Nanoparticle Surface Functionality Dictates Cellular and Systemic

Review pubs.acs.org/cm

Nanoparticle Surface Functionality Dictates Cellular and Systemic Toxicity Amir Ata Saei,† Mahdieh Yazdani,‡ Samuel E. Lohse,§ Zahra Bakhtiary,∥ Vahid Serpooshan,⊥ Mahdi Ghavami,# Mahtab Asadian,⊗ Samaneh Mashaghi,∇ Erik C. Dreaden,¶ Alireza Mashaghi,◆,○,★,⬠,⧄ and Morteza Mahmoudi*,⟠ †

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 171 77, Sweden Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States § Physical and Environment Sciences Program, Colorado Mesa University, Grand Junction, Colorado 81501, United States ∥ Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz 5166/15731, Iran ⊥ Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California 94305, United States # Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen 1165, Denmark ⊗ Faculty of Engineering & Architecture, Department of Applied Physics, Research Unit Plasma Technology (RUPT), Ghent University, Ghent 9000, Belgium ∇ School of Engineering and Applied Sciences and Department of Physics, Harvard University, 29 Oxford Street, Cambridge, Massachusetts 02138, United States ¶ Koch Institute for Integrative Cancer Research, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ◆ Leiden Academic Centre for Drug Research, Faculty of Mathematics and Natural Sciences, Leiden University, Leiden 2311 EZ, The Netherlands ○ Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts 02115, United States ★ Dana-Farber Cancer Institute, Boston, Massachusetts 02215, United States ⬠ Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ⧄ Basir Eye Health Research Center, Tehran 1418643561, Iran ⟠ Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States ‡

ABSTRACT: Engineered nanoparticles (NPs) have opened new frontiers in therapeutics and diagnostics in recent years. The surface functionality of these nanoparticles often predominates their interactions with various biological components of human body, and proper selection or control of surface functionality can greatly enhance subsequent therapeutic effects of NPs while diminishing their adverse side effects. In this review, we will focus on the effect of surface functionality on the cellular uptake and the transport of NPs by various subcellular processes.

parameters;6 however, systemic applications of NPs in the clinical setting are currently limited to a subset of NP

1. INTRODUCTION Nanoparticles (NPs) are promising tools for biomedical diagnostics and therapeutics.1−5 Depending on their specific application, varying design architectures are required (e.g., core material, size, shape, surface characteristics etc.). The biological impact of NPs in human body depends on all these © 2017 American Chemical Society

Received: May 13, 2017 Revised: July 19, 2017 Published: July 20, 2017 6578

DOI: 10.1021/acs.chemmater.7b01979 Chem. Mater. 2017, 29, 6578−6595

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Chemistry of Materials

mediating NP interactions with cellular membranes.8,26,30 The surface chemistry of many NPs, however, is challenging to characterize in detail, and NP surface chemistry will change in response to immersion in many biological systems, making precise NP surface chemistry difficult to describe at the molecular level and challenging to control in biological systems. Because of the importance of NP surface chemistry in dictating biological interactions, synthetic strategies for controlling NP functionalization have been extensively explored.31−34 Comprehensive functionalization strategies now exist to modulate the surface chemistry of a wide variety of engineered NPs,29−31,35−39 including metallic NPs, semiconductor NPs,40−42 inorganic carbon NPs (including carbon nanotubes, fullerenes, and nanodiamond),43,44 and polymeric NPs.45 The exact chemical strategy employed for attaching ligands or capping agents to the NP surface depends primarily on the nature of the NP core material. For instance, ligands for metal NPs are often bound to the NP core through a thiol bond.22 In contrast, the same functionalities might be introduced onto the surface of nanodiamond by grafting a functionalized molecule onto the NP surface through a terminal alkene or by condensation with COOH groups present on the nanodiamond surface.32 Polymers may be electrostatically adsorbed to existing capping agents on many different NP surfaces, making polymer wrapping a common functionalization technique for NPs with many different core materials.38,46−48 Once an initial ligand is attached, further surface chemistry modifications are sometimes made through traditional organic chemistry reactions (such as click chemistry49).50 Coating NPs with an inorganic shell (such as silica), is also common, and allows the attachment of functionalized molecules onto the new silica shell via silane chemistry.51,52 The sheer number of synthetic routes available to synthesize functionalized NPs makes it seem as though controlling NP surface chemistry is a simple task, when in fact, the surface of a NP is a complex milieu of chemical species that may contain a combination of adsorbed ligands, adsorbed ions, or alloyed metals. For example, the surface of anisotropic metal NPs typically display surfactant molecules, adsorbed anions (such as bromide), or even submonolayer levels of silver adsorption,53 and the exact structure and speciation of such adsorbates has only begun to be fully elucidated even for many commonly studied functionalized NPs.54 Though the characterization of NP surface chemistry is often perceived to be a simple problem, developing an accurate picture of the surface chemistry of even the “simplest” NP can be a very demanding analytical challenge. As a result of their unique size regime, most functionalized NPs can only be incompletely characterized using the tools of chemistry, materials science, and colloid analysis instrumentation. Developing a complete picture of the surface chemistry of a NP requires a suite of instruments used (ideally) in concert. Colloid-oriented analytical techniques, such as dynamic light scattering (DLS), ζ-potential analysis, differential centrifugal sedimentation (DCS), transmission electron microscopy (TEM), spectral turbidimetry, and fluorescence correlation spectroscopy (FCS) are typically used to establish NP size distribution, surface charge, diffusion coefficients, and aggregation behavior.28,34,55−57 The colloidal stabilities of the functionalized NPs are also monitored with DLS and DCS.28,58 However, colloid-oriented analytical techniques provide very little information about the deep details of NP surface chemistry. Organic chemistry analytical techniques (such as

compositions and shapes. Thus, surface characteristics stands out as one of the most important, if not the main, determinants of biological performance, as the NP surface is the most prominent and earliest point of exposure. In addition to directing their interactions with serum proteins, the interfacial properties of NPs have crucial influence on their behavior toward cells.7−15 The chemical nature of these surfaces, therefore, can often be the strongest determinant of NP interactions and behavior in a given biological system. NPs can strongly interact with different components of a cell from cytoplasmic and organelle membranes to genomic DNA, glycans, proteins, and even small molecules.16 These interactions can serve as important therapeutic properties; however, they can cause toxic effects as well. For example, the intracellular interactions of NPs can lead to the disturbance of the membrane electron/ion transports,17 induction of conformational changes in proteins,18 production of reactive oxygen species (ROS),19 and oxidative stress-mediated genotoxicity.20 In addition, biological systems often sense and react to the nanoscale particulates, which further complicates our understanding of their subsequent biological effects.21,22 These interactions can occur either at the cellular level (e.g., altered endocytic and metabolic activities)23 or tissue level (e.g., release of inflammatory cytokines).24 A detailed understanding of the role that surface functionalization plays in the biological effects of NPs is needed to facilitate efficient engineering of NPs for nanomedicine. One example that illustrates the complexity of this issue is that even the coordination of ligands on the surface of a NP can significantly enhance subsequent cytotoxicity.25 A potential strategy for understanding the role of surface functionality in NP−cell interaction would be to systematically study NPs featuring the same size and shape but differing surface functionalities. However, one of the major challenges in such investigations is the heterogeneity and often poor reproducibility of NP architectures. In addition, the multiparameter nature of such studies, makes drawing definitive conclusions rather difficult. Cell-specific responses and the formation of nonspecific protein coronas on NP surface also complicate research findings. Although a large number of studies have focused on studying the interactions of nanomaterials with cells and biological structures, the field still has a long way to go before predictive NP−cell interactions become a reality. In this paper, we review recent progress in understanding how NP surface functionality can cause cellular toxicity via damaging cell membranes, immunotoxicity, blood incompatibility and organ toxicities. We conclude by discussing a number of recent advances whereby NPs are used as “self-therapeutics” with tissue-specific toxicity.

2. SURFACE FUNCTIONALIZATION OF NANOPARTICLES The surface chemistry of functionalized NPs is one of their most influential physiochemical properties.8,26,27 Engineered NPs display chemical species adsorbed to the NP surface, either by design or incidental deposition.28 These adsorbates govern the surface charge of the NP, provide electrostatic or steric barriers to aggregation, and act as the chemical interface between the surface of the NP and its environment.8,26,28,29 NP surface chemistry has been shown to play a very influential role in governing the interaction of NPs with biomolecules in the environment, dictating the long-term stability of NPs, acting as a determining factor in NP biological fate and transport, and 6579


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reduced for these NPs. These types of functionalization strategies are vital in minimizing nonspecific protein adsorption to the NP surface, preserving the chemically engineered targeting properties of the NPs. It has previously been shown that serum protein adsorption to functionalized polystyrene NPs interferes with the NP’s ability to target specific receptors in the cell membrane.71 This is not an entirely unexpected result, because protein adsorption effectively overcoats the targeting ligands attached to the NP surface. The adsorption of proteins to the NP surface does not necessarily have such predictable effects on the interactions of NPs with cell membranes or proteins. As a result, effective strategies to use surface chemistry to control bionano interactions are emerging slowly, and surface chemistry strategies that reduce protein adsorption (or increase cellular uptake) for one NP core size and composition may actually lead to increased protein adsorption (or decreased cellular uptake) for another NP. For instance, in the case of functionalized gold NPs (AuNPs), it has been shown that the AuNPs can adsorb proteins from serum, which will actually increase their affinity for certain cell membrane receptors, leading to greater AuNP uptake by the cells.72 Protein adsorption from serum may also alter the physiochemical properties of NPs in other ways, such as initiating the widespread formation of protein−AuNP aggregates in serum,73 or in other cases, reducing the aggregation of functionalized AuNPs in serum. The precise interaction between proteins and NPs depends on a number of factors including the serum conditions and the initial NP size and surface chemistry.74 In a recent study by Saha et al.,75 it was shown that the nature and identity of the protein corona can be tuned by modulating the surface functionalities of NPs. Using a series of engineered cationic AuNPs, they monitored the uptake patterns using macrophages cells. It was observed that formation of protein corona on these NPs was dependent on hydrophobicity as well as arrangement of chemical motifs on the surface of AuNPs. The formation of biomoelcular coronas on the NP surface could specially have a detrimental effect on active targeting strategies. Salvati et al.71 have shown that silica NPs functionalized with transferrin were screened by a protein corona mask and were thus unable to recognize the cognate receptors on the targeted cells. These effects might eventually cause accumulation of these NPs in the cells that were not intended to be the target. Enshrouding NPs with polyethylene glycol (PEG) also has an interesting behavior on both protein corona formation and cellular uptake.76 Although, PEGylation generally prevents protein corona by formation of a hydration shell, which hinders protein adsorption,77 it also reduces the chance of NPs being uptaken by intended cells. This reduced uptake is the result of the PEG capping agent hindering the interaction of NP ligands with cell membranes.78 Because of the importance of NP surface chemistry in governing nanobio interactions (which will be discussed in detail below), a number of challenges exist in controlling and characterizing the surface chemistry of functionalized NPs that must be met in the coming years. Two of the principal challenges that have to be addressed are the development of new methods (or the refinement of existing methods) to generate a complete or fully accurate picture of the NP’s surface, and a detailed understanding of how the NP’s original “engineered” surface chemistry influences subsequent NP interactions with biomacromolecules and ions present in solution. The second of these goals is further complicated by

Fourier transform infrared spectroscopy (FTIR), liquid chromatography−mass spectrometry (LC-MS), thermogravimetric analysis (TGA) or 1H NMR) can be used to identify the functional groups present on the NP surface or molecules that may have been adsorbed, but most of these techniques at best only provide semiquantitative information as to the total number/density of ligands on the NP surface.59 More materials-oriented analyses, such as energy-dispersive X-ray spectroscopy (EDS) or X-ray photoelectron spectroscopy (XPS) can supplement all these techniques to provide accurate insight as to the composition of both the core and the surface absorbates (in the case of EDS) for inorganic NPs and an accurate determination of the ratio of core atoms to ligands or adsorbed species (in the case of XPS).60−62 It should be noted though that XPS provides reliable composition ratios primarily for large NPs (significantly greater than 10 nm in diameter) whose diameter easily exceeds the penetration depth of the Xrays.60−62 For smaller particles, corrections for surface curvature and other geometric effects must be applied to the raw data to accurately quantify the ligand:core atom ratio.62 Although EDS is primarily used for the identification of inorganic elements, it can be coupled to high-resolution electron microscopy techniques, which even permits the identification of adsorbed elements on specific facets of a nanocrystal.60 One additional challenge in accurately characterizing the surface chemistry of functionalized NPs (particularly in biological systems) lies in the fact that NPs surface chemistry changes and evolves as they are transported into new environments. Regardless of what their surface chemistry may have been engineered to be, functionalized NPs can acquire new adsorbates as they are transported to new environments.63,64 These new adsorbates may include proteins (from serum, forming the well-established “protein corona”),63,64 lipids (from biological fluids or possibly cell membranes),65 polysaccharides,66 natural organic matter,67 and adsorbed ions. Once adsorbed, many of these biomolecules may become a “permanent” component of the NP’s surface chemistry, modifying the NP’s surface charge, and changing its interactions with the surrounding environment.63,64 At present, the precise manner in which these changes in surface chemistry modulate NP behavior in biological systems is not entirely predictable.68 Several factors can affect the composition and arrangement of the protein corona and other adsorbates on the NP’s surface, among which are NP size, surface charge, hydrophobicity, and surface chemistry.69,70 Because the desired size and chemical composition of the NP core are typically predetermined by the desired application, the surface chemistry of the particle is the physiochemical parameter that is most typically exploited to control the particle’s biointeractions. In recent years, a host of synthetic strategies have been developed to better use surface chemistry to control the NP interactions with both plasma/ serum proteins and cell membranes, however, a comprehensive picture of how NP surface chemistry influences bionano interactions has yet to emerge. Santos et al.49 have shown that surface modifications of alkyne-terminated thermally hydrocarbonized porous silicon (THCPSi) NPs with targeting peptides and antifouling proteins not only can modulate cellular uptake but control plasma protein association. For instance, dextran surface modified THCPSi NPs can reduce adsorption of immune proteins (fibrinogen and immunoglobulin G) and thus the chance of being recognized by the immune system and consequent clearance by phagocytosis is 6580


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Figure 1. Varying cellular uptake mechanisms for NPs. From left to right: phagocytosis, macropinocytosis, clathrin-mediated endocytosis, caveolinmediated endocytosis, and nonclathrin- and noncaveolin-mediated endocytosis Reproduced with permission from reference 82. Copyright 2013 American Chemical Society.

disrupt vesicles and enter the cell nucleus hypothetically through their debated “proton sponges” capability or by yet unknown mechanisms (Figure 2).86,87

the fact that surface chemistry is often not the sole determinant of a NP’s biointeractions. Instead, surface chemistry, size, and core composition interact synergistically to determine a NP’s fate in a biological system. As a result, there is probably no “one size fits all” approach to controlling nanobio interactions through surface chemistry. Nevertheless, the surface of the particle remains the primary interface through which the NP interacts with its surroundings, and precise control of NP surface chemistry is essential in controlling the bio fate and transport of engineered NPs.

3. INTERACTION OF FUNCTIONAL NANOPARTICLES WITH CELL MEMBRANES When NPs approach the border of cells, they can interact with the plasma membrane. The mammalian cell surface is covered with a meshwork of polysaccharides named glycocalyx,79 which is the first cellular component NPs see upon interaction with a given cell. Subsequently, NPs enter the cells through several processes including phagocytosis, pinocytosis, receptor-mediated endocytosis, and energy independent direct translocation (see Figure 1 for details).10 In a majority of cases, NPs are entrapped in the vesicular structures (e.g., lysosomes, caveosomes, and early/late endosomes) and cannot reach the cytosol.80 However, some NPs can enter the cytosol through receptor-mediated uptake, passive passage or other nonspecific mechanisms.8,10 Surface functionality thus has a large influence not only on the kinetics and mechanism of NP uptake into cells23 but also on controlling their subcellular trafficking into specific organelles.16,81 3.1. Effect of Surface Charge. The surface charge of NPs plays a critical role in determining their cellular uptake mechanism. Typically, anionic and neutral NPs have low affinity to the anionic cell membrane, whereas cationic NPs are much more electrostatically attracted to the membrane, leading to higher cellular uptake. This trend is confirmed by experimental evaluations.83,84 Neutral and anionic NPs can also interact with the negatively charged membrane (mainly through nonspecific binding and aggregation of the particles on positively charged sites of cell membrane8,85) and are entrapped within vesicular structures inside the cell through pinocytosis. Cationic NPs can also enter into the cells through pinocytosis if their surface charge is not too great. This is because strongly positive NPs can hamper the membrane wrapping efficacy of the cells (as revealed by molecular dynamics simulation75). However, unlike neutral and anionic NPs, cationic NPs can

Figure 2. Importance of the surface functionality in cellular uptake. Cationic NPs have significant cellular uptake, compared to the anionic and neutral NPs mainly because of their high attractive electrostatic interactions with the negatively charged cell membrane. In addition, it is speculated that cationic NPs are capable of acting as “proton sponges” that disrupt the lysosomes and deliver the NPs into the cytosol. TEM images (the bottom right and left panels) demonstrate higher entrance of the cationic (amine amine-poly(vinyl alcohol)coated) superparamagnetic iron oxide NPs into the HeLa cells compared to the anionic ones, respectively. Reproduced with permission from reference 87. Copyright 2012 Royal Society of Chemistry.

Though NPs with various surface functionalities can enter the cells through different pinocytotic routes, cationic NPs are capable of directly crossing cell membranes and entering the cytosol. This path of entry is quite rare and is reported predominantly with cationic NPs. This direct entrance into the cells (unlike unspecific receptor-mediated uptake) can form pores in the plasma membrane. One of the more important electrostatic interactions here occurs between the cationic moieties on the particles and phosphate groups of the lipid bilayer, resulting in NP−membrane binding and potentially increasing membrane surface tension and associated pore formation. Xia et al.88 reported that the uptake rate of cationic AuNPs into SK-BR-3 cells is more than 5 times higher than that of anionic counterparts. This was attributed to the fact that half 6581


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Increasing surface charge from a cationic coverage (percentage of ammonium-functionalized ligands) of 10% to a 50% optimum enhanced membrane penetrations. However, further increase of charge densities resulted in membrane disruption.90 For instance, Li and Malmstadt91 demonstrated that relatively small cationic polystyrene (20 nm; with 80.2 mequiv of amide per g of polymer) NPs induce membrane deformation and pore formation in giant unilamellar vesicles; in contrast, larger cationic polystyrene NPs with lower positive charge densities (120 nm; with 39.7 mequiv of amide per g of polymer) formed smaller pores. In another study, Holl et al.92 examined an AFM/SLB (atomic force microscopy of supported lipid bilayers) assay across a wide range of cationic materials to study the degree of associated membrane disruption. Three general types of perturbations were observed: (i) NPs that accumulate around the edges of pre-existing defects but do not induce new defects (e.g., PAMAM G3-NH2 dendrimers); (ii) NPs that mainly disrupt the bilayer by adhering to pre-existing defects and expanding them (e.g., amine-coated AuNPs (AuNH2), the cell penetrating peptide MSI-78 and PAMAM G5NH2 dendrimers); and (iii) NPs that directly induce the formation of holes and defects in lipid bilayers (e.g., TAT sequence31 employed by HIV virus, PAMAM G7-NH2, polyethylenimine (PEI), di3ethylaminoethyl-dextran (DEAEDEX), and amine-coated silica NPs (silica-NH2)). NP surface charge can also affect the cell membrane potential. To investigate this effect, the cellular uptake of AuNPs (∼2 nm core size) with different charges (i.e., positive, negative, neutral, and zwitterionic) into cells of varying membrane potential was investigated in both malignant (ovarian cancer CP70 and A2780 cells) and nonmalignant, excitable cells (human bronchial epithelial cells (BECs) and human airway smooth muscle cells (ASM)).93 These results showed that only positive NPs induce membrane depolarization across different cell types and the uptake of cationic particles was significantly higher than AuNPs of other charges (see Figure 4). The greatest depolarization with positively charged AuNPs was observed in ovarian cancer cells (CP70, A2780), roughly comparable to that achieved with 40 mM KCl. This induced membrane depolarization was associated with an increase in intracellular uptake of cationic AuNPs or Ca2+, resulting in modulation of intracellular signaling pathways (e.g., inhibition of proliferation and reduction of viability in normal cells). The increase in intracellular Ca2+ levels varied across cell lines of different lineages. Though the addition of positively charged AuNPs resulted in an immediate increase in Ca2+ in CP70 and A2780 cells, in BEC and ASM cells, Ca2+ release was delayed. Furthermore, a temporal study showed that depolarization procedes the elevation of Ca2+ in cells, which links AuNP-induced membrane depolarization to increased Ca2+. In contrast, AuNPs with other charges had negligible effects on membrane depolarization, cell uptake, and Ca2+ release (see Figure 4d). Interestingly, pretreating cells with KCl (i.e., membrane depolarizing agent) resulted in a substantial decrease in the amount of cationic NP uptake in all cell types and the extent of membrane depolarization induced by positive NPs, suggesting that transmembrane potential is the driving force in the direct passage of cationic NPs. The series of events described in this study were mostly cell-line dependent. AuNPs (mostly positively charged) enter cells based on their membrane potential, which subsequently leads to membrane depolarization and increase in Ca2+ levels. Finally, cell cycle arrest or apoptosis occurs depending on cell type.

of cationic AuNPs diffuse into cells by pore formation, whereas anionic and neutral NPs were internalized into cells only under endocytotic pathways. As such, cationic NPs can irreparably damage the existing fine balance of ions, proteins, and macromolecules across the cell membrane and lead to disruption of cellular homeostasis. Another interesting issue in this study is that, negatively charged NPs were internalized at a slightly higher level than their neutral counterparts. This might be due to some positively charged regions on the surface that provide an opportunity for the uptake of negatively charged NPs.89 It was also found that membrane adsorption was rate-limiting for NP internalization, because neutral and negatively charged AuNPs have low adhesion to negatively charged cell surface. These structures were therefore internalized to a much lower extent.88 The highly disruptive effects of cationic NPs to cell membranes are also demonstrated in molecular dynamics simulation that was performed to probe the interaction of cationic and anionic AuNPs with electronegative and electroneutral bilayers.90 The results showed that the positively charged NPs have much stronger deleterious influence on negative bilayers (see Figure 3) compared to the negatively charged NPs. Furthermore, the levels of both membrane penetration and disruption depend on surface charge density.

Figure 3. General illustration of lipid membrane penetration mechanism by positively charged gold nanoparticles. (a) A schematic representation of functionalized AuNPs with ammonium (positive) or carboxylate groups (negative). (b) The binding occurring between the ligand terminals (at the surface of AuNPs) and the lipid head groups, which leads to the entrapment of AuNPs by a lipid shell. (c) Formation of pore and defective areas on the bilayer by penetration of the AuNPs. (d) The generated pore makes the bilayer water permeable. (e) The lower leaflet of the lipid bilayer extrudes to facilitate penetration of the AuNPs. (f) A representative scheme showing the interface between AuNP and lipid bilayer. (g) Snapshots showing the interactions of AuNPs with different cationic surface charge densities (the percentage of cationic coverage is indicated in each snapshot) with a negative bilayer. Gold core is shown in yellow, hydrophobic ligands in green, cationic ligands in red, dipalmitoylphosphatidylcholine head groups in ice blue, dipamitoylphosphatidylglycerol head groups in pink, lipids tails in sliver, and water in transparent white. Reproduced with permission from reference 90. Copyright 2010 American Chemical Society. 6582


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In addition to coincident effects, the surface charge of NPs can be leveraged for specific biomedical applications. For example, carboxymethyl chitosan/chitosan-nanoparticles have demonstrated surface charge-dependent enhancement of epithelial permeation and promotion of intestinal absorption.96 Surface charges of these NPs has the ability to open intestinal epithelial tight junctions through varying mechanisms, including the down-regulation of tight junction protein claudin-4 as well as its activating phosphorylated form. In another study, it was shown that negatively charged AuNPs have a higher percutaneous efficiency in delivering vascular endothelial growth factor with respect to the positively charged or uncharged particles.97 3.2. Effect of Surface Hydrophobicity/Hydrophilicity. The cellular membrane is the first contact point for a given NP. The nature of this interaction can affect cellular processes in myriad ways, including modulation of intracellular trafficking, intracellular signaling and lipid membrane structure and integrity. As such, investigating the interactions of NPs with cellular membranes (or their model systems) are of paramount importance. The self-assembly behavior of NPs and their interactions with lipids can be driven by hydrophobic/ hydrophilic interfacial effects. Hydrophobicity plays an important role in the interaction of NPs and membranes.98 Korgel et al.99 for example, investigated how hydrophobic AuNPs (2 nm) interact with phosphatidylcholine (PC) vesicles. The results showed two scenarios where NP−vesicle hybrids are possible depending on the synthesis method employed for fabrication of the hybrid (extrusion or dialysis): (i) the NPs can form a close-packed monolayer, associating with the hydrophobic tails of the PC vesicles and being embedded within the bilayer; and (ii) the NPs cluster on one side of the lipid bilayer and subsequently create deformations. Supported lipid bilayers (SLBs) are planar structure formed on a solid support and provide exceptional model systems for investigating the surface chemistry of the cell and its interactions with other systems. Because of the resilience and stability of SLBs, they are not easily destroyed or disturbed by physical stressors such as vibration or puncture and as such, unlike cell membranes, are suitable for long-term studies. Moreover, on the contrary to freely floating surfaces or membranes, due to the flat nature of SLBs, a large array of tools can be employed for characterization purposes. Jing and Zhu100 reported that initiating pore formation (lipid-poor regions) on L-α-phosphatidylcholine (α-PC) SLBs) with adsorbed semihydrophobic polystyrene NPs occurs above a critical NP concentration, which is independent of NP size. Furthermore, researchers found that the extent to which lipid molecules from SLB adsorb and wrap onto the NP surface is dictated by hydrophobic interactions, which can be enhanced by electrostatic interactions screened at increasing ionic strength. Binder et al.101 reported that the localization of quantum dot (QD) NPs (2 nm) in mixed lipid/polymer membranes is dependent on their hydrophobic, hydrophilic, or amphiphilic surface properties. The findings indicated that hydrophobic QD NPs can be selectively positioned within polymer domains of a mixed lipid/polymer membrane system. In contrast, amphiphilic counterparts showed no specific localization in phaseseparated lipid/polymer films. Moreover, hydrophilic NPs were found to exhibit the ability to adsorb onto lipid/polymer monolayers, showing a larger influence of molecule packing in pure lipid films in comparison with mixed monolayers. Bishop et al.102 reported that relatively large (∼6 nm) AuNPs

Figure 4. Effect of AuNPs’ surface charge on cellular membrane potential (a) Schematic showing the AuNPs of different surface charges (neutral (0AuNP), positive (+AuNP), negative (−AuNP), and zwitterionic (AuNP). The diameter (*) and surface charge (**) were measured by DLS and ζ -potential techniques, respectively. (b) Rapid and significant membrane depolarization induced by +AuNPs (1.2 μM) in 4 different cells (two ovarian cancer cell lines (CP70, A2780), human bronchial epithelial cells (BEC), and human airway smooth muscle (ASM) cells); * indicates significant AuNP effect (p < 0.05). (c) Significant uptake of +AuNPs (0.4 μM), by CP70 cells, compared to the other types of AuNPs. (d) +AuNPs (1.2 μM) produced considerable increases in [Ca2+]g3i levels in the cells loaded with the ratiometric fluorescent Ca2+ indicator fura-2; * indicates significant AuNP effect (p < 0.05). Reproduced with permission from reference 93. Copyright 2010 American Chemical Society.

Further evidence of details that transmembrane depolarization is the driving force for the translocation of cationic NPs is revealed in a coarse-grain molecular dynamic simulations performed by Lin and Alexander-Katz.94 In this simulation, the direct translocation of ammoniate functionalized Au-NP in a dipalmitoylphosphatidylcholine (DPPC) bilayer system was investigated. The DPPC system is divided into two regions, mimicking the ionic imbalances between intracellular and extracelluar regions of normal cell. The simulation revealed that AuNPs are directly translocated into the intracellular region of the DPPC system by generating nanoscale defect through the bilayer that have a small, continuous potential gradient. In contrast, the simulation performed without a transmembrane potential only showed that the NPs are attaching to the DPPC bilayer. It was also found that the nanoscale defect resealed just nanoseconds after translocation was completed as membrane stress is diminished after the large charge balance between the two regions was almost completely dissipated. During the translocation process it is found that the beginning of the hole formation caused Na+ and Cl− ion to flow in and out of the intracellular region respectively, and a subsequent inflow of Cl− ion when the AuNPs crossed the membrane, as the hydration shell of the positively charged AuNPs included Cl− ions. Electrical current flows from the simulation were also consistent with experimental measurements performed on human embryonic kidney and epidermoid carcinoma cell with cationic NPs at noncytotoxic concentration. Molecular dynamic simulations have also revealed the importance of NP’s surface charge on their interaction with single stranded DNA and RNA.95 Studies have shown that the structural stability of NPs−nucleic acid complex is more dependent on NP’s surface charge than the nucleic acid’s sequence and type. 6583


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Chemistry of Materials functionalized with mixed hydrophilic and hydrophobic ligands, symmetrically penetrate (i.e., “Janus” penetration) and strongly associate with surfactant vesicles (∼2.5 nm thickness) to form NP−vesicle complexes. The hydrophobic and hydrophilic ligands in this case are able to redistribute themselves dynamically depending on environmental conditions (i.e., hydrophobic sides of the NP surface interact with the hydrophobic core of the bilayer, whereas hydrophilic parts remain in interaction with the aqueous solution). Several theoretical studies demonstrate how the hydrophobicity of NPs can affect their interactions with the lipid bilayers. Using MD simulations, Gu et al.103 predicted that hydrophobic NPs are thermodynamically stable at the midplane of the hydrophobic core of the membrane. Moreover, the process of NP inclusion leads to deformation and heterogeneity in the distribution of lipid molecules within the bilayer (i.e., hydrophobic mismatch). In contrast, semihydrophilic NPs energetically preferred to adsorb to the surface of the bilayer, which can induce wrapping processes (i.e., endocytosis). Balazs et al.104 theoretically showed that the relative distribution of hydrophilic and hydrophobic moieties on Janus NP surfaces influences the stability of pre-existing pores of a lipid bilayer. Specifically, Janus NPs having a 60° hydrophobic part are close enough to the optimal composition for forming stable pores in amphiphilic membranes.

Figure 5. Graphs showing the induction of G2/M arrest in (a) U251and (b) IMR-90-cells after incubation with silver NPs with various concentrations. Comet analysis of the U251 cells (c) before and (d) after interactions with silver NPs. (e) Graph demonstrates the tail moments of DNA (μm). * indicates significant silver NPs effect (P < 0.05). Reproduced with permission from reference 107. Copyright 2009 American Chemical Society.

In contrast to cationic NPs, both anionic/hydrophilic and neutral NPs have the capability to be merely adsorbed onto the cell membrane and create a suitable environment conducive to endocytosis.7 This is why negatively charged particles typically induce much lower toxicity than similar positively charged particles under the same conditions (e.g., particle concentration, physicochemical properties (except for the type of functionalized terminal groups), and duration of the exposure to the cells).109,110 For example, Oh et al.111 probed cytotoxicity of cationic, anionic, and neutral functional silica− titania hollow NPs on mouse alveolar macrophages. It was found that cationic NPs exhibited the highest uptake efficiency and the most toxic effects on cells. This was attributed to the fact that cationic NPs likely have a higher uptake by cells and potential to cause significant membrane damage. The endocytosis of toxic materials can open up several intracellular toxicity mechanisms such as ROS and interference with cell signaling molecules/proteins.112,113 For instance, the effect of polystyrene NPs (60 nm) with different surface functionality, cellular uptake, subcellular localization, and ability to catalyze the formation of ROS under biotic and abiotic conditions was investigated in a lung phagocytic cell lines (RAW 264.7).114 It was revealed that cationic polystyrene NPs could induce cellular ROS production, glutathione (GSH) depletion, and toxic oxidative stress (see Figure 6). Furthermore, cationic particles could increase Ca2+ uptake, due to the membrane perturbation,93 leading to the organelle damage, mitochondrial injury, and consequently cell death. In contrast to the cationic NPs, the anionic and neutral polystyrene NPs did not show significant signs of toxicity. TEM images of the NP-treated cells revealed the existence of anionic polystyrene NPs within the cell vesicles, whereas cationic particles could enter the cell nucleus. In addition, the mitochondria maintained their normal morphology in the cells incubated with the anionic NPs. As also briefly indicated, it would be too simplistic to envision that all cells would react similarly to the same NP. NP interactions and toxicity can be cell- and even species-specific. For instance, Joris et al.115 have investigated the toxicity of gold, silver, and iron oxide NPs in cells of different lineages, namely human and murine neuroblastoma cells, neural progenitor cell line, and neural stem cells. The highest and lowest acute cytotoxicity were noted in stem cells and murine neuroblastoma cells, respectively. Moreover, the authors examined

4. FUNCTIONAL NANOPARTICLES INDUCE CELL TOXICITY NPs with various surface functionalities can cause cellular toxicity. However, cationic and hydrophobic NPs can induce significantly higher toxicities compared to the anionic- and hydrophilic-NPs. Clearly, structural alteration, pore formations, and phase transitions of the cell membrane are involved in the passive passage of NPs and thus represent important mechanisms of cytotoxicity. Such membrane perturbations can diminish the membrane’s capability to control nonspecific entrance of ions and foreign extracellular biomacromolecules to the cytosol.8 Cationic- and hydrophobic-NPs can induce various biological stresses such as disruption of mitochondrial function, activation of defensive signaling pathways (e.g., nuclear factor (erythroid-derived 2)-like 2 (NRF-2) pathway), induction of ROS, energy failure, and genotoxicity.82,105 For example, it was shown that positively charged AuNPs had both acute cytotoxicity and genotoxicity through the induction of elevated ROS.106 The observed toxicities were strongly dependent on the type of the attached hydrophobic ligands on their surface. In another study, starch-coated silver NPs could reduce the adenosine triphosphate content on normal human lung fibroblast (IMR-90) and human glioblastoma cells (U251) and induce damage to the mitochondria. In addition, the particles caused DNA damage and cell cycle arrest in G2/M phase (see Figure 5).107 This induced cytotoxicity was strongly dependent on cell lineage and the amount of G2/M phase arrest was increased in the IMR-90-cells than U251 cells (Figure 5a,b). In addition, IMR-90 cells demonstrated concentration-dependent enhancement in DNA damage up to 100 μg/mL of silver NPs, whereas U251 cells exhibited a steady increase up to 400 μg/mL (see Figure 5e for details). It is noteworthy that the different cytotoxic effects created by the same NPs on various cell types may be due to the fact that the cellular responses could be considerably different according to the cell type.108 Thus, the cytotoxic effects of one cell type are not necessarily the same in comparison with other cells. 6584


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receptor on the surface of THP-1 cells and induces the NF-κB signaling pathway leading to the release of inflammatory cytokines. Mahmoudi et al.126 reported that poly(vinyl alcohol)-coated superparamagnetic iron oxide NPs could cause irreversible denaturation of iron saturated human transferrin protein. This irreversible change in transferrin conformation from a compact to an open structure, resulted in the release of iron from transferrin. NP−proteins complexation reactions are largely driven by electrostatic and hydrophobic interactions. For example, in a study by De et al.,127 the surface distribution of charged and hydrophobic residues on the protein surface were shown to control the complexation of amino acid-functionalized AuNPs with proteins through cooperative electrostatic and hydrophobic interactions (Figure 7). Studying the binding

Figure 6. Induction of cellular ROS by NPs of varying surface chemistry. (a) TEM images of polystyrene NPs of varying surface architecture. (b) Histogram demonstrating the fold increase in mean fluorescence intensity (MFI), in dichlorofluorescein diacetate stained RAW 264.7 cells, after incubation with various polystyrene NPs (10 μg/mL). (c) Mitochondrial O2•− production in RAW 264.7 cells (population of bright-positive (M1-gated) cells, obtained by MitoSOX Red staining) after treatment with various polystyrene NPs (10 μg/ mL). (d) Histogram showing the effect of various polystyrene NPs (10 μg/mL) on mitochondrial calcium levels. (e) Immunoblotting showing the effect of various polystyrene NPs (10 μg/mL) on induction of HO-1 expression. (f) TEM images of the cells exposed to anionic and cationic polystyrene NPs (Labels: V (vesicle), M (mitochondria), and P (particles)). * Indicates significant difference (p < 0.01) with control (Φ). Reproduced with permission from reference 114. Copyright 2006 American Chemical Society.

various indicators of toxicity and noted both cell type- and species-specific responses to the iron oxide NPs with regards to generation of ROS, calcium levels, mitochondrial integrity, and cell morphology, indicating that the toxicity were induced through distinct mechanisms. Although, the dependence of NP toxicity on cell lineage is a well-documented phenomenon.116−121 predicting toxicological properties of NPs based on their chemical characteristics is challenging task.122 The later has been shown in a study by Gornati et al. in which the cytotoxic effects of three transition metal NPs (Fe, Co, and Ni) with similar chemical properties have been studied on two cell lines (SKOV-3 and U87). Though in both cell lines, Co- and Ni-NPs were more toxic, the two cell lines exhibited distinct differential gene expression patterns, predominantly in genes responsible for cellular stress or other cellular processes.122In light of these studies, care should be taken in the interpretation NP toxicity and extrapolation to differing cell lines, especially when specific surface modifications are involved. Functionalized NPs can cause the denaturation of key cell signaling molecules/proteins, leading to disruption in cellular homeostasis. It must be noted, however, that most of the studies performed on NP−protein interactions have been done in cell-free conditions and such research findings must be interpreted with care. As an example for NP-mediated denaturation of biomolecules, both cationic (with aminoethanethiol coating) and anionic (with bis(p-sulfonatopehnyl)phenylphosphine coating) AuNPs could denature cytochrome c; the absence of functional cytochrome c reduces cell energy generation and defense against detoxifying ROS and increases cytotoxicity of charged AuNPs.123,124 The neutral AuNPs, in contrast, have no effect on the conformation of cytochrome c. In addition to the intracellular proteins, interaction of surface functionalized NPs with extracellular proteins can change their conformation, disrupting protein function. Minchin et al.125 demonstrated that fibrinogen protein could be denatured after interactions with negatively charged poly(acrylic acid) coated AuNPs. The denatured fibrinogen activates MAC-1 integrin

Figure 7. Comparison of the binding modes of cytochrome c, chymotrypsin and histone with amine-functionalized AuNPs. The blue sections on the proteins represent the positively charged residues on their surface. Reproduced with permission from reference 127. Copyright 2007 American Chemical Society.

thermodynamics of these NPs with α-chymotrypsin, histone, and cytochrome c using isothermal titration calorimetry (ITC) studies revealed that the complexation of NPs with αchymotrypsin is driven by enthalpy changes, while the complexation of identical NPs with histone and cytochrome c is entropy-controlled. The complexation stability of αchymotrypsin as a model protein could be manipulated by varying ionic strength, which subsequently perturbed these electrostatic and hydrophobic interactions. Another recent study showed that the interaction of gold nanorods with model proteins such as lysozyme, cytochrome c, and bovine serum albumin can be tuned by functionalization with cationic or anionic end-functionalized PEG-thiols.128 It was demonstrated that gold nanorod−protein interactions could be almost completely reversed by addition of salt. This indicates the importance of electrostatic effects in governing these interactions. The NP protein interactions can also be 6585


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electron transfer between coenzyme Q-cytochrome c reductase and cytochrome c oxidase, which eventually perturbs the cellular process for energy production and detoxification of ROS.123

modulated by spatial orientation and relative electronic properties of functional groups on the NP surface. In a representative study,129 a new class of cationic AuNPs were fabricated that harbored benzyl moieties featuring −NO2 and −OMe groups. The binding affinities of NPs bearing electronwithdrawing groups toward green fluorescent protein (GFP) was much higher than those bearing electron-donating groups. Although the binding affinity to GFP was not altered by substituting a −NO2 group at ortho position, such an addition in meta and para positions significantly enhanced the binding affinity by ∼7.5- and ∼4.3-fold, respectively compared to the unsubstituted ring. As indicated above, NP−protein interactions have the potential to induce reversible or irreversible conformational changes of proteins that may potentially significantly alter the activities of the proteins in question.126 The properties of NP largely influence the extent to which the protein is denatured.130 Furthermore, there are some concerns that such interactions can lead to the presentation of epitopes that do not naturally occur.131 These interactions can also bring about complications such as fibrillation and aggregation of the proteins leading to amyloid formation, and loss of critical enzymatic activities. Because of the complexity of NP structure and surface properties as well as the diversity of proteins and isoforms, it is not possible to fully predict the numerous potential interactions of a given NP with proteins in most organisms. Nevertheless, studying the influence of different surface parameters on NP interaction with protein can help engineer NPs with desired functions and reduce, or if possible eliminate unfavorable interactions. In an early study by Fischer et al.,132 the interaction of anionic mercaptoundecanoic acidfunctionalized AuNPs (2 nm core) with chymotrypsin was shown to inhibit its protease activity in a two-step process. During the first step, the electrostatic interactions between the NP and protein caused a rapid but reversible inhibition of chymotrypsin activity, whereas chymotrypsin underwent an irreversible denaturation process afterward.133 The adsorbed chymotrypsin can be dissociated from the anionic NPs by cationic surfactants. Later, You et al.134 demonstrated that functionalization of AuNPs with hydrophilic amino acids destabilized α-chymotrypsin by competitive hydrogen binding and disruption of salt bridges inside the protein. The denaturation rate was significantly slower when AuNPs were functionalized hydrophobic amino acid side chains. The exterior and interior hydrophobic moieties of the NP can have contrasting effects on the complexation of amino acid functionalized gold clusters with α-chymotrypsin.135 In this study, You et al. fabricated several L-amino acid functionalized AuNPs with oligo(ethylene glycol) (OEG) tethers of varying lengths. Though the hydrophobic side chains of amino acids positively contribute to the retention of α-chymotrypsin’s structure, the interior alkyl chains of the linker promoted its denaturation. α-chymotrypsin exhibited a 80-fold range of denaturation rate constants while interacting with different NPs. The authors concluded that a rational choice of the amino acid side chains and OEG tethers helps to tune the denaturation of proteins. These types of studies can be fundamental for developing NP applications in protein stabilization, alteration, and delivery. Similarly, Hamad-Schifferli and Aubin-Tam124 showed that AuNPs with PEG ligands denatured Saccharomyces cerevisiae cytochrome c protein through attachment to the specific cysteine 102 residue. Cytochrome c denaturation leads to the malfunction of

5. INTERACTIONS OF FUNCTIONAL NANOPARTICLES WITH IMMUNE SYSTEM AND BLOOD COMPONENTS NPs can directly or indirectly interact with the immune system, leading to activation of complement system,136 release of inflammatory cytokines,125 cellular mobilization (e.g., macrophage migration),137 and differentiation and activation of immune cells (e.g., exosome-mediated maturation of dendritic cells and activation of splenic T cells).138 Such interactions can change the trafficking, distribution, uptake, and clearance of NPs, which ultimately affect NP cytotoxicity. Furthermore, circulating and resident immune cells such as monocytes, neutrophils and dendritic cells, and tissue phagocytes are programmed to engulf and eliminate NPs.139 Some NP−immune system interactions are desirable, for example when it comes to delivery of vaccines or therapeutics to immune cells for inflammatory and autoimmune disorders.140 Several physiochemical properties such as size, hydrophobicity, surface charge, solubility, and surface functional groups are known to affect NP uptake by macrophages. There is general consensus that larger particles are taken up to a larger extent than smaller particles with identical composition and surface properties.141 NPs have been shown to accumulate in distinct cell populations based on size.142 Charged NPs exhibit higher cell uptake than neutral NPs with the same size. 143 As an example, Demento et al. 144 fabricated inflammasome-activating NPs for boosting vaccine efficacy. The authors developed lipopolysaccharides-modified PLGA NPs loaded with ovalbumin antigen. LPS-modified NPs were preferentially internalized by dendritic cells and upon administration to mice, stimulating humoral and cellular immunity. Moreover, pulsing wild-type macrophages with LPS-modified NPs led to the production of proinflammatory cytokine IL-1β. The immunization of mice with a similar formulation incorporating a recombinant West Nile envelope protein, protected them against a murine model of West Nile encephalitis. Except for such specific circumstances, NP uptake by the immune system is not desired typically, because it can lead to rapid clearance of the particles. Several strategies have been adapted to produce “camouflage”69 NPs that can prevent recognition by the immune system and thus avoid phagocytosis. One classic example of NP functionalization affecting immune interaction is PEG, which is used to shield NPs76,145,146 against the mononuclear phagocyte system and to delay clearance, a feature that is utilized in well-established therapeutics such as Doxil.147 However, in certain cases, production of PEG-specific antibodies after administration of PEG-coated liposomes have been noted, which might lead to accelerated clearance of NPs from blood.148,149 That said, the role of PEG is complex and not completely understood. PEGylation can exert changes to NPs with profound consequences. For example, PEGylation affected NP mobility and diffusion in various models of the extracellular matrix.150 Furthermore, it is excruciatingly difficult to avoid unwanted changes in physiochemical properties of NPs upon PEGylation. Different PEG parameters such as molecular weight, content, density, and conformation can affect NP interactions with 6586


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8). Activation of the complement system was primarily induced by the alternative pathway rather than the lectin pathway. The

immune system, protein adsorption, stability, circulation time, cellular uptake, toxicity, and overall behavior.151,152 In turn, the NP parameters such as curvature can also affect the outcome of PEGylation.153 As such, depending on NP core size (curvature) and PEG concentration, grafting densities and hydrodynamic volumes on the NPs were shown to be affected.152 Moreover, as PEG provides a pan-cellular evasion, it might also influence the targeting efficiency of NPs.154 As another strategy to hide from the immune system, a selfprotein was attached to the surface of NPs so that NPs can camouflage themselves as endogenous, thus preventing phagocytosis.155 Masking NPs through biomimetic coating with cell membranes isolated from leukocytes156 and red blood cells157,158 has also been shown to increase NPs’ blood circulation time. In this regard, wrapping of NPs with biomimetic cancer cell membrane was shown to aid the targeting of gold nanocages to the target cells.159 A similar strategy was used for camouflaging upconversion NPs using macrophage membranes, a strategy that was further used for targeting through the natural tendency of adhesion between macrophages and cancer cells.160 Interestingly, protein corona formation has also been modulated to act a “Trojan horse” enabling targeted delivery. This has been achieved through attracting a specific class of proteins toward the NP surface. Kreuter et al.161 have shown that poly(butyl cyanoacrylate) NPs coated with polysorbate 80 are able to cross the bloodbrain barrier by mimicking lipoprotein NP characteristics through adsorption of apolipoproteins B and E. Another advantage of engineered protein coronas is the ability to optimize the subsequent cytotoxicity of NPs.162 Surface charge also has a prominent effect on NP−immune system interactions. For example, among anionic, cationic and neutral silica−titania NPs, cationic particles were shown to be most immunotoxic in mouse macrophages.163 These NPs induced cytokine production (e.g., IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α) by T helper 1 cells in mice and activated the toll-like receptor-4 to significantly higher extent than identical anionic or neutral NPs.164 The IV administration of positively charged NPs and not the neutral or negatively charged NPs was associated with induction of IFN type I response and enhanced the expression of interferon responsive genes. In another study bimodal cationic AuNPs (10−20 and 32−54 nM) were shown to activate NF-κB in THP-1 monocytes.165 Subsequent thermal proteome profiling experiments166 in living cells identified nine proteins changing their stability in response to treatment with NPs. One such protein was tumor necrosis factor (TNF)-α-induced protein 8-like protein 2 (TIPE2), which is involved in maintaining immune homeostasis by negatively regulating NF-kB functions.167 Oslakovic et al.168 have investigated whether the interaction of polystyrene NPs with blood proteins involved in coagulation can alter the naturally existing balance between pro- and anticoagulant pathways. To that end, the effect of polystyrene NPs on the generation of thrombin in plasma was studied. Though amine-modified NPs decreased the thrombin formation through binding to and depletion of factors VII and IX, carboxyl-modified NPs provided a surface for activation of the intrinsic blood coagulation pathway in plasma. SalvadorMorales et al.169 studied the effect of surface functionality of lipid−polymer hybrid NPs on complement activation. Among hybrid NPs modified with methoxyl, caboxyl, and amine surface groups, those with methoxyl and amine groups induced the lowest and highest complement activation, respectively (Figure

Figure 8. Activation of human serum complement system by lipid− polymer hybrid NPs modified with carboxyl, methoxyl, and amine groups. (a) Complement system activation at the terminal cascade, represented by the concentration of SC5b-9, a sensitive biomarker of C5a formation via both the alternative pathway and classical pathways. (b) Activation of the alternative pathway biomarker, Bb, which is the proteolytically active fragment of factor B. (c) Activation of the complement system via the classical pathway, as shown by classical pathway biomarker C4d, which measures the amount of C4dcontaining activation fragments of C4. Reproduced with permission from reference 169. Copyright 2009 Elsevier.

results of complement and coagulation activation experiments highlighted NPs with methoxyl surface groups as the best candidates for drug delivery applications, due to the lack of adverse immune reactions. In this regard, the anticoagulant activity of NPs can be used for therapeutic purposes. The effect of NP surface charge on platelet aggregation has therefore been studied. Fuentes et al.170 have investigated the antiplatelet activity of PEGylated lipid6587


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cationic NPs. Surface charge affects tissue-level distribution as well. Using a three-dimensional model of tumor tissue, it has been shown that AuNPs can move and localize differently according to their surface charge. Though cationic thioalkyl tetra(ethylene glycol)lyated trimethylammonium monolayercoated AuNPs were efficient in delivering their doxorubicin content to the majority of proliferating cells, anionic thioalkyl tetra(ethylene glycol)lyated carboxylic acid AuNPs proved to perform better in delivery of drugs deep into the tumor model as result of having quicker diffusion rates (Figure 9).176

polymer NPs with different surface charges by light transmission aggregometry and flow cytometry. PEGylated NPs were shown to inhibit platelet aggregation in a dose-dependent manner in both ADP- and collagen-induced platelet aggregation experiments. Furthermore, the NPs led to downregulation of P-selectin. Charged NPs (especially cationic ones) inhibited platelet aggregation more potently than their neutral counterparts. Besides charge, another important parameter affecting the immune interactions of NPs is the surface hydrophobicity. Moyano et al.171 studied the interaction of monolayerprotected 2 nm core diameter AuNPs with different hydrophobicities and the immunological response induced in splenocytes. The NPs were conjugated with a passivating, noninteracting tetra(ethylene glycol) spacer and further functionalized with hydrophobic groups. Hydrophobicity was directly and quantitatively associated with the induction of immunological responses, with alterations in the gene expression of cytokines such as IFN-R and TNF-γ as well as IL-2, -6, and -10 in splenocytes isolated from mice. NPs with higher hydrophobicity elicited the expression of pro-inflammatory TNF-R and anti-inflammatory IL-10. Hamad et al.136 have studied the interactions of NPs with surface projected PEG chains in mushroom−brush and brush configurations and their effect on the initiation of complement cascade, an important immunological process belonging to innate immunity. The conformational states of PEO chains, which project from block copolymer poloxamine 908 adsorbed on polystyrene NPs were shown to differentially trigger complement activation. Changing the PEO configuration from mushroom to brush switches the complement activation from the C1q-dependent classical to lectin pathway and reduces the level of complement activation products C4d, Bb, C5a, and SC5b-9. Surface charge and hydrophobicity are postulated to be the most important factors regulating NP interactions with immune cells. With expansion in our knowledge of the specific surface elements contributing to immune reactions, NPs with higher biocompatibility and lower immunotoxicity can be engineered with greater ease over time. We also envision growing interest in the use of NPs for immunotherapy of cancer and autoimmunity.172,173

Figure 9. Delivery of doxorubicin therapeutics into tumor cylindroids by AuNPs. Green cells are those taking up the NPs. Viable cells are shown with smooth uninterrupted boundaries, whereas the membrane of necrotic cells have been shown with dashed line. The diffusion and cellular uptake trajectories have been shown by dashed arrows. Reproduced with permission from reference 176. Copyright 2010 Springer.

Sukru et al. have also recently reported on the effect of surface charge on suborgan distribution of AuNPs.177 Laser ablation inductively coupled plasma mass spectrometry (LAICP-MS) was used to determine the in vivo localization of intravenously injected functionalized AuNPs in mice. Though positively charged AuNPs accumulated extensively in the kidney glomeruli, red pulp of the spleen and liver hepatocytes, negatively charged AuNPs were largely found in the red pulp of the spleen and showed a broad distribution in the liver. On the other hand, uncharged NPs accumulated in the white pulp, marginal zone of the spleen and around Kupffer cell in the liver, to a higher level than the positively or negatively charged NPs. Because positively charged NPs accumulated more in kidney glomeruli, they might have a different renal filtration rate than the other NPs tested in this study. Surface functionality also has a critical role in the fate of NPs. Zhu et al.178 have studied the clearance kinetics of positively charged 2 nm core AuNPs with both hydrophilic and hydrophobic surfaces in Japanese medaka fish. Although a hydrophilic surface facilitates NP clearance, a hydrophobic surface distributes into fish organs and kills the fish in less than 1 day. This is while the hydrophilic counterparts are mainly found in the fish intestines with no deleterious health effects. Not only hydrophobicity but type of functionality would also dictate NP distribution. It has been shown that majority of targeting ligands used for active targeting have great distribution in the liver and spleen.179 Because the formation of protein corona is governed by NP surface parameters, any changes in the surface can be associated

6. DISTRIBUTION, DIFFUSION, ACCUMULATION, AND ORGAN TOXICITY OF FUNCTIONAL NPS NP accumulation in organs and tissues is another consideration in nanotoxicology, because NP persistence in vivo can raise concerns regarding chronic toxicity. Different factors can affect biodistribution of NPs such as composition, size, core properties, surface modification (such as PEGylation and surface charge), and also targeting ligand functionalization.174 The effect of NP surface charge on accumulation in organs has been studied.175 Though neutral (TEGOH) and zwitterionic AuNPs exhibited relatively good circulation times (in order of hours) upon IV or intraperitoneal (IP) administration, cationic (TTMA), and anionic (TCOOH) NPs demonstrated relatively short half-lives. Moreover, the clearance of cationic NPs was 4.3 times faster than their anionic counterparts after IV administration. Upon IV injection, all 4 NPs accumulated in the liver and spleen cells, whereas NPs injected through IP route accumulated in pancreas. The accumulation of NPs was different in solid tumors in murine model. Neutral and zwitterionic NPs had a higher accumulation in tumors than 6588


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of different physicochemical parameters such as size, hydrophobicity, ζ-potential, and elasticity, many of which depend on the type of PEGylated AuNPs. As such, it was observed that the thicker the PEG coating (higher molecular weight), the lower the observed NP uptake is. Note that PEGylation here did not significantly influence the NP elasticity.194 Regarding biodistribution, as expected, soft NPs tend to have longer blood circulation time.186,195 For example, softer 120 nm poly(carboxybetaine) nanogels showed longer circulation and lower accumulation in the spleen, as they more readily passed through splenic sinusoids than harder ones.195 Elasticity can also affect the targeting of NPs. For instance, harder 100 nm PLGA−lipid (core−shell) spheres had higher tumor targeting efficiency and induced more tumor shrinkage in comparison with their softer counterparts.196

with a change in corona, which can also in turn alter NP biodistribution.180 Along with surface composition, blood circulation half-life and accumulation of NPs in organs can also depend on particle size and molecular weight (MW) of their functional groups. For instance, liver clearance of PEGylated NPs is known to be enhanced with PEG functionality that have a MW > 50 000 g/ mol (PEG 50,000). Consequently, the circulation half-life of PEG 6000 was less than 30 min whereas that of PEG 190,000 was extended to 1 day. A similar trend was observed for Poly(vinyl alcohol) (PVA) of different sizes, 90 min for PVA 14,800 and 23h for PVA 434,000.181 Particle size have also shown to be effective in in vivo biodistribution of NPs. A study have shown that polystyrene NPs with size of 500 nm have higher liver accumulation than the 50 nm ones.174,182 In a very recent study, AuNP were functionalized with pHresponsive alkoxyphenyl acylsulfonamide ligands.183 Because of the unique ligand structure, this NP is neutral at pH 7.4, but positively charged at tumor microenvironment (pH < 6.5), which in turn enhances cellular uptake and cytotoxicity of NPs. Particle uptake and cytotoxicity were found to increase over this pH range with no observable hemolytic activity. 6.1. Impact of NP Elasticity on Cell Internalization and Biodistribution. NPs can have different mechanical properties (for example, varying degrees of hardness) that largely tune their behavior such as cell uptake and biodistribution. For instance, liposomes are generally harder than polymeric NPs, but are softer than solid metal or carbon-based NPs.184 NP hard- or softness will affect both cellular internalization and routes of excretion for a given NP.184 The NP hard- or softness will most likely affect the internalization or distribution of a given NP.185 Hard NPs are normally internalized to a higher extent than soft NPs in immune cells.186 Several studies have shown that soft NPs have difficulty undergoing phagocytosis due to particle deformation.187−189 Therefore, surface hardness can be partially involved in NP−immune system interactions. Theoretical studies have related the low efficiency of internalization of soft NPs to their incapability of inducing full wrapping by the cell membrane.190,191 In a recent study by Li et al.,184 dissipative particle dynamics simulation showed that although the entry of solid NPs can happen through straightforward endocytosis, for soft NPs, such entrance is hampered by two effects: wrapping-induced shape deformations and nonuniform ligand distribution. As such, soft NPs have to rearrange their hydrophobic and hydrophilic segments to find a route of entry. Interestingly, in a theoretical study, the relationship between uptake efficiency and elasticity have been examined. The authors observed that soft NPs are less energetically favorable for membrane wrapping than their hard counterparts. However, when energetically favorable, observed wrapping is kinetically faster.192 Though many studies have shown that hard NPs are generally internalized to a higher degree in immune cells, some other studies have found that NPs with intermediate or optimal elasticity are more efficiently internalized. For example, 170 nM hydrogel NPs with an intermediate elasticity (Young’s moduli of 35 and 136 kPa) showed higher cellular internalization than NPs with higher or lower elasticity.193 In this study, soft NPs (18 kPa) were internalized by micropinocytosis, hard NPs (211 kPa) were taken up by a clathrin-dependent mechanism, whereas the intermediate NPs were internalized via both mechanisms and thus had a higher overall uptake. In another study, Pino et al. have compared a library of PEGylated AuNPs

7. CONCLUSION Surface functionalization of NPs is a major factor that dictates their cellular transport dynamics. However, deeper understanding of the interplay between surface functionality and biological interactions is necessary to accurately determine the effects of NPs and their byproducts (e.g., ions) on cells and their organelles. This will greatly assist with the effort to engineer safe and effective nanoproducts for a variety of applications. Here, we briefly elaborated the effects of specific NP surface characteristics such as charge and hydrophobicity on cell membranes and their corresponding cellular toxicities through different mechanisms (e.g., membrane perturbation, oxidative stress signaling, signaling protein/molecule damage, and genotoxicity). Charge seems to be one of the most important NP surface factors enhancing toxicity but also perhaps therapeutic efficacy (due to higher internalization). As such, a precise engineering of different surface factors will be required for fabricating effective nanoparticulate delivery systems. The complexity and the multiparametric nature of NP interactions necessitate the performance of precise experiments, in which different factors are well-controlled. Such experiments are possible in settings where all NP parameters and experimental conditions are kept constant, whereas a single surface feature is altered to dissect the biological effects resulting from subtle changes in NP physiochemisty. Furthermore, the standardization of NP testing and characterization procedures as well as detailed analysis and transparency of NP physiochemical properties can reduce the discrepancy among research findings in different laboratories, which is one of the biggest challenges of nanotoxicology today. Ultimately, the knowledge of the interactions between functional NPs and personalized biological systems (e.g., diseases-specific protein corona)197 will play a critical role in development of new theranostic nanosystems in the age of personalized medicine.

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AUTHOR INFORMATION

Corresponding Author

*M. Mahmoudi. Email: [email protected]. ORCID

Morteza Mahmoudi: 0000-0002-2575-9684 Notes

The authors declare no competing financial interest. 6589


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ABBREVIATIONS AFM, atomic force microscopy; AuNP, gold nanoparticle; DCS, differential centrifugal sedimentation; DEAE-DEX, di3ethylaminoethyl-dextran; DLS, dynamic light scattering; EDS, energy-dispersive X-ray spectroscopy; FCS, fluorescence correlation spectroscopy; FTIR, Fourier transform infrared spectroscopy; LC-MS, liquid chromatography−mass spectrometry; NMR, nuclear magnetic resonance; NP, nanoparticle; PEG, polyethylene glycol; PEI, polyethylenimine; QD, quantum dot; ROS, reactive oxygen species; SLB, supported lipid bilayers; TEM, transmission electron microscopy; TGA, thermogravimetric analysis; THCPSi, thermally hydrocarbonized porous silicon; XPS, X-ray photoelectron spectroscopy

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