Cellular and Subcellular Nanotechnology 2013
Cellular and Subcellular Nanotechnology 2013
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
Edited by
Mark Olsen
Department of Pharmaceutical Sciences
Midwestern University College of Pharmacy
Glendale, AZ, USA
“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.
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
ix
x Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Contributors
xiii
xiv Contributors
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
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
2 Materials
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
3 Methods
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
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 Notes
Acknowledgment
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
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.
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
2 Materials
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 Methods
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)
3.2 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
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)
% 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)
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).
Table 2
Mean endosomal pH (±s.e.m.) at various time points postinjection
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)
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
Acknowledgments
References
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Chapter 3
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
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
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.
2.4 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
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
4 Notes
Acknowledgment
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
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.
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
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
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
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
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)
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
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.
1 Introduction
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
3 Methods
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.
a b c
Endocytic vesicles
Nanoparticles with nanoparticles
Centrifuge
PBS 24 h
10 min
Wash
400 x g
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)
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
Acknowledgments
References
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.
1 Introduction
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.
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.
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.
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.
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.
3.6 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
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
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
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.
1 Introduction
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
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
2 Materials
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
3 Methods
4 Notes
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
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.
1 Introduction
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.
2 Materials
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¢
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).
2.5 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.
2.8 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.
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
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
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).
3.1.4 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.
3.3 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.
3.6 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.
3.7 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
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 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
References
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
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.
2 Materials
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
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).
3.3 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
1. GNR
5. Sonication 9. Sonication
13. PE-GNR
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. 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)
4 Notes
Acknowledgments
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Chapter 10
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
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
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
References
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adrenalin after intratracheal injection. J Exp McPhail AT, Sim GA (1968) Crotepoxide a,
Med 23(6):757–772 novel cyclohexane diepoxide tumor inhibitor
2. Pearce L (1921) Studies on the treatment of from Croton macrostachys. J Am Chem Soc
human trypanosomiasis with tryparsamide (the 90(11):2982–2983
sodium salt of N-phenylglycineamide-p-arsonic 6. Kupchan SM, Hemingway RJ, Werner D,
acid). J Exp Med 34(6 Suppl 1):1–104 Karim A, McPhail AT, Sim GA (1968)
3. Moore HF (1915) A further study of the bac- Vernolepin, a novel elemanolide dilactone
tericidal action of ethylhydrocuprein on tumor inhibitor from Vernonia hymenolepis.
Pneumococci. J Exp Med 22(5):551–567 J Am Chem Soc 90(13):3596–3597
4. Schemir FR, Gilson B, Hurst WW, Gilson JS, 7. Barry VC, Byrne J, Conalty ML, O’Sullivan JF
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intravenous solutions; their effect on the blood antimycobacterial activity of some novel meth-
and their clinical usefulness. Rocky Mt Med J ylglyoxal derivatives. Proc R Ir Acad B
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98 Puiyan Lee and Kenneth K.Y. Wong
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-
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therapeutic compounds. PLoS One 6(2): terization, and enhanced drug delivery to
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analog RC-160 on endocrine status and tumor effects of lipidic formulated gold-porphyrin
growth in the Dunning R-3327H rat prostate nanoparticles for the treatment of neuroblas-
cancer model. Prostate 20(4):297–310 toma. Nanotechnol Sci Appl 3:23–28
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Chapter 11
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.
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
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
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.
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).
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.
3.3 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
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
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
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.
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Chapter 12
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
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
2 Materials
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
3.3 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
4 Notes
References
based on ordered pores in alumina. colloidal lithography. Colloids Surf A 214. doi:dx.
Chemphyschem 10:885–889 doi.org/10.1016/S0927-7757(02)00367-9
29. Romer W, Steinem C (2004) Impedance anal- 33. Pfeiffer I, Seantier B, Petronis S, Sutherland D,
ysis and single-channel recordings on nano- Kasemo B, Zach M (2008) Influence of nano-
black lipid membranes based on porous topography on phospholipid bilayer formation on
alumina. Biophys J 86:955–965 silicon dioxide. J Phys Chem B 112:5175–5181
30. Grant AW, Hu QH, Kasemo B (2004) 34. Jonsson MP, Dahlin AB, Feuz L, Petronis S,
Transmission electron microscopy ‘windows’ Höök F (2010) Locally functionalized short-
for nanofabricated structures. Nanotechnology range ordered nanoplasmonic pores for bio-
15:1175–1181 analytical sensing. Anal Chem 82:2087–2094
31. Weiskopf D, Schmitt EK, Kluhr MH, Dertinger 35. Svedhem S, Pfeiffer I, Larsson C, Wingren C,
SK, Steinem C (2007) Micro-BLMs on highly Borrebaeck C, Hook F (2003) Patterns of
ordered porous silicon substrates: rupture pro- DNA-labeled and scFv-antibody-carrying lipid
cess and lateral mobility. Langmuir 23: vesicles directed by material-specific immobili-
9134–9139 zation of DNA and supported lipid bilayer for-
32. Hanarp P, Sutherland DS, Gold J, Kasemo B mation on an Au/SiO2 template. Chembiochem
(2003) Control of nanoparticle film structure for 4:339–343
Chapter 13
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.
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
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.
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
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 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
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
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
Acknowledgment
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,
2. Sessa G, Weissmann G (1968) Phospholipid Svedhem S (2011) Structural rearrangements
spherules (liposomes) as a model for biological of polymeric insulin-loaded nanoparticles
membranes. J Lipid Res 9:310–318 interacting with surface-supported model lipid
3. Zasadzinski JA, Viswanathan R, Madsen L, membranes. J Biomater Nanobiotechnol 2:
Garnaes J, Schwartz DK (1994) Langmuir- 180–192
Blodgett films. Science 263:1726–1733 11. Edvardsson M, Svedhem S, Wang G, Richter
4. Rodahl M, Höök F, Fredriksson C, Keller CA, R, Rodahl M, Kasemo B (2009) QCM-D and
Krozer A, Brzezinski P, Voinova M, Kasemo B reflectometry instrument: applications to sup-
(1997) Simultaneous frequency and dissipa- ported lipid structures and their biomolecular
tion factor QCM measurements of bio- interactions. Anal Chem 81:349–361
molecular adsorption and cell adhesion. 12. Frost R, Coué G, Engbersen JFJ, Zäch M,
Faraday Discuss 107:229–246 Kasemo B, Svedhem S (2011) Bioreducible
5. Keller CA, Kasemo B (1998) Surface specific insulin-loaded nanoparticles and their interac-
kinetics of lipid vesicle adsorption measured tion with model lipid membranes. J Colloid
with a quartz crystal microbalance. Biophys J Interface Sci 362(2):575–583
75:1397–1402 13. Kunze A, Sjövall P, Kasemo B, Svedhem S
6. Cho NJ, Frank CW, Kasemo B, Höök F (2010) (2009) In situ preparation and modification of
Quartz crystal microbalance with dissipation supported lipid layers by lipid transfer from
monitoring of supported lipid bilayers on vari- vesicles studied by QCM-D and TOF-SIMS.
ous substrates. Nat Protoc 5:1096–1106 J Am Chem Soc 131:2450–2451
7. Reimhult E, Zäch M, Höök F, Kasemo B 14. MacDonald RC, MacDonald RI, Menco BPM,
(2006) A multitechnique study of liposome Takeshita K, Subbarao NK, Hu L-R (1991)
adsorption on Au and lipid bilayer formation Small-volume extrusion apparatus for prepara-
on SiO2. Langmuir 22:3313–3319 tion of large, unilamellar vesicles. Biochim
8. Richter RP, Bérat R, Brisson AR (2006) Biophys Acta 1061:297–303
Formation of solid-supported lipid bilayers: 15. Frisken BJ, Asman C, Patty PJ (2000) Studies
an integrated view. Langmuir 22: of vesicle extrusion. Langmuir 16:928–933
3497–3505 16. Patty PJ, Frisken BJ (2003) The pressure-
9. Rossetti FF, Textor M, Reviakine I (2006) dependence of the size of extruded vesicles.
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.
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
2 Materials
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
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)
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
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
References
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2. Niemz MH (2004) Laser-tissue interactions. (2007) Construction and calibration of an
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101(28):10266–10271 Biochem 77:205–228
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Science 13(4507):505–513
Chapter 15
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
λ
d xyR = 0.61 . (2)
NA
Single Quantum Dot Imaging in Living Cells 151
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
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
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).
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
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)
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
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
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
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.
1 Introduction
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
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.
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.
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
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).
3.3.3 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
References
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
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
2 Materials
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
3 Methods
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
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
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)
a b
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
4 Notes
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
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,
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
7. Bauhuber S et al (2009) Delivery of nucleic 13. Lee Y et al (2007) Visualization of the degra-
acids via disulfide-based carrier systems. Adv dation of a disulfide polymer, linear poly
Mater 21:3286–3306 (ethylenimine sulfide), for gene delivery.
8. Dhananjay J et al (2009) Bioreducible poly- Bioconjug Chem 18:13–18
mers for efficient gene and siRNA delivery. 14. Kunishima M et al (1999) 4-(4,6-dimethoxy-
Biomed Mater 4:025020 1,3,5-triazin-2-yl)-4-methyl-morpholinium
9. Oupicky D et al (2001) Triggered intracellular chloride: an efficient condensing agent leading
activation of disulfide crosslinked polyelectrolyte to the formation of amides and esters.
gene delivery complexes with extended systemic Tetrahedron 55:13159–13170
circulation in vivo. Gene Ther 8:713–724 15. Breunig M et al (2008) Mechanistic investiga-
10. Felgner JH et al (1994) Enhanced gene deliv- tion of poly(ethylene imine)-based siRNA
ery and mechanism studies with a novel series delivery: disulfide bonds boost intracellular
of cationic lipid formulations. J Biol Chem release of the cargo. J Control Release
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
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.
1 Introduction
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
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
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
4 Notes
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
1. Binnig G, Quate CF, Gerber C (1986) Atomic 6. Pelling AE, Sehati S, Gralla EB, Valentine JS,
force microscope. Phys Rev Lett 56:930–933 Gimzewski JK (2004) Local nanomechanical
2. Putman CA, van der Werf KO, de Grooth BG, motion of the cell wall of Saccharomyces cere-
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.
3. Radmacher M, Tillmann RW, Fritz M, Gaub ScientificWorldJournal 6:1609–1618
HE (1992) From molecules to cells—imaging 8. Horton M, Charras G, Ballestrem C, Lehenkari
soft samples with the atomic force microscope. P (2000) Integration of atomic force and con-
Science 257:1900–1905 focal microscopy. Single Mol 1:135–137
4. Charras G, Horton MA (2001) Cellular mech- 9. Lehenkari PP, Charras GT, Nykänen A, Horton
anotransduction and its modulation: an atomic MA (2000) Adapting atomic force microscopy
force microscopy study. Biophys J 80: for cell biology. Ultramicroscopy 82:289–295
305A–306A 10. Bereiterhahn J, Voth M (1994) Dynamics of
5. Rotsch C, Jacobson K, Radmacher M (1999) mitochondria in living cells—shape changes,
Dimensional and mechanical dynamics of dislocations, fusion, and fission of mitochon-
active and stable edges in motile fibroblasts dria. Microsc Res Tech 27:198–219
investigated by using atomic force micros- 11. Brady S, Lasek R, Allen R (1982) Fast axonal
copy. Proc Natl Acad Sci USA 96: transport in extruded axoplasm from squid
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12. Heggeness MH, Simon M, Singer SJ (1978) 18. Blumenfeld R (2006) Isostaticity and con-
Association of mitochondria with microtubules trolled force transmission in the cytoskeleton:
in cultured cells. Proc Natl Acad Sci USA a model awaiting experimental evidence.
75:3863–3866 Biophys J 91:1970–1983
13. Morris R, Hollenbeck P (1995) Axonal trans- 19. Sbalzarini IF, Koumoutsakos P (2005) Feature
port of mitochondria along microtubules and point tracking and trajectory analysis for video
F-actin in living vertebrate neurons. J Cell Biol imaging in cell biology. J Struct Biol
131:1315–1326 151:182–195
14. Drubin D, Jones H, Wertman K (1993) Actin 20. Silberberg YR, Pelling AE, Yakubov GE, Crum
structure and function: roles in mitochondrial WR, Hawkes DJ, Horton MA (2008) Tracking
organization and morphogenesis in budding displacements of intracellular organelles in
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Mechanotransduction across the cell-surface Mitochondrial displacements in response to nano-
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1124–1127 22. Levy R, Maaloum M (2002) Measuring the
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transduction: all signals point to cytoskeleton, cantilevers: thermal fluctuations and other
matrix, and integrins. Sci STKE 2002:pe6 methods. Nanotechnology 13:33–37
Chapter 19
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
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
2 Materials
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
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
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
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.
4 Notes
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
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
References
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fluorescence microscopy using supported lipid Deliv 8(4):403–407
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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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and live target cells. J Control Release Parker PJ, Michell RH (1997) Osmotic stress
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Ferruti P, Duncan R (2010) Intracellular fate 8. Mullock B, Bright N, Fearon C, Gray S, Luzio
of bioresponsive poly(amidoamine)s in vitro JP (1998) Fusion of lysosomes with late endo-
and in vivo. J Control Release 142:78–88 somes produces a hybrid organelle of interme-
4. Richardson SC (2010) Tracking intracellular diate density and is NSF dependent. J Cell Biol
polymer localisation via fluorescence micros- 140:592–601
copy. In: Weissig V, D’Souza G (eds) Organelle- 9. Mallard F, Antony F, Tenza D, Salamero J,
specific pharmaceutical nanotechnology. Wiley, Goud B, Johannes L (1998) Direct pathway
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Chapter 20
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.
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)
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
Δr 2 = (xi +n − xi ) + (y i +n − y i )
2 2
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 ]
2 Materials
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
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.
2.3 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
3.3 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
3.4 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
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
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
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
References
1. Suh J et al (2003) Efficient active transport of 7. Lai SK, Hanes J (2008) Real-time multiple par-
gene nanocarriers to the cell nucleus. Proc Natl ticle tracking of gene nanocarriers in complex
Acad Sci USA 100:3878–3882 biological environments. Methods Mol Biol
2. Huang F et al (2011) Quantitative nanoparticle 434:81–97
tracking: applications to nanomedicine. 8. Kawai M et al (2009) Dynamics of different-sized
Nanomedicine 6(4):693–700 solid-state nanocrystals as tracers for a drug-deliv-
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
sional systems. Biophys J 60:910–921 9. Chen C, Suh J (2010) Real-time particle track-
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-
of calcium-induced differentiation in cultured technology. Wiley, Hoboken
epithelial cells. Biophys J 65:2021–2040 10. Wirtz D (2009) Particle-tracking microrheol-
5. Saxton MJ, Jacobson K (1997) Single-particle ogy of living cells: principles and applications.
tracking: applications to membrane dynamics. Annu Rev Biophys 38:301–326
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
tracking: applications to drug and gene deliv- study of chromosome dynamics. IEEE Trans
ery. Adv Drug Deliv Rev 57:63–78 Image Process 14:1372–1383
Chapter 21
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
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)
[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.
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)
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
NPsurface area ⎡⎣ nm 2 ⎤⎦
Number of surface sites / NP = . (8)
protein surface area ⎡⎣ nm 2 ⎤⎦
Fig. 3 KD plot—a linear fit of this plot yields a gradient of 1/KD and thus KD
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
References
resonance energy transfer-derived structure of 26. van de Hulst H (1981) Light scattering by
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(2006) Adsorption-induced conformational Mueller W, Krohne K, Maskos M (2010)
changes of proteins onto ceramic particles: Characterization of polymer nanoparticles by
differential scanning calorimetry and FTIR asymmetrical flow field flow fraction (AF-FFF).
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and the conformational analysis of biomole- Vecerova R, Pizurova N, Sharma VK, Nevecna
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15. Berova N, Nakanishi K, Woody RW (eds) synthesis, characterization, and their antibacte-
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Chapter 22
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
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 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
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
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
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
References
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Chapter 23
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.
2.4 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.
2.6 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.
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.
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.
3.3.1 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
3.5.1 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.
3.5.2 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
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
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
Acknowledgments
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
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
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.
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.
3.2 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
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
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.
3.5 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
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)
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
Acknowledgment
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
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
2 Materials
3 Methods
Prepare all solutions using ultrapure laboratory grade water or PBS
at room temperature (unless indicated otherwise).
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
3.4 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
Acknowledgments
References
1. Campbell PL, McCluskey J, Yeo JP, Toh BH 3. Raptis LH, Liu SK, Firth KL, Stiles CD,
(1995) Electroporation of antibodies into mam- Alberta JA (1995) Electroporation of peptides
malian cells. Methods Mol Biol 48:83–92 into adherent cells in situ. Biotechniques
2. Marrero MB, Schieffer B, Paxton WG, Schieffer 18(1):104, 106, 108, 110 passim
E, Bernstein KE (1995) Electroporation of pp 4. Nolkrantz K, Farre C, Hurtig KJ, Rylander P,
60c-src antibodies inhibits the angiotensin II Orwar O (2002) Functional screening of intra-
activation of phospholipase C-gamma 1 in rat cellular proteins in single cells and in patterned
aortic smooth muscle cells. J Biol Chem cell arrays using electroporation. Anal Chem
270(26):15734–15738 74(16):4300–4305
280 Seong Loong Lo and Shu Wang
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
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
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
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
3.2 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. 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. 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 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
Acknowledgment
References
1. Vives E, Brodin P, Lebleu B (1997) A trun- 7. Lee JS, Tung C-H (2011) Enhanced cellular
cated HIV-1 Tat protein basic domain rapidly uptake and metabolic stability of lipo-
translocates through the plasma membrane oligoarginine peptides. Biopolymers.
and accumulates in the cell nucleus. J Biol 96:772–779
Chem 272:16010–16017 8. O’Donnell JM, Alpert NM, White LT,
2. Frankel AD, Pabo CO (1988) Cellular uptake Lewandowski ED (2002) Coupling of mito-
of the tat protein from human immunodeficiency chondrial fatty acid uptake to oxidative flux in
virus. Cell 55:1189–1193 the intact heart. Biophys J 82:11–18
3. Green M, Loewenstein PM (1988) 9. Fernandez-Carneado J, Kogan MJ, Van Mau
Autonomous functional domains of chemically N, Pujals S, Lopez-Iglesias C, Heitz F, Giralt E
synthesized human immunodeficiency virus tat (2005) Fatty acyl moieties: improving Pro-rich
trans-activator protein. Cell 55:1179–1188 peptide uptake inside HeLa cells. J Pept Res
4. Mitchell DJ, Kim DT, Steinman L, Fathman 65:580–590
CG, Rothbard JB (2000) Polyarginine enters 10. Futaki S, Ohashi W, Suzuki T, Niwa M, Tanaka
cells more efficiently than other polycationic S, Ueda K, Harashima H, Sugiura Y (2001)
homopolymers. J Pept Res 56:318–325 Stearylated arginine-rich peptides: a new class
5. Lee JS, Tung CH (2010) Lipo-oligoarginines of transfection systems. Bioconjug Chem
as effective delivery vectors to promote cellular 12:1005–1011
uptake. Mol Biosyst 6:2049–2055 11. Koppelhus U, Shiraishi T, Zachar V, Pankratova
6. Futaki S, Suzuki T, Ohashi W, Yagami T, S, Nielsen PE (2008) Improved cellular activ-
Tanaka S, Ueda K, Sugiura Y (2001) Arginine- ity of antisense peptide nucleic acids by conju-
rich peptides. An abundant source of mem- gation to a cationic peptide-lipid (CatLip)
brane-permeable peptides having potential as domain. Bioconjug Chem 19:1526–1534
carriers for intracellular protein delivery. J Biol 12. Pham W, Kircher MF, Weissleder R, Tung CH
Chem 276:5836–5840 (2004) Enhancing membrane permeability by
292 Jae Sam Lee and Ching-Hsuan Tung
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.
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. 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).
3 Methods
All reactions are done at room temperature (unless described
differently).
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
4 Notes
References
9. Wang L, Zhao W, Tan W (2008) Bioconjugated Alexa 488 marked silica nanoparticles with an
silica nanoparticles: development and applica- endoscopic device for real time cancer visual-
tions. Nano Res 1:99–115 ization. Mater Res Soc Symp Proc 1190
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
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)
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.
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
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
Acknowledgment
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.
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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
3. Richard JP, Melikov K, Vives E et al (2003) in living cells and organisms: a review.
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
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
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.
2 Materials
3 Methods
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.
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
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nanotube-filled polymer composite with an 16. Pantarotto D, Singh R, McCarthy D, Erhardt
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Chapter 30
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
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.
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
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
2 Materials
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
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.
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
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
Vc/Vout = 2
1.2 Vc/Vout = 3 180
Vc/Vout = 4
1.0 160
Vc/Vout = 5
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
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)
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
References
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based on a coupling of concentration and Sedimentation field-flow fractionation. Anal
flow nonuniformities. Separation Sci 1(1): Chem 46(13):1917–1924
123–125 7. Caldwell KD, Gao YS (1993) Electrical field-
2. Giddings JC (1989) Field-flow fractionation of flow fractionation in particle separation. 1.
macromolecules. J Chromatogr 470(2):327–35 Monodisperse standards. Anal Chem 65(13):
3. Giddings JC et al (1980) Analysis of biological 1764–1772
macromolecules and particles by field-flow frac- 8. Giddings JC, Yang FJ, Myers MN (1976) Flow-
tionation. Methods Biochem Anal 26:79–136 field-flow fractionation: a versatile new separa-
4. Schimpf ME, Caldwell K, Giddings JC (2000) tion method. Science 193(4259):1244–1245
Field flow fractionation handbook, vol xviii. 9. Vickrey TM, Garcia-ramirez JA (1980)
Wiley-Interscience, New York, p 592 Magnetic field-flow fractionation: theoretical
5. Thompson GH, Myers MN, Giddings JC (1969) basis. Sep Sci Technol 15(6):1297–1304
Thermal field-flow fractionation of polystyrene 10. Wahlund KG, Giddings JC (1987) Properties
samples. Anal Chem 41(10):1219–1222 of an asymmetrical flow field-flow fractionation
Separation Science: Principles and Applications for the Analysis of Bionanoparticles… 341
channel having one permeable wall. Anal 23. Gaponik N et al (2010) Progress in the light
Chem 59(9):1332–1339 emission of colloidal semiconductor nanocrys-
11. Wahlund KG, Litzen A (1989) Application of tals. Small 6(13):1364–1378
an asymmetrical flow field-flow fractionation 24. Smith MH et al (2010) Monitoring the erosion of
channel to the separation and characterization hydrolytically-degradable nanogels via multiangle
of proteins, plasmids, plasmid fragments, poly- light scattering coupled to asymmetrical flow field-
saccharides and unicellular algae. J Chromatogr flow fractionation. Anal Chem 82(2):523–530
461:73–87 25. Giddings JC (1993) Field-flow fractionation:
12. Fraunhofer W, Winter G (2004) The use of analysis of macromolecular, colloidal, and
asymmetrical flow field-flow fractionation in particulate materials. Science 260(5113):
pharmaceutics and biopharmaceutics. Eur J 1456–1465
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14. Pinaud F et al (2010) Probing cellular events, fractionation (AsFlFFF) coupled to induc-
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15. Maysinger D, Lovric J (2007) Quantum dots of natural colloids and synthetic nanoparticles.
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to tail single bio-molecules inside living cells. ronmental risk assessment of engineered nano-
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(2011) Engineered nanoparticles for biomo- 30. Al Hajaj et al. (2011) ACS Nano, 5(6):
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Chapter 31
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
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
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.
3 Methods
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
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.
3.6 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
References
1. Rust MJ, Bates M, Zhuang X (2006) Sub- particles and their biosensory function. Angew
diffraction-limit imaging by stochastic optical Chem Int Ed Engl 47:9922–9926
reconstruction microscopy (STORM). Nat 9. Erogbogbo F, Yong KT, Hu R, Law WC, Ding
Methods 3:793–795 H, Chang CW, Prasad PN, Swihart MT (2010)
2. Schermelleh L, Heintzmann R, Leonhardt H Biocompatible magnetofluorescent probes:
(2010) A guide to super-resolution fluorescence luminescent silicon quantum dots coupled
microscopy. J Cell Biol 190:165–175 with superparamagnetic iron(III) oxide. ACS
3. Massignani M, Canton I, Sun T, Hearnden V, Nano 4:5131–5138
Macneil S, Blanazs A, Armes SP, Lewis A, 10. Slowing I, Trewyn BG, Lin VS (2006) Effect
Battaglia G (2010) Enhanced fluorescence of surface functionalization of MCM-41-type
imaging of live cells by effective cytosolic deliv- mesoporous silica nanoparticles on the endo-
ery of probes. PLoS One 5:e10459 cytosis by human cancer cells. J Am Chem Soc
4. Yang Z, Zheng S, Harrison WJ, Harder J, Wen 128:14792–14793
X, Gelovani JG, Qiao A, Li C (2007) Long- 11. Liong M, Lu J, Kovochich M, Xia T, Ruehm
circulating near-infrared fluorescence core- SG, Nel AE, Tamanoi F, Zink JI (2008)
cross-linked polymeric micelles: synthesis, Multifunctional inorganic nanoparticles for
characterization, and dual nuclear/optical imaging, targeting, and drug delivery. ACS
imaging. Biomacromolecules 8:3422–3428 Nano 2:889–896
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DJ (2011) Targeted delivery of nanoparticles Canton I, Presti C, MacNeil S, Du J, Blanazs
to ischemic muscle for imaging and therapeu- A, Madsen J, Armes SP, Lewis AL, Battaglia G
tic angiogenesis. Nano Lett 11:694–700 (2008) Non-cytotoxic polymer vesicles for
6. Nasongkla N, Bey E, Ren J, Ai H, Khemtong C, rapid and efficient intracellular delivery. Faraday
Guthi JS, Chin SF, Sherry AD, Boothman DA, Discuss 139:143–159, discussion 213–128,
Gao J (2006) Multifunctional polymeric micelles 419–120
as cancer-targeted, MRI-ultrasensitive drug 13. Du J, Tang Y, Lewis AL, Armes SP (2005)
delivery systems. Nano Lett 6:2427–2430 pH-sensitive vesicles based on a biocompatible
7. Little LE, Dane KY, Daugherty PS, Healy KE, zwitterionic diblock copolymer. J Am Chem
Schaffer DV (2011) Exploiting bacterial pep- Soc 127:17982–17983
tide display technology to engineer biomateri- 14. Massignani M, LoPresti C, Blanazs A, Madsen
als for neural stem cell culture. Biomaterials J, Armes SP, Lewis AL, Battaglia G (2009)
32:1484–1494 Controlling cellular uptake by surface chemis-
8. Park C, Im MS, Lee S, Lim J, Kim C (2008) try, size, and surface topology at the nanoscale.
Tunable fluorescent dendron-cyclodextrin Small 5:2424–2432, Weinheim an der
nanotubes for hybridization with metal nano- Bergstrasse, Germany
Chapter 32
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
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.
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
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).
3.3 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.
4 Notes
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
Acknowledgments
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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
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
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