curve, determine the concentration of
each purified protein (see Note 4).
310 Mi-Sook Lee and Song Her

3.2 Transduction The following quantification protocol based on the ATP-dependent


of PTD-Fluc In Vitro luciferase reaction allows the direct and real-time measurement of
transduction in live cells without interfering with surface-bound
PTDs (Fig. 1).
1. Seed HeLa cells in a 96-well black microplate in 100 μl of
DMEM complete media at 10,000 cells per well.
2. Culture the cells in a humidified atmosphere at 37°C under 5%
CO2 for 20–24 h.
3. Prepare 100 μl of fresh PTD-Fluc dissolved in DMEM com-
plete media to a final concentration of 1–500 nM. Also, prepare
fresh Fluc solution in DMEM complete media (see Note 5).
4. Remove the media from the cells and wash them once with
pre-warmed DMEM complete media, taking care not to detach
the cells.
5. Add 100 μl of PTD-Fluc or control Fluc simultaneously
using a multichannel pipette to the appropriate wells (see
Notes 6 and 7).
6. At suitable time points, wash the cells twice with 1× PBS
without Mg2+ and Ca2+ to remove surface-bound proteins (see
Note 8).
7. To image Fluc, add 100 μl of D-luciferin stock solution to each
well at a final concentration of 150 μg/ml using a multichannel
pipette.
8. Image initially at 1–5 s, 10 bin, f/4 using Living Image soft-
ware, version 3.0. The imaging times and binning can then be
adjusted accordingly (see Note 9).

3.3 Transduction The results of real-time in vivo monitoring of PTD transduction


of PTD-Fluc In Vivo are illustrated in Fig. 3.
1. Prepare 100 μl of fresh PTD-Fluc dissolved in PBS (50 μM
stock solution) to final dose range of 0.1–10 μM/kg body
weight. Also prepare fresh Fluc in PBS at the same concentra-
tion as PTD-Fluc (see Note 5).
2. Inject 100 μl of PTD-Fluc or Fluc intraperitoneally into
unanesthetized male ICR mice using a 27-gauge needle (see
Notes 10 and 11).
3. Immediately after injection of each protein, administer 30 mg
of D-luciferin/kg body weight intraperitoneally using a
27-gauge needle (see Note 12).
4. Ten minutes after the injection of D-luciferin, anesthetize the
mice in an isoflurane induction chamber for 1–2 min (mean
time until loss of the righting reflex) and then transfer to an
IVIS-200 imaging chamber (see Note 13).
5. Take a brief test image at 1 min, 10 bin, f/8. Use this image to
estimate the exposure time and binning needed for subsequent
Real-Time Quantification of PTD Transduction 311

images (usually a 1–5-min exposure). Quantify the biolumi-


nescence (p/s) within a region of interest encompassing the
specific area of bioluminescence.
6. Repeat steps 3–5 every 2 h (see Note 14).

4 Notes
1. Hexahistidine (His6)-tagged PTD-Fluc and Fluc expressed in
Escherichia coli BL21 (DE3) cells harboring pRSET-PTD-Fluc
or pRSET-Fluc should be purified using an Ni-NTA Fast Start
Kit (#30600, Qiagen) (8, 9). Although affinity tags using His6
are powerful and convenient tools for the purification of
recombinant proteins, you can also use genetically engineered
fusion partners such as glutathione S-transferase (GST), FLAG,
or maltose-binding protein (MBP) (10); however, you should
check whether the tag has a deleterious effect on the biological
properties of the recombinant protein (e.g., effects on solubil-
ity and biological activity).
2. D-Luciferin (potassium salt) can be dissolved up to a concentra-
tion of 50 mg/ml (62.8 mM); it precipitates out at 60 mg/ml.
3. To establish a standard luciferase calibration curve, make a
series of luciferase solutions (10 μl each) ranging in concentra-
tion from 1 pg/ml to 1 ng/ml in elution buffer. Alternatively,
each sample can be serially diluted by a factor of 10.
4. Keep the purified proteins (if possible, maintain at >10 μM
since a high concentration will help prevent a loss of protein
stability) in an Ni-NTA elute buffer supplemented with 10%
glycerol (#30600, Qiagen) at −80°C in small aliquots for up to
a few days. Do not thaw and refreeze; whenever possible, use
either fresh or one-time-thawed proteins.
5. Make sure that the activity and concentration of the PTD-Fluc
protein are accurate and essentially no different from those of
the control (Fluc). You should always perform a cell-free
luciferase assay (see Subheading 3.1) and check the activity and
concentration of each sample prior to running PTD transduc-
tion experiments.
6. Treating the PTD proteins at the same time is important.
PTD-Fluc may appear to be internalized very rapidly (within
minutes), although the response to treatment is dependent on
cell type. Figure 2 illustrates the real-time monitoring of PTD
transduction in living cells.
7. Check the viability of the cells under a microscope before pro-
ceeding to the next step. The criteria for determining the max-
imal PTD-Fluc concentration depend on cellular viability.
8. If you wish to test for PTD transduction in a matter of min-
utes, this step can be skipped (i.e., go on to BLI) because
312 Mi-Sook Lee and Song Her

Fig. 2 Real-time imaging and quantification of PTD-Fluc transduction. Time-


course representative image (upper panel ) and quantification of luciferase activity
in the presence of 40 nM Fluc or PTD-Fluc (lower panel ). The data represent the
mean ± SEM for each assay, performed in triplicate with three independent
experiments. Colored bars indicate the bioluminescence signal intensity (p/s/
cm2/sr) (Reproduced with permission from ref. 8)

ATP-free extracellular or surface-bound PTDs may not affect


the luciferase reaction. This may be confirmed by comparing
the results of PTD-Fluc and control (Fluc) transduction
(Fig. 2).
9. The level of bioluminescence is directly proportional to the
exposure time, depending on the level of PTD-Fluc transduc-
tion. If you are unsure of what exposure time to use, start with
low-sensitivity settings and increase as necessary (typical range,
0.5–1 min).
10. In general, the maximum intraperitoneal injection volume
should not exceed 10 ml/kg body weight for adult mice.
11. Intraperitoneal administration is the injection of a substance
into the peritoneal cavity. Note that intraperitoneal delivery is
difficult to perform correctly, as you can easily misplace the
dose into the intestine, gut, urinary bladder, muscle, or other
organs. To avoid puncturing the abdominal viscera, hold the
animal with its head tilted downward and insert the needle
quickly into the lower left of the midline umbilicus.
Real-Time Quantification of PTD Transduction 313

12. The first image capture after PTD-Fluc injection should be


delayed by approximately 15 min because it takes time to reach
peak luminescence output from intact cells after D-luciferin
injection (Fig. 3).

Fig. 3 In vivo real-time imaging and quantification of PTD-Fluc transduction. ICR


mice were injected intraperitoneally once with 10 μM/kg PTD-Fluc or Fluc. Next,
the mice were given an injection of D-luciferin at 30 mg/kg after 15 min, 1, 2, 4,
6, 8, and 10 h. Anesthesia and BLI were performed as in Subheading 3.3, steps
4 and 5. Representative images from the same mouse in the Fluc-treated group
(n = 4) and PTD-Fluc-treated group (n = 6) are displayed as pseudocolor images
of peak bioluminescence, with variations in color representing the light intensity
at a given location (upper panel ). Red represents the most intense light emission,
while blue corresponds to the weakest signal. The colored bar indicates the bio-
luminescence signal intensity (p/s/cm2/sr). The mice were imaged with an inte-
gration time of 3 min at a binning of 10. The dotted circle indicates the region of
injection. Quantification of in vivo tracking by measuring the exterior luciferase
activity (lower panel). Exterior activity was calculated by subtracting the activity
in the dotted circle from the total activity. Error bars represent the SEM.
*Significantly different from the Fluc-injected control group (p < 0.05); **p < 0.01
(Reproduced with permission from ref. 8)
314 Mi-Sook Lee and Song Her

13. The mice should be anesthetized with 3.0% isoflurane delivered


in 100% oxygen in a gas anesthesia induction chamber prior to
imaging. Once the mice have been anesthetized, they should
be moved onto the imaging stage; anesthesia should be main-
tained with 1.0–3.0% isoflurane in 100% oxygen inside the
IVIS-200.
14. One of the advantages of BLI is that a mouse can be imaged
repetitively (e.g., before and after drug administration). This
strategy allows each mouse to serve as its own control, which
reduces experimental variation.

Acknowledgment

This study was supported by a KBSI grant (T31901) to S.H.

References
1. Johnson RM, Harrison SD, Maclean D (2011) odomain-derived cell-penetrating peptides:
Therapeutic applications of cell-penetrating interaction in a membrane-mimicking environ-
peptides. Methods Mol Biol 683:535–551 ment and cellular uptake efficiency.
2. Jarver P, Mager I, Langel U (2010) In vivo Biochemistry 45:1408–1420
biodistribution and efficacy of peptide mediated 7. Greer LF 3rd, Szalay AA (2002) Imaging of
delivery. Trends Pharmacol Sci 31:528–535 light emission from the expression of luciferases
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Cell-penetrating peptides. A reevaluation of Luminescence 17:43–74
the mechanism of cellular uptake. J Biol Chem 8. Lee MS, Kwon EH, Choi HS et al (2010)
278:585–590 Quantification of cellular uptake and in vivo
4. Fischer R, Waizenegger T, Kohler K et al tracking of transduction using real-time moni-
(2002) A quantitative validation of fluorophore- toring. Biochem Biophys Res Commun
labelled cell-permeable peptide conjugates: 394:348–353
fluorophore and cargo dependence of import. 9. Choi JM, Ahn MH, Chae WJ et al (2006)
Biochim Biophys Acta 1564:365–374 Intranasal delivery of the cytoplasmic domain
5. Burlina F, Sagan S, Bolbach G et al (2005) of CTLA-4 using a novel protein transduction
Quantification of the cellular uptake of cell- domain prevents allergic inflammation. Nat
penetrating peptides by MALDI-TOF mass Med 12:574–579
spectrometry. Angew Chem Int Ed Engl 10. Murphy MB, Doyle SA (2005) High-
44:4244–4247 throughput purification of hexahistidine-
6. Balayssac S, Burlina F, Convert O et al (2006) tagged proteins expressed in E. coli. Methods
Comparison of penetratin and other home- Mol Biol 310:123–130
Chapter 29

Genotoxic Assessment of Carbon Nanotubes


Olivera Nešković, Gordana Joksić, Ana Valenta-Šobot, Jelena Cvetićanin,
Djordje Trpkov, Andreja Leskovac, and Sandra Petrović

Abstract
Carbon nanotubes are unique one-dimensional macromolecules with promising application in biology
and medicine. Since their toxicity is still under debate, here we describe an investigation of genotoxic
properties of purified single-walled carbon nanotubes (SWCNT), multiwall carbon nanotubes (MWCNT),
and amide-functionalized purified SWCNT. We used two different cell systems: cultured human lympho-
cytes where we employed cytokinesis-block micronucleus test and human fibroblasts where we investigate
the induction of DNA double-strand breaks (DSBs) employing H2AX phosphorylation assay.

Key words Carbon nanotubes, Genotoxicity, Human cells, Micronuclei, g-H2AX foci

1 Introduction

Carbon nanotubes (CNT) are unique, one-dimensional macro-


molecules, whose outstanding properties have sparked an abun-
dance of research since their discovery in 1991 (1). Single-walled
carbon nanotubes (SWCNT) are constructed of a single sheet of
graphite (diameter 0.4–10 nm), while multiwall carbon nanotubes
(MWCNT) consist of multiple concentric graphite cylinders of
increasing diameter (10–100 nm) (2). Both SWCNT and MWCNT
possess high tensile strengths, are ultralight weight, and have excel-
lent thermal and chemical stability. In combination with their
metallic and semiconductive electronic properties, this remarkable
array of features has seen a plethora of applications proposed.
One of the major areas of CNT research is the field of bio-
medical materials and devices. Many applications for CNT have
been proposed including biosensors, drug and vaccine delivery
vehicles, and novel biomaterials (2). CNT can be used as nano-
fillers in existing polymeric materials to both dramatically improve
mechanical properties and create highly anisotropic nanocomposites

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_29, © Springer Science+Business Media New York 2013

315
316 Olivera Nešković et al.

(3, 4). They can also be used to create electrically conductive


polymers and tissue engineering constructs with the capacity to
provide controlled electrical stimulation (5–7). However, before
such materials can be incorporated into new and existing biomedi-
cal devices, the toxicity and biocompatibility of CNT needs to be
thoroughly investigated. Within the realm of biotechnology, car-
bon nanotubes, a major class of carbon-based tubular nanostruc-
tures, have been utilized as platforms for ultrasensitive recognition
of antibodies (8) as nucleic acids sequencers (9) and as biosepera-
tors, biocatalysts (10), and ion channel blockers (11) for facilitat-
ing biochemical reactions and biological processes. Towards
nanomedicine, an emerging field of utilizing nanomaterials for
novel and alternative diagnostics and therapeutics has been devel-
oped. CNT have been utilized as scaffolds for neuronal and liga-
mentous tissue growth for regenerative interventions of the central
nervous system and orthopedic sites (12), substrates for detecting
antibodies associated with human autoimmune diseases with high
specificity (13), and carriers of contrast agent aquated Gd3+-ion
clusters for enhanced magnetic resonance imaging (14). When
coated with nucleic acids (DNA or RNA) or proteins, CNT have
been shown as effective substrates for gene sequencing and as gene
and drug delivery vectors to challenge conventional viral and par-
ticulate delivery systems (15–19). Consequently, efforts to take
advantage of the physical and chemical properties of CNT in bio-
logical settings must first circumvent the hydrophobicity of these
nanomaterials.
Research over the past decade has shown that CNT and fuller-
enes can be readily modified, either covalently or non-covalently,
by incorporating chemical and biological functional groups for
much enhanced solubility and bioavailability. The covalent
modification of single-walled carbon nanotubes, for example, nor-
mally involves esterification or amidation of acid-oxidized nano-
tubes and sidewall covalent attachment of functional groups
(20–24). However, these covalent schemes are often marred by
undesirable modifications to the physical and chemical properties
of SWCNT (25). Furthermore, such functionalized SWCNT often
have dangling bonds at the defective sites and are prone to gener-
ating free radicals. In comparison, the non-covalent modifications
of SWCNT employ adsorption of proteins, biopolymers and syn-
thetic polymers (DNA, RNA, polyvinyl pyrrolidone, polystyrene
sulfonate), and surfactants (sodium dodecyl sulfate or SDS) to
form supramolecular assemblies (8, 26–34). For both covalent and
non-covalent solubilization schemes, the introduction of surfac-
tants, surface charges, organic solvents, and residues may induce
additional genotoxicity. Developing well-characterized solubiliza-
tion schemes on the base of functionalized nanotubes is thus cru-
cial for facilitating the full range biological and biomedicinal
applications of nanotubes and their derivatives (35).
Genotoxic Assessment of Carbon Nanotubes 317

Structural characterization is necessary while investigating the


influence of carbon nanotubes on the living systems, since previous
investigations showed that the genotoxicity level is different
depending of the length, diameter, total surface, irregularities in
the lattice organization, purity and presence of the functional
groups, and the solubility of carbon nanotubes (36).
Considering numerous possible interactions between CNT
and biomolecules, as well as their existing and potential applica-
tions in biological sciences and other fields of science and technol-
ogy, it is of great interest to determine their influence on living
systems.
Here we present a study investigating genotoxic properties of
carbon nanotubes: purified SWCNT, MWCNT, and amide-func-
tionalized purified SWCNT on cultured human lymphocytes
employing cytokinesis-block micronucleus test and enumeration of
g-H2AX foci in human fibroblast cell line.

2 Materials

2.1 Laboratory 1. Laminar flow cabinet.


Equipment 2. Ultrasonic bath.
3. Incubator.
4. Water bath.
5. Centrifuge.
6. Vortex mixer.
7. Micropipette.
8. Microscope.
9. Microscope slide staining jar.
10. Microscope with illuminator for fluorescence microscopy.

2.2 Solutions 1. Carbon nanotubes dispersion: Disperse 5 mg of each carbon


nanotube sample (SWCNT, MWCNT, and amide-functional-
ized purified SWCNT) into 1 ml of 96% ethanol. Sterilize in an
autoclave. Sonicate prior to use for 30 min.
2. Cytochalasin B stock solution: Dissolve 10 mg cytochalasin B
into 33.3 ml DMSO in laminar hood. Filter DMSO with sin-
gle-use syringe filter with pore sizes 0.20 mm. Aliquot in 2 ml
safeseal micro tubes (see Note 1).
3. Hypotonic solution: Mix equal volumes of 0.56% KCl and
0.9% NaCl water solutions. Calculate final volume according
to number of specimens. Keep in wash bottle in water bath at
37°C (see Note 2).
318 Olivera Nešković et al.

4. Fixative solution: In 3× volume of methanol (CH3OH), add


1× volume of acetic acid (CH3COOH).
5. SØRENSEN’S phosphate buffer: (Stock solutions: (a) 0.2 M
Na2HPO4 and (b) 0.2 M NaH2PO4) To prepare 100 ml of
working buffer (0.1 M, pH 6.8), mix 24.5 ml of (a) with
25.5 ml of (b). Dilute to 100 ml with ddH2O.
6. 10% Giemsa in SØRENSEN’S buffer: To prepare 100 ml, add
10 ml Giemsa stain to 90 ml of SØRENSEN’S buffer.
7. 1% HCl ethanol solution: To prepare 1 l, add 10 ml 37% HCl
to 990 ml 95% ethanol.
8. 90% ethanol solution: To prepare 100 ml, add 10 ml ddH2O
to 90 ml 100% ethanol.
9. 70% ethanol solution: To prepare 100 ml, add 30 ml ddH2O
to 70 ml 100% ethanol.

2.3 Cell Culture 1. Human dermal fibroblasts HDMEC (PromoCell GmbH,


Components Heidelberg, Germany).
2. T25 tissue culture flasks.
3. Dulbecco’s modified Eagle’s medium (DMEM).
4. Fetal bovine serum.
5. 0.05% tripsin-EDTA.
6. Polyprep glass slides.
7. Disposable Petri dishes (100 mm).
8. Lithium heparin BD Vacutainer® tubes with BD Hemogard®
closure (Becton-Dickinson, Franklin Lakes, NJ, USA).
9. PB-MAX karyotyping medium (Invitrogen-Gibco, Paisley, UK).

2.4 Immunofluore- 1. 1× phosphate-buffered saline (PBS): To prepare 1 l, add 8 g


scence ( g-H2AX NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 to
Assay) Components 800 ml deionized H2O. Adjust pH to 7.4. Adjust volume to
1 l with deionized H2O. Sterilize by autoclaving and store at
room temperature.
2. 4% formaldehyde: 10 ml 37% formaldehyde dilute in 90 ml
PBS. Prepare fresh (see Note 3).
3. 0.2% Triton-X: Add 200 ml Triton-X (Sigma-Aldrich Co.,
Steinheim, Germany) to 100 ml deionized PBS. Store at 4°C.
4. 1× TBS-T (Tris-buffered saline Tween-20) buffer: To prepare
1 l, add 8.8 g NaCl, 0.2 g KCl, 3 g Tris-base, and 500 ml Tween
20–800 ml ddH2O. Adjust pH to 7.4. Adjust volume to 1 l with
ddH2O. Sterilize by autoclaving. Store at 4°C (see Note 4).
5. 0.5% BSA: Dissolve 0.05 g bovine serum albumin (BSA,
Sigma-Aldrich) into 10 ml deionized PBS.
Genotoxic Assessment of Carbon Nanotubes 319

6. Primary antibody: Anti-phospho-H2AX (Ser 139) mouse


monoclonal antibody (Upstate Cell Signaling Solutions), dilute
in 0.5% BSA (v/v = 1:500).
7. Secondary antibody: Antimouse fluorescein isothiocyanate
(FITC) antibody, dilute in 0.5% BSA (v/v = 1:400).
8. 4,6-Diamidino-2-phenylindole (DAPI)-containing antifade
solution (Vector Laboratories Inc., Burlingame, CA, USA).
9. Coverslips (22 × 50 × 0.13 mm).
10. Clear, nonfluorescent nail varnish.

3 Methods

3.1 Cell Culture- 1. Collect fresh blood by venepuncture in 6 ml lithium heparin


Human Blood Cells BD Vacutainer® tubes with BD Hemogard® closure. Store
blood at 4°C prior to procedure.
2. Aliquot PB-max karyotyping medium (Invitrogen-Gibco,
Paisley, UK), 4.5 ml in each 10 ml sterile tissue/culture test
tube in laminar flow hood. Add 0.5 ml of whole blood. Close
the cap of the test tube and put into the incubator.
3. Keep cell culture in an incubator at 37°C. One hour after the
stimulation of cells, add the agent of interest to cultures: dif-
ferent volumes of carbon nanotubes dispersion (25, 50, 100,
and 150 ml per 5 ml of total cell culture volume) and one
untreated specimen as a control.

3.2 Micronuclei 1. Add 0.1 ml of the cytochalasin B solution after 44 h of culture,


Preparation: and then incubate for next 28 h.
Micronucleus Assay 2. Centrifuge cell suspension the next day (after 72 h of cell har-
vesting) at 500 × g for 10 min.
3. Remove the supernatant.
4. Resuspend pellet on vortex mixer and add pre-warmed hypo-
tonic solution.
5. Keep in water bath for 5 min at 37°C.
6. Centrifuge at 500 × g for 10 min.
7. Remove supernatant up to 1 ml and add fixative solution on a
vortex mixer to 12 ml.
8. Leave samples at room temperature for 30 min.
9. Repeat steps 6 and 7 until the suspension is clear.
10. After last centrifugation, aspirate up to 0.5 ml. Resuspend pel-
let with Pasteur pipette and prepare slides (37).
320 Olivera Nešković et al.

3.3 Preparing Slides 1. Degrease slides with detergent, wash thoroughly with distilled
for Micronucleus water, and keep over night in 1% HCl ethanol solution. Prior
Assay to use, wash slides with distilled water and bi-distilled water.
2. Onto clean, dry slides, put three drops of the cell suspension.
3. Air-dry the slides.
4. Stain slides (in staining jar) with 10% Giemsa in SØRENSEN’s
buffer (pH 6.8) for 10 min.

3.4 Slide Scoring 1. Score at least 1,000 binuclear (BN) cells per sample. Analyze
for Micronucleus slides with a microscope using magnification 400× or 1,000×
Assay when necessary.
2. Score a minimum of 1,000 binucleated cells to evaluate the
percentage of cells with one, two, three, four, or more than
four micronuclei.
3. Calculate a cytokinesis-block proliferation index (CBPI) as fol-
lows: CBPI = MI + 2MII + 3(MIII + MIV)/N, where MI–MIV
represents the number of cells with one to four nuclei, respec-
tively, and N is the number of cells scored (38).

3.5 Cell Culture- 1. Incubate normal human dermal fibroblasts HDMEC in tissue
Human Fibroblasts culture flask for 48 h under standard tissue culture conditions
in DMEM, supplemented with 10% of fetal bovine serum at
37°C and in the atmosphere of 10% CO2.
2. When reach 80–90% confluence, remove growth medium from
the flask by aspiration.
3. Incubate the flask with 1 ml 0.05% Trypsin-EDTA at 37°C for
4–7 min. Examine the flasks microscopically to make sure the
cells begin to round. The cells should detach from the flask
surface after 7 min (see Note 5).
4. Tighten cap and lightly tap the side of the flask to lift the
remaining cells from the flask. Wash the sides of the flask with
growth medium to inactivate the trypsin (see Note 6). Gently
mix cells and medium. Pipette the cell suspension up and down
so as to obtain a suspension of individual cells.

3.6 Immunofluore- 1. Distribute 1 ml aliquots of the cell suspension to polyprep


scence ( g-H2AX slides (see Note 7).
Assay) 2. Transfer polyprep slides into Petri dishes (see Note 8).
3. Add appropriate dose of carbon nanotubes (0.5–30 ml per/ml)
into cell suspension seeded on the polyprep slide. Close the
dish and incubate at 37°C in a humid atmosphere for the next
24 h (see Note 9).
4. Wash slides in PBS for 5 min.
Genotoxic Assessment of Carbon Nanotubes 321

5. Fix cells in fresh 4% formaldehyde (see Note 3) for 15 min.


6. Permeabilize cells in 0.2% Triton-X at 4°C for 10 min.
7. Block reaction by transferring slides in 0.5% BSA/PBS for
30 min, at room temperature.
8. While blocking, prepare primary antibody.
9. Aspirate blocking solution and apply 100 ml diluted primary
antibody to each slide. Incubate for 1 h in a light-tight damp
container.
10. Wash slides three times in TBS-T for 3 min each.
11. Incubate slides with 100 ml secondary anti-goat antibody con-
jugated with Fluorescein isothiocyanate (FITC) for 2 h in a
light-tight damp container.
12. Wash slides three times in TBS-T for 3 min each.
13. Fix cells with 70, 90, and 100% ethanol, 5 min each, and air-
dry in the dark.
14. Counter stain cells with 15 ml 4¢,6¢-diamidino-2-phenylindole
(DAPI)-containing antifade solution and cover with coverslips.
Apply nail varnish to seal the samples.
For the best results, examine specimens immediately. For
long-term storage (several weeks), store slides at 4°C protected
from light.
15. At least 200 cells should be analyzed to evaluate the number of
g-H2AX positive foci by using the microscope with illuminator
for fluorescence microscopy and the computer software
ImageJ.

4 Notes

1. Store at −20°C.
2. Make it prior to use. Use double distilled water.
3. Formaldehyde is toxic; use only in fume hood.
4. Adjustment of pH should be performed with 1 N HCl.
5. If the cells do not become detached after 7 min, incubate an
additional 1–2 min.
6. Add growth medium at volume equal to or greater than vol-
ume of trypsin added.
7. Sterile pipette must be used.
8. One polyprep slide per Petri dish.
9. All subsequent incubations should be carried out at room tem-
perature unless otherwise noted.
322 Olivera Nešković et al.

Acknowledgments

This work was financially supported by the Ministry of Education


and Science of the Republic of Serbia, under Project No. III 45005
and Project No. 173046.

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Chapter 30

Separation Science: Principles and Applications


for the Analysis of Bionanoparticles by Asymmetrical
Flow Field-Flow Fractionation (AF4)
Alexandre Moquin, Françoise M. Winnik, and Dusica Maysinger

Abstract
Field-flow fractionation is an analytical technique that allows the separation of particles over a size range,
from a few nanometers to several microns in diameter. The separation takes place under mild conditions
and is suited for the analysis of neutral or charged particles. A single measurement yields the size and con-
centration of each component of a mixture. However, developing a suitable fractionation method can be
tedious and time-consuming. In this chapter, we present asymmetrical flow field-flow fractionation (AF4)
conditions that have proven their reliability for the analysis of quantum dots and other nanoparticles in the
5–50 nm size range. Common pitfalls are emphasized together with strategies to overcome them.

Key words Asymmetrical flow field-flow fractionation, Quantum dots, Aggregation, Nanoparticles,
Dynamic light scattering

1 Introduction

Field-flow fractionation (FFF) offers great versatility in the types of


sample to be analyzed, and it provides a full sample characterization
in a single measurement typically within 30 min (1–3). Various FFF
subtechniques are available depending on the field applied to the
sample (4–9). They each have specific advantages and drawbacks. For
example, sedimentation FFF (SdFFF), in which the field is gravita-
tional, has a much greater resolving power than the other subtech-
niques, but it is only applicable for particles larger than 100 nm that
have a density significantly different from that of the carrier liquid. In
flow field-flow fractionation (FlFFF), the field is provided by a
crossflow applied perpendicularly to the elution flow. The acronym
AF4 (asymmetrical flow field-flow fractionation) refers to FlFFF for
which the channel is of trapezoidal shape instead of an elon-
gated hexagon (10). Most commercial FlFFF systems are using
AF4, in view of its versatility, relative simplicity, and its wide range in

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_30, © Springer Science+Business Media New York 2013

325
326 Alexandre Moquin et al.

terms of particle size (11–13). In the following section, we present


the principle of the fractionation and review the importance of each
parameter as well as describe step-by-step the process to obtain a frac-
tionation of quantum dots (QDs) and optimize the fractionation
conditions.
Quantum dots are one of the most representative nanoparticles
used in the nanosciences. They are nanometer-sized semiconduc-
tor crystals, which can be made with core or core-shell architec-
ture, and have been extensively used for imaging applications both
in vitro, at the single-cell level (14–16), and in vivo (17–23), mainly
in animals bearing tumors. Their main attractiveness relies on the
fact that their luminescence emission can be tuned by controlling
the size and size distribution of the particles. Because, the emission
maxima are directly related to the sizes, it is possible to have a
qualitative appreciation of the size distribution of a QD sample by
measuring its luminescent emission spectrum. FlFFF can provide
quantitative data on the QD’s (and other nanoparticles) size distri-
bution, presence of aggregates, adsorbed proteins (opsonized par-
ticles), and nanoparticle decomposition (24).

1.1 Principle of Flow In FFF, the separation of the sample takes place inside a narrow
Field-Flow ribbonlike channel clamped between two parallel surfaces through
Fractionation which a field can be applied (Fig. 1). A carrier liquid is pumped
through the channel from the inlet (sample injection) to the outlet
(detector). A parabolic flow profile (Newtonian flow) is established
inside the channel, as in a capillary tube. Flow velocities vary from
0 on the walls to a maximum value in the center of the channel. A
field is applied perpendicularly to the flow direction, while the car-
rier liquid containing the sample flows through the channel. This
induces the transport of the sample towards one wall, creating a
concentration gradient. A diffusion flux in the opposite direction is
induced according to Fick’s law leading to a steady state where
each component of the sample reaches a position at a unique dis-
tance from the wall. Due to the parabolic flow profile, the compo-
nents are transported in the direction of the longitudinal channel
axis at varying velocities, depending on their distance from the
channel walls. Since smaller objects diffuse at a faster rate than
larger ones, the elution from the channel outlet proceeds from the
smaller species to the larger ones. In AF4, three flows are used
(Chapter 18 in (4, 10)): (1) a tip flow is introduced from the top
of the channel, and it is through this flow that the sample is injected
and carried down the channel; (2) a focus flow is introduced from
the midsection of the channel or from the output of the channel,
depending on the instrument. This flow creates a focusing zone as
it meets the tip flow at the top of the channel. The distance between
the input and the focusing zone is controlled by adjusting the rate
of each flow; (3) a crossflow is applied perpendicularly to the direc-
tion of the channel, either passively or with a pump that sucks the
Separation Science: Principles and Applications for the Analysis of Bionanoparticles… 327

Fig. 1 Schematic representation of the channel used in asymmetrical flow field-flow fractionation. The inset
illustrates the laminar flow in the channel

carrier liquid out through an ultrafiltration membrane placed on


the bottom wall of the channel.

1.2 Features The channel is held in place by two blocks. At the channel bottom,
of the Channel there is a stainless steel block fitted with a permeable frit serving as
a support for the membrane. A spacer sheet, 190–800 μm thick, is
placed on top of the membrane. A trapezoidal area is cut out within
this sheet to form the channel. The thickness of the spacer sheet
determines the thickness of the channel, although the channel is
slightly thinner than the spacer itself due to the compression
imposed on the sheet by the two blocks. The actual channel vol-
ume can be determined experimentally by measuring the elution
time of a sample which has a well-known diffusion coefficient at a
given temperature (Chapter 18 in (4)). The second block, made of
Plexiglas, forms an impermeable wall on top of the channel. The
advantage of Plexiglas is that it offers a smooth surface and is trans-
parent, allowing the user to measure the distance of the focusing
line from the top of the channel and to check for air bubbles, leaks,
disturbances in the flow, or sample adsorption on the membrane.

1.3 Features Several membranes are available commercially (Table 1). The
of the Membrane selection of the most suitable membrane for a specific sample is
328 Alexandre Moquin et al.

Table 1
List of membrane materials available for analysis of different sample types in aqueous media

Membrane name Use MW cutoff (kDa) Supplier Catalog number


Regenerated cellulose Analysis of proteins/ 1/10/30 Postnova Z-AF4-
(RC) peptides/antibodies/ MEM-612
viruses/nanoparticles/
carbon nanomaterials

RC amphiphilic Analysis of amphiphilic 10 Postnova Z-MEM-


or cationic polymers AQU-631
Polyethersulfone (PES) Negatively charged 0.3/1/5/10/20 Postnova Z-AF4-
samples/biopolymers/ MEM-611
polyelectrolytes
Cellulose triacetate Proteins/peptides/ 5/10/20 Postnova Z-AF4-
(CTA) antibodies/viruses MEM-613
PVDF hydrophilic Large hydrophilic 30/200 Postnova Z-AF4-
particles/nanopar- MEM-614
ticles/carbon
nanomaterials

based on the following considerations: (1) the membrane material


must be compatible with the solvent (no swelling or dissolution);
(2) the sample analyzed should not adsorb on the membrane; (3) the
membrane surface should be flat, smooth, and devoid of ridges;
(4) thick membranes should be avoided as they may protrude
into the channel upon compression of the spacer, thereby causing
channel clogging; the optimal membrane thickness is 250 μm;
and (5) the molecular weight cutoff (MWCO) of the membrane
is commonly set at 10 kDa. The use of membranes with smaller
MWCO values results in an increase in the system pressure.
If membranes of low MWCO are needed, the crossflow value
should be decreased, keeping the ratio of the crossflow to detector
flow (Vc/Vout) constant. Membranes with higher MWCO values
may lead to significant loss of small species in a sample. The use
of an inappropriate membrane may lead to loss of signal upon
sample injection, an indication that the sample is adsorbed onto
the membrane. This problem can be solved by conditioning the
membrane: several injections of the same sample are performed
until the membrane surface is saturated, such that the subsequent
runs will lead to full sample recovery. It is also possible to choose
a carrier liquid with a different ionic strength and pH or with an
Separation Science: Principles and Applications for the Analysis of Bionanoparticles… 329

added surfactant. For charged samples, a membrane with the


same charge should be used, to prevent electrostatically induced
adsorption of the sample to the membrane.

2 Materials

2.1 Apparatus AF4 experiments were carried out on an AF2000-MT (Postnova


Analytics GmbH, Landsberg, Germany), equipped with a channel
oven for temperature control in the mid-temperature range (MT:
5–80°C), which was left at ambient temperature for this experi-
ment. Three pumps were used: two isocratic pumps (Postnova,
PN1130) to provide the tip flow and focus flow, while a Kloehn
syringe pump (Postnova, PN1610) provided the crossflow. The
degasser (Postnova, PN7505) was placed between the carrier liq-
uid reservoir and the isocratic pumps. Two 0.1 μm VVPP filters
(Durapore, VVLP04700, Millipore) were placed in-line after each
pump. They were changed regularly, as soon as the pressure
increased above 20 bar for a flow rate of 1 mL/min of aqueous
carrier liquid (see Note 1).
The AF4 was equipped online with a UV absorbance detector
from Shimadzu (SPD-20A, sold by Postnova PN3211, operating
from 190 to 700 nm), measuring absorbance at the wavelength of
300 nm, which is strongly absorbed by quantum dots. A
spectrofluorometric detector, also from Shimadzu (RF-10AXL, sold
by Postnova PN3410), was placed after the UV absorbance
detector.
For the negatively charged QDs, a polyethersulfone membrane
(Z-AF4-MEM-611-10KD) was installed in the channel.

2.2 Reagents 1. Deionized water was obtained from a Millipore Milli-Q water
and Chemicals system (18.2 MΩ cm, 25°C) and was further filtered using
0.1 μm VVPP filters for aqueous solutions. The 10 mM NaCl
solution was prepared from the same Milli-Q water and was
filtered using the 0.1 μm VVPP filter. The carrier liquid was
degassed by sonication for 15 min.
2. Bovine serum albumin was purchased from Sigma-Aldrich
(³98%, A7906) as lyophilized powder.
3. Chemicals used to prepare the QDs were purchased from
Sigma-Aldrich and PCI Synthesis and were of technical grade.
The end product was purified by precipitation in methanol and
resuspension in chloroform, before surface ligand exchange
using 3-mercaptopropionic acid (MPA). After suspension in
water, the QDs were separated from the excess MPA by pre-
cipitation in the presence of 1:1 (v/v) water to ethanol and
resuspension in deionized water.
330 Alexandre Moquin et al.

2.3 Sample If possible, the sample should be prepared in the same liquid as the
Preparation (Rewrite: carrier solution. It should be freshly prepared prior to analysis. The
Shorten and Adapt to volume to be injected is determined by the volume of the injection
Materials + Add a loop. It can be tuned by modifying the length or the inner diam-
Section in Met.) eter of the loop tubing. In general, volumes between 3 and 100 μL
are selected. The sample load per injection should be small (1–100 μg/
injection) (25). This will decrease particle-particle and/or particle-
wall interactions that can occur with sample overload, resulting in
reduced resolution or sample loss. The optimal sample load has to
be adjusted to the sensitivity of the detectors in order to obtain
acceptable signal to noise ratios (see Note 2 about Sample over-
loading). Surfactants may be used to increase the solubility and
improve the dispersion of the sample (4). Certain samples can also
be evenly dispersed using vigorous mechanical agitation or sonica-
tion; however, care must be taken to avoid damaging or causing
changes to the surface of the samples.
We have chosen as an example, a QD sample with MPA as a
surface ligand. The CdSe/CdZnS QDs were prepared by follow-
ing the protocol presented by Pons et al. (26). QDs have been
analyzed previously by FFF in environmental studies (27, 28), as
proof of concept (29, 30), or in Zattoni et al. (31) for polymeric
coated QDs.
Two modes exist in FlFFF: the normal mode, for small parti-
cles with diameter between 2 nm and 1 μm, and the steric mode
for particles larger than the micrometer. Depending on the size of
the particles contained in the sample to be analyzed, the method
will have to be adapted. A higher sample load (50–1,000 μg) is
usually required in the steric mode.

2.4 Eluent Virtually any liquid compatible with the sample and the channel/
tubing materials can be used in AF4. Stainless steel channels are
also available for fractionation analysis requiring organic solvents.
All liquids should be degassed and filtered with 0.1–0.2 μm filters
to remove any particulate material before entering the pumps. The
use of an online degasser is recommended to remove residual
microbubbles, which can cause baseline noise in light scattering
detection.
Aqueous buffers should be prepared from deionized water to
which salts are added. Bactericides, such as sodium azide (0.01–
0.02%, 0.05% in case of long-term disuse), may be added to pre-
vent bacterial growth (see Note 3). The ionic strength of the buffer
has to be selected carefully as it affects sample retention time, sta-
bility against aggregation, and adsorption on the membrane. If the
ionic strength is too low, repulsion forces between charged
particles will cause the particles to equilibrate further from the
membrane, and consequently, the sample will elute too quickly.
Hupfeld et al. have studied how the ionic strength and osmotic
pressure of the carrier liquid affects the retention time of liposomes
(32). The pH of the solution affects the retention time of samples
Separation Science: Principles and Applications for the Analysis of Bionanoparticles… 331

carrying pH-sensitive groups (carboxylic acids, amines, etc.). For


protein analysis, the buffer pH has to be different from the isoelec-
tric point of the protein in order to avoid adsorption of the protein
on the membrane. Ethanol is not recommended to use in the long
term as it may cause cracks in the Plexiglas plate (see Note 4).
Viscosity should also be considered when choosing a proper
solvent, as the relationship between the hydrodynamic radius of
the particles and the retention time is dependent on the viscosity.
It is recommended to use solvents of low viscosity (see Note 5).

2.5 Miscellaneous Filters and Tubings: Filters should be placed in-line after each pump
Instrumental Features to prevent contamination of the sample by particulate matter from
the pumps. The filters should be changed frequently (when the
pump pressure reaches 20 bar for 1 mL/min, see Note 5). Selection
of the proper tubing is important in the FlFFF system, since the
inner diameter of the tubing regulates the pressure in different
regions of the system. Tubing with an inner diameter of 0.01 in is
usually satisfactory. The length of the tubing should be kept as
small as possible, mainly in the critical regions such as the tubing
linking the injector to the channel, the channel to the detectors,
and in between detectors. The dead volume contained in this tub-
ing is responsible for band broadening because in the absence of
field, the sample diffuses freely. One should keep in mind that small
inner diameters can introduce shear stress on the sample. For anal-
ysis of shear sensitive samples, it is possible to use larger diameter
tubing, at the expense of resolution. PEEK (polyetheretherketone)
tubing is used for all of the applications of FlFFF in aqueous media.
For organic solvents, such as tetrahydrofuran, PEEK cannot be
used; stainless steel is recommended in this case.
Detectors: For complete characterization of a sample, it is useful to
have more than one detection method. The concentration of the
eluting sample is determined with a UV/Vis absorption detector or
a refractive index detector (see Note 6). A multiangle light scat-
tering (MALS) detector yields the molecular weight and the root-
mean-square (rms) radius of each sample component as it elutes
from the channel. A photon correlation spectroscopy (PCS) or
dynamic light scattering (DLS) detector measures the diffusion
coefficient of the eluting components. The diffusion coefficients
can also be calculated from the species retention times using the
FFF theory. Therefore DLS detection provides an independent
method to confirm the validity of the theory. Other detectors, such
as a fluorescence detector, can be linked to the instrument for
enhanced characterization of samples subjected to analysis. Finally,
an added feature of FlFFF is the ability to collect fractions as they
elute out of the channel. The collected fractions can be analyzed by
complementary techniques, such as scanning electron microscopy,
transmission electron microscopy, inductively coupled plasma, and
mass spectrometry, or used for in vitro biological experiments.
332 Alexandre Moquin et al.

3 Methods
Have ready before sample analysis:

3.1 Channel The channel should be prepared in advance with the appropriate
Preparation membrane and spacer for the sample to be analyzed. It is recom-
mended to change the membrane after 30 runs.
1. Rinse all the elements and soak the membrane and the frit for
a few minutes in Milli-Q water, before assembling the
channel.
2. Place the frit in the bottom block and lay the membrane above
it with the smooth side facing up, making sure it stays aligned
with the frit.
3. Install the spacer above the membrane.
4. Close the channel with the block of Plexiglas.
5. Use a torque wrench to bolt the channel together. The torque
wrench is used to provide a precise and uniform pressure
throughout the channel. Tighten the bolts from the center of
the channel moving outwards in a spiral fashion. The amount
of torque to apply to the bolts depends on the channel type
and should be specified in the manufacturer’s guide (it varies
from 2 to 9 N.m).
After the channel is assembled and placed in a vertical position:
1. Connect the focus flow tubing, and pump the filtered carrier
liquid into the channel at a rate of 1–2 mL/min to fill the
channel and to flush air bubbles towards the remaining chan-
nel openings.
2. Connect the tubings located at the bottom of the channel.
This will force the carrier liquid to move up the channel, evac-
uating the large air bubbles with it.
3. Once the carrier liquid reaches the top of the channel, connect
the tip flow input tubing to the channel, and pump liquid
through it, thereby filling the remainder of the channel until
carrier liquid comes out from the crossflow output. Once the
carrier liquid reaches the top of the channel, the crossflow
tubing can be connected to the channel. Crossflow should be
switched on at 1–1.5 mL/min with a tip flow of 2 mL/min.
This will remove the remaining air bubbles in the channel
through the membrane and the frit. Carrier liquid should be
pumped through the channel at 1 mL/min for 1–2 h to
equilibrate the channel and remove any air bubbles trapped in
the channel. If the frit has been allowed to dry completely,
carrier liquid should be pumped continuously through the
channel for 2–4 days approximately, with a flow rate of
1–2 mL/min.
Separation Science: Principles and Applications for the Analysis of Bionanoparticles… 333

Table 2
Set of flow rates used for the analysis of mercaptopropionic-coated CdSe/
CdZnS quantum dots

Vc/Vout 2 3 4 5
Detector flow Vout (mL/min) 0.5 0.5 0.5 0.5
Crossflow Vc (mL/min) 1.0 1.5 2.0 2.5
Focus flow Vfoc (mL/min) 1.3 1.8 2.3 2.8
Tip flow during focusing (mL/min) 0.2 0.2 0.2 0.2
The crossflow was linearly decreased for all methods from the initial value to 0 mL/min
over a period of 20 min, before being let constant at 0 mL/min for 10 min. The channel
used had a length of 27.5 cm, a width of 2.0 cm, and a thickness of 350 micrometers.

Setting Up the Instrument for Sample Fractionation, Sample


Preparation, and Sample Injection
Setting up the instrument (before the sample preparation and
separation):
1. Turn on the detectors before the sample preparation (you need
about 1 h to establish the baseline conditions).
2. Set the tip flow rate (same as detector flow rate to be used: i.e.,
0.5 mL/min) using AF2000 software (Postnova).
3. Open a “New Run” in the same software and create the method
using the flow rates presented in Table 2.
4. Preset the focusing flows according to the selected method.
5. Open the light scattering software.
6. Create a new template and put all parameters for the sample
and separation conditions (as requested by the light scattering
software and seen on the monitor).
7. Keep the instrument on “stand by” while you prepare your
sample.

3.2 Sample 1. Have QDs (prepared commercially or in-house, at least 35 μL


Preparation and per injection and concentration above 1 mg/mL) in the
Injection medium as the carrier liquid.
2. If required, sonicate or vortex the sample to obtain a homoge-
neous suspension.
3. Use Hamilton microsyringe to withdraw the sample (at least
10 μL more than the volume of the sample loop) from the
tube. Remove any air bubble from the syringe.
4. The inject lever on the instrument should be in the “Inject”
position. Insert the syringe through the septum and release
2–3 μL of the sample (to eliminate any risk of injecting air
bubbles in the sample loop).
334 Alexandre Moquin et al.

5. Change the lever to the “Load” position and inject your sample
(add 10 μL more than the sample loop volume to fully load it).
6. Change the lever position back to the “Inject” position. This
will start the sample fractionation.

3.3 Sample Analysis 1. The signal from the detectors (UV or refractive index,
fluorescence, light scattering) will appear on the monitor.
2. During the elution, the user can already start analyzing the elu-
tion profile and determine how to improve the elution.
(a) The first thing to look at is the following: Is there a reason-
able signal? If no, then the reason might be because of
total adsorption of the sample on the membrane; if that is
the case, see Note 7. Another possibility is that the sample
load is too small. Third possibility is the detectors are not
sensitive enough.
(b) If there is not enough separation between the main peak
eluting while the crossflow is kept constant and the resid-
ual peak eluting after the crossflow has been decreased, the
user can either increase the time the crossflow is main-
tained constant or can decrease the Vc/Vout.
3. Once the run is finished (with or without the rinsing step), it is
good to either rerun the same method using a blank injection,
to rinse the channel and verify if there was any sample adsorp-
tion during the previous run which could have detached dur-
ing the subsequent run, thereby contaminatng it. The other
option is to open the purge valve and flush the channel with a
fast axial flow rate (2 mL/min tip flow) for 10 min. One of the
advantages of FFF techniques is the absence of stationary
phase; therefore, there is no risk of interaction between the
sample and the stationary phase, and flow rates can be changed
rapidly without risking erosion of the stationary phase
material.
4. For the next run, the user can start by opening the same tem-
plate as the previous run and modifying the ratio of crossflow/
outflow, either decreasing it if the sample was retained too much
in the channel or increasing it if the sample eluted too rapidly.
In the first step, outflow rate should be kept the same. Later on,
to improve the resolution, the outflow rate can be increased; it
is better to start with low outflow rates (<0.5 mL/min). Once
an appropriate ratio of crossflow/outflow rate has been determined,
for which there is no sign of peak splitting and the retention time
is short enough, then it is interesting to increase the crossflow
as much as possible in between runs to determine the best one,
as this will increase the retention level (tr/t0) and lead to a bet-
ter resolution between the components (see Note 8).
Separation Science: Principles and Applications for the Analysis of Bionanoparticles… 335

Vc/Vout = 2 Vc/Vout = 2
a b

Hydrodynamic radius (nm)


60
1.40min Vc/Vout = 3 Vc/Vout = 5
1.0 2.06min 1.0
UV absorbance (a.u.)

UV absorbance (a.u.)
Vc/Vout = 4 50
0.8 2.26min Vc/Vout = 5 0.8
40
0.6 0.6
2.41min 30
0.4 0.4
20
0.2 0.2
10
0.0 0.0
0
1 2 3 4 5 6 0 2 4 6 8 10 12 14
Time (min) Time (min)

Fig. 2 (a) AF4 fractogram of the UV/Vis signal at λ = 300 nm plotted vs. time using increasing crossflow/outflow
ratios. (b) Plot of ratios 2 and 5 showing the UV absorbance on the left y-axis and the hydrodynamic radius (Rh)
on the right y-axis plotted as a function of elution time

Another way to visually determine at which crossflow/outflow


rate the components will elute out, one can position the time of
elution on the method template and see during the crossflow
decrease, at which crossflow value the samples start to elute out (see
Notes 9–12).
5. As a result of the QD separation by AF4, one can obtain the
fractogram illustrated in Fig. 2:
The fractogram of QD-MPA was obtained using deionized
water as a carrier liquid and a 10 kDa cut-off polyethersulfone
(PES) membrane as an accumulation wall. The ratio of crossflow
to channel flow was increased from 2 to 5 to observe the effect on
retention time. The UV signals were all normalized to the signal
from the Vc/Vout = 2 condition. By integrating the UV signal as a
function of elution time for each peak, we determined that the
areas were all equal, meaning that there was no loss of sample
material in between the different flow rate conditions presented in
Table 2.
In the first step, 5 mg/mL solution was injected, with an injec-
tion loop of 21.5 μL, which represents a mass of 107.5 μg. The
detector flow rate was set at 0.5 mL/min to avoid excessive flow
rates in the channel. Since the QDs are relatively small particles
(8–16 nm in diameter), we used a higher crossflow rate than the
detector flow rate. We tested four Vc/Vout ratios: 2, 3, 4, and 5, Vc
being the crossflow rate and Vout being the flow rate at the output
of the channel, also called flow rate at the detectors. The fractograms
presented in Fig. 2 show the relative retention time for the various
crossflow to detector flow ratios. Retention time was increased
progressively with the increase of crossflow/channel flow ratio.
Bellow values of Vc/Vout = 3, the peak of the QDs coelutes with the
336 Alexandre Moquin et al.

Vc/Vout = 2
1.2 Vc/Vout = 3 180
Vc/Vout = 4
1.0 160
Vc/Vout = 5

Rayleigh ratio (a.u.)


140

rms radius (nm)


0.8
120
0.6 100
80
0.4 60
0.2 40
20
0.0 0

0 2 4 6 8 10 12 14 16 18 20
Time (min)

Fig. 3 AF4 fractogram showing the Rayleigh ratio signal as a function of elution
time for increasing ratios of Vc/Vout. The rms radius (geometrical radius) is reported
on the right y-axis

void peak. By increasing to 4 and 5, we obtain good separation


between the main peak and the void. However, a shoulder to the
right appears.
By looking at the QELS and MALS signals (Figs. 2 and 3,
respectively), we observe that this shoulder corresponds to larger
aggregates which have formed because of the strong flow rates in
the channel. The Rh plot (Fig. 2) shows aggregates between 10
and 50 nm in hydrodynamic radius. This corresponds to a few QDs
agglomerating together under the influence of the strong crossflow
rates. In Fig. 3, showing the Rayleigh ratio as a function of elution
time, a secondary peak appears for the Vc/Vout ratios 4 and 5, even
though the UV/Vis signal intensity is low, because of the stronger
scattering intensity of larger particles. The agglomerated QD par-
ticles formed are large enough to give precise information on the
rms radius of the eluting particles, as seen in Fig. 3. Non-
agglomerated QDs were too small compared to the wavelength of
the laser, to give useful information on the rms size. They were
smaller than the limit of detection of the MALS detector.
Aggregates, of up to 200 nm in geometric diameter, are formed
under the influence of the crossflow rate.
The effect of increasing the rate of the crossflow for a given
detector flow on the nanoparticle recovery was also assessed, by
integrating the elution peak of the UV/Vis signal as a function of
elution time to obtain the area under the curve (as seen in Fig. 2).
Results show that the recovery, as determined by the area under
the curve, was the same over several injections using the same
sample concentration and while increasing the crossflow/outflow
ratio. Therefore, the increased flow rates in the channel induced
Separation Science: Principles and Applications for the Analysis of Bionanoparticles… 337

Vc/Vout = 2 in ddH2O
Vc/Vout = 2 in 10mM NaCl
1.0
0.0020

UV absorbance (a.u.)
0.8
0.0015

0.0010
0.6
0.0005

0.4 0.0000

1 2 3 4 5 6 7
0.2

0.0

0 1 2 3 4 5 6 7
Time (min)

Fig. 4 Effect of 10 mM NaCl on the fractionation of QD-MPA on a polyethersulfone


membrane. The UV/Vis absorbance at 300 nm is plotted as a function of elution
time

aggregation of the QD-MPA; however it did not lead to increased


sample adsorption to the membrane material.
As observed earlier, the Vc/Vout of 4 and 5 lead to a shoulder.
The Rayleigh ratio shows a more important signal compared to the
UV/Vis detector, as the signal read corresponds to larger particle,
which scatter more light even at lower concentration. The rms
radius is sensitive to particles which are approximately 1/10th of
the wavelength of the laser used. Here, individual QD-MPA are
not seen, as they are too small (10–16 nm); however, the crossflow-
induced aggregation leads to a strong and useful signal.
Keeping the same membrane material (PES), we changed the
carrier liquid, to assess the effect of ionic strength on the stability
of these small ligand-coated QDs. The carrier liquid was changed
to a solution of sodium chloride (10 mM, in deionized water),
which was properly filtered on a 0.1 μm VVPP (Durapore) filter
and degassed. The same fractionation method was used (Vc/Vout = 2,
detector flow = 0.5 mL/min) for both carrier liquid conditions.
Figure 4 shows that there is almost complete adsorption of the
QD sample to the membrane material. The sizes using the sodium
chloride carrier liquid cannot be reported accurately because of the
too low concentration of the sample exiting the channel, but from
the peak shape, it doesn’t appear that the salts caused aggregation
of the eluting QDs.
Through this case study, we have briefly observed the effect of
crossflow/detector flow ratio on the retention of small spherical
nanoparticles; we have seen that by increasing this ratio, we were
able to shift the elution of the particles to longer retention times.
338 Alexandre Moquin et al.

However, we have also observed the limits of increasing the


crossflow/detector flow, as this affected the stability of the particles
being analyzed. This effect was accentuated by the nature of the
sample to be analyzed, which relies uniquely on electrostatic repul-
sion between particles for its stability in suspension. The higher
crossflows were able to force the particles in vicinity which acceler-
ated their precipitation. However, it didn’t seem to affect nega-
tively the particle-wall interactions, as no loss of material was
observed when increasing the crossflow rates. The strength of the
hyphenated technique is that whatever happens in the channel is
picked up by the changes in elution time but also the light scattering
detectors, which provide crucial information for the thorough
characterization of the state of the sample. In this case study, we
were able to quantify and characterize the sizes of the aggregates
formed in the channel, through the combined use of the UV/Vis,
MALS, and the QELS detectors.
The importance of the carrier liquid composition on the analy-
sis was also underlined. In this study, the addition of a salt to the
carrier liquid induced particle-wall interactions. By screening the
surface charges of the QD-MPA as well as the PES ultrafiltration
membrane (pI ~ 2.4) (33), the salts in the carrier liquid caused the
QDs to come in proximity to the membrane. This is why the user
should always do verifications of possible interactions when study-
ing charged samples, as the carrier liquid composition, the mem-
brane material, and the surface properties of the sample play a role
in the good process of the fractionation. The interactions may not
always cause complete adsorption of the sample to the membrane
material; they may slow the elution resulting in overestimation of
the size if using the retention time theory alone. This is an example
why the hyphenation of AF4, by mounting light scattering detec-
tors, offers such a powerful solution to the thorough analysis of
samples of broad size distribution, as the in-line detectors do not
rely on the retention time to measure accurately the sizes. The
fractionation is useful too to separate the species by size and pres-
ent them as monodispersed samples to the light scattering detec-
tors, therefore solving the problem of sizes being biased by the
presence of a few large particles.

4 Notes
1. If available, read the pressure at the tip flow pump and the
system pressure when the tip flow rate is set at 1 mL/min. For
a given carrier liquid, the pressure should not vary too much.
If it has increased significantly, this is sign that there is blockage
in the channel or, more frequently, that the filter placed after
the pump needs to be replaced.
2. Sample overloading usually occurs for sample loads above
100 μg. It is caused by the relatively small volume in which
Separation Science: Principles and Applications for the Analysis of Bionanoparticles… 339

the separation occurs (usually in a 5 μm high layer above the


channel wall). Each particle requires a certain volume to reach
equilibrium; if the sample is too concentrated, then the parti-
cles cannot reach equilibrium because of steric interference
with each other, leading to cases of precipitation and aggrega-
tion if the particles are attracted to one another. For charged
particles in which there is significant particle-particle repul-
sion, the particles need even more volume to equilibrate;
therefore, the sample loads have to be smaller (about 1 μg).
Signs of overloading are peak fronting and digitation. To test
for sample overloading, several runs with varying sample
loads are necessary; if the retention time does not vary with
the sample load, then there is no overloading.
3. The amount added must be taken into consideration in the
calculation of the ionic strength. Solutions containing sodium
azide should be disposed of appropriately.
4. Ethanol should be avoided as a carrier liquid if it is used for pro-
longed periods of time as it can cause the Plexiglas to crack.
5. If buffers are used, the usual procedures should be used to
flush the pump pistons to remove the crystalline materials,
from the salts, that may damage the pump seals.
6. Refractive index detectors are sensitive to pressure and cannot
withstand pressures in excess of 100 psi; therefore, they should
be placed last in-line of the detectors. Large inner-diameter
tubing should be used as output to reduce as much as possible
back pressure.
7. To determine if there is sample adsorbing to the membrane,
inject the same sample using the same method and measure the
concentration eluting out; if there is increase or decrease of sig-
nal intensity and area under the peak, then it is possible that the
sample is interacting with the membrane. Possible solutions:
Membrane pretreatment; changing the membrane; changing
the salt concentration of the carrier solution; injecting more
sample to have enough at the detectors: use preconcentration
of the sample by injecting several times the same sample during
the focusing period, or use a slot pump to remove the excess of
solvent contained above the sample layers.
8. Increasing the crossflow rate/outflow rate ratio will also lead
to a proportional increase of system pressure.
9. It is important not to decrease the crossflow rate too quickly,
as it may cause sample to be released by the channel prema-
turely, because of disturbance caused in the channel laminar
flow by rapid crossflow decrease. This can also cause the system
pressure to increase rapidly, causing significant pressure drop
once the crossflow reaches zero, which often causes a small
depression in the signal read at the detectors, as well as the
340 Alexandre Moquin et al.

unknown complex effects occurring in the channel when the


system’s pressure changes abruptly.
10. It is usually not recommended to keep the sample retained in
the channel for too long with a too high crossflow rate/outflow
rate, if it is not required by the sample size, as sample can inter-
act with itself (particle-particle) causing aggregation and
appearance of larger particles or can interact with the mem-
brane, causing sample loss due to reversible/irreversible
adsorption and therefore poor recovery and signal intensity.
11. If bubbles appear in the syringes of the crossflow pump, this may
be caused by two problems. Either, upon using high crossflow
rates, there is cavitation occurring in the syringes which may be
caused by an air leak. The solution to this is to change the whole
syringe by ordering a new part or to find the leaky gasket and
change it. The other possibility is that air bubbles were intro-
duced in the channel and they have been pumped out of the
channel by the crossflow pump. This may be because of a too
weak axial flow rate and a too strong crossflow rate. If this is the
case, the system pressure will indicate null pressure in the system,
which means that solvent is backing up from the output back
into the channel and out by the crossflow pump. To resolve this
problem, it is necessary either to disassemble the channel and
rewet the membrane and frit which may be dry or to pump
solvent at a high flow rate while opening the purge valve
12. If an acute peak is observed during the elution of the sample, this
may have been caused by the passage of an air bubble through
the detectors; this usually means that the bubble will be seen on
the signals of several detectors in the order of their placement
in-line of the instrument. If this peak reappears when repeating
the same run, then it’s possible that it is caused by a drop in the
pressure from the channel to the detectors and can be solved by
using a small length of thinner tubing between the channel and
the detector, therefore compensating for the drop of pressure.

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Chapter 31

Polymersomes-Mediated Delivery of Fluorescent Probes


for Targeted and Long-Term Imaging in Live Cell Microscopy
Irene Canton and Giuseppe Battaglia

Abstract
Fluorescent microscopy becomes an essential tool for live imaging analysis of complex biological pathways
and events as it enables noninvasive real-time/real-space imaging. The design of fluorescent probes to
provide dynamic information and long-term tracking of samples without altering physiological and struc-
tural integrity is critical in live imaging. In recent years, nanotechnology has produced a new generation of
imaging probes with promising applications in live imaging. In particular, we describe the use of pH-sen-
sitive amphiphilic block copolymer PMPC25-PDPA70. This polymer forms biomimetic nanometer-sized
vesicles (known as polymersomes) that are readily uptaken by a wide variety of cell types. The pH sensitiv-
ity confers much needed endolysomal escape capability without inducing cellular toxicity or stress. Two
different characteristic compartments in the polymersomes (hydrophilic core and hydrophobic membrane)
allow for encapsulation of different labeling cargoes such as lipidic cell membrane probes, quantum dots,
fluorescent dyes, and fluorescent biomolecules such as nucleic acid and protein probes.

Key words Live imaging, Polymersomes, Fluorescent probes, Endocytic uptake, Intracellular delivery

1 Introduction

The use of fluorescence microscopy has become essential in biology


and biomedical sciences for the study of functional and structural
aspects at the cellular, tissue, or whole animal level. Due to recent
technical advances (1, 2) and compared to many other imaging
techniques, fluorescence microscopy today achieves high spatial res-
olution; it is very sensitive and specific with safe and relatively easy
detection procedures. One of the biggest advantages is perhaps that
it allows compatibility with living specimens, thus providing real-
time dynamic studies of biological events. However, live imaging is
a fairly recent technology prompted by the introduction of video
signal techniques in the 1980s. Fluorescence imaging has tradition-
ally been performed on fixed samples, and at present, improvements
are very much needed, especially in the design of fluorescent probes
and chemo-sensors suitable to perform live imaging in cells.

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_31, © Springer Science+Business Media New York 2013

343
344 Irene Canton and Giuseppe Battaglia

The ability to bring together multi-functionalities within a sin-


gle particle gives an advantage to nanotechnology in the creation of
such revolutionary imaging devices. These functionalities mainly
include coatings for biocompatibility (3) and/or prolonged half-life
(4), targeting sequences (5) combined with drug encapsulation for
theragnostic applications (6), powerful bioimaging fluorescent dyes
(7), activating fluorescent probes in response to bio-stimuli (8), and
also combination of fluorescent and magnetic labels (9).
To gain the most relevant information in live cellular imaging,
the probe has often to target the inside of cells. Small hydrophobic
molecules can permeate cell membranes with relative ease, but
hydrophilic molecules and especially large macromolecules such as
proteins and nucleic acids require a vector to assist their transport
across the cell membrane. Nanotechnologists have exploited endo-
cytosis as a successful and reliable gate to enter cells in live bioimag-
ing (10, 11). Understanding the uptake process of the fluorescent
nanoparticle from the particular endocytic mechanism to intracel-
lular dynamics of fluorescent nanoparticles is imperative to its bio-
logical application. Furthermore, to enable rational design of
nanovectors, interactions of the probe with the cell as well as with
the biological environment that surrounds the cells need to be
properly understood (i.e., effect of cell seeding density, effect of cell
trypsinization on receptor-mediated uptake, or presence of serum
in the media). Indeed, there are a number of underlying mecha-
nisms that govern the success/failure of the fluorescent probe.
Efficient uptake characterization protocols must demonstrate three
important parameters in nanoformulations: how fast the signal is
detected, how much of the formulation is required to obtain a good
enough signal, and how long can be this signal tracked. The ulti-
mate challenge is to unify all these parameters into an efficient intra-
cellular delivery protocol that additionally causes no unwanted
effects on the cells (i.e., toxicity and cellular stress).
We have developed an efficient nanovector for the delivery of
both hydrophilic and hydrophobic fluorescent imaging compounds
(Massignani PLoS ONE) as well as bioactives within cells (12).
This is based on the self-assembly of pH-sensitive poly(2-methacry
loxyethylphosphorylcholine)-block-(2-(diisopropylamino)ethyl-
methacrylate) (PMPC-PDPA) (13). The PMPC block is highly
biocompatible, while the PDPA block imparts pH sensitivity to the
copolymer. At physiological pH, the copolymer forms colloidally
stable nanometer-sized vesicles, whereas below the pKa of the
PDPA block, rapid dissociation of the vesicles occurs, as the ter-
tiary amine groups on the PDPA chains become protonated. We
have demonstrated that PMPC-PDPA polymersomes are efficiently
uptaken by cells and the cargo is able to escape the endocytic path-
way. The delivery of the fluorophores and bioactives is achieved
without affecting the metabolic activity of the cell or triggering
proinflammatory pathways (14).
Polymersomes-Mediated Delivery of Fluorescent Probes for Targeted and Long-Term… 345

2 Materials
Follow these general aseptic guidelines when using PMPC-PDPA
vesicles for intracellular DNA delivery into mammalian cells. Always
use sterile solutions and, when possible, endotoxin/RNAse-free
reagents/materials. This is especially important when encapsulat-
ing RNA (RNAse-free) or when using highly sensitive stimulation
experiments (cell cultures and/or animal models).
Use low-passage cells, and ensure that cells are healthy and
greater than 90%viable. Transfect cells with PMPC-PDPA poly-
mersomes (both fluorophore and/or bioactive-containing poly-
mersomes) at 70–80%confluence. To increase accuracy and reduce
assay variability, we recommend performing triplicate wells for each
sample condition.

2.1 Polymer Film 1. PMPC25-PDPA70 is poly(2-(diisopropylamino) ethyl


Production methacrylate)-b-poly(2-methacryloyloxy-ethyl phosphoryl-
choline) (PMPC25-b-PDPA70) diblock copolymer (13).
2. Chloroform to methanol solution (2:1).
3. Glass vials (28 ml clear universal vial capped, Reagecon
Diagnostics Limited, Shannon, Co. Clare, Ireland).

2.2 Polymersome HCl 1 M (sterile).


Formation NaOH 1 M (sterile).
PBS pH 2 (sterile).
Sepharose 4B GPC column (sterile).

3 Methods

Procedures are to be carried out at room temperature unless oth-


erwise stated. Figure 1 illustrates few examples of probes that can
be delivered by PMPC-PDPA polymersomes:

3.1 Polymer Film This step is necessary to facilitate subsequent solubilization of the
Making and polymer in aqueous solution. The formation of a thin film is
Encapsulation of required to successfully solubilize the polymer. Maintaining poly-
Water-Insoluble Agents mer to solvent mixture ratios is crucial to obtain a thin film as well
via Stirring Method as the diameter of the glassware used to evaporate the polymer to
solvent mixture. It is recommended to scale up accordingly.
For a 20 mg polymer film:
1. In a clean glass vial weight 20 mg of PMPC25-PDPA70.
2. In a fume cupboard, mix 6 ml of chloroform and 3 ml of
methanol (ratio 2:1) and add it to the polymer glass vial (see
Note 1) gently stirring until the polymer is totally dissolved.
The solution must be clear at this point; if it appears cloudy,
346
Irene Canton and Giuseppe Battaglia

Fig. 1 Confocal micrographs of live primary human dermal fibroblast after 24 h incubation with PMPC-PDPA polymersomes loaded with (a) Rhodamine B octadecyl
ester perchlorate, (b) BODIPY TR ceramide, (c) fluorescein 1,2-dihexadecylphosphatidylethanolamine (DHPE), (d) labeled NBD cholesterol, (e) DNA-staining mem-
brane-impermeable propidium iodide, (f) FITC-labeled antibody antitubulin, (g) labeled nucleic acids, or (h) quantum dots
Polymersomes-Mediated Delivery of Fluorescent Probes for Targeted and Long-Term… 347

discard the solution due to the presence of impurities.


Concentrations of polymer in methanol to chloroform solvent
mixture above 4 mg/ml will produce thicker films upon evap-
oration and will be difficult to solubilize.
3. Water-insoluble dyes or agents to be encapsulated are then
added to the polymer to solvent solution. In general, a 5%molar
ratio (dye to polymer) is necessary.
4. Evaporate the solution overnight under vacuum to obtain a
uniform film (see Note 2).
5. To form the polymersomes and encapsulate hydrophobic dyes,
add 0.1 ml pH 7.4 PBS/mg of polymer in the film and stir
with a magnetic stirrer for 10 days. This ensures complete and
homogeneous solubilization.
6. Once a day for ten consecutive days, sonicate the sample for
30 min to allow “onion-like” structures (multilamellar struc-
tures) to break.

3.2 Hydrophilic Dyes 1. Dissolve the film completely in sterile PBS pH 2, making a
and Bioactive Agents 10 mg/ml polymer solution (see Note 3). Ensure a pH of 2 in
Encapsulation: the polymer solution to optimize the solubilization by adding
Polymersome dropwise 1 M HCL.
Formation via pH 2. Once the polymer film is completely dissolved, filter sterilize
Switch the solution using a 200 nm diameter pore filter (see Note 4).
3. Increase the pH of the solution slowly to pH 6 (see Note 5) by
adding dropwise sterile NaOH 1 M with the help of a 10 μl
Gilson pipette while stirring vigorously with a vortex to avoid
supramolecular aggregations and precipitation of the polymer.
4. When encapsulating water-soluble dyes or agents, add the solu-
tion of the dye or agent at this point (see Note 6). We recom-
mend the following concentrations of hydrophilic agents:
(a) Hydrophilic dyes (i.e., cascade blue, propidium iodide):
5–10%molar ratio (dye to polymer).
(b) DNA: 5 μg/mg polymer in the solution.
(c) siRNA/ODNs (with/without fluorescent labels): 4 μg/
mg polymer in the solution.
(d) Protein (including antibodies): 5 μg/mg of polymer in the
solution.
5. Increase further the pH to 7 to allow the formation of vesicles
and the encapsulation of dyes/agents by adding dropwise ster-
ile NaOH 1 M with the help of a 10 μl Gilson pipette while
stirring vigorously with a vortex.
6. Sonicate the sample for 30 min to allow “onion-like” struc-
tures to break (see Note 7).
348 Irene Canton and Giuseppe Battaglia

3.3 Separation of 1. Harvest the vesicles from the solution by molecular-size fractioning
Encapsulated Fraction in aseptic conditions with a clean and sterile GPC column filled
by Size Exclusion 2/3 of the length with Sepharose 4B (see Note 8).
Chromatography 2. Once the PBS has completely entered the column, add your
sample onto the sepharose carefully (see Note 9) (also, save a
small volume of sample aside, for calculating the encapsulation
efficiency).
3. Straight after the sample has gone inside the column, top the
bed volume up with sterile PBS and start collecting the frac-
tions (see Note 10).
4. An increased turbidity of the collected fractions will reveal
presence of polymersomes. Pull the turbid samples together.
Annotate the final volume of polymersomes.

3.4 Calculation Calculate the size and distribution of the polymersomes using
of Polymersome Size dynamic light scattering (see Note 11). It is always recommend-
Distribution and Size able to use TEM as a complementary technique to confirm poly-
Control Procedures mersome morphology.

3.5 Calculation of 1. Measure the dye/agent concentration by fluorescence or to


the Bioactive/Agent identify the ex/em spectra at pH 6.
Encapsulation 2. Create a standard curve of the agent dissolved in polymersome
Efficiency solution (pH 6) (see Note 12).
3. Measure the absorbance or fluorescence of the samples col-
lected (before column (BC) and after column (AC)) and calcu-
late the concentration from the standard curve data.
4. Calculate the % encapsulation efficiency:
(Concentration AC)/(Concentration BC) × 100

3.6 Cellular Uptake Polymersomes should be added directly in normal cell medium.
of Polymersomes Cell types that grow in suspension (i.e., lymphocytes) as well as
adherent monolayers can actively uptake PMPC-PDPA polymer-
somes, provided that cells are viable and able to do endocytosis.
1. Seed cells on required well size tissue culture plates at an appro-
priate density to achieve 80–90%confluent monolayers after
1–2 days (see Note 13).
2. Add then the polymersomes containing the desired cargo onto
the cells typically in a 1:10 dilution (v/v) directly into the cell
monolayers (see Note 14).
3. Incubate the cells at 37°C in a humidified CO2 incubator for
24 h (see Note 15).
4. Afterwards, image the cells in imaging medium (when using
imaging dyes) or assay the cells for transfection efficiency
(plasmidic, siRNA, and protein cargoes).
Polymersomes-Mediated Delivery of Fluorescent Probes for Targeted and Long-Term… 349

4 Notes

1. Plasticware made of polystyrene will dissolve in a solution with


a high concentration of chloroform. This will add impurities to
the film that will sometimes form a cloudy precipitate. Use
chloroform-resistant plastic or preferably glassware.
2. Ensure that all solvent has evaporated prior to solubilization of
the film in aqueous solution. Failure to remove solvent will
produce precipitates and reduce significantly encapsulation
efficiency of hydrophobic compounds.
3. The concentration of 10 mg/ml is required to maintain the
colloidal stability of the vesicles; above this concentration, the
vesicles aggregate.
4. From this point onwards, the solution is sterile and aseptic
techniques should be followed. The procedures should be car-
ried out in a class II sterile cabinet. Use of sterile or aseptic
accurate pH meter is also necessary from this point.
5. pH 6 is an adequate (not too low) pH for ensuring stability
when encapsulating hydrophilic and bioactive cargoes that are
normally maintained at physiological pH. Molecules that are
negatively charged (i.e., nucleic acids) at pH 6 can interact
with the positive tertiary amine groups, and this can result in
increased encapsulation efficiency. However, strong interac-
tions polymer/encapsulates can also promote precipitation of
the sample. It is recommended to work at lower polymer/
encapsulate concentration if precipitation events occur.
6. We recommend adding the hydrophilic cargoes to the polymer
solution at pH 6 in a maximum volume of 10%(v/v). This is to
avoid dilution of the polymer and maximize encapsulation
efficiency.
7. The sonication time may be tailored to the particular bioactive
to encapsulate. Some cargoes are much more sensitive than
others to sonication, and standardization assays testing stability
of the cargo over sonication time are strongly recommended.
In any case, we recommend also maintaining the sonication
bath at a constant room temperature (or below room tempera-
ture if the samples are sensitive) to avoid overheating and deg-
radation of bioactive samples/dyes.
8. GPC columns can sometimes resist high-pressure sterilization
via autoclave (consult with manufacturer). Otherwise, cleaning
the columns profusely with sterilizing agents (laboratory use
surfactants) followed by overnight sterilization in 70%EtOH is
recommended. Several washes with sterile deionized water
should be performed in a laminar flow class II cabinet.
Sepharose 4B is not autoclavable; it should be sterilized by
350 Irene Canton and Giuseppe Battaglia

suspension (or washing) in 70%EtOH overnight at 4°C.


The next day, wash the sepharose 4B in sterile PBS (at least 10
volumes) to ensure that no ethanol remains. Adapt the length
of the column to the volume of sample intended to put through.
Follow manufacturer’s instructions for best results.
9. During the size exclusion chromatography procedure, ensure
that the surface on the sepharose in the column does not dry
out; this will result in clotting of the sample onto the column.
10. Do not add PBS to push the sample through until your sample
has completely entered the column. This will result in unwanted
dilution of your sample and will decrease the efficiency of
recovery.
11. Size distribution can be controlled using an extruder with a
defined set of pore size membrane. For optimal cellular uptake,
polymersomes should not be bigger in size than 200 nm.
Hydrophilic agents cannot be encapsulated inside hydropho-
bic cores of polymeric micelles; in these cases the lower limit
should be thoroughly controlled, rejecting column fractions
with size below 60 nm.
12. Work within the correct maxima wavelengths of ex/em (equally
for UV/Vis absorption) for your dye/bioactive agent to calcu-
late the encapsulation efficiency. You can use additional dyes to
enhance detection or modify your agents (i.e., DAPI to label
DNA, PicoGreen® to label double stranded RNA, bicin-
choninic acid to detect protein via UV/Vis absorption). To
allow for effective detection, the standard curve should start at
the same theoretical concentration of the cargo in the sample
before passing through the column (original sample) and
should contain serial dilutions down to 1:100 of that original
concentration in the sample.
13. We strongly recommend allowing cells to grow for 2 days in
the tissue culture plates before adding the polymersomes to
the culture, especially after the use of enzymatic products (i.e.,
trypsin) to detach the monolayers. An easy way to increase cel-
lular uptake is starving the cells overnight (no serum for 16 h)
prior to adding the polymersomes in serum containing medium.
This, in general, increases cellular uptake of polymersomes.
14. The 1:10 dilution is an arbitrary value based on the nontoxic
effect of these nanoparticles on most cell types. We advise to
perform toxicity curves for each cell type using MTT tests or
similar. As the nanoparticles are in PBS solution, concentra-
tions above 10%in the medium may produce stress due to the
PBS effect. If required to go above 10 %, it is necessary to add
a toxicity control with the same volume of PBS and no
polymersomes.
Polymersomes-Mediated Delivery of Fluorescent Probes for Targeted and Long-Term… 351

15. Removal of the polymersome medium is not required; however


growth medium may be changed after 24 h of cell contact with
polymersomes. When using plasmidic cargoes, it is also recom-
mended assaying the target gene 24–48 h after transfection
(48–72 h after incubation).

References

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circulating near-infrared fluorescence core- SG, Nel AE, Tamanoi F, Zink JI (2008)
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characterization, and dual nuclear/optical imaging, targeting, and drug delivery. ACS
imaging. Biomacromolecules 8:3422–3428 Nano 2:889–896
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tic angiogenesis. Nano Lett 11:694–700 (2008) Non-cytotoxic polymer vesicles for
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32:1484–1494 Controlling cellular uptake by surface chemis-
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nanotubes for hybridization with metal nano- Bergstrasse, Germany
Chapter 32

Protocol for the Preparation of Stimuli-Responsive Gold


Nanoparticles Capped with Elastin-Based Pentapeptides
Vincent Lemieux, P. Hans H.M. Adams, and Jan C.M. van Hest

Abstract
Stimuli-responsive materials are playing an increasingly important role in a wide range of applications such
as drug delivery, diagnostics, sensors, and tissue engineering. Among them, gold nanoparticles responding
to changes in their surrounding environment are of particular interest due to their size-related optical
properties. Here, we present a novel strategy for the preparation of gold nanoparticles exhibiting a stimuli-
responsive behavior. We rely on the use of a ligand consisting of only a single repeat of the elastin-based
pentapeptide VPGVG. In this contribution, we describe a protocol for the solid-phase peptide synthesis of
thiol-terminated VPGVG ligand, and for the preparation of gold nanoparticles covered with the pentapep-
tide through a ligand-exchange reaction.

Key words Elastin, VPGVG, Gold nanoparticles, LCST, Solid-phase peptide synthesis, Ligand exchange

1 Introduction

Recently, nanomaterials sensitive to changes in their environment have


been at the core of many research programs in the materials science
community. Among this class of materials, gold nanoparticles (Au
NPs) with surface coatings responsive to external stimuli such as light
exposure and variations in pH or temperature have received much
attention. This is not surprising since they are promising candidates in
the development of many new applications in the field of sensory sci-
ences and for the controlled release of active agents (1–6).
To date, the primed strategy for the preparation of thermosensi-
tive particles is the grafting of linear thermally responsive polymers
onto the surface of Au NPs (1–4). By far the most commonly used
polymer is poly(N-isopropylacrylamide) (pNIPAM). When heated at
around 32°C, aqueous solutions of pNIPAM undergo a phase transi-
tion leading to the precipitation of the polymeric chains (7). However,
even on gold surfaces, this lower critical solution temperature (LCST)
can only be varied within a 10°C range, limiting the number of poten-
tial applications (5). In order to broaden the scope of thermally

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7_32, © Springer Science+Business Media New York 2013

353
354 Vincent Lemieux et al.

responsive nanoparticles, novel strategies are required to obtain nano-


particles with flexibly tunable transition temperatures. This could be
achieved by interfacing nanoparticles with stimuli-responsive polypep-
tides/proteins such as elastin-like polypeptides (ELPs).
ELPs are synthetic polypeptides derived from the structural pro-
tein elastin, whose most prominent amino acid sequence is VPGVG
(where V = valine, P = proline, and G = glycine) (8). When heated,
synthetic polymers made of the VPGVG sequence, poly(VPGVG),
undergo a transition from a hydrophilic random coil conformation
to a hydrophobic b-spiral; the ELPs then aggregate and precipitate
(9). Linear poly(VPGVG) is not the only ELP that exhibits this
LCST behavior. The phenomenon has also been observed with
polymers having VPGVG side chains (10–13) and even with single
repeats (14) of the pentapeptide. Moreover, the transition tempera-
ture of these elastin-based structures can be tuned within a wide
temperature range by varying various parameters such as the molec-
ular weight, concentration, and pH (15, 16). Replacing the fourth
residue with any other amino acid (VPGXG), except proline, also
has an important influence on the LCST (15). The idea of coating
gold nanostructures with ELPs has been exploited in the past (17,
18). Thermally and optically responsive gold nanoassemblies have
been prepared using cysteine-containing ELPs. However, the prepa-
ration of such polypeptides involves genetically encoded recombi-
nant methods and is nontrivial to most chemists.
We recently described a novel strategy to prepare thermally
responsive materials based on the use of gold nanoparticles capped
with a layer of only a single repeat unit of thiol-functionalized
VPGVG pentapeptide (19) (Fig. 1). This simple 5-mer peptide
offered the advantages of a facile, efficient, multi-gram compatible
synthesis, and ease of derivatization. In this contribution, we pres-
ent the extended protocol for the preparation of these thermore-
sponsive gold nanoparticles, including the solid phase peptide
synthesis of the thiol-terminated VPGVG ligand and the prepara-
tion of DMAP-capped nanoparticles used for the ligand-exchange
reaction. Figure 2 shows an overview of the protocol.

2 Materials

All solvents and reagents were purchased from Aldrich and used as
received unless indicated otherwise.

2.1 Components for 1. Resin: p-alkoxybenzyl alcohol “Wang” resin with a loading of
the Preparation of the 1.14 mmol/g.
Elastin-Based Ligand 2. Solvent: Dimethylformamide (DMF).
Using Solid-Phase
3. Protected amino acids: 9-Fluorenylmethoxy carbamate protected
Peptide Synthesis
valine (Fmoc Val-OH, >99%), glycine (Fmoc Gly-OH, >99%),
and proline (Fmoc Pro-OH, >99%).
Elastin-Based Stimuli-Responsive Gold Nanoparticles 355

Fig. 1 Schematic of the stimuli-responsive gold nanoparticles and molecular structure of the thiol-terminated
VPGVG ligand

Fig. 2 Overview of the steps for the preparation of VPGVG-capped gold nanoparticles

4. Terminal thiol unit: 3-Mercaptopropionic acid (³99%).


5. Coupling reagents: 1-Hydroxybenzotriazole hydrate (HOBt,
>98%) and N,N-di-isopropylcarbodiimide (DIPCDI, >98%).
6. Deprotection solution: Dimethylformamide (DMF) (J.T. Baker)
solution containing 20% v/v piperidine.
7. Cleavage reagents and scavenger: Trifluoroacetic acid (TFA, 99%)
(Acros); ethane dithiol (EDT, >99%).

2.2 Components for 1. Solution A: Ninhydrin, ethanol.


the Kaiser (Ninhydrin) 2. Solution B: Phenol, ethanol.
Tests
3. Solution C: Potassium cyanide (KCN), pyridine (seccosolv).
356 Vincent Lemieux et al.

2.3 Components for 1. Gold source: Hydrogen tetrachloroaurate trihydrate


the Preparation of the (HAuCl4·3H2O, 99.9 + %).
Gold Nanoparticles 2. Reducing agent: Sodium borohydride (AF granules, 10–40
mesh, 98%).
3. Ligands: Tetraoctylammonium bromide (TOAB, ³98%);
4-(dimethylamino)pyridine (DMAP, 99%).
4. Solvents: Toluene; deionized water with a typical resistivity of
18.2 MW/cm was obtained using a Labconco Water Pro PS
purification system.
5. Dialysis membrane: Spectra/Por molecular porous membrane
tubing with a MWCO of 12,000–14,000 g/mol.

3 Methods

3.1 Synthesis 1. Prepare a suspension of Wang resin (30 g) in DMF (300 mL)
of Fmoc-Glycine- and cool in an ice bath.
Functionalized Resin 2. Add Fmoc Gly-OH (13.5 g, 45 mmol), HOBT (9.20 g,
60 mmol), and DIPCDI (4.30 g, 34.2 mmol).
3. Shake the mixture for 6 h.
4. Filter the functionalized resin and wash repeatedly with dichlo-
romethane (DCM) (3 × 50 mL), DMF (3 × 50 mL), and iso-
propyl alcohol (3 × 50 mL).
5. Resuspend the resin in DCM (300 mL) and cool in an ice
bath.
6. Add benzoyl chloride (10.2 mL) and pyridine (8.4 mL) in
order to cap the unfunctionalized groups.
7. Shake the mixture for 30 min
8. Filter, and wash repeatedly with DCM (3 × 50 mL), DMF
(3 × 50 mL), and isopropyl alcohol (3 × 50 mL).
9. Dry the resin in air and then under vacuum.
10. Determine the loading of the resin (see Note 1).

3.2 Synthesis The Fmoc-VPGVG resin was synthesized by standard solid-phase


of Fmoc-VPGVG- methods using an Fmoc-glycine-functionalized “Wang” resin
Functionalized Resin (20, 21).
1. Mix the Fmoc-Gly functionalized resin (5.0 g, loading
0.64 mmol/g) with DMF (45 mL) and allow the resin to swell
for 20 min.
2. Filter the resin.
3. Add a DMF solution containing 20% v/v piperidine (45 mL).
4. Shake the mixture for 20 min to remove the Fmoc group.
Elastin-Based Stimuli-Responsive Gold Nanoparticles 357

5. Perform a Kaiser test (22) to verify the presence of free primary


amines (positive result) (see Note 2). Repeat steps 2–5 if the
Kaiser test is negative.
6. Filter, and wash repeatedly with DMF (3 × 45 mL).
7. Coupling of the next amino acid: Add to the resin a solution of
Fmoc Val-OH (3.26 g, 9.6 mmol), a 1 M HOBt solution in DMF
(11.5 mL, 11.5 mmol), and a 1 M DIPCDI solution in DMF
(10.6 mL, 10.6 mmol). Dilute to about 45 mL with DMF.
8. Shake the mixture for 45 min.
9. Filter, and wash repeatedly with DMF (3 × 45 mL).
10. Perform a Kaiser test to verify the completeness of the reaction
(negative result) (see Note 2). Repeat steps 7–10 if the Kaiser
test is positive.
11. Repeat steps 2–10 with the following three amino acids in this
order: Fmoc Gly-OH (2.85 g, 9.6 mmol), Fmoc Pro-OH
(3.24 g, 9.6 mmol), and Fmoc Val-OH (3.26 g, 9.6 mmol).
12. Wash repeatedly with DCM (3 × 40 mL), DMF (3 × 40 mL),
and isopropyl alcohol (3 × 40 mL).
13. Dry the resin in air and then under vacuum.
14. Determine the loading of the resin (see Note 1).

3.3 Synthesis of 1. Mix the Fmoc-VPGVG functionalized Wang resin (2.0 g, load-
Thiol-Functionalized ing 0.52 mmol/g) with DMF (45 mL) and allow it to swell for
VPGVG Peptide 20 min.
2. Filter the resin.
3. Add a DMF solution containing 20% v/v piperidine (45 mL).
4. Shake the mixture for 20 min to remove the Fmoc group.
5. Perform a Kaiser test to verify the presence of free primary
amines (positive result) (see Note 2). Repeat steps 2–5 if the
Kaiser test is negative.
6. Filter, and wash repeatedly with DMF (3 × 45 mL).
7. Coupling of the thiol-containing acid: add to the resin a solu-
tion of 3-mercaptopropionic acid (0.26 mL, 3.0 mmol), a 1 M
HOBt solution in DMF (3.6 mL, 3.6 mmol), and a 1 M
DIPCDI solution in DMF (3.3 mL, 3.3 mmol). Dilute to
about 45 mL with DMF.
8. Shake the mixture for 60 min.
9. Filter, and wash repeatedly with DMF (3 × 45 mL).
10. Perform a Kaiser test to verify the completeness of the reaction
(negative result) (see Note 2). Repeat steps 7–10 if the Kaiser
test is positive.
11. Wash repeatedly with DCM (3 × 40 mL), DMF (3 × 40 mL),
and isopropyl alcohol (3 × 40 mL).
358 Vincent Lemieux et al.

12. Allow the resin to dry in air.


13. Cleave the thiol-VPGVG peptide from the resin: Add to the
resin 10 mL of a solution containing 95% of TFA, 2.5% of
water, and 2.5% of EDT to reduce any disulfide complexes
involving the VPGVG peptide.
14. Stir the mixture for 60 min.
15. Precipitate the peptide by adding a third of the TFA solution
(3.333 mL) dropwise to diethyl ether (~40 mL).
16. Centrifuge and decant.
17. Resuspend the solid in diethyl ether, and add dropwise a fur-
ther 3.33 mL of TFA solution.
18. Repeat steps 16 and 17 with the last remaining portion of TFA
solution (3.33 mL).
19. Resuspend the solid in diethyl ether (~40 mL).
20. Centrifuge and decant.
21. Repeat steps 19 and 20 twice.
22. Dry the white solid in air.
23. Purify the peptide by column chromatography using silica gel
and CHCl3/MeOH/water (65:25:4) as the eluent. (Rf = 0.24).
24. From 2.0 g of Fmoc-VPGVG functionalized Wang resin,
461 mg of peptide was obtained (see Note 3).

3.4 Synthesis of The DMAP-stabilized gold nanoparticles were prepared according


DMAP-Stabilized Gold to the method proposed by Curasso (23) as described by Lennox
Nanoparticles (24, 25).
1. Mix an aqueous solution of hydrogen tetrachloroaurate trihy-
drate (30 mM, 30 mL, 0.9 mmol) with a solution of tetraoc-
tylammonium bromide (TOAB) (25 mM, 80 mL, 2.0 mmol)
in toluene.
2. Stir vigorously the biphasic mixture until all the tetrachloroau-
rate has transferred into the toluene layer, giving a deep orange
organic layer and colorless aqueous layer.
3. Add to the stirring mixture a freshly prepared aqueous solution
of sodium borohydride (0.4 M, 25 mL, 10 mmol) over a
period of 2 s. The organic phase should immediately become
deep red as the TOAB-capped gold nanoparticles are formed.
4. Stir the mixture for 90 min.
5. Separate the organic layer from the aqueous layer and wash it
three times with deionized water (3 × 100 mL).
6. Dry the solution over anhydrous sodium sulfate, and filter it.
7. To this deep red colored solution, add an aqueous solution of
DMAP (0.1 M, 80 mL, 8 mmol). The color of the aqueous
Elastin-Based Stimuli-Responsive Gold Nanoparticles 359

phase progressively becomes deep ruby as the phase transfer of


the particles takes place and the ligand exchange from TOAB
to DMAP occurs.
8. Stir the mixture for 60 min.
9. Isolate the aqueous layer containing the nanoparticles.
10. Adjust the concentration of the gold nanoparticles with an
aqueous solution of DMAP (0.1 M, 97 mL) to obtain a solu-
tion with a gold content of 1 mg/mL (see Note 4).
11. Store the DMAP-stabilized gold nanoparticles solution at 4°C
(see Notes 5 and 6).

3.5 Synthesis of 1. Add the thiol-functionalized VPGVG peptide (15.5 mg,


VPGVG-Capped Gold 0.03 mmol) to a stirring aqueous solution of the DMAP-
Nanoparticles stabilized gold nanoparticles (1 mg/mL gold content,
10.8 mL, 0.054 mmol of Au).
2. Stir the solution overnight at ambient temperature.
3. Place the solution in a dialysis bag and dialyze the solution against
deionized water for 24 h, changing the water periodically.
4. Freeze-dry the solution to obtain the VPGVG-capped gold nano-
particles as a deep purple solid (yield: 14.8 mg) (see Notes 7–9).

4 Notes

1. The loading of the resin can be evaluated by mass difference


before and after the peptide coupling.
2. The presence or absence of terminal amino groups on the resin
can be confirmed by a Kaiser (ninhydrin) test. Prepare three
stock solutions: (1) 500 mg of ninhydrin in 10 mL ethanol, (2)
80 g phenol in 20 mL ethanol, and (3) 2 mL 0.001 M solution
of KCN diluted to 100 mL with pyridine. Add 10–20 mg of
resin to a 12 × 75 mm test tube. Rinse the resin with DMF,
centrifuge, and decant. Repeat twice more. Add 2–3 drops of
each stock solution to the resin in the test tube. Place the test
tube in a boiling water bath for 5 min. If the resin beads remain
white/yellow (negative test), the reaction is complete. If the
resin beads become dark blue (positive test), some amino
groups are deprotected.
3. Characterization of the thiol-terminated VPGVG ligand: 1H
NMR (DMSO-d6, 400 MHz): d 12.50 (bs, 1H), 8.30
(t, J = 5.8 Hz, 1H), 8.18 (t, J = 6.0 Hz, 1H), 8.08 (d, J = 8.5 Hz,
1H), 7.61 (d, J = 9.0 Hz, 1H), 4.30 (m, 2H), 4.18 (m, 1H),
3.80–3.40 (m, 6H), 2.63 (t, J = 7.5 Hz, 2H), 2.44 (q, J = 7.5 Hz,
2H), 2.22 (t, J = 7.5 Hz, 1H), 2.10–1.90 (m, 4H), 1.90–1.75
(m, 2H), 0.90 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 6H),
360 Vincent Lemieux et al.

0.82 (d, J = 6.7 Hz, 3H). 13C NMR (DMSO-d6, 100 MHz): d
171.82, 171.16, 170.59, 170.35, 169.87, 168.38, 59.87,
59.22, 55.82, 47.50, 43.24, 42.16, 38.85, 30.86, 30.11, 29.12,
24.52, 20.14, 19.12, 18.90, 18.73, 18.53. IR (solid): n 3,284
(NH); 3,071, 2,965, 2,924, 2,872 (CH); 1,622 (C = O amide
I); 1,530 (amide II); 1,443, 1,410, 1,313, 1,233, 1,208, 1,037
(unassigned). LC-MS for C22H37N5O7S in order of decreasing
intensity: 538.5 (M + Na+), 516.4 (M + H+), 554.4 (M + K+),
560.5 (M − H+ + 2 Na+), 279.2 (M + 2 Na+).
4. The molecular weight of gold is 196.97 g/mol. Since 30 mL
of 30 mM solution of hydrogen tetrachloroaurate trihydrate
were added, 0.9 mmol of gold atoms are present, and thus,
0.9 mmol × 196.97 g/mol (177.3 mg) of gold is present in
solution. To obtain a concentration of about 1 mg/mL of
gold, the total volume of the solution should be adjusted to
177 mL. The volume of the solution should be 80 mL at this
point, and hence, 97 mL of DMAP solution (0.1 M) has to be
added.
5. The DMAP-Au NPs can be kept at 4°C for several months
without apparent degradation.
6. See ref. 23–25 for full characterization details.
7. Upon freeze-drying, VPGVG–Au NPs were obtained as a
black/deep purple hygroscopic powder that could be readily
redissolved in water at temperatures below the LCST to form
clear red solutions. Samples of VPGVG–Au NPs were stable
both in solution and in powder form (freeze-dried), and could
be kept at room temperature under ambient atmosphere. The
solutions remained clear red, and no precipitate formed even
after several months.
8. Characterization of the VPGVG-AuNPs: 1H NMR (DMSO-
d6 + TFA for solubility, 400 MHz) d 8.30 (t, J = 5.8 Hz, 1H),
8.18 (t, J = 6.0 Hz, 1H,), 8.08 (d, J = 8.5 Hz, 1H), 7.61 (d,
J = 9.0 Hz, 1H), 4.30 (m, 2H), 4.18 (m, 1H), 3.80–3.40 (m,
6H), 2.10–1.90 (m, 4H), 1.90–1.75 (m, 2H), 0.90
(d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 6H), 0.82 (d, J = 6.7 Hz,
3H). IR (solid): n 3,284 (NH); 3,071, 2,964, 2,924, 2,872
(CH); 1,621 (C = O amide I); 1,529 (amide II); 1,442, 1,394,
1,311, 1,237, 1,200, 1,033 (unassigned). Transmission elec-
tron microscopy (TEM): An average diameter of 3.2 nm was
obtained from the analysis of several micrographs where the
diameter of at least 200 particles was measured. TGA (on 1.727
and 2.525 mg samples): weight loss of 30% between 250 and
500°C. The number of thiol-VPGVG ligands on the surface of
each Au NP is evaluated to be approximately 211, assuming the
presence of 1,289 gold atoms in the core of a nanoparticle with
an average diameter of 3.2 nm (26).
Elastin-Based Stimuli-Responsive Gold Nanoparticles 361

9. Neutral solutions of VPGVG–Au NPs show no LCST behavior.


This is anticipated since the end-group of the VPGVG ligand
is a free carboxylic acid, which is expected to be largely depro-
tonated at pH around 7. At low pH, the carboxylic acid moi-
eties are protonated, giving the nanoparticles a more
hydrophobic character. Under these conditions, a clear hydro-
philic–hydrophobic transition is observed as the solutions
become turbid upon heating. Summary of the LCSTs: pH 2.1:
14°C, pH 2.8: 25°C, pH 3.0: 31°C, pH 3.3: 41°C, pH 3.6: no
LCST. See ref. 19 for a more detailed description.

Acknowledgments

The authors thank the Netherlands Organisation for Scientific


Research (NWO) and the Fonds québécois de la recherche sur la
nature et les technologies (FQRNT) for their financial support.
Christine Lavigueur is acknowledged for the design of Fig. 1.

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INDEX

A B
Ablation laser .......................................... 140, 141, 143, 147 Basic peptide ...................................................................282
Acidification ....................................................................195 BBSA. See Biotin-amidocaproyl Bovine Serum
Acid-oxidized nanotubes .................................................316 Albumin (BBSA)
ACQ. See Aggregation caused quenching (ACQ) BCECF. See Bis (2-carboxyethyl)-5-(and-6)-
Acridine orange ............................................... 26, 27, 29, 30 carboxyfluorescein (BCECF)
Actin filaments .................................................. 60, 185, 251 Bessel function.................................................................150
Acute cardiogenic shock ..................................................103 Beta (β)-galactosidase..............................................276–279
Adjuvant therapy .............................................................103 Bioavailability ..........................................................282, 316
Adriamycin ......................................................................103 Biocompatible polymers .................................. 197, 203–204
Adsorption ..............................................114, 115, 120, 136, Bioconstructs .......................................................................1
225, 226, 227, 229, 231, 316, 327, 329–331, Biological labeling ...........................................................149
334, 337, 338, 340 Biological sensing ..............................................................81
Adsorption equilibrium constant .............................230–232 Bioluminescence imaging (BLI) ............................. 308, 309,
AF4. See Asymmetrical flow field-flow 311, 313, 314
fractionation (AF4) Biomarker ............................................................................ 1
AFM. See Atomic force microscopy (AFM) Biosensor ..........................................................113, 114, 315
Aggregation caused quenching (ACQ) ........................... 164 Biotin-amidocaproyl Bovine Serum Albumin
Aggregation-induced emission (AIE) (BBSA) .........................................114–116, 118, 120
luminogens ..................................................163–169 Biotinylated ligands .........................................................156
AIE luminogens. See Aggregation-induced emission Bis (2-carboxyethyl)-5-(and-6)-carboxyfluorescein
(AIE) luminogens (BCECF)............................................................... 10
ALBR. See Anionic ligand binding receptor (ALBR) BLI. See Bioluminescence imaging (BLI)
Allergic-type immune reactions ......................................275 Blue copper proteins ........................................................261
Aluminum oxide surfaces ................................................114 BODIPY FL L-cysteine ......................... 172–176, 180, 181
Anionic ligand binding receptor (ALBR) ............. 14–16, 21 Brij 78.......................................................................... 95, 96
Anisotropic nanocomposites............................................315 Brilliant Blue R250 ........................................................... 53
Anisotropy ......................................................................... 69 Brownian motion............................................. 153, 192, 233
Annexin V Alexa fluor 488 ...............................141, 145, 147 BSA-TxR. See Texas red conjugated BSA (BSA-TxR)
Anthracycline ....................................................................99
Antibody-bead conjugates .................................................43 C
Anti-neoplastic agent ........................................................99 Caenorhabditis elegans ........................................... 10, 15, 139
Antioxidant treatments ....................................................101 Cancer .............................................. 1–7, 82, 83, 93, 94, 101
Apoptosis...........................100, 108, 110, 111, 140, 145, 147 Cancer drugs .....................................................................93
Argan oil.................................................................. 103, 105 Carbon nanotubes (CNT) ................................. 27, 315–321
Asymmetrical flow field-flow fractionation Carbon nanotubes, multi-walled ................. 27, 30, 315, 317
(AF4) ........................................................... 325–340 Carboxyfluorescein ............................................................10
Atomic force microscopy (AFM) ....................114, 116, 118, Carboxyl-functionalized QDs .........................................250
120–121, 123, 128, 129, 185–188, 190, 191 Cardiolipin ......................................................................101
AuNP. See Gold nanoparticle Cardiomyocytes ........................ 37, 58, 59, 63, 100, 101, 104
Au tip .............................................................................. 265 Cardiomyopathy .............................................. 100, 101, 103
Autocorrelation function ...................................................76 Cardiotoxicity ............................................................99–112
Autofluorescence .......................................................20, 206 Ca2+ release ..................................................................58, 59
Azurin ......................................................261–264, 266–269 Ca2+ sparks ...................................................................58, 59

Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology,
vol. 991, DOI 10.1007/978-1-62703-336-7, © Springer Science+Business Media New York 2013

363
CELLULAR AND SUBCELLULAR NANOTECHNOLOGY
364 Index

Cationic polymers.....................................82, 84, 85, 89, 328 Diabetes............................................................................. 93


Caveolin ...........................................................251, 255, 258 DIC. See Differential interference contrast (DIC)
CdSe/ZnS core-shell .......................................................250 Differential interference contrast (DIC)...........141, 145, 243
CD spectroscopy. See Circular dichroism (CD) spectroscopy Diffraction pattern...........................................................150
Cell death .................................................................. 25, 101 Diffusivity................................................................ 212, 213
Cell imaging ............................................ 163–169, 195, 263 1,2-dioleoyl-3-trimethylammonium-propane
Cell-mediated nanoparticle delivery ..................................41 (DOTAP) ...................................................... 53, 171
Cell-penetrating peptides (CPPs) .......................... 249, 259, Directionality...................................................................212
281–283, 290, 291 Dissociation constant ......................................................225
Cell signaling receptors ...........................................237–245 1,2-Distearoyl phosphatidyl ethanolamine-methyl-
Cellular sub-domains...................................................57–63 polyethyleneglycol-2000
Cell viability assay ................................... 104, 106–107, 141 (mPEG2000-DSPE) ................................................ 52
Centroid position ....................................................151, 157 Disulfides.......................................... 172, 173, 177, 178, 358
Cetyl alcohol................................................................95, 96 DLS. See Dynamic light scattering (DLS)
Chaotropic effects............................................................101 D-luciferin....................................................... 308–311, 313
Chemical nose .................................................................1–7 DMAP-stabilized gold nanoparticles ......................358–359
Chemotherapy ............................................. 93–97, 101, 103 DNA adducts ..................................................................101
Chlorpromazine ..............................................................251 DNA icosahedra ..........................................................65–79
Circular dichroism (CD) spectroscopy ....................225, 226 DNA scaffold ....................................................................66
Clathrin ............................................................251, 255, 258 Donor/acceptor (D/A) ratio ........................................16, 20
Clathrin-coated vesicles...................................................251 DOTAP. See 1,2-dioleoyl-3-trimethylammonium-
CNT. See Carbon nanotubes (CNT) propane (DOTAP)
Coelomocytes ..............................................................12–22 Dounce homogenizer ...........................42, 43, 46, 48, 50, 53
Colloidal lithography ...............................................114–119 Doxil® ............................................................................... 94
Colloidal stabilities ....................................................82, 349 Doxorubicin (DOX) ............................................ 94, 99–112
Confocal microscopy .............. 33, 34, 58, 185, 213, 250, 288 Drosophila melanogaster..................................................... 139
Congestive heart failure ...........................................100, 103 Drug delivery, in vivo.......................................................152
Controlled drug release......................................................47 Drug delivery system ...................................................93–95
Convolution.......................................................................34 Drug encapsulation .............................................94–95, 344
Core-shell ........................................................ 293–305, 326 3D topography images.....................................................116
CPPs. See Cell-penetrating peptides (CPPs) Dynamic light scattering (DLS)........................6, 38, 68, 69,
Crossflow .................................. 325, 326, 328, 329, 331–340 72, 76–77, 94, 96, 106, 123, 132, 134, 233, 303, 331,
Crossflow rate .......................................... 335, 336, 338–340 348
Crosslinking ............................................ 172–174, 176, 181
CSA. See Cyclosporine (CSA) E
Cyclic voltammetry .........................................................262 E. coli ...........................................................................10, 311
Cyclosporine (CSA) ........................................ 101–108, 110 Elastin ..................................................................... 354–355
Cycophilin ....................................................................... 101 Elastin-based pentapeptides ....................................353–361
CytD. See Cytochalasin D (CytD) Electrochemical scanning tunneling microscopy
Cytochalasin D (CytD) ....................................... 60, 62, 251 (ECSTM).................................................... 261–273
Cytochrome c ..................................................................261 Electrochemical scanning tunneling spectroscopy
Cytokinesis-block micronucleus test ...............................317 (ECSTS) ..................................................... 261–273
Cytoskeletal network .......................................................211 Electron microscopy .................................. 33, 34, 37, 58, 79
Cytoskeleton.........................................58, 60, 185, 186, 192 Electron transport chain ..................................................101
Cytosolic glutathione.......................................................172 Electrophoresis ................................................ 253, 257, 258
Cytotoxicity .............81, 83, 86, 111, 128, 163, 281, 284, 286 Embryogenesis ....................................................................9
Encapsulation volume .......................................................65
D
Endocytic pathway .............................. 20, 21, 237–245, 344
D-alpha (α)-tocopherol ...........................................103, 105 Endocytic vesicles ............................................ 17, 18, 41–46
D-alpha (α)-tocopherol polyethylene glycol Endocytosis ..............................9, 14, 16, 17, 22, 25, 44, 205,
1000 succinate .....................................................103 237, 255, 344, 348
D/A ratio. See Donor/acceptor (D/A) ratio Endoplasmic reticulum ......................................................58
Deconvolution ..................................226, 231, 234, 240, 244 Endosomal markers ............................................... 17, 19, 45
Delivery systems ........................................ 33, 172, 212, 316 Endosomal maturation ................................................10, 14
CELLULAR AND SUBCELLULAR NANOTECHNOLOGY
Index
365

Endosomes Gene expression level ......................................................172


early ....................................................14, 19, 21, 45, 238 Genomics ........................................................................ 275
late ..............................................................14, 19, 21, 45 Genotoxic assessment ..............................................315–321
nanoparticle-containing.........................................42–45 Genotoxicity ............................................................316, 317
recycling...............................................................45, 238 GFP. See Green fluorescent protein (GFP)
Epifluorescence illumination ...........................................145 Glued nanoparticles.................................................212, 221
Equilibrium constant ...............................................225–234 Glycine receptors .............................................................152
Extrusion ..................................................120, 129–132, 135 GNPs, encapsulation of .....................................................74
GNRs, polyelectrolyte-coated
F (PE-GNRs) ..........................................82, 83, 85–89
Gold nanoparticle
Fatty acid ..............................................94–96, 103, 282–286
cationic ..........................................................................4
Fatty acyl chloride ...................................................284–286
functionalized ................................................................2
Feature-point tracking ..................................... 186, 188, 190
Gold nanorods (GNRs)............................................... 81–89
Femtosecond near-IR pulse laser .....................................142
Gold nanorod synthesis ...............................................82, 84
Fermi level ............................................................... 261, 262
Gold (III) porphyrin....................................................96, 97
FFF. See Field-flow fractionation (FFF)
Gold porphyrin nanoparticles......................................93–97
Field-flow fractionation (FFF) .................325, 330, 331, 334
Gold sol ................................................................. 34, 37, 38
Filament-based displacement ..........................................192
G-protein coupled receptors (GPCRs) ................... 238, 245
Filipin .............................................................................. 251
Green fluorescent protein (GFP)........................ 1–7, 10, 12,
Firefly luciferase-tagged PTD (PTD-Fluc) ............ 308–313
15–19, 58, 59, 180, 200, 222
FITC. See Fluorescein isothiocyanate (FITC)
Growth defects ....................................................................9
Fixed cells .................................................196, 197, 199–201
Flow cytometry................................................. 26, 171–183, H
283, 284, 287, 289, 307
Flow field-flow fractionation ...................................326–327 Half icosahedra ................................................ 70, 71, 73, 74
Fluorescein isothiocyanate (FITC) Hematological malignancies ............................................100
dextran .........................................................................75 Hemi-icosahedra .........................................................66, 70
labeling ..............................................................284, 286 Hermaphrodites .............................................. 15–17, 19, 21
Fluorescence ..............................................1, 4, 6, 10, 17, 20, Hexaphenylsilole .............................................................164
25–27, 30, 34, 48, 59, 72, 77–79, 104, 105, 107–112, Hydrodynamic radius (Rh) ................................ 77, 335, 336
145, 147, 154, 156, 163, 164, 166, 172, 173, 175,
177–179, 181–183, 185, 186, 212, 222, 242, 243,
I
245, 250, 254, 255, 256, 283, 287–289, 294, 303, ImageJ .........................................................13, 20, 157, 159,
307, 331, 334, 343, 348 186, 188, 189, 191, 212, 216–218, 220, 240, 243,
Fluorescence recovery after photobleaching (FRAP) ...... 212 244, 321
Fluorescent probes ....................................... 1, 172, 343–351 Imaging ........................................... 13, 27, 48, 81, 116, 141,
Fluorescent silica nanoparticles (FSNPs) ................ 163–169 149, 163, 185, 195–209, 212, 240, 249, 262, 279,
Fmoc solid-phase chemistry ............................................285 293, 307, 326, 343–351
Föerster resonance energy transfer (FRET) ...................... 34 Imaging, ratiometric ..........................................................10
Force transmission ...................................................186, 191 Immunoisolation .........................................................41–46
Fractionation of (QD) quantum dots ......................326, 337 Immunostaining ........................................ 48, 199, 202–203
Fractogram ..............................................................335, 336 Intracellular barriers ................................................206, 275
FRAP. See Fluorescence recovery after Intracellular delivery .................103, 275–279, 281–291, 344
photobleaching (FRAP) Intracellular sub-domains ......................................33–39, 57
Free radical formation .....................................................100 Inverted fluorescence microscope ............................154, 166
FRET. See Föerster resonance energy transfer (FRET) Ionophores...................................................................17, 58
FSNPs. See Fluorescent silica nanoparticles (FSNPs) Ischemia-reperfusion injury .............................................103
Fullerenes ........................................................................ 316 I-switch ............................................................10–18, 20, 21

G J
Galacto-Light Plus™ .............................................. 276, 278 J-aggregates .....................................................................108
Gamma-aminobutyric acid A receptor (GABAA) ........... 152 JC-1 ..........................................................104–105, 108–111
Gene carriers ...........................................................171–183 Jurkat cells ................................................283, 284, 286–289
CELLULAR AND SUBCELLULAR NANOTECHNOLOGY
366 Index

K MDC. See Monodansylcadaverine (MDC)


Mean square displacement (MSD)......................... 157, 158,
Kaiser test kit ...........................................................284, 286 160, 212–216, 218–222
Knockout ........................................................................... 20 Mechanical stress.............................................................185
Mechanotransduction ......................................................185
L
Membrane-bound endosomes .........................................238
Lactobacillus delbruecki ........................................................ 10 Membrane disruption techniques ....................................140
Langmuir-Blodgett films.................................................127 Membrane permeabilization........................................57, 58
Laser ...................................... 13, 69, 76, 121, 123, 139–148, 2-(6-Mercaptoalkyl) hydroquinone ........................ 263, 264,
185, 222, 283, 284, 288, 291, 336, 337 266–267, 269–270
Laser-induced necrosis ....................................................140 3–mercaptopropionic acid ............................... 329, 355, 357
Laser surgery ...........................................................139, 146 Metabolic stability ................................... 282, 283, 288–289
Lauroyl chloride ..............................................................284 Metastasis ............................................................................ 9
LC-MS/MS. See Liquid chromatography-mass Microelectrophoresis .......................................................303
spectrometry (LC-MS/MS) Microinjection .......................................................12, 15–17
Ligand-exchange reaction ...............................................354 Micromanipulators ..........................................................139
Ligand-qdot nanoconjugates ...................................155, 156 Micro/nanofabrication.....................................................114
Linear discriminant analysis ................................................5 Microscopy, ratiometric .....................................................20
LIPEX™ extruder ........................................... 104, 106, 110 Mitochondria ............................................25, 34, 37, 59, 62,
Lipid peroxidation ...........................................................100 63, 99–112, 145, 147, 185, 186, 188, 190, 191
Lipid raft ......................................................................... 249 Mitochondria, fluorescently-labeled ................................185
Lipofectamine® ................................................197, 201, 251 Mitochondrial Ca2+ homeostasis .......................................99
Lipo-oligoarginine-based delivery ...........................281–291 Mitochondrial damage ......................................................99
Lipo-oligoarginine peptide (LOAP) ....................... 283–291 Mitochondrial displacements ..................................185–192
Lipophilic carrier systems ..................................................93 Mitochondrial inner membrane ........................................34
Lipophilic formulations .....................................................94 Mitochondrial intermembrane space .................................34
Lipoplexes ....................................................................... 171 Mitochondrial matrix ........................................................34
Liposomes ..........................................94, 102, 127–136, 331 Mitochondrial membrane permeability transition
Liquid-air interface .........................................................127 (mMPT) ...................................................... 101, 102
Liquid chromatography-mass spectrometry Mitochondrial polarization assay .............................104–105
(LC-MS/MS).......................................47, 48, 51, 52 Mitochondrial redox state................................................101
Listeria ................................................................................ 10 Mitochondrial respiration................................................100
Listeria innocua ................................................................... 10 Mitochondriotropic STTP ligands .................................105
Live cell imaging .............. 156, 197, 204, 205, 212, 216–217 Mito Tracker Red .............................141, 145, 147, 186, 187
Live cell microscopy ................................................343–351 mMPT. See Mitochondrial membrane permeability
LOAP. See Lipo-oligoarginine peptide (LOAP) transition (mMPT)
Luciferase assay ............................................. 83, 87–89, 308 Modular self-assembly strategy .........................................65
Luciferase assay, cell-free .........................................309, 311 Molar ellipticity ...............................................................230
Lysosomal membrane Molecular cargo ...........................................................65–79
damage ..................................................................26, 30 Monodansylcadaverine (MDC) ...................................... 251
integrity .................................................................25–30 Monodisperse .................................................. 180, 293–305
Lysosomal storage disease....................................................9 MSD. See Mean square displacement (MSD)
Lysosomes MTT cell proliferation assay .............................................83
integrity .................................................................25–30 Multi-photon absorption.................................................142
visualization .................................................................30 Multiwalled carbon nanotubes
(MWCNT) ......................................27, 30, 315, 317
M Mutants ......................................................10, 12, 14, 20–21
Macrophage ..........................................27, 30, 45, 48, 51, 53 MWCNT. See Multiwalled carbon nanotubes
Macropinocytosis ....................................................251, 255 (MWCNT)
Magnetic beads............................................................42, 43 Myocardial apoptosis .........................................................99
MALDI mass spectrometry ............................................290 Myocardial protection ...............................................99–112
MALDI-TOF ..........................165, 284, 286, 289, 291, 307 Myocytes, ventricular....................................... 36, 58, 60, 62
Mass spectrometry....................................46, 49, 52, 54, 332 Myristic acid ....................................................................282
MATLAB .................157, 158, 160, 190, 212, 216, 218–221 Myristoyl chloride ...........................................................284
CELLULAR AND SUBCELLULAR NANOTECHNOLOGY
Index
367

N O
Nanocapsules .....................................................................66 Obesity .............................................................................. 93
Nanocarriers ................... 33, 53, 81, 101, 102, 152, 211–222 Oil-based nanoemulsions ................................................101
Nanoconjugates .......................................................155, 156 Oligoarginine ..................................................................282
Nanoconjugates, target-specific .......................................156 Optical trap ..................................................... 139, 142–145
Nanodrugs ....................................................................... 128 Optical tweezers .........................10, 139–141, 143, 144, 147
Nanoelectronic biosensing ...............................................249 Opticution ....................................................................... 140
Nanoemulsions (NEs) ....................................... 96, 101–111 Organelle
Nanoemulsions, mitochondria-targeted ..........................101 removal ......................................................................140
Nanofillers ....................................................................... 315 trapping .............................................................144–145
Nanoformulations................................................ 48, 53, 344 Organo-functional silanes ...............................................293
Nanomachine ....................................................................10 Oscillatory motion...........................................................128
Nanomechanical forces ............................................185–192 Oxygen scavengers.............................................................99
Nanomechanical properties .............................................128
Nanomechanics ...............................................................128 P
Nano-objects ................................................... 33–39, 57–63 Palmitoyl chloride............................................................284
Nano-objects, electronopaque ...........................................34 ParticleTracker .........................157, 159, 160, 186, 188, 189
Nanoparticle-lipid membrane interaction ...............127–136 Particle trajectories .......................................... 186, 212–214
Nanoparticle-protein interaction .............................225–234 Payload ...................................................................... 81, 283
Nanoparticles (NP) PDI. See Polydispersity index (PDI)
cationic ......................................................................6, 7 PDMS wells. See Polydimethylsiloxane wells (PDMS wells)
cell-mediated delivery ..................................................41 PEI. See Poly (ethylene imine) (PEI)
crystalline antiretroviral ................................... 45, 48, 52 Peptide-based carriers ..............................................275–279
drug-loaded .................................................................41 Perimembrane ...................................................................57
Nanosecond UV pulse laser .............................................142 Perinuclear region ............................................................212
Nanosensor ....................................................................9–22 pH
Nanosensor, DNA .........................................................9–22 changes, spatiotemporal ...........................................9–22
Nanostructures surfaces ........................... 114, 115, 118–119 clamping .................................................... 12–13, 16–17
Nanosurgery environmental ................................................................9
laser............................................................................ 140 homeostasis..................................................................14
single-cell...........................................................139–148 mapping .................................................................17–21
Nanotherapeutics.............................................................212 organellar .......................................................................9
Nano-therapy ..................................................................103 responsive probes .........................................................10
NCI. See N-cyano imidazole (NCI) sensitive amphiphilic block copolymere.....................343
N-cyano imidazole (NCI) ..........................68, 70, 73–75, 79 sensitive probes ............................................................10
Near-field scanning optical microscopy (NSOM) ........... 149 sensor ..................................................................... 10, 14
Necrosis ................................................................... 140, 145 sensor, FRET-based...............................................10, 14
Nerve growth factor (NGF) ............................................ 152 triggered nanomachine ..................................................9
NEs. See Nanoemulsions (NEs) Pharmaceutical nanocarriers ....................................211–222
Neuromuscular junction ....................................................10 pHluorin ............................................................................ 10
Neurons ............................................................. 10, 152, 186 Phosphorylation assay .....................................................315
Newtonian flow ...............................................................326 Photobleaching..................... 6, 145, 152, 163, 204, 222, 238
NGF. See Nerve growth factor (NGF) Photon yield ....................................................................151
Ninhydrin test ......................................... 284, 290, 355, 359 Photostable nanoparticles ................................................239
Nona-arginine .................................................................250 Photothermal therapy ........................................................81
Non-invasive real-time/real-space imaging .....................343 PicoSPM microscope ......................................................123
Nonradiative relaxation channel ......................................164 Piezoelectric properties....................................................128
Nonviral gene delivery .......................................................81 Piezo voltage ...................................................................268
Non-viral vectors .............................................................171 Pit-spanning phospholipid bilayers .........................113–123
NP. See Nanoparticles (NP) Pitted surfaces .........................................................114, 122
NSOM. See Near-field scanning optical microscopy Place exchange reaction ...................................................2, 6
(NSOM) Plasma membrane permeability.......................................281
Nystatin ........................................................................... 251 Plasmid DNA...........................81–83, 86–89, 174, 201, 222
CELLULAR AND SUBCELLULAR NANOTECHNOLOGY
368 Index

Plasmid DNA, delivery ...............................................81–89 Quartz crystal microbalance with dissipation monitoring
Platonic solids....................................................................65 technique (QCM-D) ...........................113–118, 120,
P
LL. See Poly (L-lysine) (PLL) 121, 123, 127–136
pNIPAM. See Poly(N-isopropylacrylamide) (pNIPAM) Quencher ..........................................................69, 72, 77–79
Point-spread function (PSF).............................150, 151, 157 Quenching
Polarized light .................................................................226 intensity based .................................................72, 77–79
Poloxamer-188 .................................................................. 52 lifetime based .........................................................78–79
Poly (ethylene imine) (PEI).................................... 171–173,
176–179, 181, 183 R
Poly (L-lysine) (PLL) .......................................171–173, 175, Rab-GTPase proteins ......................................................195
177, 180, 181, 183 Radiative decay ................................................................164
Poly(N-isopropylacrylamide) (pNIPAM)........................ 353 Rayleigh distance .............................................................150
Polycations............................................... 171–173, 175, 181 Reactive oxygen species (ROS)........................................ 100
Polydimethylsiloxane wells (PDMS wells) ...... 140–142, 144 Real-time monitoring ..............................................307–314
Polydispersity index (PDI) ................................ 94, 106, 132 Real-time movement .......................................................150
Polyelectrolytes .............................82–86, 115, 117, 119, 122 Real-time particle tracking ......................................211–222
Polyhedral .......................................................................... 65 Receptor recycling ...........................................................237
Polymer-gold nanorod assemblies ...............................81–89 Redox couple ...................................................................263
Polymersome ........................................... 344–348, 350, 351 Redox metalloproteins .....................................................261
Polymersomes-mediated delivery ............................343–351 Redox molecules ......................................................261, 262
Polyplexes .........................................171, 172, 177–179, 182 Reduction sensitive gene carriers .............................171–183
Polystyrene particle suspension.........115, 117, 119, 121, 122 Renal toxicity ...................................................................101
Polyvinylpyrrolidone (PVP)........................34, 35, 37, 38, 62 Reporter gene assay .................................................276, 278
Proapoptotic agents .........................................................102 Resonance frequency ............................... 116, 128, 132, 135
Programmed cell death ......................................................99 Reynold’s lead citrate stain ................................................35
Prostate cancer cell lines ....................................................82 Rh. See Hydrodynamic radius (Rh)
Protein Richardson Piper media ..................................................197
adsorption ..........................................................226, 227 RNA interference (RNAi) ..............................14, 20, 21, 255
corona ........................................................................ 225 ROS. See Reactive oxygen species (ROS)
delivery ...................................................... 275–277, 307
structure ......................................225, 226, 230, 231, 233 S
trafficking ..................................................................149
Saponin .......................................... 58–60, 62, 199, 208, 209
Protein-based therapeutics ..............................................275
Saponin extraction buffer ................................ 199, 205, 206
Protein-binding biofunctional shell .........................293–305
SEC. See Size exclusion chromatography (SEC)
Protein transduction domain (PTD) ....................... 307–314
Secondary antibody hybridization ...................................202
Proteomics ............................................................... 275, 291
Sedimentation FFF (SdFFF) .......................................... 325
Pseudocoelom ..............................................................15, 21
Self-assembling complex .................................................275
PSF. See Point-spread function (PSF)
Semiconductor quantum dots ..................................163, 249
PTD. See Protein transduction domain (PTD)
Sensing techniques, antibody-based ....................................1
PTD-Fluc. See Firefly luciferase-tagged PTD (PTD-Fluc)
Sensor arrays........................................................................1
Pt/Ir tip ................................................................... 264–265
Sensor surface ................................................. 113, 119, 121,
PVP. See Polyvinylpyrrolidone (PVP)
128, 132–133, 135, 136
Q Sentinel lymph node mapping.........................................249
Separation science ...................................................325–240
QCM-D. See Quartz crystal microbalance with dissipation Serotonin ..................................................152, 239, 240, 245
monitoring technique (QCM-D) SHRIMP. See Single-molecule high resolution imaging
QD/CPP complex ...........................................................259 with photobleaching (SHRIMP)
Qdots. See Quantum dots (Qdots) Signal down-regulation ...................................................238
Qdot streptavidin conjugate ....................................154, 156 Silica
Quantum dot labeling .............................................155–156 core-shell nanoparticles .....................................293–305
Quantum dots (Qdots) ....................149–160, 163, 239–243, nanoparticles.......................163–169, 294–296, 298–303
245, 249–259, 326, 329, 330, 333, shells .................................................................. 168, 169
336–338, 346 Silicon dioxide (SiO2)
Quartz crystal microbalance ........................... 113–118, 120, evaporation source .....................................................117
121, 123, 127–136 surface ................................................................ 113–123
CELLULAR AND SUBCELLULAR NANOTECHNOLOGY
Index
369

Silicon nitride surfaces.....................................................114 STPP. See Stearyl-triphenylphosphonium cations (STPP)


Silver enhancement ......................................... 34, 37, 38, 60 Strep-qdots ......................................................................156
Silver grains ........................................................... 34, 38, 60 Subcellular compartments ...........................................47–54
Single mitochondria capillary electrophoresis .................145 Subcellular markers .................................................197, 199
Single-molecule .................................................... 65, 66, 71, Subcellular trafficking........................................................48
150–153, 156–160, 261, 262 Submicron particles .........................................................293
Single-molecule dynamics ...............................................150 Sub-pixel resolution.........................................................157
Single-molecule fluorescent microscopy ..........................149 Superresolution structured illumination microscopy
Single-molecule high resolution imaging with (SSIM)................................................................... 34
photobleaching (SHRIMP) ................................. 149 Supported lipid bilayers ....................113, 124, 125, 137, 209
Single-molecule investigation ..................................261–273 Supramolecular assemblies ..............................................316
Single-molecule tracking .........................................152, 157 Surface modification ................................... 81, 99, 121, 128,
Single particle tracking (SPT) ................................. 157, 186 293, 295–303
Single QD nanoparticles .........................................237–245 Surface plasmon resonance ..............................................128
Single quantum dot imaging ...................................149–160 Surface-to-volume ratio.....................................................81
Single walled carbon nanotubes (SWCNT) ............ 315–317 Surfactants .................................................45, 52, 84, 95–97,
SiO2. See Silicon dioxide (SiO2) 105, 316, 329, 330, 349
siRNA. See Small interfering RNA (siRNA) Suspended lipid membrane .............................................114
Site-specific delivery ..........................................................94 SWCNT. See Single walled carbon nanotubes (SWCNT)
Size distribution ......................................106, 107, 123, 132, Sylgard elastomer kit ...............................................140, 141
135, 293, 303, 326, 338, 348, 350 Synaptic vesicles ........................................................10, 152
Size exclusion chromatography (SEC) ............69, 72, 75–76, Synap-topHluorins ............................................................10
174, 181, 348, 350 Synthetic nona-arginine (SR9) ........................ 250, 253–256
Small interfering RNA (siRNA) .............172, 174, 251–253,
255–258, 347, 348 T
Small ligand coated-QD .................................................337 Tat protein .......................................................................281
SNARE. See Soluble NSF attachment protein TEM. See Transmission electron microscopy (TEM)
receptor (SNARE) TEMPO dextran ...............................................................69
Soft tissue sarcoma ............................................................99 Texas red conjugated BSA (BSA-TxR) ............203, 204, 209
Sol–gel particles...............................................................293 Texas red conjugated WGA (WGA-TxR) ............. 198–201,
Sol–gel reaction ...............................................................163 203, 204, 209
Solid-phase peptide synthesis .......................... 250, 354–355 Theragnostic applications ................................................344
Soluble NSF attachment protein receptor (SNARE) ........ 10 Thermal drift ........................................... 123, 267, 268, 273
Sonicator ...................................... 43, 45, 48, 50, 82, 85, 106 Thermally responsive polymers .......................................353
Spatial resolution ............................................. 139, 149, 343 Thermosensitive particles ................................................353
Spectroscopy-like imaging.......................................262, 263 Thiol-functionalized VPGVG pentapeptide ...................354
Speed-vac concentrator .....................................................46 Time-lapse fluorescence imaging ............................186, 242
Spermatogenesis ..................................................................9 Time-lapse images........................................... 153, 155, 156
Spin-coated single quantum dots ............................154–155 TiO2 ............................................................................ 27, 30
SPT. See Single particle tracking (SPT) Titania ............................................................................. 128
Squamous epithelial cells ................................. 201, 206, 207 Titanium (Ti) evaporation source ...................................117
SR9. See Synthetic nona-arginine (SR9) TLL switch. See Transistor-transistor logic (TLL) switch
SSIM. See Superresolution structured illumination Tocopherol...............................................................103, 105
microscopy (SSIM) TPP. See Triphenylphosphonium (TPP)
Stearyl-triphenylphosphonium cations (STPP) ...... 102, 106 Tracking construction ......................................................157
STED. See Stimulated emission depletion microscopy Tracking resolution .................................. 212, 216, 221, 222
(STED) Trajectory construction ............................................157, 160
Stern–Volmer constant ......................................................77 Trajectory, five frame ...............................................214, 215
Stimulated emission depletion microscopy (STED) ....... 149 Transducer ........................................................................... 1
Stimuli-responsive gold nanoparticles .....................353–361 Transgene expression ..................................... 83, 86–88, 207
Stochastic optical reconstruction microscopy Transistor-transistor logic (TLL) switch .........................142
(STORM) ........................................................... 149 Transmembrane transporting ............................................60
STORM. See Stochastic optical reconstruction microscopy Transmission electron microscopy (TEM) ................... 6, 68,
(STORM) 69, 71, 74–75, 94–96, 164–166, 332, 348, 360
CELLULAR AND SUBCELLULAR NANOTECHNOLOGY
370 Index

Trap-position sensing ......................................................142 Viscoelasticity ..................................................................185


Triphenylphosphonium (TPP) ........................................ 102 Viscoelastic properties .....................................................191
TTL switch. See Transistor-transistor logic switch Voltage dependent anion channel (VDAC)....................... 63
Tumor .......................................................9, 10, 94, 100, 326 Voltammetry ....................................................................262
Tumor-targeting ligands ....................................................94 VPGVG-capped gold nanoparticles........................355, 359
Tumor-targeting nanocarrier ...........................................152
Tunneling current ............................................ 262, 268–270 W
Tweezers, optical ........................10, 139–141, 143, 144, 147 Western blot .................................................... 252, 256–258
WGA. See Wheat germ agglutinin (WGA)
U
WGA-TxR. See Texas red conjugated WGA (WGA-TxR)
Ultramicrotome ...........................................................35, 37 Wheat germ agglutinin (WGA) ............................. 200, 203
Ultraviolet laser ...............................................................141 Worms ............................................................. 15–17, 19–22
UV/Vis ..................46, 96, 175–177, 234, 331, 335–338, 350
Y
V
Yeast ................................................................................ 186
VDAC. See Voltage dependent anion channel (VDAC)
Vectashield® .................................................................... 206 Z
Velocity ............................... 58, 153, 191, 212, 218, 305, 326 Zebrafish ......................................................................... 139
Vertebrate neurons...........................................................186 Zeta potential ................... 106, 107, 130, 131, 166, 302, 303
Vesicular acidification ......................................................195 Zwitterionic phospholipid vesicles ..................................113
Viral vectors.....................................................................141

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