Review

pubs.acs.org/CR

Chemical Basis of Interactions Between Engineered Nanoparticles

and Biological Systems
Qingxin Mu,†,‡ Guibin Jiang,§ Lingxin Chen,∥ Hongyu Zhou,†,⊥ Denis Fourches, Alexander Tropsha,#
and Bing Yan*,†
†
School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
§
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese
Academy of Sciences, Beijing, 100085, China
∥
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China
⊥
Department of Surgery, Emory University School of Medicine, Atlanta, Georgia 30322, United States
#
Laboratory for Molecular Modeling, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina
27599, United States
4.1.1. Kinetics and Thermodynamics of the
Cellular Uptake of Nanoparticles 7754
4.1.2. Determinants of the Cellular Uptake of
Nanoparticles 7754
4.2. Cellular Substructural and Functional Alter-
ations by Nanoparticles 7755
4.2.1. Nanoparticle-Induced Cell Membrane
Disruption 7755
4.2.2. Ion Channel Inhibition by Nanoparticles 7755
4.2.3. Cytoskeleton Alterations by Nanopar-
ticles 7756
4.2.4. Mitochondrial Alterations by Nanopar-
CONTENTS ticles 7756
1. Introduction 7741 4.2.5. Nuclear Alterations by Nanoparticles 7757
2. Factors Affecting Nanoparticle−Biological Sys- 4.3. Oxidative Stress and Nanoparticles 7757
tem Interactions 7742 4.3.1. Oxidative Stress Induced by Nanopar-
2.1. Nanoparticles 7742 ticles 7757
2.2. Biological Factors 7742 4.3.2. Reduction of Oxidative Stress by Nano-
3. Nanoparticle−Biomolecule Interactions 7743 particles 7758
3.1. Nanoparticle−Phospholipid Interactions 7743 4.4. Nanoparticle-Induced Systematic Cellular
3.1.1. Interaction of Nanoparticles with Phos- Biochemical Perturbations 7758
pholipids in Pulmonary Surfactant Sol- 4.4.1. Perturbation of Cell Signaling Pathways
utions 7743 by Nanoparticles 7758
3.1.2. Interactions Between Nanoparticles and 4.4.2. Global Biochemical Alterations by Nano-
Model Membranes 7744 particles 7760
3.2. Nanoparticle−Protein Interactions 7745 5. Chemical Approaches To Regulate and Under-
3.2.1. Determination of Nanoparticle−Protein stand Nanoparticle−Biological System Interac-
Interactions 7745 tions 7761
3.2.2. Chemical Origin of Nanoparticle−Pro- 5.1. One-at-a-Time Nanoparticle Surface Chem-
tein Interactions 7746 istry Modification 7761
3.2.3. Interactions Between Nanoparticles and 5.1.1. Modulation of the Surface Chemistry of
Proteomes 7749 Nanoparticles 7761
3.3. Nanoparticle−DNA Interactions 7751 5.1.2. Modulation of the Surface Charge of
3.3.1. Determination of Nanoparticle−DNA Nanoparticles 7761
Interactions 7751 5.1.3. Modulation of the Surface Hydropho-
3.3.2. Current Understanding of Nanopar- bicity of Nanoparticles 7762
ticle−DNA Interactions 7751 5.1.4. Changes in the Interactions of Nano-
3.4. Adsorption of Crucial Small Molecules by particles with Biological Systems 7763
Nanoparticles 7753
4. Nanoparticle−Cell Interactions 7753
4.1. Cellular Uptake and Intracellular Transport Received: May 29, 2013
of Nanoparticles 7753 Published: June 13, 2014

© 2014 American Chemical Society 7740 dx.doi.org/10.1021/cr400295a | Chem. Rev. 2014, 114, 7740−7781
Chemical Reviews Review

Figure 1. Interactions of nanoparticles with biological systems at different levels. Nanoparticles enter the human body through various pathways,
reaching different organs and contacting tissues and cells. All of these interactions are based on nanoparticle−biomacromolecule associations.

5.2. Combinatorial Chemistry for Nanoparticle ture, chemicals and coatings, catalysts, environmental protec-
Modifications 7763 tion, computer memory, biomedicine, and consumer products.
5.2.1. Parallel Chemistry for Nanoparticle Driven by these demands, the worldwide ENM production
Modifications 7763 volume in 2016 is conservatively estimated in a market report
5.2.2. Nanocombinatorial Chemistry To Mod- by Future Markets to be 44 267 tons or ≥$5 billion.6
ify Nanoparticles 7766 As the production and applications of ENMs rapidly expand,
5.3. Computational Approaches To Elucidate their environmental impacts and effects on human health are
Nano−Bio Interactions 7766 becoming increasingly significant.7 Due to their small sizes,
5.3.1. Quantitative Structure−Activity Rela- ENMs are easily made airborne.8 However, no accurate method
tionships (QSAR) 7767 to quantitatively measure their concentration in air currently
5.3.2. QNAR Modeling: Applications to the exists. A recently reported incident of severe pulmonary fibrosis
Rational Design of Nanoparticles with caused by inhaled polymer nanoparticles in seven female
Desired Bioactivity Profiles 7769 workers obtained much attention.9 In addition to the release of
6. Concluding Remarks 7770 ENM waste from industrial sites, a major release of ENMs to
Author Information 7771 environmental water occurs due to home and personal use of
Corresponding Author 7771 appliances, cosmetics, and personal products, such as shampoo
Present Address 7771 and sunscreen.10 Airborne and aqueous ENMs pose immediate
Notes 7771 danger to the human respiratory and gastrointestinal systems.
Biographies 7771 ENMs may enter other human organs after they are absorbed
Acknowledgments 7772 into the bloodstream through the gastrointestinal and
Abbreviations 7772 respiratory systems.11,12 Furthermore, ENMs in cosmetics and
References 7773 personal care products, such as lotion, sunscreen, and shampoo,
may enter human circulation through skin penetration.13 ENMs
are very persistent in the environment and slowly degraded.
1. INTRODUCTION The dissolved metal ions from ENMs can also revert back to
As defined by the European Commission, a nanomaterial is a nanoparticles under natural conditions.14 ENMs are stored in
natural, an incidental, or a manufactured material containing plants, microbes, and animal organs and can be transferred and
particles in an unbound state or as an aggregate or agglomerate accumulated through the food chain.15,16 In addition to
in which ≥50% of the particles in the number size distribution accidental entry of ENMs into human and biological systems,
have one or more external dimensions in the size range from 1 ENMs are also purposefully injected into or enter humans for
to 100 nm. In specific cases and where warranted by concerns medicinal and diagnostic purposes.17 Therefore, interactions of
for the environment, health, safety, or competition, the number ENMs with biological systems are inevitable.
size distribution threshold of 50% may be replaced with a In addition to engineered nanomaterials, there are also
threshold between 1% and 50%.1 Engineered nanomaterials naturally existing nanomaterials such as proteins and DNA
(ENMs) refer to man-made nanomaterials. Materials in the molecules, which are key components of biological systems.
nanometer range often possess unique physical, optical, These materials, combined with lipids and organic and
electronic, and biological properties compared with larger inorganic small molecules, form the basic units of living
particles, such as the strength of graphene,2 the electronic systems cells.18 To elucidate how nanomaterials affect organs
properties of carbon nanotubes (CNTs),3 the antibacterial and physiological functions, a thorough understanding of how
activity of silver nanoparticles,4 and the optical properties of nanomaterials perturb cells and biological molecules is required
quantum dots (QDs).5 The unique and advanced properties of (Figure 1). Rapidly accumulating evidence indicates that ENMs
ENMs have led to a rapid increase in their application. These interact with the basic components of biological systems, such
applications include aerospace and airplanes, energy, architec- as proteins, DNA molecules, and cells.19−21 The driving forces
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for such interactions are quite complex and include the size, 2. FACTORS AFFECTING
shape, and surface properties (e.g., hydrophobicity, hydrogen- NANOPARTICLE−BIOLOGICAL SYSTEM
bonding capability, π bonds, and stereochemical interactions) INTERACTIONS
of ENMs.22−25
Evidence also indicates that chemical modifications on a 2.1. Nanoparticles
nanoparticle’s surface alter its interactions with biological The extremely large surface area of nanoparticles results in
systems.26−28 These observations not only support the strong adsorption of various molecules to reduce nanoparticle
hypothesis that basic nano−bio interactions are mainly surface energy. Pristine nanoparticles are nearly nonexistent.
physicochemical in nature but also provide a powerful approach Interactions between nanoparticles and biological systems
to controlling the nature and strength of a nanoparticle’s (nano−bio interactions) are actually driven by modified
interactions with biological systems. Practically, a thorough nanoparticles in biological media, such as nanoparticle
understanding of the fundamental chemical interactions complexes or their aggregates.32 The real identity of nano-
between nanoparticles and biological systems has two direct particles in biological systems is determined by their intrinsic
impacts. First, this knowledge will encourage and assist properties, such as their size, shape, surface charge, and
experimental approaches to chemically modify nanoparticle hydrophobicity (Scheme 1). Therefore, a rigorous character-
surfaces for various industrial or medicinal applications. Second, ization of nanoparticles is essential prior to investigating nano−
a range of chemical information can be combined with bio interactions.
computational methods to investigate nanobiological properties
and predict desired nanoparticle properties to direct experi- Scheme 1. Factors Influencing Nanoparticle−Biomolecule
ments.29−31 Interactions
The literature regarding nanoparticle−biological system
interactions has increased exponentially in the past decade
(Figure 2). However, a mechanistic understanding of the

Figure 2. Analysis of literature statistics indicates growing concern for

the topics that are the focus of this review. The number of publications
and citations was obtained using the keywords “nanoparticles” and
“biological systems” in the subject area of “Chemistry” when searching A very large collection of methods has been used to
the Thomson Reuters (ISI) Web of Knowledge for the period 1995− characterize nanoparticle physical and chemical properties.
2013. Transmission electron microscopy (TEM), scanning electron
microscopy (SEM), and atomic force microscopy (AFM)
provide information on nanoparticle morphology.33 Dynamic
chemical basis for such complex interactions is still lacking. This
light scattering (DLS) provides information on the hydro-
review intends to explore such an understanding in the context
dynamic radii of nanoparticles in solution.34 The surface charge
of recent publications.
properties of nanoparticles can be determined with ζ-potential
A breakthrough technology cannot prosper without wide
measurements.35 Auger electron spectroscopy, X-ray photo-
acceptance from the public and society, that is, it must pose
electron spectroscopy (XPS), time-of-flight mass spectrometry
minimal harm to human health and the environment.
(TOF MS), and elemental analyses reveal chemical composi-
Nanotechnology is now facing such a critical challenge. We
tion details of nanoparticles.36,37 Surface ligands or adsorbed
must elucidate the effects of ENMs on biological systems (such
molecules can be identified with magic angle spinning nuclear
as biological molecules, human cells, organs, and physiological
magnetic resonance (MAS NMR), liquid chromatography mass
systems). Accumulating experimental evidence suggests that
spectroscopy (LC-MS), and Fourier-transform infrared spec-
nanoparticles interact with biological systems at nearly every
troscopy (FTIR).38−40 Surface-enhanced Raman spectroscopy
level, often causing unwanted physiological consequences.
(SERS) is also among the most frequently applied analytical
Elucidating these interactions is the goal of this review. This
method for characterizing nanoparticles.41
endeavor will help regulate the proper application of ENMs in
various products and their release into the environment. A 2.2. Biological Factors
more significant mission of this review is to direct development In biological systems, nanoparticles are surrounded by excessive
of “safe-by-design” ENMs, as their demands for and amounts of biological molecules, which may result in the
applications continue to increase. saturation of the nanoparticle surface. This phenomenon occurs
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whether the nanoparticles are present in the body or in a nucleic acids, such as DNA.53 Furthermore, nanoparticles in the
cellular environment. Such biologically modified nanoparticles blood or cells also adsorb small molecules, such as amino acids,
are the real participants in nano−bio interactions.32 These biotin, and folic acid. Depletion of small metabolites may also
modifications also affect cell binding events and internalization contribute to the toxicity of nanoparticles.54 In this section, we
of nanoparticles. Inside the cell, nanoparticles tend to localize in discuss interactions of nanoparticles with proteins, lipids, DNA,
certain cellular organelles and affect specific cell regions.42−44 and small molecules. We focus on the characterization and
In biological fluids, nanoparticles interact with phospholipids, mechanistic elucidation of these interactions.
proteins, DNA, and small metabolites. Phospholipids are the 3.1. Nanoparticle−Phospholipid Interactions
main components in the pulmonary system and cell
membranes, consisting of a negatively charged phosphate 3.1.1. Interaction of Nanoparticles with Phospholipids
group (head) and a hydrophobic carbon chain (tail).18 in Pulmonary Surfactant Solutions. Phospholipids and
Nanoparticles may interact with the charged head and/or the proteins constitute a pulmonary surfactant system. Inhaled
hydrophobic tail depending on their surface hydrophobicity. nanoparticles inevitably interact with these components.
Nanoparticles spontaneously adsorb proteins in the blood- Interactions between nanoparticles and the pulmonary
stream or biological fluid. Proteins are large biomolecules of surfactant system determine nanoparticles’ pulmonary toxicity
folded amino acid residues with different sizes, shapes, and net or drug delivery efficacy. Therefore, understanding these
charges at physiological pH. Protein surfaces also exhibit interactions and effectively regulating these interactions are
varying hydrophobicity depending on the nature of the exposed imperative for safe nanotechnology and better nanomedicine.
amino acid residues.18 All of these properties may affect the Pulmonary surfactant solutions have been used to mimic the
stoichiometry and orientation of their bindings to nano- human pulmonary surfactant system for mechanistic studies.
particles. Nanoparticles may also bind DNA molecules in The nature of nanoparticle−phospholipid interactions can be
biological systems. DNA molecules carry negative charges from investigated after isolating and identifying nanoparticle-bound
the phosphate groups and base π systems.18 Single- or double- surfactant molecules and characterizing the properties of
strand DNA molecules can bind to nanoparticles through nanoparticle−surfactant complexes. Nanoparticle-bound lipid
electrostatic, π−π stacking, and hydrophobic interactions molecules can be isolated by size exclusion chromatography55
(section 3.3). Therefore, molecular interactions of nano- or thin layer chromatography50 and structurally identified by
particles in biological environments involve interactions with NMR55 or MS.51 Nanoparticle−phospholipid interactions can
proteins, DNA, phospholipids, small molecules, and inorganic also be measured by identifying alterations in nanoparticle
ions. Such interactions are further complicated by the kinetics physicochemical properties. Surfactant molecule coatings alter
and thermodynamics of the nanoparticle associations with each nanoparticle sizes and dispersion states, which can be measured
component. using DLS,56 TEM,56,57 or SEM.58 Surfactant molecules can
The organ from which a cell originates influences nano- also alter the surface charge status of nanoparticles, as
particle−cell interactions. First, different cell types have determined with ζ-potential analysis.56 Binding of surfactant
different shapes. Human erythrocytes exhibit a smooth, molecules onto the surfaces of nanoparticles may alter their
spherical shape, while ependymal cells demonstrate a long, surface plasma resonance (SPR) absorption and can also be
slender shape.45,46 These differences produce different cellular determined using UV−vis absorption spectroscopy.56 The
responses when internalizing foreign matter, such as nano- surface properties of pulmonary surfactant solutions, such as
particles. Second, plasma membranes from different cell types equilibrium and dynamic surface tension behavior under the
have different lipid compositions, which influence the physical pulsation of samples, are also used to evaluate the effects of
properties of membranes and make them function differently. nanoparticles.57,59
Third, different types of cells express different membrane Dipalmitoylphosphatidylcholine (DPPC, the most abundant
receptors. For example, macrophages express a large number of surfactant molecule in a pulmonary surfactant mixture; also
scavenger receptors that help internalize foreign matter termed Survanta) is strongly adsorbed onto the surface of
nonspecifically.47 Fourth, different cellular organelles have nanoparticles (e.g., carbon-based nanoparticles), forming a rigid
various impacts on nanoparticles. Lysosomes are acidic monolayer. The binding of long aliphatic hydrocarbon chains to
organelles, while the cytoplasm is nearly neutral. Nanoparticles carbon nanoparticle surfaces results in increased nanoparticle
in lysosomes may undergo degradation, which may result in the surface charge, size, and stability. Adsorption onto a hydro-
release of ions and a toxic state.48 However, drug cargos may be phobic surface is also energetically favorable for DPPC.56
preferably released in acidic environments for therapeutic Although proteins bind to multiwalled CNTs (MWNTs) with
purposes.49 different binding patterns depending on the surface function-
alization of the CNTs, the surfactant lipid may nonspecifically
3. NANOPARTICLE−BIOMOLECULE INTERACTIONS adsorb onto different MWNTs.50 Adsorption of lipids onto the
Nanoparticles enter the human body via multiple routes, surface of carbon-based nanoparticles may affect their in vitro
encountering various biomolecules regardless of the pathway by and in vivo behavior. When coated with DPPC, nanoscale
which they enter the human body. Airborne nanoparticles may carbon black and crocidolite asbestos increase reactive oxygen
enter the human body through inhalation. In the lungs, species (ROS) generation in primary bronchial epithelial cells
nanoparticles exhibit strong interactions with the lung and in A549 alveolar epithelial carcinoma cells, while fetal
surfactant system, which consists of proteins and phospholi- bovine serum protein-coated nanoparticles protect cells from
pids.50,51 Engineered medical nanoparticles are generally oxidative insult.60 However, in another study, DPPC-coated,
administered into the circulation system through intravenous single-walled CNTs (SWNTs) did not generate cytotoxic or
injection. Injected nanoparticles first interact with blood fibrogenic effects in vitro. Lung fibrosis in mice can be induced
proteins before being distributed in various organs.52 Some by SWNTs with or without DPPC coating, indicating that
nanoparticles may also enter the cell nucleus and interact with DPPC does not significantly mask the bioactivity of SWNTs in
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vivo.58 Surfactants and the nanoparticle system can also affect membranes or liposomes. A quartz crystal microbalance
each other. Phospholipids have been shown to stabilize (QCM) with dissipation monitoring can be used for real-time
colloidal gold nanoparticles (GNPs) by forming lipid bilayers monitoring of nanoparticle−lipid membrane interactions.74
on the particle surface, while the presence of GNPs reduces the Fluorescence microscopy,65,75 fluorescence spectroscopy,68
surface activity of surfactants, such as their ability to form a fluorescence correlation spectroscopy (FCS),73 Föster reso-
surface film or the ability of this film to reach low surface nance energy transfer,73,76 NMR,77 and liposomal leakage
tension during compression.57 The size of particles also affects assay78 have all been used to study the biophysical changes and
their interactions with lipids. TiO2 nanoparticles but not disruptions of model lipid membranes or liposomes by
microsized particles increase the adsorption surface tension of nanoparticles.
the surfactant in a dose-dependent manner. These phenomena Hydrophobic69,70 and electrostatic interactions74,78 may
are also observed during surface area cycling in the presence of serve major roles in nanoparticle−lipid membrane interactions.
nanoparticles. TEM observations have demonstrated that The interactions of nanoparticles with the lipid membranes lead
lamellar body-like structures are deformed and decreased in to changes in both components, such as disruption of the lipid
size, with formation of unilamellar vesicles.61 Nanoparticles membrane and modification of the nanoparticle surface
enter the human body through respiratory, ingestion, and properties. Guanidinylated dendrimers that carry a positive
dermal systems and are distributed in many internal organs surface charge first adhere to the liposomal membrane through
after absorption. Therefore, nanoparticles tend to interact with binding to negatively charged phosphate groups. The
many cell types. Since DPPC is also a key membrane dendrimer surfaces then become hydrophobic due to charge
component of many cells, studies using DPPC as a model neutralization. The dendrimers are consequently transported
system have broad implications. through the hydrophobic liposomal membrane.68 Other
The binding events of proteins to nanoparticles most likely polycationic polymers (i.e., polyamidoamine dendrimers,
affect the surfactant interactions of nanoparticles. Interactions pentanol-core polyamidoamine dendrons, polyethylenimine,
of polymeric nanoparticles with lipids in human plasma have and diethylaminoethyl dextran) also induce the disruption of
been investigated using size exclusion chromatography, NMR, supported bilayers, including formation of holes, membrane
and enzymatic assays. Nanoparticles bind cholesterol, trigly- thinning, and membrane erosion.79 Polycationic polymers
cerides, and phospholipids in human plasma. The binding disrupt the liquid phase but not the gel phase of
events depend on the nanoparticle surface properties. Higher dimyristoylphosphatidylcholine bilayers.77 Electrostatic inter-
cholesterol content is present in the pellets of hydrophobic actions can also occur between protein-coated CNTs and
nanoparticles when compared to those of more hydrophilic liposomes, leading to liposome leakage.78 Surface-charged
nanoparticles.55 polystyrene nanoparticles bind to liposomes and induce surface
3.1.2. Interactions Between Nanoparticles and Model reconstruction of the phospholipid membranes, indicating a
Membranes. Model membranes are organized lipids that potential structural perturbation of the cellular membrane by
mimic the arrangement of lipids in natural cell membranes. nanoparticles (Figure 3).73 Nanoparticle-induced lipid bilayer
Interactions between nanoparticles and model membranes have rearrangements are limited to bubble-like liposomes not
been investigated using biophysical approaches to evaluate the supported bilayers.76 QDs can insert into lipid membranes
cellular translocation of nanoparticles and the potential effects and induce a current burst under an electric field, indicating
of nanoparticles on the integrity of the cell membrane. Lipid their potential to perturb neuronal and muscle cells.80,81
monolayers, bilayers, and liposomes are three widely used Cationic phosphorus dendrimers can alter the thermotropic
model membranes.62 behavior of lipid bilayers by reducing the cooperativity of the
The surface pressure−area (π−A) isotherm is an indicator of phospholipids; this effect is membrane surface charge depend-
the monolayer properties of an amphiphilic material and ent.71 A model has been proposed to describe the behavior of
obtained by measuring the surface pressure as a function of the nanoparticles at the air−liquid interface of alveoli (Figure 4).
water surface area available to each molecule.63 The isotherm is Nanoparticles inhaled into the alveolar space during breathing
recorded by compressing the film at a constant rate while may interact with continuous surfactant films. The nano-
continuously monitoring the surface pressure. Although π−A particles then become wetted and lined with phospholipid
isotherm measurements have been used to study the properties bilayers.57
of model lipid monolayers in the presence of nanoparticles, this In addition to the lipid membranes properties, factors that
approach is limited to monolayer membranes that lack influence nanoparticle−lipid interactions include nanoparticle
anchoring membrane proteins.64 AFM can be used to measure dose, composition, size, and surface chemistry. Hydrophobic
the effects of nanoparticles on morphological changes of polyorganosiloxane nanoparticles do not substantially affect the
Langmuir−Blodgett (LB) films. AFM measures subtle structural organization of a model pulmonary surfactant film at
morphological details of soft matter at subnanometer levels, low concentrations (e.g., 10 μg/mL) but significantly affect
enabling observation of the formation of lipid bilayers and their such properties at high concentrations (e.g., 3000 μg/mL).82
disruption by nanoparticles.65−68 A liquid crystal-based system Polystyrene nanoparticles with a diameter of 20 nm
has been developed to study the molecular interactions demonstrate greater interactions with the endothelial model
between protein-coated nanoparticles and a liquid crystal- membrane than those with a diameter of >60 nm.64
supported model membrane. The spatial and temporal Perturbations from nanoparticles may cause pore formation
organization of the lipid monolayer is coupled to the in lipid bilayers.65,66,79 Studies of polycationic polyamidoamine
orientation of the liquid crystal, permitting direct observation dendrimers and supported dimyristoylphosphatidylcholine
of the dynamic characteristics of the lipid monolayer.69,70 bilayers have demonstrated that G7 dendrimers (8.0−8.2
Differential scanning calorimetry (DSC)68,71,72 and isothermal nm) but not G3 dendrimers (3.5−4.1 nm) induce formation of
titration calorimetry (ITC)73 can also be used to measure holes in lipid bilayers. G5 dendrimers (4.3−6.6 nm) have a
nanoparticle-induced thermodynamic changes in supported reduced effect when compared to G7 dendrimers but had no
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Figure 3. Nanoparticle-induced surface reconstruction of phospholipid

membranes. Phospholipid bilayer vesicle with bound nanoparticles is
shown. Binding of anionic nanoparticles to the lipid bilayer in the fluid
phase causes a transition to gel phase at the site at which the
nanoparticle binds. Binding-induced reorientation of the phosphocho-
line (PC) headgroup causes lipids in the fluid phase to exhibit a lower Figure 4. Potential inhibitory effects of GNPs on pulmonary
density (A) than in the gel phase (B). In the PC headgroup, P− and N+ surfactants in the alveolar space. (a) Continuous surfactant film
are denoted by blue and red, respectively. Reprinted with permission (monolayer and underlying multilayer) forms after the rupture of
from ref 73. Copyright 2011 National Academy of Sciences, USA. pulmonary surfactant vesicles at the air−liquid interface as the
hypothetical rate-limiting intermediate structure between bilayer
vesicles and the interfacial monolayer. (b) Disrupted interfacial
effect when their amine end groups were replaced with surfactant film due to entrapment of GNPs (from the air phase as
acetamide groups, indicating the role of surface chemistry.67 pollutants) by pulmonary surfactant is depicted. (c) Self-aggregation of
The density of the functional groups also affects the lipid-capped GNPs in the presence of surfactant protein B is shown in
interactions. Lightly guanidinylated dendrimeric derivatives red. Reprinted with permission from ref 57. Copyright 2008 Cell Press.
act as “molecular glue” by adhering to liposomes, while highly
guanidinylated dendrimeric derivatives induce liposome
mechanisms have been studied, and the results have yielded
fusion.68 The crucial role of nanoparticle surface hydro-
explanations for some experimental outcomes.87,88,90,93,95−98
phobicity has also been demonstrated. Hydrophobic, choles-
In summary, investigation of nanoparticle−lipid membrane
terol-coated GNPs interact with DPPC vesicles and decrease
interactions through molecular simulations, biophysical charac-
their melting point, while hydrophilic nanoparticles increase the
terization, and cellular studies may collectively unravel the
melting point of DPPC vesicles.72 The properties of model
mechanisms of these processes and elucidate regulation
membranes, such as membrane surface charge71 and cholesterol
strategies. Such developments will benefit nanotechnology-
content, also affect these interactions. A high cholesterol
based drug delivery and rational reduction of nanotoxicity.
content in the phospholipid/cholesterol monolayer makes the
lipids more susceptible to disruption by protein-coated 3.2. Nanoparticle−Protein Interactions
GNPs.69 The interactions between nanoparticles and proteins are
Development of efficient nanocarrier systems based on complex. Nanoparticles are generally surrounded by plasma
model membrane studies requires a good correlation between proteins upon entering the circulatory system. As nanoparticles
model systems and cellular studies. The effects of the size and enter cells and are distributed into different organs, they are
surface chemistry of polymeric nanoparticles in model also in close contact with cellular proteins. Protein binding-
membranes correlate well with cell membrane disruption and induced changes in the surface properties of nanoparticles affect
cellular uptake.83−86 their biodistribution and clearance.99 Moreover, conformational
The interactions of nanoparticles with model membranes changes in bound proteins may expose unexpected epitopes to
have also been investigated by molecular simulation ap- activate cell signaling.100 The interactions of nanoparticles with
proaches. Although computational methods cannot fully proteins are dynamic processes and governed by properties of
mimic complex biological systems, they facilitate an enhanced both components. The physicochemical changes in nano-
mechanistic understanding of their interactions and improved particles or proteins also allow characterization of the
property predictions. The interactions of carbon nanoparticles, thermodynamic and kinetic processes of their interactions.
polymers, and nanocrystals with lipid bilayers have been 3.2.1. Determination of Nanoparticle−Protein Inter-
simulated by all-atom and coarse-grained approaches.87−94 The actions. Similar to drug−protein or protein−protein inter-
influences of nanoparticle size, surface chemistry, hydro- actions, nanoparticle−protein interactions are also character-
phobicity, shape, and bilayer properties on the interaction ized by binding affinity, stoichiometry, and kinetic properties.
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Determining these properties helps us understand the chemical

and dynamic nature of these interactions. Modulating these
properties by modified nanoparticles may prevent unwanted
interactions or enhance specific binding events.
3.2.1.1. Determination of Binding Affinity and Stoichiom-
etry and Identification of Binding Sites. Steady-state and time-
resolved fluorescence spectroscopy are used to study nano-
particle−protein binding affinities, complex formation, and
binding-induced protein conformational changes.19 The
dependence of protein fluorescence intensity or lifetime on
quencher (nanoparticle) concentration follows the Stern−
Volmer equation, eq 1
F0/F = τ0/τ = 1 + K sv[Q ] (1)
where F0, F, τ0, and τ represent the initial or modified
fluorescence intensity or lifetime, respectively. Ksv is the Stern− Figure 5. Two-dimensional (2D) [15N−1H]-HSQC NMR spectra of
Volmer constant, and [Q] is the quencher (nanoparticle) free human ubiquitin (red) and human ubiquitin plus GNPs (yellow)
concentration.101 at pH 7.7, showing chemical shift perturbation for some NH groups
upon interaction with GNPs. Black arrows indicate the NH backbone
Fluorescence polarization permits the detection of interact-
groups with the largest chemical shift perturbations (Q2, L15, and
ing macromolecules in homogeneous solutions and can E18) in this spectral window. Reprinted with permission from ref 104.
quantify nanoparticle−protein interactions based on the change Copyright 2010 American Chemical Society.
in rotational freedom of the fluorescent molecule upon complex
formation.23,101 An advantage of fluorescence polarization is
that it avoids complex separation, which may induce cleavage followed by matrix-assisted laser desorption-ionization
denaturation of proteins or interrupt reaction equilibria. The time-of-flight mass spectrometry (MALDI TOF-MS). The
binding ratio can be determined on the basis of fluorescence nanoparticle binding site on HSA was therefore identified.
polarization measurements. Identification of binding sites helps determine the binding
Stepwise photobleaching has been used to characterize specificity and predict possible biological consequences of
nanoparticle−protein interactions. Both protein and nano- nanoparticle−protein interactions.105
particles are labeled with fluorescence dyes. The protein− DLS can be used to estimate nanoparticle−protein binding
nanoparticle binding ratio is estimated by counting the steps of stoichiometry. Adsorption of proteins causes a small increase in
the nanoparticle photobleaching. The distribution of photo- the dynamic radius of nanoparticles. In one study, adsorption of
bleaching steps is quantified to obtain the average number of hexahistidine-tagged cytochrome P450BSβ molecules to the
proteins per nanoparticle. The photobleaching counts can be surface of QDs was investigated by DLS. The measurements
obtained by ensemble or single-molecule measurements.102 revealed that six P450BSβ molecules bind to the surface of each
FCS measures the fluctuations of fluorescence intensity due QD.106
to the Brownian motion of fluorescent particles in a small 3.2.1.2. Measuring Nanoparticle−Protein Binding Kinetics.
space. The hydrodynamic particle radii (RH) can be calculated SPR technology is based on the change in the oscillation of
through the average diffusion time (τD), the diffusion surface plasmon waves caused by adsorption of molecules onto
coefficient (D), and the Stokes−Einstein relationship. Using a metal surface.107 Using this approach, polymeric nano-
this approach, the KD of human serum albumin−nanoparticle particle−plasma protein binding kinetics have been inves-
binding (∼5.1 μM) and the protein−nanoparticle thickness tigated. Nanoparticles are anchored on the gold surface of the
(∼3.3 nm) were estimated.103 sensor chip, and proteins are injected to flow over the
The binding sites of nanoparticles on a protein can be nanoparticle-modified surface.108 A QCM can be used to
identified using NMR. 2D [15N−1H]-Heteronuclear single- measure the resonant frequency shift, which can be correlated
quantum coherence (HSQC) NMR was used to identify a with a mass change at the oscillating quartz surface. Either
specific binding domain of GNPs on the surface of human proteins or nanoparticles can be immobilized onto the gold
ubiquitin. Addition of GNPs changed the positions of certain surface on the quartz crystal. The binding partner (nano-
peaks, while others were unaffected (Figure 5), indicating particles or protein) is injected and flowed through a flow
specific interactions. In the preassigned peak map, the largest chamber over the quartz surface, and the frequency is
chemical shift changes were from the NH backbone of Q2, L15, monitored.109,110 Using either SPR or QCM, the real-time
and E18. On the basis of a combination of the chemical shift and quantitative binding profile can be obtained, and the
changes within the protein crystal structure, a domain association and dissociation constants can be determined by
containing Q2−I3 and L15−E18 was proposed to be the fitting to the Langmuir adsorption isotherm or Scatchard
specific binding site for GNPs in GNP−ubiquitin interac- equation.
tions.104 3.2.2. Chemical Origin of Nanoparticle−Protein
The binding sites of nanoparticles on a protein can also be Interactions. Interactions of nanoparticles with proteins are
identified using cross-linking chemistry and MS. As shown in dominated by many chemical processes. The properties of the
Figure 6, the peptides located very close to poly(acrylic acid)- nanomaterials significantly influence these processes. Next, we
coated Fe3O4 nanoparticles in the binding site of human serum discuss the mechanisms of interactions between proteins and
albumin (HSA) were cross-linked onto the nanoparticle surface different nanomaterials.
with 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydro- 3.2.2.1. Carbon Nanoparticles. Carbon nanoparticles, such
chloride. The attached peptides were identified by protein as CNTs, fullerene, and graphene, contain only sp2 carbons,
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Figure 6. Identification of peptides associated with nanoparticle−protein interactions by cross-linking and MS. (A) Schematic illustration of the
detection principle. (B) MALDI-MS spectra of released peptides when human serum albumin was (a) cross-linked and (b) adsorbed to the
poly(acrylic acid)−Fe3O4 nanoparticles. Analysis of the supernatant from the cross-linked and non-cross-linked samples is shown in c and d,
respectively. A peak of m/z 1640 in Ba indicates a peptide cross-linked onto the nanoparticle surface; in the supernatant, the peak intensity is
significantly reduced (Bc). Arrows in Bc indicate 3 of 13 identified peptides with consistently reduced peak intensity in supernatant samples.
Reprinted with permission from ref 105. Copyright 2011 American Chemical Society.

while nanodiamonds contain exclusively sp3 carbons.111−114 and heavy chains.139 The binding sites of C60 in human and
The uniqueness of sp2 carbon nanomaterials is due to their bovine serum albumin (BSA) and HIV−protease are quite
overwhelming π−π systems.114 Strong interactions between similar but different from that in the C60 antibody.132 Amino
these materials and proteins are derived from π−π stacking, acid-modified C60s can penetrate the lipid membrane, bind to
hydrophobic, and electrostatic interactions.19,115,116 Interac- the hydrophobic domains of proteins, and alter the function of
tions between aromatic groups on carbon nanomaterials and membrane-bound enzymes.140
aromatic or amine residues in proteins play a vital role in Graphene is another hexagonal carbon nanostructure. In
binding.117 Therefore, nanoparticle−protein binding events contrast to CNTs and fullerenes, graphene consists of planar
depend on the carbon nanoparticles, the nature of proteins, and atomic thin layers.2,141 Because of the sp2 carbon structure, the
microenvironmental factors, such as pH and ionic driving forces for interactions between proteins and graphenes
strength.19,115,118−122 Charge transfer between CNTs and are mainly hydrophobicity and π−π stacking. Molecular
streptavidin has been observed, indicating interactions between dynamics simulation predicted that human insulin can be
the amine groups of the protein and the aromatic surface of the adsorbed onto graphene surfaces and that some proteins would
CNTs.117 TEM and AFM observations have indicated that the be destroyed or partially destroyed.142 Water-soluble graphene
bound proteins were found to form well-organized structures oxide nanosheets (GO) are oxidation products of graphene
on the nanotube surface depending on the nanotube’s diameter nanosheets. GOs have a stronger serum protein adsorption
and the bound proteins (Figure 7).52,123 CNT-bound proteins capability than SWNT or MWNT. The thickness of the GO
generally form multiple layers on the nanotube surface.124 This layer increased from ∼1.0 to 4.0−18.0 nm after protein
property can be used for nanotube debundling, stabilization, binding.143 The function of proteins adsorbed on the GO
separation, and protein sensing.115,120,125−127 The bound surface can be inhibited (e.g., the enzymatic activity of
proteins often undergo changes in structure and activity. chymotrypsin144). The GO−protein binding equilibrium can
Molecular simulation studies have demonstrated that proteins be reached in 30 min, and protein adsorption mitigates the
undergo stepwise conformational changes after binding to cytotoxicity of GO.143
CNTs.128−130 For example, both soybean peroxidase and α- In contrast to sp2 nanostructures, diamond nanoparticles
chymotrypsin bind to SWNTs; soybean peroxidase retains 30% consist exclusively of sp3 bonds. These nanoparticles bind
activity, while α-chymotrypsin retains only 1% activity. This protein molecules through hydrophobic interactions and
difference in activity occurs because soybean peroxidase physical adsorption and are used in tissue engineering (e.g.,
partially maintains its secondary structure after binding, while immobilization of bone morphogenetic protein 2 (BMP-2) to
α-chymotrypsin completely loses its secondary structure.118 promote bone formation145) or as mass spectrometric
CNTs enhance the rate of protein fibrillation by decreasing the matrices.146
lag time preceding nucleation, indicating the potential of CNTs 3.2.2.2. GNPs. GNPs most likely interact with proteins
to induce amyloid formation, as well as assembly of novel through electrostatic, hydrophobic, and sulfur−gold interac-
nanoparticles.131 tions.23,147−151 GNPs with citrate as a stabilizing agent can bind
The interactions of C60 with proteins have been studied to proteins according to the Langmuir isotherm.151,152 The size
using biochemical, structural, and molecular simulation of nanoparticles influences the mechanism of protein binding.
approaches.132−137 The possible chemical interactions respon- One study demonstrated that electrostatic interaction is the
sible for C60−protein binding events include hydrophobicity, major binding force for nanoparticles with a diameter of 16 nm,
surface curvature, π−π stacking, uneven charge distribution, site while hydrophobic interaction is the major force for nano-
fit, and solvent displacement.135 Molecular dynamic studies particles with a diameter of 2−4 nm.148 Because the gold
have demonstrated that interactions such as hydrophobicity and surface forms strong bonds with thiol groups in cysteine
π−π stacking represent the dominant factors.138 The binding residues, the GNPs bind BSA and heparin-binding growth
site of C60 on its antibody has been identified using X-ray factors.151,153 The latter activity may be used to inhibit tumor
crystallography. C60 binds to the interface of the antibody light angiogenesis.154 Perturbation of the conformation and activity
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Figure 7. Interactions between bovine fibrinogen, immunoglobulin, transferrin, bovine serum albumin, and SWNTs. Molecular modeling
illustrations of proteins (in bead representation) binding to SWNTs after incubation for 10 min (A) and 5 h (B). (C) Locations of the most
preferred binding sites on proteins for SWNTs. Residues highlighted in the van der Waals representation correspond to tyrosine (red) and
phenylalanine (green). Other regions of the protein are presented in pink. (D) Detailed orientations of the aromatic rings of the tyrosine and
phenylalanine residues interacting with the six-membered rings of SWNTs are represented in silver. Tyrosine residues are rendered as licorice
representations (red), while phenylalanine residues are shown in green. Adapted from ref 52 with permission. Copyright 2011 National Academy of
Sciences, USA.

of bound proteins is influenced by the size and surface modified GNPs can also tune the enzymatic activity of bound
properties of the nanoparticles, the characteristics of the α-chymotrypsin.159 The nanoparticle size influences the
protein, and the ionic strength and pH of the solu- denaturation kinetics of BSA on GNPs.157 Negatively charged
tion.148,155−158 Cytochrome c is adsorbed onto anionic poly(acrylic acid)-conjugated GNPs bind to and induce the
nanoparticles followed by conformational changes and unfolding of fibrinogen. The unfolded fibrinogen promotes
proteolysis.147 Chymotrypsin binds to GNPs and undergoes a MAC-1 receptor activation and inflammation.160
two-stage change: fast, reversible inhibition followed by slow, 3.2.2.3. Magnetic Iron Oxide Nanoparticles (MNPs).
irreversible inactivation.150 The nanoparticle surface chemistry Adsorption of BSA molecules onto MNPs fits well to the
can regulate GNP−protein interactions.155,159 Amino acid- Langmuir isotherm.161 The adsorption kinetics fit to a linear
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driving force mass-transfer model. Similar to other nano- activity in bound lysozyme172 and induce a greater decrease in
particles, iron oxide nanoparticles also induce protein the thermodynamic stability of RNase A.173 In another study,
conformational changes and alter protein activity. Human silica nanoparticles were shown to promote α-helical structures
transferrin molecules are adsorbed onto the surface of MNPs in a designed peptide of low helical content in solution.174
and undergo conformational changes from a compact to an Consequently, the protein conformational changes altered their
open structure. This transformation induces the release of cell binding capability and reduced proliferation of human
iron.162 cells.175
3.2.2.4. Quantum Dots (QDs). Electrostatic interactions, 3.2.2.6. Polymeric Nanoparticles. Adsorption of proteins
sulfur−metal bond formation, and spatial effects are believed to onto polymeric nanoparticles also depends on the hydro-
be major driving forces for QD−protein binding.163−165 CdS phobicity and surface curvature of nanoparticles. Interactions of
nanoparticles are trapped in the cylindrical cavity of chaperonin proteins with N-isopropylacrylamide/N-tert-butylacrylamide
proteins and become thermally stable and tolerant to copolymer nanoparticles with different monomer ratios and
electrolytes.163 The binding between QDs and HSA occurs particle sizes demonstrate that proteins tend to bind to more
near a protein pocket centered at Lys199.164 Calculations reveal hydrophobic nanoparticles by forming a single layer on the
negative enthalpy (ΔH) and positive entropy (ΔS) values for surface.176 Surface chemistry also influences protein binding.
this interaction, indicating that electrostatic interactions play a For instance, in dextran phenoxy/poly(ethylene oxide)
major role in the binding reaction. The stoichiometry of bound copolymer-modified polystyrene latex nanoparticles, BSA can
protein molecules on each QD is approximately 6. The bound deform and penetrate the poly(ethylene oxide) layer to bind to
proteins undergo substantial secondary and tertiary structural the nanoparticle surface. In contrast, a densely packed dextran
changes.164 In another study, the electrostatic adsorption of phenoxy layer prevents nonspecific adsorption.177 Similar to
hemoglobin (Hb) to CdS nanoparticles was shown to be CNTs and QDs, polymer nanoparticles also promote protein
energetically favorable. Raman spectroscopic results indicated fibrillation by enhancing the nucleation of protein fibrils from
that the sulfur atoms of the cysteine residues form direct human β2-microglobulin.131 Promotion of fibrillation may be a
chemical bonds with the CdS QD surface. Although higher common property for a large number of nanoparticles. Another
structures are disturbed, the spin state of the heme iron is not study concluded that the first layer of bound proteins is much
affected.166 In addition to native proteins, denatured BSA more stable than the secondary layer on the nanoparticle
molecules also bind to the surface of CdTe QDs and form a surface. The first layer was hence called the “hard corona”,
shell-like complex.167 The protein shell structure results in the indicating nearly irreversible binding, and the secondary layer
removal of Te atoms from the CdTe surface and the formation was termed the “soft corona”, indicating greater dynamic
of a thermodynamically stable nanostructure. Similar to CNTs, exchange with the surrounding milieu.178
QDs also enhance protein fibrillation.131 The above studies demonstrate that protein binding is a
3.2.2.5. Silica Nanoparticles. Electrostatic interactions may nature of all nanoparticles, regardless of material. The material
also be a driving force for protein adsorption by silica origin, size, shape, and surface chemistry of nanoparticles
nanoparticles, as determined by DSC.168 A decrease in ionic determine the chemical or physical forces involved in the
strength is accompanied by a reduction in protein binding interactions of nanoparticles with proteins. These interactions
enthalpy, indicating that there is a strong electrostatic attraction can be modified through adjusting the physicochemical
in silica nanoparticle/lysozyme and silica nanoparticle/RNase properties of the nanoparticles.
binding.168 Hydrophobic interactions and hydrogen bonding 3.2.3. Interactions Between Nanoparticles and Pro-
are also driving forces in silica nanoparticle−peptide binding.22 teomes. Although the elucidation of the interactions between
The bound proteins on silica nanoparticles assume a specific nanoparticles and single proteins facilitates the understanding
orientation. Adsorption of human carbonic anhydrase II onto of the chemical nature of such interactions, in biological
negatively charged silica nanoparticles appears to be specific to systems, nanoparticles encounter proteomes, not a single
limited regions at the N-terminal domain of the protein.169 In protein. Blood and cellular proteomes consist of thousands of
this study, the orientation of the bound proteins is also pH proteins with diverse functions, sequences, molecular weights,
dependent. At pH 6.3, a histidine-rich area around residue 10 is isoelectric points, surface hydrophobicities, and concentrations.
the dominant binding region. At higher pH values (e.g., pH The protein corona composition is determined by the
9.3), the protein is adsorbed near a region close to residue 37, thermodynamic and kinetic properties of the interactions
which contains several lysine and arginine residues. This between the nanoparticles and proteins.108 The selectivity in
absorption behavior indicates that specific binding may be a protein binding is influenced by factors such as the size and
result of electrostatic interactions between the positively surface chemistry of the nanoparticles and the properties of the
charged areas on the protein surface and the negatively charged proteins.179−182
silica surface. The binding of protein onto nanoparticles occurs Binding between polymer nanoparticles and plasma proteins
in two steps: rapid formation of a protein−particle complex is highly dynamic. Abundant yet low-affinity HSA molecules
followed by an irreversible protein conformational change.170 quickly bind to the surface of nanoparticles and are soon
Nanoparticle-induced protein conformational changes vary replaced by the high-affinity and slow-exchanging apolipopro-
depending on the nature of proteins. Unlike the “hard” (very teins AI, AII, AIV, and E. These proteins remain associated with
stable) protein carbonic anhydrase II, the “soft” (less stable) the particles.108,182,183 Although formation of a protein
protein carbonic anhydrase I undergoes a larger structural monolayer is an acceptable model, nanoparticles can also
rearrangement when adsorbed onto silica nanoparticles, as attract additional layers of proteins. By analyzing the adsorption
shown by NMR.171 The size of silica nanoparticles determines of fetal bovine serum proteins onto iron oxide nanoparticles
the surface curvature, which influences protein adsorption and using gel electrophoresis and LC-MS/MS, proteins that
the stability of the adsorbed proteins. For instance, larger strongly bind to the surface of nanoparticles were identified.
nanoparticles cause a greater loss of α helicity and enzymatic These proteins include complement factor H, antithrombin,
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Figure 8. PEG density-based determination of the composition of the adsorbed serum protein corona on the surface of GNPs. Adsorbed serum
proteins were isolated from 15 nm GNPs grafted with PEG at varying densities, purified, digested with trypsin, and characterized by LC-MS/MS.
Proteins were clustered into one of four groups, A, B, C, or D (represented by the colored bars), based on the correlation of their relative abundance
across PEG densities. PEG/nm2: number of PEG molecules per square nanometer. Reprinted from ref 181 with permission. Copyright 2012
American Chemical Society.

complement factor I, α-1-antiproteinase, and apolipoprotein E. and E. The reduced protein binding on nanoparticles coincides
These surface-bound proteins serve as linkers to further bind with decreased nanoparticle internalization by cells.180 Stepwise
other serum proteins, such as BSA molecules, which adsorb increases in PEG density on nanoparticle surfaces shift the
with lower affinity onto iron oxide nanoparticles.184 serum protein binding pattern in addition to the binding
Like single-protein bindings, proteome binding by nano- amount (Figure 8). An increase in PEG density reduces the
particles is strongly affected by the surface chemistry of internalization of GNPs into cells, similar to polymeric
nanoparticles. Low-density lipoprotein, very-low-density lip- nanoparticles.181 Nanocapsules with longer PEG chains and
oprotein, and high-density lipoprotein bind strongly to QDs higher PEG density also reduce complement system activation
with cholesterol and atheronal-B modifications when compared and uptake into J774A1 macrophage-like cells.185 Polysacchar-
to amine-modified QDs.179 Poly(ethylene glycol) (PEG)- ide modifications on the surface of poly(isobutylcyanoacrylate)
modified nanoparticles reduce protein binding. PEG chain nanoparticles reduce plasma protein binding and complement
length and density on polymeric nanoparticle surfaces regulate system activation.186 MNPs with different sizes and surface
the amount of human plasma protein adsorption but not the chemistries bind different serum proteins without changing the
binding pattern. The main bound proteins identified by 2D protein secondary structure.187 The PEG coating on these
polyacrylamide gel electrophoresis analyses are albumin, nanoparticles reduces both cell uptake and cytotoxicity. Smaller
fibrinogen, IgG, Ig light chains, and the apolipoproteins AI PEG-coated nanoparticles bind relatively more serum proteins
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and display reduced cellular uptake. The composition of cell nanoparticle−DNA complexes can be isolated by anion
culture media also affects protein adsorption and cellular effects. exchange chromatography.193
Using GNPs as a model, Dulbecco’s modified Eagle’s medium Circular dichroism (CD) can be used to detect DNA
elicited formation of a protein corona, while use of Roswell conformational changes induced by nanopar-
Park Memorial Institute medium resulted in different dynamics ticles.53,191,197,202,208−213 B−A and B−Z transitions can be
with reduced protein coating. Protein−nanoparticle complexes identified by the appearance of characteristic CD bands.197,211
formed in the latter medium are more abundantly internalized NMR provides more detailed structural information than
by cells and cause greater cytotoxicity than those formed in the CD.208 Conformational information can also be obtained by
former medium.188 Heat inactivation of serum affects protein surface-enhanced infrared spectroscopy, which measures the
corona formation and cellular uptake of protein−nanoparticles vibrational modes of DNA molecules.214 The structural stability
complexes because heating induces changes in serum of DNA can also be measured by its m elting
composition and protein conformation.189 curve.53,197,206,208,210,212 DNA degradation can be evaluated
by gel electrophoresis.198,201,208,209,215−217 In addition to
3.3. Nanoparticle−DNA Interactions experimental approaches, molecular simulations can predict
Nanoparticles bind nucleic acids (e.g., DNA) in biological nanoparticle−DNA binding patterns and nanoparticle-induced
systems. The features of intermolecular forces involved in DNA DNA conformational changes.218,219
binding are similar to those involved in nanoparticle−protein 3.3.2. Current Understanding of Nanoparticle−DNA
binding. Such interactions can be exploited for biomedical Interactions. 3.3.2.1. Carbon Nanoparticles. Due to the large
applications, including DNA loading on nanoparticles for gene surface area of CNTs, DNA molecules bind to the surfaces of
transfer and siRNA delivery. Nanoparticle−DNA interactions nanoparticles almost spontaneously.218 This binding enhances
depend on the properties of both the DNA molecules and the the aqueous solubility of CNTs.193,220 Due to the unique shape
nanoparticles. and electronic properties of CNTs, the interaction modes
3.3.1. Determination of Nanoparticle−DNA Interac- between DNA and CNTs are different than those between
tions. Nanoparticle−DNA interactions can be detected DNA and other nanoparticles. DNA molecules can adsorb and
through determining complex formation, binding-induced wrap onto the nanotube through π−π stacking and hydro-
DNA conformational changes, and DNA degradation. phobic interactions or even insertion into the central
The binding of DNA molecules to nanoparticles alters the cavity.190,199,218,219 Such interactions also cause conformational
properties of both components. For example, binding alters changes in DNA molecules.190,197,208,211,214
nanoparticle dispersibility and surface charge. Such changes can Both experimental evidence and molecular simulation
be detected with DLS and ζ-potential measurements.53,190,191 indicate that DNA molecules form a helical structure before
The nanoparticle−DNA complexes have altered electron wrapping up CNTs.193,199 Optical absorbance measurements
have revealed that anisotropic hypochromicity results from
absorption spectroscopic patterns, which can also be measured
single-strand DNA/CNT interactions through π−π stacking
using UV−vis spectroscopy.192−194 DNA binding may increase
interactions.194 Due to π−π stacking interactions, DNA bases
the near-IR fluorescence of some nanoparticles. This increase
tend to orient themselves (Figure 9) facing the tube, as
can be used to monitor the binding process.190 DNA molecules,
when bound to nanoparticles, can be detected using XPS.20,195
By enhancing adenine and guanine ring breathing vibrations,
surface-enhanced Raman spectroscopy (SERS) has been used
to monitor DNA binding onto GNPs.196 AFM detects
subnanometer-scale phenomena and is used for the ultra-
structural observation of nanoparticle−DNA com-
plexes.190,193,197−199 Linear dichroism monitors the differential
absorption of polarized light parallel and perpendicular to the
orientation direction and enables determination of the
approximate orientations of the DNA molecules on
CNTs.200,201
Fluorescence spectroscopy can be employed to determine
DNA/nanoparticle binding affinity. Because of the weak
intrinsic fluorescence emission of DNA, ethidium bromide
(EB) or the low toxicity dye SYBR Green is used to label DNA
molecules.53,202 The resonance light-scattering method has Figure 9. Orientation of the bound DNA bases relative to the SWNT
been used to detect metal ions and biomolecules.203,204 axis. Green arrows and wavelengths indicate the directions and
Resonance light scattering from nanoparticles and DNA wavelengths of optical dipole transitions, respectively. Reprinted with
molecules is weak, but the scattering intensity is enhanced permission from ref 194. Copyright 2007 American Chemical Society.
dramatically when nanoparticles and DNA bind to each other,
permitting quantification of nanoparticle−DNA interac- determined by investigating the interactions between CNTs
tions.191,205 Real-time polymerase chain reaction (RT-PCR) and DNA homopolymers.194 Although one study demonstrated
can be used to amplify short DNA fragments attached to that binding of CNTs with DNA is independent of DNA
nanoparticles to quantify their surface coverage.206 The sequence,198 the sorting and separation of CNTs by DNA has
adsorption and desorption processes of nanoparticle−DNA been shown to be affected by DNA sequence.199 The
complexes can also be measured using electrochemical separation of CNTs by d(GT)n (n = 10−45) is more efficient
approaches, such as cyclic voltammetry.207 In some cases, the than separation by other sequences present in a single-strand
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DNA library. DNA molecules with GT-rich sequences wrap visible light is present.209,217 The magnitudes of the binding
individual nanotubes more effectively and form a helical energy of bases with graphene are similar to those found in
structure.199 Linear dichroism revealed that the DNA molecules SWNTs. Interactions are also driven by hydrophobic
lay flat on the nanotube surface with the backbone wrapping interactions and π−π stacking.221,222 The binding of a DNA
around the nanotube at an oblique angle of 45°.200 duplex to graphene oxide causes a partial deformation of the
DNA/CNT binding induces DNA conformational changes. double helix.223 Therefore, a high binding affinity to DNA
Binding of SWNTs to DNA induces B−A and B−Z (Figure molecules may be a common feature of carbon-based
10) transitions.197,211,214,218 The B−Z transition modulates the nanomaterials.
3.3.2.2. GNPs. Interactions between GNPs and DNA
molecules are mainly driven by electrostatic interac-
tions.192,216,224 Formation of a complex between DNA and
tiopronin-modified GNPs occurs in three steps:53 the first step
is diffusion-controlled formation of an external adduct; the
second step is formation of DNA−GNP complex I based on
their binding affinity; and the third step is formation of a more
compact DNA−GNP complex II after DNA conformational
changes. DNA−GNP interactions are influenced by the
properties of the DNA and nanoparticles and the micro-
environment.202,225 Small Au55 clusters (size ≈ 1.8 nm) attach
to the major grooves of B−DNA and cause a B−A transition.
Au55 nanoclusters are then degraded to 13-atom nanoclusters
due to shrinkage of the major grooves. Au13 nanoclusters and
A−DNA ultimately form a wire-like structure.24 In another
study, GNPs with a diameter of 4.4 nm and coated with
trimethyl(mercaptoundecyl)ammonium monolayer assembled
into a one-dimensional (1D) chain along a DNA strand
through electrostatic interactions between the cationic nano-
particle surface and the anionic DNA bases.192 The surface
coverage of thiol-capped oligonucleotides bound to GNPs
decreases from 30 to 13 pmol/cm3 when the nanoparticle
diameter is increased from 13 to 30 nm.206 In addition to
nanoparticle size, nanoparticle surface chemistry, salt concen-
tration, and sonication affect DNA/GNP interactions.225
In addition to GNP-induced DNA conformational changes,
nanoparticles also induce other changes in DNA molecules.
Figure 10. Illustration of DNA undergoing a conformational transition Nanoparticles promote relaxation of supercoiled DNA under X-
from B form (top) to Z form (bottom) on a carbon nanotube. ray radiation,226 inhibit hybridization and transcription,20,227
Reprinted with permission from ref 211. Copyright 2006 American induce double-strand separation,228 and enhance Raman signals
Association for the Advancement of Science. from double-stranded DNA molecules.196 These altered
properties can be employed for DNA detection,229 protection
dielectric environment of the SWNTs and decreases their near- of DNA from DNAase I digestion,230 inhibition of the
IR emission. These changes have been detected in whole blood, amplification of mismatched primer−template pairs, and
tissue, and living cells.211 SWNTs inhibit DNA duplex enhancement of the specificity of allele-specific PCR.231
association and induce telomeric i-motif formation by binding Interactions between DNA and GNP can also be used to
to the 5′-end major groove.208 In addition to CNT-induced build novel nanostructures. For example, hybridization of
DNA conformational changes, the binding of SWNTs affects complementary DNA is used to construct discrete GNP−QD
DNA hybridization. DNA molecules adsorbed to SWNTs nanostructures.232 Organized DNA motifs are used to make
display much slower hybridization kinetics than free DNA.190 ordered GNP arrays.233
Preadsorption on the SWNT surface causes a decrease in the 3.3.2.3. Other Nanoparticles. Other nanoparticles also
free energy of the DNA, which increases the energy required interact with DNA molecules through electrostatic and
for conversion of the DNA strands into the transition state. The hydrophobic interactions. Linear QD chains are formed along
process can be fitted well with a two-step Langmuir model.190 double-stranded DNA through electrostatic interactions.234
The binding of DNA to CNTs can also alter the optical Plasmid DNA bound to the QD surface can be released by
properties of SWNTs. Racemic SWNTs exhibit circular intracellular glutathione (GSH) during transfection.195 Calf
dichroism only when wrapped by DNA molecules. This thymus DNA molecules are adsorbed onto zirconia nano-
induced circular dichroism occurs when transition dipole particles.207 Titanium oxide nanoparticles are widely used in
moments of optically active electronic transitions in the DNA sunscreen products, and their potential toxicity has received
molecule are coupled to transition dipole moments of the considerable attention.235 These nanoparticles bind to DNA
SWNTs.213 The altered CD spectra can be used to characterize molecules and cause DNA degradation due to generation of
DNA−SWNT wrapping. hydroxyl radicals on the nanoparticle surfaces.236,237 This DNA
Other carbon nanoparticles also exhibit strong interactions damages induce oxidative stress in living cells and animals.238
with DNA molecules. For example, graphene oxide nanosheets Similar to titanium oxide nanoparticles, copper nanoparticles
and fullerene can cleave DNA strands when copper ions or cause DNA degradation through generation of singlet oxy-
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gen.239 Ag and Pt nanoparticles inhibit DNA hybridization by molecules may influence proper cellular functions and cause
disrupting hydrogen bonding between DNA double strands. toxicity.54,244,246 Small molecules are adsorbed onto nano-
This inhibition is weakened as the nanoparticle size increases.20 particles through molecular interactions, such as hydrophobic,
Binding of calf thymus DNA to Ag nanoparticles induces π−π stacking, and electrostatic interactions.54,247 Although the
conformational changes in DNA molecules.210 chemical bases of nanoparticle/protein interactions and nano-
Both cationic and anionic polymer nanoparticles bind DNA particle/amino acids interactions are similar, the latter is
molecules and cause DNA conformational changes.191,212 simpler and faster and does not involve conformational
Cationic poly-L-lysine-modified silica nanoparticles inhibit alterations.
DNA transcription, and this inhibition is size dependent SWNTs adsorb various amino acids (e.g., Arg, His, Met, Phe,
(Figure 11). DNA chains are adsorbed irreversibly to the and Tyr), vitamins (folate, riboflavin, and thiamine), and
phenol red in cell culture media.54 An analysis of the structures
of the adsorbed molecules indicates that π−π interactions and
electrostatic interactions are likely the driving forces for
adsorption. The SWNT-induced depletion of amino acids
and vitamins in cell culture medium reduces cell viability.
Spectroscopic studies have revealed that both SWNTs and
MWNTs affect the cell culture medium in this manner.244,248 A
study of the adsorption of folic acid and vitamin B1 by carbon
fibers with modified nanopores revealed that the pore structure
and surface modifications influenced the adsorption capacity
and adsorption rate.245
Computational chemistry reveals that the curvature of
nanoparticles and the polarizability of amino acids determine
binding strength.247 Doping the carbon surface with calcium
atoms results in a dramatic enhancement of binding of collagen
amino acids (Gly, Pro, and Hyp) to graphene. Electronic charge
transfers from the calcium atoms (donor) to graphene
(acceptor), and the carboxyl group of the amino acid
(acceptor) may contribute to this enhancement.249

4. NANOPARTICLE−CELL INTERACTIONS
The cell is the elemental unit of all living organisms.
Nanoparticles perturb living systems by interacting and altering
live cells. Such perturbations occur at various levels. Nano-
particles are first adsorbed to the cell surface and then
internalized. As the nanoparticles are endocytosed and
Figure 11. TEM of T4 DNA complexes with L (large, 40 nm), M transported within cells, the cells undergo structural and
(medium, 15 nm), and S (small, 10 nm) nanoparticles, and schematic
representations of the complexes. Reprinted with permission from ref
functional changes. Some crucial processes, such as oxidative
240. Copyright 2007 Cell Press. balance, are perturbed. Eventually, global alterations occur that
involve cell signaling, genomic, proteomic, and metabonomic
processes.
surface of nanoparticles (40 nm), inhibiting polymerase 4.1. Cellular Uptake and Intracellular Transport of
function. However, smaller nanoparticles (10 nm) form chains Nanoparticles
along DNA strands and might eventually dissociate from the Because of their sizes and surface properties, nanoparticles can
DNA strand. This allows the DNA molecules to become enter various organisms and cells, including mammalian cells,
accessible to the polymerase, and transcription proceeds.240 plant cells, bacteria, fungi, and viruses.250−254 These processes
Interactions between nanoparticles and DNA molecules can be energy-dependent intake or energy-independent
share some common features with nanoparticle−protein insertion.255,256 The former is also referred to as endocytosis,
interactions. In both cases, biomolecules are adsorbed to the a process by which cells take up molecules or particles by
nanoparticle surface and binding induces conformational engulfing them.257 Nanoparticles enter cells by taking
changes in these molecules. Nanoparticle−DNA interactions advantage of various known endocytotic pathways, such as
can also be used to develop advanced nanoparticle−DNA clathrin-mediated endocytosis, caveolae-mediated endocytosis,
hybrids to control cellular processes for biomedical and phagocytosis, and macropinocytosis.256,258−261 Direct penetra-
biosensing purposes. tions occur for 1D nanostructures or strongly positively charged
3.4. Adsorption of Crucial Small Molecules by nanoparticles.255,262 After entering cells, nanoparticles may
Nanoparticles localize in endosomes, lysosomes, the cytoplasm, mitochondria,
Due to their high surface energy, nanoparticles also adsorb the endoplasmic reticulum, or the nucleus, depending on the
various small molecules in biological systems. These molecules nature of the nanoparticle.256,258−261,263−266 Various imaging
include but are not limited to carbohydrates, vitamins, techniques have been developed to monitor the cell uptake
hormones, amino acids, and nucleic acid bases.54,241−245 process, in addition to conventional TEM and confocal laser
These small molecules normally play essential roles in cell scanning microscopy (CLSM) analyses.267,268 For example, X-
signaling and cell physiology. A sudden depletion of these ray fluorescence microscopy is used to determine chemical
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element distribution of nanoparticles in cell.269 Magneto- endocytosis is limited because the adhesion energy is too low to
photoacoustic imaging permits the differentiation of mem- compensate for the bending energy of the cell membrane.
brane-adhered or endocytosed nanoparticles.270 Dynamic When the nanoparticles are too large (greater than 60 nm), the
colocalization microscopy permits the spatiotemporal charac- free receptors on the cell membrane are depleted and
terization of internalized nanoparticles.271 Raman spectral nanoparticles may be only partially wrapped without
imaging maps vibrational bands of nanoparticles in live cells endocytosis. Here, size refers to the apparent size after
in the absence of an external label.272 Atomic force microscopy aggregation. A further understanding of the kinetics and
(AFM) measures the force between nanoparticles and the cell thermodynamics of the cellular uptake of nanoparticles will
surface, indicating receptor binding and binding assist future nanoparticle design and applications.
strength.273−275 4.1.2. Determinants of the Cellular Uptake of Nano-
4.1.1. Kinetics and Thermodynamics of the Cellular particles. Many physicochemical and biological factors govern
Uptake of Nanoparticles. Endocytosis of nanoparticles is a the cellular uptake of nanoparticles. These factors include the
dynamic process, and endocytosis and exocytosis processes size, shape, surface chemistry, surface charge, and mechanical
eventually reach thermodynamic equilibrium. Endocytosis properties of nanoparticles as well as cell type. The
likely involves at least two steps: binding of the nanoparticles combination of these factors affects the cell surface binding
to the cell surface followed by internalization of the strength, 273,275 cellular entrance route, 283,284 uptake
nanoparticles into cells (Figure 12).276 Binding parameters amount,278,285−290 and intracellular distribution.283,291,292
SWNTs with lengths of 100−200 nm can be internalized
into cells through clathrin-coated pits. SWNTs with lengths of
50−100 nm enter cells through caveolae-mediated endocytosis,
and some can enter the nucleus. The shortest (less than 50 nm)
SWNTs can enter cells through direct insertion, which is
energy independent.283 Studies of GNPs (4−17 nm) in HeLa
cells have revealed that the uptake force increases with the size
of the nanoparticles.275 The shape of the nanoparticle may also
play a role. Gold nanospheres enter fibroblasts and tumor cells
more efficiently than nanorods,293−295 while nanorods are
internalized more efficiently than nanospheres in blood
phagocytes.296 Particle replication in nonwetting template
(PRINT) nanoparticles with a higher aspect ratio enter HeLa
cells more readily than those with a lower aspect ratio.297 The
surface charge on nanoparticles can play a significant role in
cellular uptake. Due to the presence of phosphate groups on
lipids, cell membranes are negatively charged. Nanoparticles
with positive charges display strong interactions with cells
compared with those with negatively charged or neutral
surfaces.292,298
Furthermore, the structure, density and distribution of
Figure 12. Receptor-mediated cellular uptake and translocation of
surface chemical groups all play significant roles in determining
nanoparticles. the cellular uptake of nanoparticles. For peptide-functionalized
GNPs, an aromatic structure at the end of the peptide enhances
their cellular uptake.290 A higher density of oligonucleotide
derived from the binding of anionic MNPs to RAW loading on the surface of GNPs generates higher cellular
macrophages and HeLa cells reveal that macrophages and uptake, while a higher density of PEG reduces GNP
tumor cells behave equally with respect to cell surface binding, uptake.181,287 GNPs that have striations on the surface with
although they differ with respect to internalization capability. alternating anionic and hydrophobic groups can penetrate the
Adsorption onto the cell surface is a slow step, and plasma membrane without disrupting it.299 The nanoparticle
internalization is fast.277−279 Nanoparticles are eventually surface ligand induces protein corona misfolding and therefore
exocytosed from living cells. The exocytosis rate of SWNTs indirectly enhances cellular uptake.300 A molecular simulation
is reported to closely match their endocytosis rate with study has proposed that the effective surface charge density
negligible temporal offset.280 However, the uptake of dye- determines cell uptake.301 An experimental study of a surface-
labeled polystyrene nanoparticles is reported to increase with modified GNP array indicated that the effective surface charge
time and be essentially irreversible. The fluorescence signal density determines the electrostatic interactions between the
decreases when cells divide.268 This controversy suggests that positively charged nanoparticles and the negatively charged cell
cell uptake kinetics may depend on both nanoparticle membrane (Figure 13).302
properties and cell type. Endocytosis of nanoparticles is also Shape is another significant factor in the cellular uptake
under thermodynamic control. Thermodynamic studies have process. For 1D nanotubes, the uptake process is spontaneous
revealed that adhesion strength governs receptor-mediated cell when the orientation favors insertion, while 2D nanosheets
uptake. Endocytosis is determined by the enthalpic and prefer to adhere to the cell surface.255,303 A nanosphere and
entropic limits of the adhesion strength.281 On the basis of nanodisk cell uptake comparison study demonstrated that
the energy requirements for a receptor-mediated uptake nanodisks mainly adhere to the cell surface, while nanospheres
process, the optimal size of nanoparticles is 25−30 nm.282 are easily internalized into cells (Figure 14).25 A similar
When the nanoparticles are too small (less than 20 nm), conclusion was reached in a subsequent study. Nonspherical
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nanoparticles in physiological environments also impacts

cellular uptake. More nanoparticles are taken up in an upright
cell culture configuration than in an inverted one, and
nanoparticles with faster sedimentation rates display greater
differences in uptake between the two configurations,
suggesting that sedimentation needs to be considered for
cellular uptake studies of heavy or large nanoparticles.278,308,309
The different protein binding properties of nanoparticles often
indicate different nanoparticle−cell interactions.287,293,309 The
pericellular matrix enhances the retention and cellular uptake of
nanoparticles due to the restriction and deceleration of
diffusion by the entrapment and accumulation of nano-
particles.310 In summary, multiple factors collectively determine
the cellular uptake of nanoparticles and their subsequent
bioactivity in cells.
4.2. Cellular Substructural and Functional Alterations by
Nanoparticles
Nanoparticles are translocated to different compartments in live
cells and may interact with relevant subcellular structures and
affect their function. Cell membranes, ion channels, the
cytoskeleton, mitochondria, and the nucleus have all been
reported to be affected by nanoparticles.
4.2.1. Nanoparticle-Induced Cell Membrane Disrup-
tion. The cellular uptake of nanoparticles results in disruption
of the plasma membrane. Membrane disruption likely involves
several physicochemical interactions. Electrostatic interactions
of positively charged nanoparticles with negatively charged
groups on the membrane surface alter cell membranes.
Membrane penetration of positively charged nanoparticles
into the cytoplasm causes formation of holes within 1−100 ms,
followed by a slow resealing in tens of seconds.311 Interactions
between positively charged nanoparticles with negatively
charged membrane also causes membrane depolarization and
perturbation of membrane potential. Furthermore, an increase
in [Ca2+] influx inhibits cellular proliferation.312 Membrane
disruption of red blood cells and perturbation of their
hemolytic activity by amorphous and mesoporous silica
nanoparticles have been attributed to silanol groups that are
accessible to the membrane.313 Nanoparticle-induced gener-
ation of ROS may also cause membrane damage. A water-
Figure 13. (A) Schematic illustration of the effect of GNP surface soluble fullerene suspension disrupts cellular functions through
charge on their cellular uptake by a typical mammalian cell. (B) lipid peroxidation by ROS. Upon addition of an antioxidant, the
Electrostatic attraction between nanoparticles and cells. (a) Surface oxidative damage can be completely prevented.314 Nanoparticle
charge density of a GNP library. (b) Cellular uptake of these GNPs in
HeLa cells, as determined by ICP-MS. Reprinted with permission from
shape may also serve a role in membrane perturbation. Needle-
refs 301 and 302. Copyright 2010 and 2012, respectively, American shaped nanoparticles, rather than sphere or flat nanoparticles,
Chemical Society. cause a transient disruption of the endothelial cell mem-
brane.315 Stimulations such as near-infrared (NIR) radiation
cause cavitation of the membrane, followed by an increased
polymeric nanoparticles exhibit reduced cellular uptake influx of [Ca2+] and a degradation of the actin network.316 By
compared to their spherical counterparts. The larger average expanding our understanding of the various mechanisms of
curvature radius of adsorbed nonspherical particles may be interactions between nanoparticles and the cell membrane,
responsible for this difference.304 However, 1D superparamag- reduction of membrane disruption will be achieved through the
netic iron oxide nanoworms showed enhanced tumor-targeting regulation of size, shape, surface geometry, surface charge, and
efficiency compared to spherical nanoworms.305 This con- surface chemistry.46,313,317,318
troversy indicates effects of nanoparticle shape on cell uptake 4.2.2. Ion Channel Inhibition by Nanoparticles. Ion
are material dependent. channels consist of pore-forming proteins that help establish
The mechanical properties of nanoparticles also influence the and control a small voltage gradient across the cell
rate, route, and amount of cellular uptake.306,307 Soft nano- membrane.319 When nanoparticles reach the cell surface, they
particles enter cells through macropinocytosis, while stiff may affect ion channel function by interacting with the
nanoparticles enter through clathrin-mediated endocytosis. extracellular domains of ion channel proteins. SWNTs
Nanoparticles with an intermediate stiffness have multiple reversibly block the K+ channel in a diameter-dependent
internalization routes.306 The aggregation and sedimentation of manner, most likely by interacting with extracellular domains of
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Figure 14. Changing polystyrene nanoparticles from a three-dimensional (3D) spherical shape to a 2D disk shape promotes their cell surface
binding, with significant reduction of cell uptake. (A) Experimental evidence of a nanodisk reducing cellular uptake. (B) Model for reduced cellular
uptake but enhanced cell surface binding by nanodisks. Reprinted with permission from ref 25. Copyright 2012 American Chemical Society.

the channel.320 In contrast to pristine SWNTs, SWNTs iron oxide nanoparticles and gelatin nanoparticles cause a
modified with 2-aminoethylmethane thiosulfonate inhibit the reorganization of the F-actin and β-tubulin cytoskeletons in
K+ channel irreversibly, suggesting possible bonding between human fibroblasts after endocytosis. Because the actin
functional groups on SWNTs and the cysteine groups in ion cytoskeleton plays important roles in receptor-mediated
channels.321 Carboxylated MWNTs suppress the K+ channel in endocytosis, the different nanoparticle-induced alterations in
PC12 cells without induction of ROS.322 Molecular simulations cytoskeleton organization may indicate different mechanisms of
indicate that there are multiple C60 binding sites on K+ internalization.327,328 Gold nanorods coated with hexadecyl-
channels. The energetically favorable bindings between C60 trimethylammonium bromide cause actin aggregation and β-
and K+ channels cause ion channel inhibition and cytotox- tubulin redistribution in the cytosol. Such effects reduce
icity.323 Although direct interactions between nanoparticles and micromotion (fluctuations in cell shape) in MDCK-II kidney
ion channels are observed, impurities may also perturb channel cells. Coating of nanorods with PEG diminishes such
proteins. Contamination with yttrium, which is used as a perturbations.326 High concentrations of surface-modified iron
catalyst in the production of CNTs, has been shown to inhibit oxide nanoparticles affect cytoskeleton and focal adhesion
ion channels.324 Thus, nanoparticle−ion channel interactions formation and maturation. The reduced expression and
must be studied with care. decreased activity of focal adhesion kinase in turn alters the
4.2.3. Cytoskeleton Alterations by Nanoparticles. The actin cytoskeleton. Large endosomes that trap nanoparticles
cytoskeleton maintains the cell shape and serves significant may also affect the normal localization of actin fibers and
roles in intracellular transport, force generation, cell motility, disrupt the actin cytoskeleton through steric hindrance.329
and division.18 The internalization of nanoparticles results in 4.2.4. Mitochondrial Alterations by Nanoparticles.
direct or indirect cytoskeletal alterations. The loss of proper Mitochondria serve as “cellular power plants” by supplying
cytoskeletal function further leads to reduced cell motility, chemical energy (ATP) to cells.18 They are also involved in a
division, and proliferation.325−329 SWNTs induce actin range of other processes, such as signaling and cell differ-
bundling in both isolated actin filaments and HeLa cells. This entiation. Nanoparticles enter the mitochondria and may affect
induces actin-related cell division defects and retards cell their functions.330−333 SWNTs have been identified inside
proliferation. A weak binding enthalpy between SWNTs and mitochondria.333 GNPs (3 nm) enter mitochondrial intermem-
actin filaments has been speculated to arise because both brane spaces through voltage-dependent anion channels.334
structures are anisotropic and similar in size. Such interactions The direct interaction of nanoparticles with mitochondria has
induce dramatic anisotropic actin bundle formation both in been detected using SERS. SERS revealed that GNPs (13 nm)
cells and ex vivo.325 Nanoparticles may also affect the may bind to proteins, lipids, and carbohydrates present on the
cytoskeleton through indirect interactions. Surface-modified mitochondrial membrane in live cells and in isolated
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mitochondria. The bands at 886, 875, and 1070 cm−1 are due to causes mitochondrial damage, an upregulation of stress-related
C−C and PO2− vibrations. Changes in these bands indicate genes, and cell necrosis.330 Generation of ROS by GNPs upon
interactions between nanoparticles and membrane lipids. short-term (4 h) exposure can be counterbalanced by cells;
However, these interactions do not cause damage to the however, longer exposure (48 h) can overwhelm antioxidant
mitochondria.331 GNPs with a much smaller size (1.4 nm) defenses.350 GNPs also catalyze nitrogen monoxide (NO)
cause indirect damage by increasing the permeability of the production from endogenous S-nitrosothiols in serum through
inner mitochondrial membrane through generation of oxidative formation of Au−thiolate on the surface of nanoparticles.342
stress.330 Second, TiO2 nanoparticles, which are widely used in
4.2.5. Nuclear Alterations by Nanoparticles. The cosmetics, cause oxidative stress with or without light
nucleus maintains the integrity of genes and controls cellular activation. TiO2 nanoparticles absorb UV light and catalyze
activity cells by regulating gene expression. The nuclear DNA damage in human cells.238 In the dark, TiO2 nano-
structures include the nuclear envelope, which consists of a particles induce ROS generation, a decrease in GSH,
double lipid bilayer, the nuclear lumina, which consists of upregulation of stress-related genes, activation of JNK and
intermediate filaments and membrane-associated proteins, the P53, and apoptosis.351,352 TiO2 nanoparticles generate O2• in
chromosome, which consists of DNA and histones, and the sunlight and HO• free radicals in the dark.353 Third, carbon
nucleolus, which consists of rRNA, rDNA, and proteins.49 black (CB) nanoparticles induce oxidative stress by producing
Because the nuclear envelope keeps many macromolecules highly diffusible 4-hydroxynonenal. This species mediates
from entering the nucleus, nanoparticles entry into the nucleus cytotoxicity through formation of 4-hydroxynonenal−protein
is limited unless the nanoparticles are extremely small (<5 nm) adducts and GSH modification.354 CB nanoparticles enhance
or engineered with nuclear targeting molecules. 335−338 the oxidative stress induced by Fe2O3 nanoparticles in cell
Unmodified QDs (3.2 nm) are able to accumulate in the culture through coexposure. However, surface-oxidized CB
nucleus and exhibit strong affinity to core histones and histone- nanoparticles have no such effects, indicating that the oxidative
rich cell organelles. Histone binding is driven by electrostatic stress is mediated by the surface reactivity of CB.348 Fourth,
interactions between negatively charged QDs and positively CNTs induce ROS-related inflammasome activation.355 Metal-
charged core histone proteins. The interaction causes a red shift lic impurities from the CNT synthesis process, if present, may
of the steady-state photoluminescent emission of the QDs and be responsible for generation of oxidative stress.356 Surface
a decrease in the fluorescence lifetime.335 GNPs (2.5 nm) may chemistry modifications by PEG reduce ROS and cytotox-
disturb the decondensation of nuclear chromatin in mouse icity.357 Fifth, fullerene (C60) causes membrane damage and
sperm, indicating their potential spermatotoxicity. Such effects cytotoxicity through ROS generation and lipid peroxidation. An
are likely driven by interactions between GNPs with DNA in antioxidant, L-ascorbic acid, completely prevents such oxidative
the nucleus.337 Elucidation of nanoparticle−nucleus interac- damages.314 Sixth, other nanoparticles, including ZnO, silica,
tions provides crucial insight into the biological activity of polystyrene, nickel ferrite, silver, and nano-Co, have also been
nanoparticles; however, further investigation is still warranted. reported to induce oxidative stress, mitochondrial damage, and
apoptosis.346,347,358−361
4.3. Oxidative Stress and Nanoparticles
A nanoparticle’s potential to generate oxidative stress
Cells maintain a homeostatic oxidative state and counterbalance depends on the nature of the materials. At the same dose,
external stimuli-induced oxidative stress through several different nanomaterials cause different levels of oxidative stress.
regulation mechanisms.339 When these regulating mechanisms For example, ZnO, CuO, and CdS nanoparticles generate a
are overwhelmed, the excessive ROS or reactive nitrogen higher level of ROS than that of TiO2, MgO, SiO2, CNTs, and
species (RNS) can damage cellular components, affect Fe3O4 nanoparticles.362−366 CeO2 can even protect cells from
biomolecule function, and ultimately induce cell death through oxidative damage.362 Due to the significance of nanomaterials’
apoptosis or necrosis. Nanoparticles may affect the cellular intrinsic properties, a predictive paradigm for oxidative stress
oxidative state in various ways. Generation of ROS in cells is has been developed based on metal oxide nanoparticle band
considered a major factor in nanoparticle-induced cytotox- gaps.367 Critical physicochemical effectors are discussed below.
icity.340 However, nanoparticles with reductive properties may The crystal structure of nanoparticles appears to play a role
act as free radical scavengers to remedy oxidative damage in in ROS generation. Rutile TiO2 nanoparticles cause generation
cells and tissues.341 of ROS and cell apoptosis, while anatase TiO2 nanoparticles
4.3.1. Oxidative Stress Induced by Nanoparticles. induce necrosis with reduced ROS generation.368 The size and
Production of nanoparticle-induced ROS is triggered by surface area of nanoparticles affect ROS generation. Smaller
electronically active nanoparticle surfaces, photoactivation, nanoparticles with larger surface areas generate much more
impurities, dissolution of metal ions, and interactions of ROS than larger nanoparticles.369−371 Surface chemistry can
nanoparticles with biomolecules.342,343 Nonfluorescent dichlor- regulate oxidative stress generation. Cellular ROS generation by
ofluorescin can be oxidized to fluorescent dichlorofluorescein surface-modified silicon nanoparticles occurs in the following
inside the cell by various oxidants, and this reaction can be used order: positively charged > neutral > negatively charged.372
to detect the total oxidative level in live cells.344 ROS Surface-modified ZnO nanoparticles generate ROS in the
generation can also be measured by free radical content, following order: uncoated > oleic acid coated > poly-
GSH level, mitochondrial membrane potential, stress-related (methacrylic acid) coated > protein coated.373 Nanoparticles
gene/protein expression, microarray, and stress-specific re- with hydrophobic molecules on the surface cause oxidative
porter assays.330,340,345−349 stress in the cell. GNPs coated with longer hydrophobic alkyl
Most nanoparticles cause oxidative stress. Although the chains cause much more ROS generation and cytotoxicity than
outcome is similar, the mechanisms may be material dependent. those with shorter hydrophobic chains.374 PEG modification on
First, GNPs with a diameter of 1.4 nm induce oxidative stress the surface reduces oxidative stress caused by SWNTs.357 Some
and reduce intracellular antioxidant levels. The oxidative stress environmental factors may impact cellular ROS production. For
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instance, exposure to light enhances generation of ROS in cells protein kinases that respond to external stimuli, such as
incubated with TiO2 nanoparticles.364 Fe2O3 or CB nano- mitogen, heat shock, and oxidative stress. The MAPK signaling
particles alone do not cause cellular oxidative stress. However, pathway regulates cell proliferation, apoptosis, morphogenesis,
coapplication of these nanoparticles induced a 2-fold increase in cell cycle, and immunity.387
the oxidation of cellular proteins. Fe2O3 nanoparticles in There are three groups of kinases in MAPK signaling. The
endosomes or lysosomes are acidified to release Fe3+ ions, first group, extracellular signal-related kinase (ERK), is activated
which do not generate hydroxyl radicals in the presence of by growth factors, mitogen, and G-protein-coupled receptors
H2O2. However, CB nanoparticles reduce Fe3+ ions to Fe2+ (GPCRs). Carbon nanoparticles activate Erk1/2 in multiple cell
ions. Fe2+ ions can permeate the vesicle membrane, take part in lines.388 ERK is also activated by silica and iron oxide
the Fenton reaction, and induce protein oxidation.375 In nanoparticles.389,390 Activation of Erk1/2 by iron oxide
addition to these physicochemical factors, cells from different nanoparticles promotes neurite outgrowth in PC12 cells.390
organs have different capabilities of internalizing nanoparticles The second group, p38 MAPK, and the third group, JNK, are
and resisting oxidative stress.376 activated by stress, GPCRs, and inflammatory cytokines. GNPs
4.3.2. Reduction of Oxidative Stress by Nanoparticles. can promote osteogenic differentiation through activation of
Several nanoparticles have free radical scavenging capabilities, p38 MAPK.21 TiO2 nanoparticles activate JNK and p53 in
which originate from their chemical structures. These effects neuronal cells and cause G2/M cell cycle arrest and
may be potentially applied in antioxidative therapy. Cerium apoptosis.351 Silica nanoparticles activate p38 and JNK and
oxide (ceria) nanoparticles, fullerene and its derivatives, CNTs, reduce cell viability.391 Not all nanoparticles activate MAPK
and heavy metal (Au and Pt) nanoparticles have been reported signaling. Carboxyl-modified fullerene nanoparticles enter
to reduce ROS through various mechanisms.341,377−386 oxidative stress-damaged cerebral microvessel endothelial
Ceria nanoparticles inhibit inflammatory mediator produc- cells, maintain cell integrity, and inhibit apoptosis. Such effects
tion and reduce cell apoptosis. These nanoparticles protect rats are the result of inhibiting the JNK pathway by nano-
against oxidative damage by scavenging free radicals.378,381,384 particles.392
When ceria nanoparticles are doped with samarium, which 4.4.1.2. Nuclear Factor Kappa B (NF-κB) Pathway. The
decreases Ce3+ without affecting the oxygen vacancy (defect in NF-κB pathway is an important cellular signaling pathway in
oxides) contents, their antioxidant and antiapoptotic abilities response to external stimuli. Under normal circumstances, NF-
are reduced. This decrease indicates that the Ce3+/Ce4+ redox κB forms a stable complex with IκB in the cytoplasm. When
reaction and not oxygen vacancy is responsible for the cells are exposed to stimuli, such as stress, free radicals, or
antioxidant activity of ceria nanoparticles.381 Fullerene and its irradiation, the kinase IKK is phosphorylated (activated) by
derivatives have the ability to quench superoxide radicals.377,379 receptors. The activated IKK subsequently phosphorylates IκB.
CNTs are able to scavenge free radicals and retard the Phosphorylated IκB undergoes ubiquitous degradation, and
oxidation reaction.380,385 Doping of boron on CNTs enhances NF-κB is released and translocated to the nucleus to initiate
the efficiency of radical scavenging, indicating that electron transcription of inflammatory factors.393 Silver nanoparticles,
affinity may be responsible for the effect. Au and Pt CNTs, silica nanoparticles, TiO2, and double hydroxide
nanostructures have also been reported to display antioxidant (Mg(OH)2:Al(OH)3 1:1) nanoparticles have been reported
activities.382,383,386 Pt nanoparticles exhibit antioxidant activity to activate NF-κB signaling and induce generation of pro-
by scavenging O2•− and HO• with a second-order reaction inflammatory cytokines including COX-2, TNF-α, IL-6, IL-12,
rate.382 Au- and Pt-supported nanodiamonds exhibit a high and inducible nitric oxide synthase.47,389,394−398 The chemical
antioxidant activity, as indicated by their peroxidase activity and basis for the activation of NF-κB signaling by different
the ability to trap carbon radicals.383 Apoferritin-coated Pt nanoparticles is that all of these nanoparticles generate ROS
nanoparticles enter cells through receptor-mediated endocy- in cells. Activation of NF-κB finally leads to a reduction of cell
tosis, decrease hydrogen peroxide-induced oxidative stress, and proliferation, inflammation, and apoptosis. Surface modifica-
reduce cytotoxicity.386
tions on MWNTs direct the receptor recognition from mainly
4.4. Nanoparticle-Induced Systematic Cellular Biochemical mannose receptors to scavenger receptors. Activation of NF-κB
Perturbations through the mannose receptor is alleviated, and the
Cells have complex biochemical networks that maintain immunotoxicity of MWNTs is reduced. 47 This study
homeostasis during proliferation, differentiation, and apoptosis. demonstrates that activation of cell signaling by nanoparticles
Any extrinsic factors, such as nanoparticles, may interfere with can be regulated through modulating receptor recognition
these processes. Therefore, understanding nanoparticle-induced events by surface chemistry modifications.
cellular biochemical perturbations to elucidate the chemical 4.4.1.3. DNA Damage and Repair. The cellular ROS
mechanisms of nanoparticle−biological system interactions is generated by nanoparticles may cause DNA damage and
imperative. activate DNA repair signals.399 Such damage may induce G2/M
4.4.1. Perturbation of Cell Signaling Pathways by cell cycle arrest, apoptosis, mutagenesis, and tumorigene-
Nanoparticles. Nanoparticles perturb many cellular signaling sis.400−404 Therefore, an understanding of nanoparticle-induced
pathways. These pathways are involved in important biological DNA damage and its repair is critical. MWNTs increase the
processes that determine cell fate. The delineation of these expression of the base excision repair protein OGG1, the
mechanisms will facilitate an understanding of the cytotoxicity double-strand break repair protein Rad 51, the phosphorylation
of nanoparticles and development of safe nanomedicines and of p53 by the checkpoint protein kinase ChK2, the
nanomaterials. phosphorylation of histone H2AX at serine 139, and SUMO
4.4.1.1. Mitogen-Activated Protein Kinase (MAPK) Signal- modification of XRCC4 (in response to DNA double-strand
ing. MAPK signaling is a significant regulatory mechanism in breakage) in mouse embryonic stem cells.402 Similarly,
eukaryotic cells. The MAPKs are serine/threonine-specific nanodiamonds and silver nanoparticles also induce DNA
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Figure 15. Schematic diagram showing the molecular interactions involved in MWNT 1-enhanced cell differentiation and apoptosis inhibition.
Reprinted with permission from ref 406. Copyright 2012 Nature Publishing Group.

damage, as demonstrated by increased expression of OGG1 and chemical modifications of CNTs can tune cell differentiation
Rad51 and phosphorylation of p53 and H2AX.400,401,403 to different levels through the regulation of CNT/BMPR2
4.4.1.4. Bone Morphogenetic Protein (BMP) and Trans- binding and BMP signaling.406
forming Growth Factor beta (TGF-β) Signaling. BMP Graphene nanosheets activate MAPK and TGF-β signaling
receptors belong to the TGF-β receptor superfamily.405 pathways in murine RAW 264.7 macrophages. Graphenes cause
CNTs suppress BMP signaling pathways and affect cell an increase in cellular ROS and activation of phosphorylation of
function (Figure 15).406−408 Suppression of BMP signaling ERK, JNK, and P38, indicating activation of MAPK signaling.
reveals a nonapoptotic mechanism for the perturbation of cell Furthermore, expression of TGF-β receptor mRNA and
proliferation by CNTs. A global examination of gene expression phosphorylation of Smad2 are also enhanced, indicating
and Western blotting revealed that the Id (inhibitor of DNA activation of TGF-β signaling. The ROS and activated TGF-β
binding) genes and proteins are suppressed by SWNTs and signaling pathways further induce cellular apoptosis.409
MWNTs. Id genes are direct targets of Smad-dependent BMP 4.4.1.5. Other Signaling Pathways. GNPs bind to heparin-
receptor signaling through activation of Smad 1/5/8 proteins. binding growth factors but not to nonheparin-binding growth
The activated Smad proteins translocate into the nucleus to factors and selectively inhibit heparin-binding growth factor-
initiate transcription. The phosphorylation and nuclear trans- induced cell proliferation. This interaction is due to direct
location of Smad 1/5/8 are inhibited by CNTs. Id genes and binding of gold surfaces with sulfur or amine groups present in
proteins are subsequently inhibited. Inhibition of the Id protein the heparin-binding domain. Inhibition of heparin-binding
negatively regulates the cell cycle transition from G1 to S phase growth factors causes a decrease in vascular endothelial growth
and cell proliferation.408 A further study revealed that factor (VEGF)-165-induced cell proliferation in vitro and
suppression of BMP signaling is due to binding of CNTs to angiogenesis in vivo.153,154 GNPs with a poly(acrylic acid)
BMP receptor type II (BMPR2) but not BMP receptor type I coating selectively bind fibrinogen and induce protein unfolding
or the BMP ligand.406 to expose the C terminus of the γ chain. The newly exposed
BMP regulates a class of bHLH transcription factor proteins. epitope interacts with the Mac-1 receptor and activates its
Id proteins are also bHLH proteins but lack the DNA binding signaling. Mac-1 activation induces the release of inflammatory
domain. Id proteins function as negative regulators of cytokines, which is an alternative mechanism for the
transcription. This regulation is significant in cell differentiation, inflammatory response of cells to nanoparticles.160 Silver
tumorigenesis, and apoptosis. Suppression of BMP signaling in nanoparticles suppress glial cell line-derived neurotrophic factor
mesenchymal stem cells leads to inhibition of proliferation, signaling in spermatogonial stem cells and reduce cell
myogenic differentiation, and reduced apoptosis. Surface proliferation. This inhibition may be due to interactions
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between nanoparticles and Fyn protein, a kinase in glial cell upregulate the inflammatory response and cell adhesion genes
line-derived neurotrophic factor signaling.410 Graphene oxide but not oxidative stress genes in human keratinocytes.417 These
nanosheets activate the toll-like receptor signaling cascades and effects indicate that TiO2 nanoparticles do not cause oxidative
trigger ensuing cytokine responses, which subsequently induce damage in the dark. Genomic expression profiles reveal that
autophagy.411 GNPs of smaller sizes (1.4 nm) upregulate heat shock and
The above reports indicate that nanoparticles may affect stress-related genes and lead to cell necrosis. However, larger
oxidative stress-dependent and -independent pathways. BMP, GNPs (15 or 18 nm) affect fewer genes, indicating their
growth factors, and Mac-1 cascades are affected by direct superior biocompatibility.330,418 The toxicity of some metallic
interactions between signaling proteins and nanoparticles nanoparticles may be due to the release of the metal ions.
(BMPR-CNT, VEGF-GNP, Mac-1 receptor-GNP, respec- Genomic transcriptional profiling reveals that silver nano-
tively). These interactions do not necessarily induce oxidative particles affect 128 genes in human epithelial cells, similar to
stress and apoptosis but may affect proliferation or differ- the response of cells exposed to silver ions.413 Treatment of
entiation. Other signaling pathways such as MAPK, NF-κB, and epithelial cells with copper oxide nanoparticles upregulates
DNA damage are responsible for nanoparticle-induced genes in MAPK and downregulates cell cycle genes, also similar
oxidative stress. These events eventually cause cell death. All to cells treated with copper ions.419 These discoveries suggest
of these events are determined by both the nature of the that the release of metal ions from nanoparticles must be
nanoparticles and their surface chemistry. Control of nano- considered when evaluating the bioactivity of metallic nano-
particle-induced signaling events and related cellular outcomes particles.
are crucial subjects for future investigations. 4.4.2.2. Proteomic Expression. Protein synthesis is down-
4.4.2. Global Biochemical Alterations by Nanopar- stream of gene transcription and controlled by a number of
ticles. In contrast to small molecule drugs, for which the regulatory factors. Evaluation of proteomic expression provides
intracellular targets and mechanisms of action have been at least more relevant information than genomic transcriptome
partially elucidated, knowledge of the molecular events involved analyses. The global protein expression profile is generally
in nanoparticle−biological system interactions is limited. examined using 2D gel electrophoresis combined with
Nanoparticle-induced global biochemical alterations may MS.420−422 Generally, total proteins extracted from cells are
provide useful insights into the molecular mechanisms and separated by 2D gel electrophoresis, and the protein spots on
chemical basis of nanoparticle−biological systems interactions. the gel are excised and digested, followed by MS analysis. MS
The replication, transcription, and translation of genes and data are analyzed by searching databases to identify proteins.
synthesis of bioactive small molecules are biochemical Another approach is the isobaric tags for relative and absolute
processes that are controlled by tens of thousands of signals quantitation (iTRAQ) technique.423 iTRAQ uses liquid
in response of intracellular demands or extracellular stresses. chromatography to separate isotopically labeled peptides and
Mapping these global profiles will facilitate identification of MS/MS analysis to identify and quantitate peptides. Protein
nanoparticle-generated biological outcomes and eventual microarrays can be used for high-throughput evaluation of
regulation of these outcomes through modulation of the protein expression/phosphorylation.420 Antibodies against
physicochemical properties of nanoparticles. protein targets can be spotted on nitrocellulose membranes,
4.4.2.1. Genomic Transcriptional Profile. Unlike small enabling detection of multiple proteins in a single experiment.
molecules, which may significantly regulate >5000 genes in Using the above technologies, proteomic expression/phosphor-
living cells, most nanoparticles usually affect much fewer ylation profiles can be obtained. A total of 51 proteins in human
(<500) genes, indicating that they may be less active and more hepatic carcinoma HepG2 cells are significantly affected by
selective toward cells.330,408,412−415 The biological activity of SWNTs, as detected by iTRAQ-coupled LC-MS/MS analyses.
nanoparticles can be investigated via genomic transcriptional These proteins are involved in metabolic pathways, redox
profiling. Carboxylated SWNTs inhibit Id genes in HEK293 regulation, signaling pathways, cytoskeleton formation, and cell
cells in a whole genome background. BMP signaling, which growth.423 Using 2D gel electrophoresis with MALDI-TOF
targets Id genes, was identified through multiple assays.408 MS, 45 altered proteins were identified in human monoblastic
Pristine SWNTs or MWNTs affect a large number of genes leukemia U937 cells in response to MWNTs. These proteins
related to the cell cycle, signaling transduction, apoptosis, participate in metabolism, biosynthesis, stress response, and
oxidative stress, metabolism, transport, and immune responses differentiation processes.422 Additionally, 2D gel electro-
in human cells.398,414,416 By contrast, multiwalled carbon nano- phoresis/MS analyses and protein phosphorylation antibody
onions affect genes that fall into the category of external stimuli array analyses revealed that GNPs activate endoplasmic
other than the immune response.416 PEG-modified SWNTs reticulum (ER) stress response in human chronic myelogenous
affect fewer genes, indicating a reduced biological activity.357 leukemia K562 cells.420 A similar analysis indicated that TiO2
PEG-modified, silica-coated QDs have been reported to affect nanoparticles significantly alter 46 proteins in human bronchial
38 genes at low doses (8 nM) and 12 genes at high doses (80 epithelial BEAS-2B cells. These proteins are involved in the
nM), with four shared genes. These genes are related to stress response, metabolism, adhesion, cytoskeletal dynamics,
carbohydrate binding, intracellular vesicle localization, and cell growth, cell death, and signaling pathways. Nanoparticles
vesicular proteins involved in stress responses.412 Fe3O4 MNPs perturb approximately 20 proteomic pathways, consistent with
alter 711, 545, and 434 genes in mouse macrophage cells 4, 24, pathways from genomic data analyses.421
and 48 h after treatment. MAPK, TLR, and JAK-STAT 4.4.2.3. Metabonomic Alterations. Nanoparticles cause
signaling pathways are also affected by Fe3O4 nanoparticles. physiological changes in living systems and generate altered
Bioinformatic analyses have revealed that these nanoparticles metabolic profiles. Therefore, investigation of global metabo-
activate inflammatory and immune responses and inhibit nomic profiles provides insight into the physiological processes
biosynthesis and metabolism.415 In the absence of illumination, that are perturbed by nanoparticles. Nanoparticle-induced
anatase TiO2 nanoparticles with diameters of 7, 20, and 200 nm metabonomic alterations are studied in live animals or
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cells.424−427 Analytical methods that include 1H NMR, MS, example, dithiocarbamate moieties are designed to replace
HPLC-MS, and gas chromatography−mass spectrometry (GC- trioctylphosphine oxide capped on CdSe/ZnS QDs,430 while
MS) have been used in metabonomic analyses.424 A global (3-carboxypropyl)trimethylammonium chloride is used to
metabolism study in live cells using high-resolution MAS NMR replace lauric acid on Fe3O4 nanocrystals.431 This ligand
spectroscopy demonstrated that silver nanoparticles decrease exchange step is essential to apply these nanoparticles in
the levels of GSH, lactate, taurine, and glycine and increase the biological systems. The covalent linkage strategy requires the
levels of most amino acids, choline analogues, and pyruvate. aid of coupling reagents, such as 1-ethyl-3-(3-(dimethylamino)-
These results indicate that the depletion of GSH by propyl) carbodiimide and N-hydroxysuccinimides, or Cu2+-
nanoparticles may induce conversion of lactate and taurine to mediated click chemistry432 to react with functional groups on
pyruvate, providing a biochemical mechanism for oxidative nanoparticles. Some nanoparticles with an aliphatic shell can be
stress.425 HR-MAS 1H NMR was also used to study the encapsulated in polymer coatings or micelles through an
metabonomic changes in RAW264.7 mouse macrophages intercalating effect with amphiphilic polymers.433 In addition,
following treatment with MNPs. The nanoparticles caused a the polymer coating can be achieved through a layer-by-layer
decrease in the levels of triglycerides, essential amino acids, and technique by sequentially trapping nanoparticles in layers of
choline metabolites while increasing glycerophospholipids, polymers of alternating charge.434
tyrosine, phenylalanine, lysine, glycine, and glutamate. These 5.1.2. Modulation of the Surface Charge of Nano-
changes reflect phagocytosis and cell membrane perturba- particles. An acidic group is often used to generate
tion.427 nanoparticles with negative charges on their surfaces. Similarly,
In general, interactions between nanoparticles and biological an amine group is used to generate nanoparticles with a
systems include direct and indirect effects. Direct effects, such positively charged surface. Surface-charged nanoparticles differ
as the binding of nanoparticles to membrane phospholipids, greatly in their electrostatic and electrodynamic properties,
receptors, cytosol proteins, and DNA molecules, are modulated which can be experimentally determined by ζ-potential
by the physicochemical properties of the biomacromolecules measurements. Polystyrene nanoparticles with different surface
and nanoparticles and their microenvironment. Direct inter- functional groups, such as carboxylic acid, PEG, amine, and
actions are much more specific and material dependent amidine groups, have ζ-potential values ranging from −50 to 40
compared to indirect interactions and may have therapeutic mV. The binding of serum proteins alters their ζ-potential
potential. However, investigation of direct interactions is values and narrows them to a range from −40 to −20 mV.435
challenging because of the complex cellular background. BSA, myoglobin, and cytochrome c are adsorbed to
Indirect effects, such as signaling pathway activation by mercaptoundecanoic acid-protected GNPs, as shown by a
nanoparticle-induced ROS, cause cytotoxicity. Future inves- quartz crystal microbalance study. All three proteins form
tigations will enhance and regulate direct interactions while adsorption layers consisting of an irreversibly adsorbed layer
minimizing indirect interactions. In this endeavor, chemical and a reversibly adsorbed layer.109 These data suggest that the
technology may be very powerful for modulating the biological charged surface of a nanoparticle is quite complicated in
activities of nanomaterials, as discussed below. solution. Multiple layers of molecules are adsorbed and
dissociated constantly. The charged surface of nanoparticles
5. CHEMICAL APPROACHES TO REGULATE AND also affects the conformation of the adsorbed proteins.
UNDERSTAND NANOPARTICLE−BIOLOGICAL Cytochrome c maintains its conformation when adsorbed to
SYSTEM INTERACTIONS neutral GNPs, and it is denatured on the surface of charged
GNPs.436 GNPs surface modified with charged and neutral
Interactions between nanoparticles and biological systems are
amino acids affect the conformation and activity of α-
complex. The biological activities of nanoparticles depend on
chymotrypsin differently. Charged GNPs exhibit more effects
their size, shape, structure, and surface properties. As
than neutral particles.437 To understand the influence of surface
nanoparticle sizes fall into the nanometer range, the surface-
exposed atoms increase exponentially. Therefore, changing the charge density on protein binding, a series of polystyrene latex
properties of the surface molecules may significantly alter the nanoparticles with the same size were prepared using different
biological activities of nanoparticles. In this section, we explore amounts of Na−styrenesulfonate comonomer. As the charge
various surface chemistry modification methods, the biological density increased, there was no change in the bound protein
impacts of such modifications, and computational models to species, while the amount of adsorbed protein increased.438
predict the relationships between nanoparticle surface chem- The different electrostatic and electrodynamic properties of
istries and their biological activities. nanoparticles also modulate their interactions with cells. Such
alterations include effects on cell internalization, cell function,
5.1. One-at-a-Time Nanoparticle Surface Chemistry and cell death. A better understanding of these effects will not
Modification only help us design nanomaterials for biomedicinal applications
5.1.1. Modulation of the Surface Chemistry of but will also elucidate mechanisms of nanotoxicity. Due to the
Nanoparticles. Various molecules have been used to modify presence of phosphate groups from lipids, cell membranes are
nanomaterial surfaces via coating chemistry and, consequently, negatively charged. Positively charged nanoparticles generally
their biological activity. Surface modification strategies include exhibit higher cell binding and cellular uptake than negatively
covalently or noncovalently linking small organic ligands or charged nanoparticles. To understand the electrostatic and
biomolecules to nanoparticles or encapsulating nanoparticles in electrodynamic interactions between nanoparticles and cells
surface coatings, such as polymers, micelles, liposomes,428 with minimal secondary interactions, a nanoparticle library with
graphene oxide,141 and silica shells.412,429 The ligand exchange a continuous change in the surface charge density was
method is effective for transferring nanoparticles synthesized in synthesized and investigated. The nanoparticle/cell interaction,
organic solvents to aqueous media by displacing hydrophobic however, did not exhibit a continuous change. Instead, a sharp
ligands on nanoparticles with water-soluble ligands. For increase in cell binding was observed as the positive surface
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charge density increased. The effective surface charge density

on the outermost layer of the nanoparticle determined the
electrostatic attraction between the nanoparticles and the
cells.439 Nanoparticles with different charges induce different
perturbations of the cell membrane potential. Positively
charged nanoparticles depolarize the membrane, and negatively
charged nanoparticles have a negligible effect. Membrane
potential perturbations result in an increase in [Ca2+] influx,
which in turn inhibits the proliferation of cells (Figure 16).312 A

Figure 17. Schematics illustrating the interactions between cells and

nanoparticles with different types of surface charges: (A) negatively
and neutrally charged nanoparticles. (B) Positively charged nano-
Figure 16. Schematic depicting the surface charge effects of GNPs on particles. Reprinted with permission from ref 440. Copyright 2009
cellular function. Reprinted with permission from ref 312. Copyright American Chemical Society.
2010 American Chemical Society.

method has been developed to differentiate GNPs bound to the charged nanoparticles is a common property regardless of the
cell surface from internalized GNPs. Neutral and negatively shape, size, and chemical composition of the nanoparticles.447
charged GNPs displayed lower cell membrane binding and cell 5.1.3. Modulation of the Surface Hydrophobicity of
internalization compared to positively charged nanoparticles.440 Nanoparticles. The hydrophobicity of nanoparticles influen-
The surface charge effect seems applicable to a variety of ces their ability to interact with biomolecules and cells. To
nanomaterials. Positively charged GNPs, MNPs, silica nano- investigate the correlation between plasma protein adsorption
particles, and QDs all exhibit higher cellular uptake efficiency and the surface hydrophobicity of nanoparticles, latex nano-
(Figure 17).440 The internalization of negatively charged particles with varied surface hydrophobicity were synthesized
nanoparticles also occurs and is likely due to other interactions and examined. The number of bound proteins increased with
and subsequent endocytosis.441,442 QDs with a carboxylic acid increasing particle hydrophobicity, as determined by 2D
surface coating are recognized by lipid rafts but not by clathrin polyacrylamide gel electrophoresis analyses and ITC. A clear
or caveolae in human epidermal keratinocytes. QDs are difference in protein affinity for polymeric particles of different
internalized into early endosomes and then transferred to late hydrophobicity has been observed.108,448
endosomes or lysosomes. In addition, endocytic interfering A set of polymethacrylate nanoparticles (140 nm) with
agents have been used to investigate the mechanism by which fluorescence labels was investigated to reveal the relationship
QDs enter cells. The cellular uptake of QDs with negative between particle hydrophobicity and cellular uptake. The
surface charges is primarily regulated by pathways associated monomer chain length controls the hydrophobicity of the
with G-protein-coupled receptors, low-density lipoprotein modified nanoparticles, as evaluated by measuring the
receptors, and scavenger receptors.443 interfacial tensions between the monomers and water. The
In a kinetics study, positively charged nanoparticles displayed length of the monomer chains influences the cellular uptake of
a higher rate of endocytosis than negative charged materials.444 nanoparticles. Increasing the side chain length increases cellular
Positively charged core−shell silica nanoparticles coated with uptake significantly. This phenomenon can be explained by the
polyethylenimine have been shown to escape from endosomes increased hydrophobicity and softness of the particles.449
and enter the cytoplasm and nucleus.445 Positively charged The surface hydrophobicity of nanoparticles is also modified
nanoparticles destabilize cell membranes and form holes or by peptide−GNP hybrids. By altering the end amino acids of
expand holes at pre-existing defects.446 The holes led to the peptide ligands, the uptake of GNPs can be dramatically
internalization of nanoparticles into cells as well as diffusion of increased or suppressed. Nanoparticles with tryptophan end
cytosolic proteins out of cells. Further studies have demon- groups enhance cellular uptake due to the membrane anchoring
strated that formation of holes in lipid bilayers by positively and permeation effects of the aromatic group.450
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5.1.4. Changes in the Interactions of Nanoparticles foundation for designing nanomedicines for cancer diagnosis
with Biological Systems. 5.1.4.1. Making Toxic Nano- and therapy. Detection of circulating breast cancer cells in the
particles More Biocompatible. Nanoparticles may induce blood was achieved using a combination of an epithelial cell-
toxicity in biological systems because of their specific specific, antibody-conjugated magnetic nanoprobes and anti-
properties.451,452 First, the large surface area to volume ratio HER2 antibody-conjugated SERS tags.476 Because the HER2
leads to very strong nonspecific interactions with biomolecules. receptors are highly expressed on the breast cancer cell
These interactions will alter the conformation of proteins or membrane, the SERS tags recognized these tumor cells. Thus,
DNA, resulting in their malfunction.453 Second, some nano- the cancer cells were detected rapidly with good sensitivity. An
particles are composed of heavy metals, such as CdTe and antiprostate antigen was conjugated to a multifunctional
CdHgTe QDs. Under some conditions, the free metal ions may nanomedicine composed of loaded drugs, NIR QDs, and a
be released into biological systems and induce toxicity.454 polystyrene matrix with internally embedded superparamag-
Third, nanoparticles may induce ROS, such as free radicals netic Fe3O4 nanoparticles. Using this multifunctional nano-
(hydroxyl radical, •OH; superoxide, •O2−) and singlet oxygen particle, specific detection of LNCaP prostate cancer cells was
(1O2) (e.g., QDs455 and Ag nanoparticles456). ROS are known achieved.477
to cause irreversible damage to nucleic acids, enzymes, and After being administered into the blood, most unmodified
cellular components that included the mitochondria, plasma, nanomaterials are taken up by the mononuclear phagocyte
and nuclear membrane. Fourth, some capping ligands derived system within minutes or hours.478 Surface modification of
from the nanoparticle synthesis system may induce cytotox- nanomaterials is generally regarded as a practical strategy to
icity.457 Surface chemistry modifications may diminish the prevent protein adsorption and, consequently, avoid triggering
above side effects and greatly improve the biocompatibility of immune responses.474 Various biocompatible polymer surface
potentially toxic nanoparticles. For example, the biocompati- coatings, such as PEG,479 hyaluronic acid,480,481 cyclodex-
bility of QDs has been improved after surface modification with tran,482,483 and polymers based on phosphorylcholine,484
dendrimers,458,459 PEGylated dihydrolipoic acid,460,461 triblock provide effective barriers against protein bindings and therefore
copolymers,462 cyclodextran,463 and alkyl-modified poly(acrylic reduce both protein adsorption and cellular uptake. These
acid).464 The coating of SWNTs with polyvinylpyrrolidone modifications are widely used to impart stealth properties to
polymers,465 phospholipid derivatives466 on silver nanopar- nanoparticles in biomedical applications. These surface
ticles, or human serum protein467 also prevents their toxicity. modifications also change the in vivo pharmacokinetics of
Hexadecyltrimethylammonium bromide (CTAB) released from nanoparticles.485 QDs modified with mPEG 5000 display a
CTAB-coated gold nanorods is highly cytotoxic at a nano- significantly prolonged in vivo circulation time in addition to
particle concentration as low as 0.05 mM (20% cell viability). reduced nonspecific accumulation in various organs.486 After
However, when toxic CTAB was replaced by PEG-SH, the cell surface PEGylation, the blood half-life of gold nanorods is
viability when subjected to nanoparticles was 95%, even at a increased to 24 h.457,487 PEG-terminated phospholipid was
concentration of 0.5 mM.457 Similar effects have also been used to encapsulate QDs488 and iron oxide nanocrystals489 for a
reported after replacement or overcoating of CTAB with longer blood circulation time in vivo. A mPEG-alkyl-thiol
poly(sodium 4-styrenesulfonate), 468 copolymer poly- polymer with an alkyl linker between the PEG and the thiol
(diallyldimethylammonium chloride)/polystyrene sulfonic moiety yielded a much stronger GNP surface protection
acid,469 or poly(allylamine hydrochloride).470 compared to commonly used methoxy-PEG-thiol in the
5.1.4.2. Regulating Interactions with Cells. The interaction presence of cysteine and cystine molecules at the physiological
of nanoparticles with cells is affected by the surrounding concentrations.490 Surface coating of lipid molecules has also
biomolecules in physiological media. When bare nanomaterials improved the pharmacokinetics of the silica nanoparticles.491
(particularly inorganic nanoparticles) encounter biological
5.2. Combinatorial Chemistry for Nanoparticle
macromolecules, they may form a nanoparticle−macromolecule
Modifications
(mainly protein) corona hybrid nanostructure.453 Moreover, a
secondary protein corona on the hybrid nanoparticles may be The discovery and optimization of novel nanoparticles with
further formed via protein−protein interactions. Some of these unique properties by conventional methods has always been
interactions do not occur in the native system in the absence of challenging and time consuming. High-throughput approaches,
the nanoparticles, and an immunological response may be including parallel and combinatorial synthesis, are more
triggered. As a result, the nanohybrids are detected as foreign efficient than conventional linear synthesis, and in recent
materials by the immune system. The nanohybrids are rapidly years, these techniques have shown an increasing impact in
cleared from the bloodstream and translocated to the liver and nanotechnology.
spleen,471 potentially limiting the biological applications of 5.2.1. Parallel Chemistry for Nanoparticle Modifica-
nanoparticles. Fortunately, the fate of nanoparticles in tions. A parallel chemistry approach provides the opportunity
biosystems can be regulated. Two main requirements, cell to rapidly synthesize a large amount of nanoparticles with
targeting and in vivo stealth properties, can be modulated by similar surface chemistries. However, this approach has
proper nanoparticle surface chemical modifications. limitations, mostly based on the lack of a general method to
When a nanoparticle is presented to a cell, the cell first modify nanoparticles rapidly and the availability of small
recognizes the coating molecules at the outermost layer of the molecules that can be used.
nanoparticle. For targeting purposes, linking specific ligands to 5.2.1.1. Parallel Nanoparticle Libraries for Regulating
nanoparticles is necessary. Certain molecules, such as antibod- Gene Delivery. Numerous biomaterials have been studied as
ies, aptamers, or small-molecule ligands, can bind to receptors potential nonviral gene delivery vectors to improve DNA
overexpressed in cells in a specific “lock−key” man- stability and the efficiency of cellular uptake. The tested
ner.428,472−475 After modification, nanohybrids are able to materials include inorganic surfactants, cationic lipids, poly-
target specific cancer cells. This targeting strategy lays the saccharides, cationic polymers, and dendrimers. These materials
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Figure 18. (A) Model of the cross-linked dextran coating modified with small molecules. Different classes of small molecules with amino, sulfhydryl,
carboxyl, or anhydride functionalities were anchored onto nanoparticles and stored in multiwell plates for testing. (B) Part of a heat map
representing the cellular uptake of different nanoparticle preparations. Columns from right to left: 1, pancreatic cancer cells (PaCa-2); 2, macrophage
cell line (U937); 3, resting primary human macrophages; 4, activated primary human macrophages; 5, human umbilical vein endothelial cells
(HUVEC). Each column represents mean values from six different experiments. Red indicates the lowest accumulation, and green indicates the
highest accumulation. Reprinted with permission from ref 28. Copyright 2005 Nature Publishing Group.

display small cargo capacity, inefficient uptake, poor biostability, human endothelial cells.496 To investigate a larger PBAE
and low safety. Using high-throughput synthesis and screening chemical space, libraries of end-modified PBAEs were also
techniques, libraries of poly-β-amino esters (PBAEs) of >2000 synthesized using various end-capping reagents.497,498 Two
members were synthesized to evaluate their gene delivery modified polymers with greatly improved transfection efficiency
efficiency.492−494 A total of 46 PBAEs were superior to both in vitro and in vivo were discovered.
polyethylenimine. A second-generation library of 486 polymers 5.2.1.2. Parallel Nanoparticle Libraries for Regulating Cell
was synthesized to optimize the polymers and investigate their Targeting. MNPs that target tissues of interest have been
structure−function relationships.495 The best-performing poly- developed by conjugating antibodies, peptides,499 or small
mer nanoparticles were <150 nm and had positive surface molecules to the surface of MNPs.26,28 Small molecules with
charges. PBAEs have been used to deliver DNA to primary anhydride, amine, hydroxyl carboxyl, thiol, and epoxy handles
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Figure 19. (A) Surface molecular compositions of combinatorial MWNT library members. (B) Multiassay score of the f-MWNT library from four
protein binding assays, cytotoxicity, and immune response assays (at 50 μg/mL) were ranked for all library members. Sum of their ranks was
designated the multiassay score and is shown as vertical bars in the graph. Structures of the optimal hits are shown. Reprinted with permission from
ref 27. Copyright 2008 American Chemical Society.

were coupled to the MNPs via different synthetic routes. The Cellular uptake was rapidly profiled in vitro via binding of those
nanoparticles were printed on glass slides and showed different nanoparticle imaging probes in primary cell isolates. This
binding properties toward different targeting proteins using approach selects nanoparticles that display the desired targeting
fluorescence detection. A collection of MNPs comprising 146 effects across all tested members of a class of cells and decreases
nanoparticles modified by different synthetic small molecules the likelihood that an idiosyncratic cell line will unduly skew the
was synthesized and screened against different cell lines (Figure
screening results. The identified nanoparticle imaging probes
18). In this screen, nanoparticles with high specificity for
endothelial cells, pancreatic cancer cells, or activated human were validated in vivo via intravital microscopy using pancreatic
macrophages were identified.28 cancer mouse model.500 Although the molecular target of the
5.2.1.3. Parallel Nanoparticle Libraries for Imaging. probes remains to be discovered, the specific localization of
Weissleder et al. developed fluorescent nanoparticles derivat- these probes validates their respective use as in vivo neural
ized with small molecules using a parallel chemistry approach. tracers and cell-discriminating agents.
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5.2.1.4. Parallel Nanoparticle Libraries for Biosensors. Six 5.2.2.2. Combinatorial Modifications To Regulate Cell
functionalized poly(p-phenyleneethynylene)s were used to Targeting. Recently, we reported a dual-ligand GNP library to
construct a protein biosensor array containing six nanoprobes. discern the slight difference between cells that have different
These highly fluorescent polymer nanoparticles possessed surface receptor profiles surrounding a common primary
various charge characteristics and molecular weights. In a receptor.505 This library used folic acid as the primary targeting
proof-of-principle experiment, 17 protein analytes were ligand. The secondary ligand was one of the ligands from a
successfully discriminated by their different fluorescence combinatorial library of 42 molecules. Cell recognition was
properties after nanoparticle binding. However, those probes investigated in four cell lines with various expressions of folate
were not tested in real samples, such as blood serum, receptor (FR) and totally unknown secondary receptor profile.
containing complex mixtures of proteins with potentially Cell binding and uptake in different cells were quite different,
interfering species.501 Parallel modifications of magnetic suggesting different receptor profiles surrounding the FRs.
nanoparticles aided in designing biomimetic nanoparticle Furthermore, multivalent bindings of dual ligands can differ-
biosensors with selectivity for specific cell types. Attachment entiate cells with high and low expression of FRs and enhance
of fluorescent molecules to magnetic nanoparticles permits the the contrast between these cells in radiation-induced cell death.
sorting or targeting of cells using both fluorescence-based This enhanced cell recognition and discrimination of cells with
technologies and MR imaging.26,499 The conjugation of a lipoic seemingly a common receptor provide an opportunity to treat
acid-containing NIR active tricarbocyanine library to GNPs patients at a personalized medicine level with a much better
yielded probes with a strong SERS enhancement with 12-fold safety profile.505
higher sensitivity than the current standard, 3,3′-diethylthia- 5.2.2.3. Combinatorial Modifications To Regulate Cell
tricarbocyanine iodide. By further conjugating the probe to a Differentiation. Biomaterials can drive stem cells to an
scFv anti-HER2 antibody, this ultrasensitive SERS bioprobe appropriate differentiation level and decrease the apoptosis of
was used for in vivo cancer imaging with potential for use as a transplanted cells. We observed that carboxylated MWNTs
noninvasive diagnostic tool for HER-2-positive cancers.502 promote myogenic differentiation of mouse myoblast cells
5.2.2. Nanocombinatorial Chemistry To Modify Nano- C2C12 and inhibit cell apoptosis under differentiation
particles. Compared with a parallel chemistry approach, conditions.506 We explored the tuning of cell differentiation
combinatorial chemistry can provide a large number of different using combinatorial surface modifications on MWNTs. Differ-
but structurally related molecules or materials. A series of ent surface modifications conferred different modulations of
nanoparticles with different surface coatings but the same core binding to BMP receptor type II and caused different levels of
structure and different diversity points can be synthesized suppression of the BMP signaling pathway. Modified MWNTs
rapidly using nanocombinatorial chemistry. The number of fine tuned the downstream signaling through a series of bHLH
compounds synthesized increases exponentially with the proteins and myogenic differentiation. These results demon-
number of diversity points. Furthermore, because the nano- strated control of biological activities through surface-modified
particles obtained by nanocombinatorial chemistry have surface nanomaterials and opened an avenue for potential application
coatings with similar structures, structure−activity relationships of nanomaterials in regenerative medicine.507
can be obtained, which is critical for computer-aided design for 5.3. Computational Approaches To Elucidate Nano−Bio
predicting nano−bio interactions. Interactions
5.2.2.1. Combinatorial Surface Modifications To Regulate The recent emergence and proliferation of experimental studies
Biocompatibility. Drug-loaded nanoparticles with targeting on the biological and toxicological activities of nanoparticles has
ligands can grant them therapeutic, targeting, or imaging created new challenges for computational scientists interested
functions. However, achieving a low blood clearance rate, low in rationalizing and exploiting these data by generating
acute toxicity, and a low immune response remains challenging. predictive models. Similar to data-rich areas of chemistry and
To discover biocompatible MWNTs without a priori knowl- biology, where development of high-throughput technologies
edge of the related targets, our laboratory developed a and accumulation of large amounts of data have spurred the
computer-assisted, nanocombinatorial synthetic library strategy. establishment of cheminformatics and bioinformatics, we are
Combinatorial modifications of nanotube surfaces allowed us to beginning to witness the appearance of nanoinformatics, which
map the unknown chemical space more effectively and rapidly. naturally complements nanotechnology. For instance, a group
Computer-aided design was used to select surface molecules to of academic, governmental, and industrial scientists interested
present the most diverse structural and physicochemical in informatics opportunities in nanotechnology met at the first
properties. As a result of multiple biological screenings, we Nanoinformatics conference in 2010 (http://
identified nanomaterials with the most optimal biocompatibility nanotechinformatics.org/2010/overview). Presentations and
and preliminary structure−activity relationship (Figure 19).27 idea exchanges at the conference led to creation of the
Furthermore, to test the improved molecular recognition after Nanoinformatics 2020 Roadmap (http://eprints.internano.org/
surface modifications on MWNTs, the library was screened 607/) as the “first broad-based community effort to articulate
against α-chymotrypsin to compare the MWNT interactions the comprehensive needs and goals in nanoinformatics”.
with the enzyme catalytic site and other binding sites. Four of One of the most important components of nanoinformatics,
80 MWNTs were found to significantly affect enzymatic which resonates with similar components of bio- and
catalysis with much less nonspecific bindings.503 The combina- cheminformatics, concerns the understanding of the relation-
tional chemistry modifications on MWNTs have also regulated ships between the structure and the physical and/or biological
immune perturbations in mice and macrophages. In another properties of nanoparticles. To this end, given that sufficient
example, a modified nanotube deterred the mannose surface and organized data collections are available, well-established
receptor from binding to the scavenge receptor and reduced methods of quantitative structure−activity relationship (QSAR)
immunotoxicity.504 modeling, widely used in cheminformatics,508 could be
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Figure 20. General workflow for development of validated and externally predictive QSAR models.

expanded toward nanoparticles, as we have begun to advocate have similar biological activities. The detailed description of
for recently.29,31 Herein, we provide a brief overview of the major tenets of QSAR modeling is beyond the scope of this
quantitative nanostructure−activity relationships (QNAR) paper; the overview of many popular QSAR modeling
modeling approach, which should help experimental scientists techniques, including statistical and data mining techniques
studying nanoparticle−biological systems interactions ration- and descriptor calculation approaches, can be found in many
alize their data as well as employ QNAR models to enable reviews and monographs.509,510
rational design of novel nanoparticles with the desired For brevity, we shall comment on the most critical general
properties. We describe major principles and best practices in aspects of model development and, most importantly,
QSAR modeling, which serves as a basis for QNAR, and validation, which are especially significant in the context of
present a few recent examples of QNAR studies to illustrate the using QSAR models for identifying novel hit compounds by
concept and its applicability to the computational modeling of virtual screening. Additional essential information concerning
nanoparticles. both common errors and established practices in the QSAR
5.3.1. Quantitative Structure−Activity Relationships modeling field can be found in other critical essays on the
(QSAR). QSAR modeling has been a widely and successfully subject.511,512 We emphasize such aspects because they are
used approach in medicinal chemistry and drug discovery for generic for any QSAR-based approach application (i.e., QNAR)
nearly 50 years. The QSAR approach can be generally to enable rigorous model development.
described as an application of data analysis methods and The strategy for developing extensively validated and
statistics to developing models for accurate prediction of externally predictive QSAR models is summarized in Figure
biological activities or properties of compounds based on their 20 (described in more detail elsewhere513). We begin by
structures. Any QSAR method can be generally defined as an carefully curating chemical structures and, if possible, associated
application of mathematical and statistical methods to the biological activities to prepare the data set for subsequent
problem of finding empirical relationships (QSAR models) of calculations. Then, a fraction of compounds (generally 10−
the form Pi = k(D ̂ 1, D2, ..., Dn), where Pi are the biological 20%) is selected randomly as an external evaluation set. A more
activities (or other properties of interest) of the molecules, D1, rigorous n-fold external validation protocol can be employed
D2, ..., Dn are calculated (occasionally experimentally measured) when the data set is randomly divided into n nearly equal parts.
structural properties (molecular descriptors) of the compounds, The n − 1 parts are then systematically used for model
and k̂ is some empirically established mathematical trans- development, and the remaining fraction of compounds is used
formation that should be applied to descriptors to calculate the for model evaluation. The remaining subset of compounds (the
property values for all molecules. The goal of QSAR modeling modeling set) is divided into multiple training and test sets.
is to develop a computational means of predicting compound The training sets are used to establish correlations between
target properties (e.g., specific bioactivities or toxicities) from values of chemical descriptors and those of biological activities
their chemical structure, as characterized with computed or other target properties using different machine learning
chemical descriptors or, in some cases, by experimental physical techniques; the models are validated internally for their ability
chemical characteristics. All QSAR approaches directly or to accurately predict the target properties of compounds in the
indirectly imply simple similarity principles, which for some test sets. All of the models that display high prediction accuracy
time have provided a foundation for experimental medicinal for both the training and the test sets form an ensemble that is
chemistry: compounds with similar structures are expected to applied to predict target properties for compounds in the
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Chemical Reviews Review

Figure 21. Applications of QNAR methods to nanomaterials with either diverse cores (characterized by experimental properties) or similar cores but
diverse surface modifiers (characterized by computed chemical descriptors of modifying organic molecules). Adapted with permission from ref 31.
Copyright 2010 American Chemical Society.

Figure 22. Unsupervised hierarchical clustering of CNTs uncovered common subgroups for both protein binders and nonbinders. (A) Heat map
represents protein binding activities of the CNTs, which are sorted according to the dendrogram. Cyan color indicates high activity, while yellow
represents low activity. (B) Cyan cluster represents four compounds consisting of a common substituent causing similar (high) protein binding
responses as measured in four different assays. (C) Yellow cluster represents five compounds consisting of another common substructure highlighted
in the yellow square. These compounds have similar (low) activity in four protein binding assays. BSA, bovine serum albumin; CA, carbonic
anhydrase; CT, α-chymotrypsin; HB, hemoglobin.

external evaluation set. The critical step of the external employ the models for virtual screening of available chemical
validation is related to the use of the applicability domain,514 databases (e.g., ZINC515) to identify putative active compounds
which is defined uniquely for each model used in the consensus
(ensemble) prediction of the external set. If external validation and work with collaborators who could experimentally validate
demonstrates the significant predictive power of the models, we such hits.
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Chemical Reviews Review

We shall emphasize two of the most important aspects of the broader swath of biology, the Shaw group measured the effects
QSAR modeling workflow that directly pertain to QNAR of nanoparticles in four diverse cell line at four doses and using
modeling. First, the most critical difference between any four assays that interrogate different aspects of cellular
chemical data sets that the QSAR modeling strategy is applied physiology. Thus, the biological effects of each nanomaterial
to is the type of descriptors used to characterize the can be described as a 64-feature vector (4 cell lines × 4 assays ×
compounds. Thus, selection of the most appropriate 4 doses).
descriptors of nanoparticles is the main challenge for In theory, 64 independent QNAR models could be
developing QNAR strategies. Second, models resulting from developed with each model attempting to reproduce the
the predictive QSAR modeling workflow (Figure 1) can be biological response induced by 44 nanoparticles for a given
used in virtual screening to prioritize selection of chemicals for assay in a particular cell line at a given dose; however, we were
experimental validation. In fact, it is increasingly critical to unable to develop statistically significant models. Therefore, we
include experimental validation as the ultimate assertion of the combined the entire 64-dimensional vector for each nano-
model-based prediction. This paradigm fully applies to the use particle into a single averaged response. We then defined two
of QNAR modeling in nanotechnology as described in the next binary classes using an arbitrary threshold at Zmean = −0.40 to
section. enable a binary classification of nanoparticles. Two groups, each
5.3.2. QNAR Modeling: Applications to the Rational containing the same number of nanoparticles, were thus
Design of Nanoparticles with Desired Bioactivity defined: 22 nanoparticles belonged to class 1 (Zmean ≥ −0.40),
Profiles. Evaluation of both the desired and the undesired and the remaining 22 were placed in class 0 (Zmean < −0.40).31
biological effects of nanoparticles is of critical importance for The WinSVM program (a Windows implementation of a
the future of nanotechnology. Predictive QNAR modeling can support vector machine) developed in house was used to derive
be used, by analogy with QSAR, to establish statistically QNAR classification models. A rigorous 5-fold external cross-
significant relationships between the measured biological effects validation was followed to validate the models; under this
of nanoparticles and their physical, chemical, and geometrical procedure, the program splits the entire data set five times into
properties. As follows from the previous section, modeling of a modeling set including 80% of nanoparticles and the external
the biological effects of nanoparticles is possible if they can be validation set, comprising the remaining 20% of nanoparticles.
characterized by descriptors. Figure 21 illustrates two different Only the modeling set (which is divided additionally into
categories of nanoparticle data sets: those comprising nano- multiple training and test sets) was used to build and validate
particles with diverse metal cores and organic decorations for models, and models with reasonable training and test set
which only experimentally measured properties (i.e., size, shape, prediction power were selected for predicting the class
zeta potential, morphology, surface area, chemical reactivity, membership of the external set compounds. Importantly,
chemical composition, and aspect ratio) can be used as each nanoparticle was included in a validation set only once,
descriptors and those involving nanoparticles possessing the allowing us to calculate the overall external prediction accuracy
same core (e.g., CNTs) but different surface-modifying organic for the whole set.
molecules for which descriptors can be calculated for a single The results demonstrated that the SVM models yielded an
representative of the surface-modifying molecule. Several external prediction as high as 73% for the entire set of 44
commercially available software packages, such as Dragon,516 nanoparticles. Within five independent external folds, pre-
MOE,517 and ISIDA,518 could compute hundreds to thousands diction performances led from 56% to 88% accuracy.
of theoretical descriptors for any single compound directly from Apparently, the combination of biological profiles into one
its molecular structure. To illustrate the possible applications of single binary classification scheme allowed us to detect the
the QNAR method, we describe several case studies for which overall biological signal from noise and thus obtain QNAR
we constructed and validated models (Figure 22). models with both good internal fitness and external predictive
5.3.2.1. Modeling of Cellular Effects Induced by Diverse power.
Nanoparticles Using Experimentally Measured Properties as 5.3.2.2. Modeling of Nanoparticle Uptake in PaCa2
Descriptors. In a study, 51 diverse nanoparticles were tested in Cancer Cells Using Computational Descriptors. Whether
various cell-based assays.30 All nanoparticles were tested in vitro the multivalent attachment of small organic molecules to the
against four cell lines in four different assays at four different same nanoparticles can increase their specific binding affinity to
concentrations, resulting in a 51 × 64 biological data matrix of certain cells without a priori knowledge of the cell target was
experimental results. Each cell of this matrix reported the investigated.519 A combinatorial library of 109 nanoparticles
biological activity profile induced by a given nanoparticle at a was synthesized in which a superparamagnetic nanoparticle
certain concentration in a particular assay for a given cell line. (cross-linked iron oxide with amine groups, CLIO-NH2) was
Assay response values were expressed in units of standard decorated with different synthetic small molecules. Nano-
deviations of the distribution obtained when control cells were particles were screened against different cell lines, including
treated with phosphate buffered saline (PBS) alone: ZNP = (μNP PaCa2 human pancreatic cancer cells, U937 macrophage cell
− μPBS)/σPBS, where μPBS is the mean of the control tests with lines, resting and activated primary human macrophages, and
PBS and σPBS is its standard deviation. The authors also human umbilical vein endothelial cells (HUVECs). In contrast
reported four experimentally measured descriptors for 44 of 51 to the other cell lines, the uptake of the nanoparticles in PaCa2
tested nanoparticles: size, two types of relaxivities, and zeta pancreatic cancer cells was diverse and highly dependent on
potential. surface modification, enabling application of the QSAR
We demonstrated the feasibility of deriving robust QNAR modeling approach to these data. We represented each
models using the following four experimental descriptors: size, individual nanoparticle by the structure of a single, organic
two types of relaxivities, and zeta potential. Four of such molecule decorating its surface. Thus, each nanoparticle was
structural descriptors were available for 44 of the 51 represented by a unique set of descriptor values determined for
nanoparticles. To capture nanoparticles’ activity across a the conjugated small molecule.
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Chemical Reviews Review

Using this representation, we developed statistically signifi- will enable the development of new and highly valuable QNAR
cant models by relying on chemical descriptors and nano- models capable of rationally guiding further experimental
particle cellular uptake. The models’ external prediction power studies toward safer and more effective nanoparticles.
was shown to be as high as R2 = 0.72 and a mean absolute error
of 0.18 under a 5-fold external validation procedure.31 This 6. CONCLUDING REMARKS
study suggests that the cellular behavior of a series of A double-edged sword, nanotechnology provides us with
nanoparticles sharing a common core can be reasonably well enormous opportunities while posing a potential threat to
predicted using QNAR models built with the descriptors of human health. Therefore, understanding how engineered
surface modifying ligands only. nanoparticles interact with biological systems is urgent and
5.3.2.3. QNAR Models as Tools for Designing CNTs with critical. Nanoparticles are known to cause organ damage in
Desired Biological Properties. CNTs are among the most well- experimental animals and even in humans. Such damage is the
known nanoparticles, particularly in the area of drug direct or indirect result of perturbations of cells and biological
delivery.520−523 We utilized a combinatorial synthesis technique molecules such as proteins, lipids, DNA, and small molecules
to decorate the CNTs’ large surface area with a series of by nanoparticles. The nature and magnitude of nanoparticle
congeneric organic molecules and investigated the overall interactions with biological molecules are determined by both
toxicity of this series of nanoparticles. A total of 84 CNTs were the biological molecules and the engineered nanoparticles
tested in several in vitro toxicological assays, including protein themselves.
binding assays (BSA, carbonic anhydrase, chymotrypsin, and The fundamental physicochemical properties of nano-
hemoglobin), acute toxicity assays (survival percentage), and particles, such as their surface charge, hydrophobicity, and
immune toxicity assays (secretion of nitric oxide), all at two chemical bonding interactions, play significant roles in nano−
different doses (7.5 and 15 μg/mL). All of the CNTs shared bio interactions. Nanoparticles with different physicochemical
the same core structure, and thus, the variability of their properties exhibit different interactions with biological systems.
biological/toxicological behavior could be interpreted only For example, they may be surrounded by protein coronas of
based on the surface modifications.27 different nature, bind to different cell membrane proteins, enter
Each CNT was represented with a unique organic molecule or perturb cells differently, or exhibit diverse compatibility with
from its coating surface, similar to the study described above, physiological systems. A systematic understanding of such
and chemical descriptors were calculated for each molecule. We chemical interactions will help elucidate the QNAR of nano−
assigned a binary toxicity class (1 as toxic and 0 as nontoxic) to bio interactions. Such an understanding also facilitates the
each CNT based on the acute toxicity assay and the toxicity generation of nanomaterials through safe-by-design processes.
measured for pristine CNTs (CNTs with no surface modifiers). On the basis of these considerations, intentional surface
Only strong and weak CNT toxicants were considered for the modification of nanomaterials by one-at-a-time, parallel, or
modeling: 38 strong toxicants (class 1) vs 35 weak toxicants combinatorial synthesis has been shown to alter nano−bio
(class 0) were subjected to QNAR modeling using the Random interactions. Such effects have encouraged the development of
Forest approach. Similarly to other case studies, we realized a 5- biocompatible or biotargeting nanoparticles. Another significant
fold external cross-validation. The results were surprisingly development is that nano−bio interactions can now be
good with an overall external prediction accuracy as high as predicted by computational chemistry. These experimental
74% (n = 73, sensitivity = 79%, specificity = 69%). We also and theoretical approaches have strengthened our under-
developed QNAR models using protein-binding profiles that standing of the chemical basis of nano−bio interactions and
yielded 75% external prediction accuracy for classifying strong provide unprecedented methodologies to proactively design
vs weak CNT protein binders. and manufacture safe nanomaterials for a sustainable nano-
These models have been employed for the virtual screening technology era.
of a large library of 240,000 potential CNT surface modifiers in However, investigation of nano−bio interactions is by no
order to identify those with desired properties (low toxicity, means complete. Many challenges remain. First, we must
low binding affinity for carbonic anhydrase). Ten compounds understand the mechanisms of nano−bio interactions with
that were predicted to be toxic and ten compounds that were greater clarity via better characterization of experimental
predicted to be nontoxic were selected for the experimental systems, such as nanoparticles. Due to their chemical nature
validation. The preliminary results suggest that all predictions and their interactions with microenvironmental molecules, the
for nontoxic compounds were accurate, and for 6 out 10 chemical identity, biological identity, or cellular identity of
compounds predicted to be toxic, the experimental results were nanoparticles may differ. Comprehensive characterization will
in agreement with the computational annotation. improve our understanding of such interaction mechanisms,
We conclude that analogous to conventional applications of and QNAR studies can be conducted with better accuracy.
QSAR modeling for analysis of bioactive organic molecules, Moreover, diverse findings from different laboratories can be
QNAR models can be useful for predicting activity profiles of compared to form a systematic knowledge base. Second, at the
novel nanoparticles from either their experimental or their cellular level, an interaction with a receptor protein leads to a
computationally derived descriptors and for designing and network of cellular signaling and release of numerous molecules
manufacturing safer nanoparticles with desired properties. for signal propagation, feedback, and adaptation. Therefore, an
However, a shortage of systematic experimental data that in-depth understanding of nano−bio interactions at the cellular
characterize the constitutional, physical, and chemical proper- level relies on future development of systems biology and
ties of nanoparticles remains. In addition, no standard set of related knowledge bases. Third and most significant, at the
biological assays has been clearly defined to objectively and physiological level, the impact of nanoparticles on human
relevantly evaluate the in vitro (and in vivo) effects of tested health is an end result of complicated interplays of complex
CNTs. Development of systematized data repositories through interactions. For example, nanoparticles always adsorb environ-
collective efforts of experimental and computational scientists mental pollutants and perturb cellular nutrient molecules and
7770 dx.doi.org/10.1021/cr400295a | Chem. Rev. 2014, 114, 7740−7781
Chemical Reviews Review

redox balance, yielding multipronged effects. Furthermore, such Guibin Jiang received his Ph.D. degree from the Chinese Academy of
toxicant exposures are due to various sources and last a lifetime, Sciences in 1991 and carried out postdoctoral research at University of
beginning with a fertilized egg. Therefore, research aimed at Antwerp, Belgium, from 1994 to 1996. He is now a professor at the
understanding nano−bio interactions is an integral part of Research Center for Eco-Environmental Sciences, Chinese Academy
exploration in chemical biology and toxicology, having unique of Sciences, and Associate Editor for the Journal of Environmental
features. Therefore, it relies on the future progress of the Sciences and Technology published by the American Chemical Society.
chemical and biological sciences. He is interested in understanding the environmental processing and
toxicology of persistent toxic substances, environmental applications,
AUTHOR INFORMATION and impacts of nanomaterials.

Corresponding Author
*Phone: +86-531-88380019. Fax: +86-531-88380029. E-mail:
[email protected].
Present Address
‡
Clinical Research Division, Fred Hutchinson Cancer Research
Center, Seattle, WA 98109, and Department of Materials
Science & Engineering, University of Washington, Seattle, WA
98195, United States.
Notes
The authors declare no competing financial interest.
Biographies
Lingxin Chen received his Ph.D. degree from the Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, China, in 2003. After
2 years of postdoctoral experience at the Department of Chemistry,
Tsinghua University, China, he joined first as a BK21 researcher and
then as a research professor at the Department of Applied Chemistry,
Hanyang University, Ansan, Korea, in 2006. Now he is a professor in
the Yantai Institute of Coastal Zone Research under the program so-
called One Hundred Outstanding Young Chinese Scientists, Chinese
Academy of Sciences, China. His research interests include the study
of novel properties of materials such as functionalization nanoparticles
for developing nanometer biochemical analysis methods, molecular
imprinting-based sample pretreatment technology−chromatography
(including microfluidic chip) combined technology, and the microbial
degradation pollutant mechanism.
Qingxin Mu received his Ph.D. degree from Shandong University,
China, in 2010. From 2007 to 2013, he worked at St. Jude Children’s
Research Hospital and University of Kansas as a research scholar and
postdoctoral fellow, respectively. He is currently an NIH T32
postdoctoral research fellow at Clinical Research Division, Fred
Hutchinson Cancer Research Center and Department of Materials
Science & Engineering, University of Washington. His research
interests include surface engineering of nanoparticles, bioactivity
investigation of engineered nanoparticles and their applications in
targeted drug delivery and tumor imaging.

Hongyu Zhou received his Ph.D. degree in Medicinal Chemistry from

Shandong University, China, in 2009. He was a postdoctoral
researcher from 2009 to 2012 under the supervision of Dr. Bing
Yan in the Department of Chemical Biology & Therapeutics at St.
Jude Children’s Research Hospital in Memphis, TN. He is currently a
postdoctoral fellow in the Department of Surgery at Emory University.
His research focuses on development of novel surface-modified
nanoparticle combinatorial libraries to regulate the biological proper-
ties of nanoparticles and nanoparticle-based drug delivery systems and
their applications in targeted cancer therapy and imaging.

7771 dx.doi.org/10.1021/cr400295a | Chem. Rev. 2014, 114, 7740−7781

Chemical Reviews Review

Denis Fourches is Research Assistant Professor at the UNC Eshelman Bing Yan received his Ph.D. degree from Columbia University in 1990
and carried out postdoctoral research at the University of Cambridge,
School of Pharmacy, UNCChapel Hill. He received his Ph.D. U.K., and University of Texas Medical School in Houston from 1990
to 1993. From 1993 to 2005 he worked at Novartis, Discovery
degree in Chemistry in 2007 from the University of Strasbourg, Partners International, and Bristol-Myers Squibb. He has been a full
France. He came to UNCChapel Hill in 2008 as a postdoctoral faculty member at the Department of Chemical Biology and
Therapeutics, St. Jude Children’s Research Hospital, in Memphis,
fellow and became an assistant professor in 2010. His research TN, from 2007 to 2012, and Professor at Shandong University, China,
from 2005 to present. He served as Associate Editor for the Journal of
interests are in the areas of cheminformatics, computer-assisted drug
Combinatorial Chemistry published by the American Chemical Society
design, computational toxicology, and structural bioinformatics. from 2005 to 2012 and was book series editor for Critical Reviews in
Combinatorial Chemistry published by the Francis & Taylor Group. He
is interested in understanding the biological activities of nanoparticles,
regulation of such activities by nanoparticle modifications, and
medicinal applications of nanotechnology.

ACKNOWLEDGMENTS
This work was supported by the National Basic Research
Program of China (2010CB933504) and the National Natural
Science Foundation of China (21077068 and 21137002).

ABBREVIATIONS
AFM atomic force microscopy
ATP adenosine-5′-triphosphate
BMP bone morphogenetic protein
BMPR2 bone morphogenetic protein receptor type
Alexander Tropsha is K.H. Lee Distinguished Professor and Associate
II
Dean for Research at the UNC Eshelman School of Pharmacy, BSA bovine serum albumin
CB carbon black
UNCChapel Hill. He received his Ph.D. degree in Chemical CD circular dichroism
CLSM confocal laser scanning microscopy
Enzymology in 1986 from Moscow State University, Russia. He came
CNTs carbon nanotubes
to UNCChapel Hill in 1989 as a postdoctoral fellow and became an CTAB hexadecyltrimethylammonium bromide
DLS dynamic light scattering
assistant professor in 1991. His research interests are in the areas of DPPC dipalmitoylphosphatidylcholine
computer-assisted drug design, computational toxicology, cheminfor-
DSC differential scanning calorimetry
EB ethidium bromide
matics, and structural bioinformatics. His has authored or coauthored ENMs engineered nanomaterials
ER endoplasmic reticulum
more than 160 peer-reviewed research papers, reviews, and book FCS fluorescence correlation spectroscopy
chapters and coedited two monographs. His research is supported by FTIR Fourier-transform infrared spectroscopy
GC-MS gas chromatography−mass spectrometry
multiple grants from the NIH, NSF, EPA, and private companies. He GNPs gold nanoparticles
GO graphene oxide
is a member of editorial boards of several scientific journals and an GPCRs G-protein-coupled receptors
elected member of the Board and vice-chair of the international GSH glutathione
Hb hemoglobin
Cheminformatics and QSAR Society. HSA human serum albumin
7772 dx.doi.org/10.1021/cr400295a | Chem. Rev. 2014, 114, 7740−7781
Chemical Reviews Review

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