Nanoparticle Surface Functionality Dictates Cellular and Systemic

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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 Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.7b01979 • Publication Date (Web): 20 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017

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Chemistry of Materials

Nanoparticle Surface Functionality Dictates Cellular and Systemic Toxicity 1

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Amir Ata Saei , Mahdieh Yazdani , Samuel E. Lohse , Zahra Bakhtiary , Vahid Serpooshan , Mahdi Ghavami , Mahtab Asadian , 8 9 10,11,12,13,14 15,16 , and Morteza Mahmoudi * Samaneh Mashaghi , Erik C. Dreaden , Alireza Mashaghi 1

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

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Department of Chemistry, University of Massachusetts Amherst, Amherst, MA 01003, USA

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Physical and Environment Sciences Program, Colorado Mesa University, CO, USA

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Research center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

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Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA

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Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark

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Faculty of Engineering & Architecture, Department of Applied Physics, Research Unit Plasma Technology (RUPT), Ghent University, Ghent, Belgium

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School of Engineering and Applied Sciences and Department of Physics, Harvard University, 29 Oxford Street, Cambridge, Massachusetts 02138, USA 9

Koch Institute for Integrative Cancer Research, Department of Chemical Engineering, Massachusetts Institute of Technology, MA, USA

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Leiden Academic Centre for Drug Research, Faculty of Mathematics and Natural Sciences, Leiden University, Leiden, The Netherlands

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Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, MA, USA

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Dana-Farber Cancer Institute, Boston, MA, USA

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Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

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Basir Eye Health Research Center, Tehran, Iran

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Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

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Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

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.

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 parameters;6 however, systemic applications of NPs in the clinical setting are currently limited to a subset of NP compositions and shapes. Thus, surface characteristics stands out as one of the most important, if not the main, determinants of

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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, 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 stressmediated 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

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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 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.

2. Surface Functionalization of Nanoparticles

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 which 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 sub-monolayer 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

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

While 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

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 non-specific 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 is 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.


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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, colloidoriented analytical techniques provide very little information about the deep details of NP surface chemistry. Organic chemistry analytical techniques (such as 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 X-rays.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 NP 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 Since 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 which is most typically exploited to control the particle’s bio-interactions. 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 bio-nano 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 reduced for these NPs. These types of functionalization strategies are vital in minimizing non-specific 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, since 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 bio-nano 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



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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

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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 cells,23 but also on controlling their subcellular trafficking into specific organelles.16, 81

Due to the importance of NP surface chemistry in governing nano-bio 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 the fact that surface chemistry is often not the sole determinant of a NP’s bio-interactions. 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 nano-bio 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 glycocalyx79, 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

Figure 1. Varying cellular uptake mechanisms for NPs. From left to right: phagocytosis, macropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and non-clathrin- and non-caveolinmediated endocytosis Reproduced with permission from Reference.82


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

Figure 2. The 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 one, respectively. Reproduced with permission from Reference.87 While 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 receptormediated 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 five times higher than that of anionic counterparts. This was attributed to the fact that half of cationic AuNPs diffuse into cells by pore formation, while 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 positivelycharged regions on the surface that provide an opportunity for the uptake of negatively-charged NPs. 89 It was also found that membrane adsorption was ratelimiting for NP internalization, since 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. Increasing surface charge from a cationic coverage (percentage of ammoniumfunctionalized 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 Malmstadt 91 demonstrated that relatively small cationic polystyrene (20 nm; with 80.2 mEq. 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 mEq. 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 G3NH2 dendrimers); ii) NPs that mainly disrupt the bilayer by adhering to pre-existing defects and expanding them (e.g. amine-coated AuNPs (Au-NH2), the cell penetrating peptide MSI-78 and PAMAM G5-NH2 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, polyethyleneimine (PEI), di3ethylaminoethyl-dextran (DEAE-DEX) and aminecoated silica NPs (silica-NH2)).



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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 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 non-malignant, 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). Greatest depolarization with positively-charged AuNPs was observed in ovarian cancer cells (CP70, A2780), roughly comparable to that achieved with 40 mM KCl.

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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. While 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 pre-treating 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. 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 AlexanderKatz.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


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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.

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 In addition to coincident effects, the surface charge of NPs can be leveraged for specific biomedical applications. For example, carboxymethyl chitosan/chitosannanoparticles have demonstrated surface chargedependent 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 selfassembly 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. Due to 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 Zhu 100 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 phase-separated 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 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



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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, semi-hydrophilic NPs energetically prefered 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. 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 non-specific 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 (Figures 5a and 5b). In addition, IMR-90 cells demonstrated concentrationdependent enhancement in DNA damage up to 100µg/mL of silver NPs, whereas U251 cells exhibited a steady increase up to 400µg/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.

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Figure 5. Graphs showing the induction of G2/M arrest in (a) U251- and (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 50,000 gr/mole (PEG 50,000). Consequently, the circulation half-life of PEG 6000 was less than 30 min while that of PEG 190,000 was extended to a day. A similar trend was observed for Poly(vinyl alcohol) (PVA) of different sizes, 90 min for PVA 14800 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

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, while 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 In a very recent study, AuNP were functionalized with pH-responsive alkoxyphenyl acylsulfonamide ligands.183 Due to 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 was found to increase over this pH rangewith no observable hemolytic activity. 6.1. The Impact of NP elasticity internalization and biodistribution

on

cell

NPs can have different mechanical properties (for example varying degrees of hardness) which 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



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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 while 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 non-uniform 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, while 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 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 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

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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 wellcontrolled. Such experiments are possible in settings where all NP parameters and experimental conditions are kept constant, while 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 labs, 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., diseasesspecific protein corona) 197 will play a critical role in development of new theranostic nanosystems in the age of personalized medicine.

AUTHOR INFORMATION Corresponding Author *EMAIL: [email protected]

Author Contributions The manuscript was written through contributions of all authors.

ABBREVIATIONS AFM, atomic force microscopy; AuNP, Gold nanoparticle, DCS, differential centrifugal sedimentation; DEAE-DEX, di3ethylaminoethyl-dextran; DLS, dynamic light scattering; DNA, deoxyribonucleic acid; 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, polyethyleneimine; 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.

REFERENCES 1. Bansal, A.; Zhang, Y., Photocontrolled Nanoparticle Delivery Systems for Biomedical Applications. Acc. Chem. Res. 2014, 47, 3052-3060.


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2. Dreaden, E. C.; Alkilany, A. M.; Huang, X.; Murphy, C. J.; El-Sayed, M. A., The golden age: gold nanoparticles for biomedicine. Chem. Soc. Rev. 2012, 41, 2740-2779. 3. Pankhurst, Q. A.; Connolly, J.; Jones, S.; Dobson, J., Applications of magnetic nanoparticles in biomedicine. J. Phys. D: Appl. Phys. 2003, 36, R167–R181. 4. Lim, W. Q.; Gao, Z., Plasmonic nanoparticles in biomedicine. Nano Today 2016, 11, 168-188. 5. Le, N. D. B.; Yazdani, M.; Rotello, V. M., Array-based sensing using nanoparticles: an alternative approach for cancer diagnostics. Nanomedicine 2014, 9, 1487-1498. 6. Henriksen-Lacey, M.; Carregal-Romero, S.; LizMarzán, L. M., Current Challenges toward In Vitro Cellular Validation of Inorganic Nanoparticles. Bioconjugate Chem. 2017, 28, 212-221. 7. Mukherjee, S.; Ghosh, R. N.; Maxfield, F. R., Endocytosis. Physiol. Rev. 1997, 77, 759-803. 8. Verma, A.; Stellacci, F., Effect of surface properties on nanoparticle–cell interactions. Small 2010, 6, 12-21. 9. Stark, W. J., Nanoparticles in biological systems. Angewandte Chemie, International Edition 2011, 50, (6), 12421258. 10. Krug, H. F.; Wick, P., Nanotoxicology: an interdisciplinary challenge. Angew. Chem. Int. Ed. 2011, 50, 12601278. 11. Nel, A.; Xia, T.; Mädler, L.; Li, N., Toxic potential of materials at the nanolevel. Science 2006, 311, 622-627. 12. Xia, X.-R.; Monteiro-Riviere, N. A.; Riviere, J. E., An index for characterization of nanomaterials in biological systems. Nat. Nanotechnol. 2010, 5, 671-675. 13. Pelaz, B.; Charron, G.; Pfeiffer, C.; Zhao, Y.; de la Fuente, J. M.; Liang, X. J.; Parak, W. J.; del Pino, P., Interfacing engineered nanoparticles with biological systems: anticipating adverse nano–bio interactions. Small 2013, 9, 1573-1584. 14. Roiter, Y.; Ornatska, M.; Rammohan, A. R.; Balakrishnan, J.; Heine, D. R.; Minko, S., Interaction of nanoparticles with lipid membrane. Nano Lett. 2008, 8, 941-944. 15. Hu, M.; Chen, J.; Li, Z.-Y.; Au, L.; Hartland, G. V.; Li, X.; Marquez, M.; Xia, Y., Gold nanostructures: engineering their plasmonic properties for biomedical applications. Chem. Soc. Rev. 2006, 35, 1084-1094. 16. Chakraborty, A.; Jana, N. R., Design and Synthesis of Triphenylphosphonium Functionalized Nanoparticle Probe for Mitochondria Targeting and Imaging. J. Phys. Chem. C 2015, 119, 2888-2895. 17. Teodoro, J. S.; Simões, A. M.; Duarte, F. V.; Rolo, A. P.; Murdoch, R. C.; Hussain, S. M.; Palmeira, C. M., Assessment of the toxicity of silver nanoparticles in vitro: a mitochondrial perspective. Toxicol. in Vitro 2011, 25, 664-670. 18. Tsai, D.-H.; DelRio, F. W.; Keene, A. M.; Tyner, K. M.; MacCuspie, R. I.; Cho, T. J.; Zachariah, M. R.; Hackley, V. A., Adsorption and conformation of serum albumin protein on gold nanoparticles investigated using dimensional measurements and in situ spectroscopic methods. Langmuir 2011, 27, 2464-2477. 19. Celardo, I.; Pedersen, J. Z.; Traversa, E.; Ghibelli, L., Pharmacological potential of cerium oxide nanoparticles. Nanoscale 2011, 3, 1411-1420. 20. Singh, N.; Manshian, B.; Jenkins, G. J.; Griffiths, S. M.; Williams, P. M.; Maffeis, T. G.; Wright, C. J.; Doak, S. H., NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials. Biomaterials 2009, 30, 3891-3914. 21. Nel, A. E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E. M.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M., Understanding biophysicochemical interactions at the nano–bio interface. Nat. Mater. 2009, 8, 543-557. 22. Omlor, A. J.; Le, D. D.; Schlicker, J.; Hannig, M.; Ewen, R.; Heck, S.; Herr, C.; Kraegeloh, A.; Hein, C.; Kautenburger, R.;

Kickelbick, G.; Bals, R.; Nguyen, J.; Dinh, Q. T., Local Effects on Airway Inflammation and Systemic Uptake of 5 nm PEGylated and Citrated Gold Nanoparticles in Asthmatic Mice. Small 2017, 13, in press. 23. Kim, S. T.; Saha, K.; Kim, C.; Rotello, V. M., The Role of Surface Functionality in Determining Nanoparticle Cytotoxicity. Acc. Chem. Res. 2013, 46, 681-691. 24. Deng, Z. J.; Liang, M.; Monteiro, M.; Toth, I.; Minchin, R. F., Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation. Nat. Nanotechnol. 2011, 6, 39-44. 25. Yeh, Y.-C.; Saha, K.; Yan, B.; Miranda, O. R.; Yu, X.; Rotello, V. M., The role of ligand coordination on the cytotoxicity of cationic quantum dots in HeLa cells. Nanoscale 2013, 5, 12140-12143. 26. Albanese, A.; Tang, P. S.; Chan, W. C., The effect of nanoparticle size, shape, and surface chemistry on biological systems. Ann. Rev. Biomed Eng. 2012, 14, 1-16. 27. Barkalina, N.; Jones, C.; Townley, H.; Coward, K., Functionalization of mesoporous silica nanoparticles with a cellpenetrating peptide to target mammalian sperm in vitro. Nanomedicine 2015, 10, 1539-1553. 28. Murphy, C. J.; Buriak, J. M., Best Practices for the Reporting of Colloidal Inorganic Nanomaterials. Chem. Mater. 2015, 27, 4911-4913. 29. Daniel, M.-C.; Astruc, D., Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem. Rev. 2004, 104, 293-346. 30. Boisselier, E.; Astruc, D., Gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity. Chem. Soc. Rev. 2009, 38, 1759-1782. 31. Dahl, J. A.; Maddux, B. L.; Hutchison, J. E., Toward greener nanosynthesis. Chem. Rev. 2007, 107, 2228-2269. 32. Hong, G.; Diao, S.; Antaris, A. L.; Dai, H., Carbon Nanomaterials for Biological Imaging and Nanomedicinal Therapy. Chem. Rev. 2015, 115, 10816–10906. 33. Liang, Y.; Ozawa, M.; Krueger, A., A general procedure to functionalize agglomerating nanoparticles demonstrated on nanodiamond. ACS Nano 2009, 3, 2288-2296. 34. Gambinossi, F.; Mylon, S. E.; Ferri, J. K., Aggregation kinetics and colloidal stability of functionalized nanoparticles. Adv. Colloid Interface Sci. 2015, 222, 332-349. 35. Alkilany, A. M.; Lohse, S. E.; Murphy, C. J., The gold standard: gold nanoparticle libraries to understand the nano–bio interface. Acc. Chem. Res. 2012, 46, 650-661. 36. Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M., Gold nanoparticles in chemical and biological sensing. Chem. Rev. 2012, 112, 2739-2779. 37. Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A., Gold nanoparticles for biology and medicine. Angew. Chem. Int. Ed. 2010, 49, 3280-3294. 38. Palui, G.; Aldeek, F.; Wang, W.; Mattoussi, H., Strategies for interfacing inorganic nanocrystals with biological systems based on polymer-coating. Chem. Soc. Rev. 2015, 44, 193227. 39. Slowing, I. I.; Vivero-Escoto, J. L.; Wu, C.-W.; Lin, V. S.-Y., Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv. Drug Deliv. Rev. 2008, 60, 1278-1288. 40. Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H., Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 2005, 4, 435-446. 41. Cassette, E.; Helle, M.; Bezdetnaya, L.; Marchal, F.; Dubertret, B.; Pons, T., Design of new quantum dot materials for deep tissue infrared imaging. Adv. Drug Deliv. Rev.s 2013, 65, 719731.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

42. Karakoti, A. S.; Shukla, R.; Shanker, R.; Singh, S., Surface functionalization of quantum dots for biological applications. Adv. Colloid Interface Sci. 2015, 215, 28-45. 43. Pentecost, A.; Gour, S.; Mochalin, V.; Knoke, I.; Gogotsi, Y., Deaggregation of nanodiamond powders using saltand sugar-assisted milling. ACS Appl. Mater. Interfaces 2010, 2, 3289-3294. 44. Hsu, M.-H.; Chuang, H.; Cheng, F.-Y.; Huang, Y.-P.; Han, C.-C.; Chen, J.-Y.; Huang, S.-C.; Chen, J.-K.; Wu, D.-S.; Chu, H.-L., Directly thiolated modification onto the surface of detonation nanodiamonds. ACS Appl. Mater. Interfaces 2014, 6, 7198-7203. 45. Liu, X.; Basu, A., Core functionalization of hollow polymer nanocapsules. J. Am. Chem. Soc. 2009, 131, 5718-5719. 46. Yang, J. A.; Murphy, C. J., Evidence for patchy lipid layers on gold nanoparticle surfaces. Langmuir 2012, 28, 54045416. 47. Mehtala, J. G.; Zemlyanov, D. Y.; Max, J. P.; Kadasala, N.; Zhao, S.; Wei, A., Citrate-stabilized gold nanorods. Langmuir 2014, 30, 13727-13730. 48. Gole, A.; Murphy, C. J., Polyelectrolyte-coated gold nanorods: synthesis, characterization and immobilization. Chem. Mater. 2005, 17, 1325-1330. 49. Wang, C.-F.; Mäkilä, E. M.; Bonduelle, C.; Rytkönen, J.; Raula, J.; Almeida, S.; Närvänen, A.; Salonen, J. J.; Lecommandoux, S.; Hirvonen, J. T.; Santos, H. A., Functionalization of Alkyne-Terminated Thermally Hydrocarbonized Porous Silicon Nanoparticles With Targeting Peptides and Antifouling Polymers: Effect on the Human Plasma Protein Adsorption. ACS Appl. Mater. Interfaces 2015, 7, 20062015. 50. Gole, A.; Murphy, C. J., Azide-derivatized gold nanorods: functional materials for “click” chemistry. Langmuir 2008, 24, 266-272. 51. Halas, N. J., Nanoscience under glass: the versatile chemistry of silica nanostructures. ACS Nano 2008, 2, 179-183. 52. Jana, N. R.; Earhart, C.; Ying, J. Y., Synthesis of watersoluble and functionalized nanoparticles by silica coating. Chem. Mater. 2007, 19, 5074-5082. 53. Lohse, S. E.; Burrows, N. D.; Scarabelli, L.; Liz-Marzán, L. M.; Murphy, C. J., Anisotropic noble metal nanocrystal growth: the role of halides. Chem. Mater. 2013, 26, 34-43. 54. Park, J.-W.; Shumaker-Parry, J. S., Structural study of citrate layers on gold nanoparticles: role of intermolecular interactions in stabilizing nanoparticles. J. Am. Chem. Soc. 2014, 136, 1907-1921. 55. Warheit, D. B., How meaningful are the results of nanotoxicity studies in the absence of adequate material characterization? Toxicol. Sci. 2008, 101, 183-185. 56. Dawson, K. A., Leave the policing to others. Nat. Nanotechnol. 2013, 8, 73-73. 57. Dawson, K. A.; Anguissola, S.; Lynch, I., The need for in situ characterisation in nanosafety assessment: funded transnational access via the QNano research infrastructure. Nanotoxicology 2012, 7, 346-349. 58. Richman, E. K.; Hutchison, J. E., The nanomaterial characterization bottleneck. Acs Nano 2009, 3, 2441-2446. 59. Marbella, L. E.; Millstone, J. E., NMR techniques for noble metal nanoparticles. Chem. Mater. 2015, 27, 2721-2739. 60. Jackson, S. R.; McBride, J. R.; Rosenthal, S. J.; Wright, D. W., Where’s the Silver? Imaging Trace Silver Coverage on the Surface of Gold Nanorods. J. Am. Chem. Soc. 2014, 136, 5261-5263. 61. Baer, D. R.; Gaspar, D. J.; Nachimuthu, P.; Techane, S. D.; Castner, D. G., Application of surface chemical analysis tools for characterization of nanoparticles. Anal. Bioanal. Chem. 2010, 396, 983-1002.

Page 16 of 21

62. Torelli, M. D.; Putans, R. A.; Tan, Y.; Lohse, S. E.; Murphy, C. J.; Hamers, R. J., Quantitative Determination of Ligand Densities on Nanomaterials by X-Ray Photoelectron Spectroscopy. ACS Appl. Mater. Interfaces 2015, 7, 1720-1725. 63. Walczyk, D.; Bombelli, F. B.; Monopoli, M. P.; Lynch, I.; Dawson, K. A., What the cell “sees” in bionanoscience. J. Am. Chem. Soc. 2010, 132, 5761-5768. 64. Giudice, M. C. L.; Herda, L. M.; Polo, E.; Dawson, K. A., In situ characterization of nanoparticle biomolecular interactions in complex biological media by flow cytometry. Nat. Commun. 2016, 7, 13475. 65. Kapralov, A. A.; Feng, W. H.; Amoscato, A. A.; Yanamala, N.; Balasubramanian, K.; Winnica, D. E.; Kisin, E. R.; Kotchey, G. P.; Gou, P.; Sparvero, L. J., Adsorption of surfactant lipids by single-walled carbon nanotubes in mouse lung upon pharyngeal aspiration. ACS Nano 2012, 6, 4147-4156. 66. Wan, S.; Kelly, P. M.; Mahon, E.; Stöckmann, H.; Rudd, P. M.; Caruso, F.; Dawson, K. A.; Yan, Y.; Monopoli, M. P., The “Sweet” Side of the Protein Corona: Effects of Glycosylation on Nanoparticle–Cell Interactions. ACS Nano 2015, 9, 2157-2166. 67. Nason, J. A.; McDowell, S. A.; Callahan, T. W., Effects of natural organic matter type and concentration on the aggregation of citrate-stabilized gold nanoparticles. J. Environ. Mon. 2012, 14, 1885-1892. 68. Murphy, C. J.; Vartanian, A. M.; Geiger, F. M.; Hamers, R. J.; Pedersen, J.; Cui, Q.; Haynes, C. L.; Carlson, E. E.; Hernandez, R.; Klaper, R. D., Biological Responses to Engineered Nanomaterials: Needs for the Next Decade. ACS Cent. Sci. 2015, 1, 117-123. 69. Blanco, E.; Shen, H.; Ferrari, M., Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 2015, 33, 941-951. 70. Müller, J.; Bauer, K. N.; Prozeller, D.; Simon, J.; Mailänder, V.; Wurm, F. R.; Winzen, S.; Landfester, K., Coating nanoparticles with tunable surfactants facilitates control over the protein corona. Biomaterials 2017, 115, 1-8. 71. Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Bombelli, F. B.; Hristov, D. R.; Kelly, P. M.; Åberg, C.; Mahon, E.; Dawson, K. A., Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat. Nanotechnol. 2013, 8, 137-143. 72. Walkey, C. D.; Olsen, J. B.; Song, F.; Liu, R.; Guo, H.; Olsen, D. W. H.; Cohen, Y.; Emili, A.; Chan, W. C., Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS Nano 2014, 8, 2439-2455. 73. Zhang, D.; Neumann, O.; Wang, H.; Yuwono, V. M.; Barhoumi, A.; Perham, M.; Hartgerink, J. D.; Wittung-Stafshede, P.; Halas, N. J., Gold nanoparticles can induce the formation of protein-based aggregates at physiological pH. Nano Lett. 2009, 9, 666-671. 74. Yang, J. A.; Lohse, S. E.; Murphy, C. J., Tuning cellular response to nanoparticles via surface chemistry and aggregation. small 2014, 10, 1642-1651. 75. Saha, K.; Rahimi, M.; Yazdani, M.; Kim, S. T.; Moyano, D. F.; Hou, S.; Das, R.; Mout, R.; Rezaee, F.; Mahmoudi, M.; Rotello, V. M., Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality and Protein Corona. ACS Nano 2016, 10, 4421-4430. 76. Pelaz, B.; del Pino, P.; Maffre, P.; Hartmann, R.; Gallego, M.; Rivera-Fernández, S.; de la Fuente, J. M.; Nienhaus, G. U.; Parak, W. J., Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake. ACS Nano 2015, 9, 6996-7008. 77. Harris, J. M.; Chess, R. B., Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2003, 2, (3), 214-221. 78. Treuel, L.; Brandholt, S.; Maffre, P.; Wiegele, S.; Shang, L.; Nienhaus, G. U., Impact of Protein Modification on the


Page 17 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Protein Corona on Nanoparticles and Nanoparticle–Cell Interactions. ACS Nano 2014, 8, 503-513. 79. Frey, A.; Giannasca, K. T.; Weltzin, R.; Giannasca, P. J.; Reggio, H.; Lencer, W. I.; Neutra, M. R., Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting. J. Exp. Med. 1996, 184, 1045-1059. 80. Conner, S. D.; Schmid, S. L., Regulated portals of entry into the cell. Nature 2003, 422, 37-44. 81. Ray, M.; Tang, R.; Jiang, Z.; Rotello, V. M., Quantitative Tracking of Protein Trafficking to the Nucleus Using Cytosolic Protein Delivery by Nanoparticle-Stabilized Nanocapsules. Bioconjugate Chem. 2015, 26, 1004-1007. 82. Rauch, J.; Kolch, W.; Laurent, S.; Mahmoudi, M., Big signals from small particles: regulation of cell signaling pathways by nanoparticles. Chem. Rev. 2013, 113, 3391-3406. 83. Cho, E. C.; Xie, J.; Wurm, P. A.; Xia, Y., Understanding the role of surface charges in cellular adsorption versus internalization by selectively removing gold nanoparticles on the cell surface with a I2/KI etchant. Nano Lett. 2009, 9, 1080-1084. 84. Villanueva, A.; Canete, M.; Roca, A. G.; Calero, M.; Veintemillas-Verdaguer, S.; Serna, C. J.; del Puerto Morales, M.; Miranda, R., The influence of surface functionalization on the enhanced internalization of magnetic nanoparticles in cancer cells. Nanotechnology 2009, 20, 115103. 85. Wilhelm, C.; Billotey, C.; Roger, J.; Pons, J. N.; Bacri, J. C.; Gazeau, F., Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating. Biomaterials 2003, 24, 1001-1011. 86. Hu, Y.; Litwin, T.; Nagaraja, A. R.; Kwong, B.; Katz, J.; Watson, N.; Irvine, D. J., Cytosolic delivery of membraneimpermeable molecules in dendritic cells using pH-responsive core-shell nanoparticles. Nano Lett. 2007, 7, 3056-3064. 87. Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M. L.; Stroeve, P.; Mahmoudi, M., Toxicity of nanomaterials. Chem. Soc. Rev. 2012, 41, 2323-2343. 88. Cho, E. C.; Xie, J.; Wurm, P. A.; Xia, Y., Understanding the role of surface charges in cellular adsorption versus internalization by selectively removing gold nanoparticles on the cell surface with a I2/KI etchant. Nano Lett. 2009, 9, 1080-1084. 89. Ghinea, N.; Simionescu, N., Anionized and cationized hemeundecapeptides as probes for cell surface charge and permeability studies: differentiated labeling of endothelial plasmalemmal vesicles. J. Cell Biol. 1985, 100, 606-612. 90. Lin, J.; Zhang, H.; Chen, Z.; Zheng, Y., Penetration of lipid membranes by gold nanoparticles: insights into cellular uptake, cytotoxicity, and their relationship. ACS Nano 2010, 4, 5421-5429. 91. Li, S.; Malmstadt, N., Deformation and poration of lipid bilayer membranes by cationic nanoparticles. Soft Matter 2013, 9, 4969-4976. 92. Leroueil, P. R.; Berry, S. A.; Duthie, K.; Han, G.; Rotello, V. M.; McNerny, D. Q.; Baker, J. R.; Orr, B. G.; Banaszak Holl, M. M., Wide varieties of cationic nanoparticles induce defects in supported lipid bilayers. Nano Lett. 2008, 8, 420-424. 93. Arvizo, R. R.; Miranda, O. R.; Thompson, M. A.; Pabelick, C. M.; Bhattacharya, R.; Robertson, J. D.; Rotello, V. M.; Prakash, Y.; Mukherjee, P., Effect of nanoparticle surface charge at the plasma membrane and beyond. Nano Lett. 2010, 10, 25432548. 94. Lin, J.; Alexander-Katz, A., Cell membranes open “doors” for cationic nanoparticles/biomolecules: insights into uptake kinetics. ACS Nano 2013, 7, 10799-10808. 95. Nash, J. A.; Tucker, T. L.; Therriault, W.; Yingling, Y. G., Binding of single stranded nucleic acids to cationic ligand

functionalized gold nanoparticles. Biointerphases 2016, 11, 04B305. 96. Wang, J.; Kong, M.; Zhou, Z.; Yan, D.; Yu, X.; Cheng, X.; Feng, C.; Liu, Y.; Chen, X., Mechanism of surface charge triggered intestinal epithelial tight junction opening upon chitosan nanoparticles for insulin oral delivery. Carbohydr. Polym. 2017, 157, 596-602. 97. Chen, Y.; Wu, Y.; Gao, J.; Zhang, Z.; Wang, L.; Chen, X.; Mi, J.; Yao, Y.; Guan, D.; Chen, B.; Dai, J., Transdermal Vascular Endothelial Growth Factor Delivery with Surface Engineered Gold Nanoparticles. ACS Appl. Mater. Interfaces 2017, 9, 51735180. 98. Rozenberg, B.; Tenne, R., Polymer-assisted fabrication of nanoparticles and nanocomposites. Prog. Polym. Sci. 2008, 33, 40-112. 99. Rasch, M. R.; Rossinyol, E.; Hueso, J. L.; Goodfellow, B. W.; Arbiol, J.; Korgel, B. A., Hydrophobic gold nanoparticle selfassembly with phosphatidylcholine lipid: Membrane-loaded and Janus vesicles. Nano Lett. 2010, 10, 3733-3739. 100. Jing, B.; Zhu, Y., Disruption of supported lipid bilayers by semihydrophobic nanoparticles. J. Am. Chem. Soc. 2011, 133, 10983-10989. 101. Olubummo, A.; Schulz, M.; Lechner, B.-D.; Scholtysek, P.; Bacia, K.; Blume, A.; Kressler, J.; Binder, W. H., Controlling the Localization of Polymer-Functionalized Nanoparticles in Mixed Lipid/Polymer Membranes. ACS Nano 2012, 6, 8713-8727. 102. Lee, H.-Y.; Shin, S. H. R.; Abezgauz, L. L.; Lewis, S. A.; Chirsan, A. M.; Danino, D. D.; Bishop, K. J. M., Integration of Gold Nanoparticles into Bilayer Structures via Adaptive Surface Chemistry. J. Am. Chem. Soc. 2013, 135, 5950-5953. 103. Li, Y.; Chen, X.; Gu, N., Computational investigation of interaction between nanoparticles and membranes: hydrophobic/hydrophilic effect. J. Phys. Chem. B 2008, 112, 16647-16653. 104. Alexeev, A.; Uspal, W. E.; Balazs, A. C., Harnessing Janus nanoparticles to create controllable pores in membranes. ACS Nano 2008, 2, 1117-1122. 105. Bhabra, G.; Sood, A.; Fisher, B.; Cartwright, L.; Saunders, M.; Evans, W. H.; Surprenant, A.; Lopez-Castejon, G.; Mann, S.; Davis, S. A., Nanoparticles can cause DNA damage across a cellular barrier. Nat. Nanotechnol. 2009, 4, 876-883. 106. Chompoosor, A.; Saha, K.; Ghosh, P. S.; Macarthy, D. J.; Miranda, O. R.; Zhu, Z. J.; Arcaro, K. F.; Rotello, V. M., The role of surface functionality on acute cytotoxicity, ROS generation and DNA damage by cationic gold nanoparticles. Small 2010, 6, 2246-2249. 107. AshaRani, P. V.; Low Kah Mun, G.; Hande, M. P.; Valiyaveettil, S., Cytotoxicity and Genotoxicity of Silver Nanoparticles in Human Cells. ACS Nano 2009, 3, 279-290. 108. Mahmoudi, M.; Saeedi-Eslami, S. N.; Shokrgozar, M. A.; Azadmanesh, K.; Hassanlou, M.; Kalhor, H. R.; Burtea, C.; Rothen-Rutishauser, B.; Laurent, S.; Sheibani, S.; Vali, H., Cell "vision": complementary factor of protein corona in nanotoxicology. Nanoscale 2012, 4, 5461-5468. 109. Dawson, K. A.; Salvati, A.; Lynch, I., Nanotoxicology: nanoparticles reconstruct lipids. Nat. Nanotechnol. 2009, 4, 8485. 110. Hoffmann, F.; Cinatl, J.; Kabičková, H.; Kreuter, J.; Stieneker, F., Preparation, characterization and cytotoxicity of methylmethacrylate copolymer nanoparticles with a permanent positive surface charge. Int. J. Pharm. 1997, 157, 189-198. 111. Oh, W.-K.; Kim, S.; Choi, M.; Kim, C.; Jeong, Y. S.; Cho, B.-R.; Hahn, J.-S.; Jang, J., Cellular Uptake, Cytotoxicity, and Innate Immune Response of Silica−Titania Hollow Nanoparticles Based on Size and Surface Functionality. ACS Nano 2010, 4, 53015313.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

112. Ishii, D.; Kinbara, K.; Ishida, Y.; Ishii, N.; Okochi, M.; Yohda, M.; Aida, T., Chaperonin-mediated stabilization and ATP-triggered release of semiconductor nanoparticles. Nature 2003, 423, 628-632. 113. Wagner, S. C.; Roskamp, M.; Pallerla, M.; Araghi, R. R.; Schlecht, S.; Koksch, B., Nanoparticle-Induced Folding and Fibril Formation of Coiled-Coil-Based Model Peptides. Small 2010, 6, 1321-1328. 114. Xia, T.; Kovochich, M.; Brant, J.; Hotze, M.; Sempf, J.; Oberley, T.; Sioutas, C.; Yeh, J. I.; Wiesner, M. R.; Nel, A. E., Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Lett. 2006, 6, 1794-1807. 115. Joris, F.; Valdepérez, D.; Pelaz, B.; Soenen, S. J.; Manshian, B. B.; Parak, W. J.; De Smedt, S. C.; Raemdonck, K., The impact of species and cell type on the nanosafety profile of iron oxide nanoparticles in neural cells. J. Nanobiotechnol. 2016, 14, 69. 116. Blechinger, J.; Bauer, A. T.; Torrano, A. A.; Gorzelanny, C.; Bräuchle, C.; Schneider, S. W., Uptake kinetics and nanotoxicity of silica nanoparticles are cell type dependent. Small 2013, 9, 3970-3980. 117. Lanone, S.; Rogerieux, F.; Geys, J.; Dupont, A.; MaillotMarechal, E.; Boczkowski, J.; Lacroix, G.; Hoet, P., Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines. Part. Fibre Toxicol. 2009, 6, 14. 118. Chung, T.-H.; Wu, S.-H.; Yao, M.; Lu, C.-W.; Lin, Y.-S.; Hung, Y.; Mou, C.-Y.; Chen, Y.-C.; Huang, D.-M., The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3-L1 cells and human mesenchymal stem cells. Biomaterials 2007, 28, 2959-2966. 119. Chang, J.-S.; Chang, K. L. B.; Hwang, D.-F.; Kong, Z.-L., In vitro cytotoxicitiy of silica nanoparticles at high concentrations strongly depends on the metabolic activity type of the cell line. Environ. Sci. Technol. 2007, 41, 2064-2068. 120. Sohaebuddin, S. K.; Thevenot, P. T.; Baker, D.; Eaton, J. W.; Tang, L., Nanomaterial cytotoxicity is composition, size, and cell type dependent. Part. Fibre Toxicol. 2010, 7, 22. 121. Mahmoudi, M.; Laurent, S.; Shokrgozar, M. A.; Hosseinkhani, M., Toxicity evaluations of superparamagnetic iron oxide nanoparticles: cell “vision” versus physicochemical properties of nanoparticles. ACS Nano 2011, 5, 7263-7276. 122. Gornati, R.; Pedretti, E.; Rossi, F.; Cappellini, F.; Zanella, M.; Olivato, I.; Sabbioni, E.; Bernardini, G., Zerovalent Fe, Co and Ni nanoparticle toxicity evaluated on SKOV-3 and U87 cell lines. J. Appl. Toxicol. 2016, 36, 385-393. 123. Min, L.; Jian-xing, X., Detoxifying function of cytochrome c against oxygen toxicity. Mitochondrion 2007, 7, 1316. 124. Aubin-Tam, M.-E.; Hamad-Schifferli, K., Gold nanoparticle-cytochrome c complexes: the effect of nanoparticle ligand charge on protein structure. Langmuir 2005, 21, 1208012084. 125. Deng, Z. J.; Liang, M.; Monteiro, M.; Toth, I.; Minchin, R. F., Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation. Nat. Nanotechnol. 2011, 6, 39-44. 126. Mahmoudi, M.; Shokrgozar, M. A.; Sardari, S.; Moghadam, M. K.; Vali, H.; Laurent, S.; Stroeve, P., Irreversible changes in protein conformation due to interaction with superparamagnetic iron oxide nanoparticles. Nanoscale 2011, 3, 1127-1138. 127. De, M.; You, C.-C.; Srivastava, S.; Rotello, V. M., Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study. J. Am. Chem. Soc. 2007, 129, 10747-10753.

Page 18 of 21

128. Scaletti, F.; Feis, A.; Centi, S.; Pini, R.; Rotello, V. M.; Messori, L., Tuning the interactions of PEG-coated gold nanorods with BSA and model proteins through insertion of amino or carboxylate groups. J. Inorg. Biochem. 2015, 150, 120– 125. 129. Ekmekci, Z.; Saha, K.; Moyano, D. F.; Tonga, G. Y.; Wang, H.; Mout, R.; Rotello, V. M., Probing the protein– nanoparticle interface: the role of aromatic substitution pattern on affinity. Supramol. Chem. 2015, 27, 123-126. 130. Lynch, I.; Salvati, A.; Dawson, K. A., Proteinnanoparticle interactions: what does the cell see? Nat. Nanotechnol. 2009, 4, 546-547. 131. Lynch, I., Are there generic mechanisms governing interactions between nanoparticles and cells? Epitope mapping the outer layer of the protein–material interface. Stat. Mech. Appl. 2007, 373, 511-520. 132. Fischer, N. O.; McIntosh, C. M.; Simard, J. M.; Rotello, V. M., Inhibition of chymotrypsin through surface binding using nanoparticle-based receptors. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5018-5023. 133. Fischer, N. O.; Verma, A.; Goodman, C. M.; Simard, J. M.; Rotello, V. M., Reversible “irreversible” inhibition of chymotrypsin using nanoparticle receptors. J. Am. Chem. Soc. 2003, 125, 13387-13391. 134. You, C.-C.; De, M.; Han, G.; Rotello, V. M., Tunable inhibition and denaturation of α-chymotrypsin with amino acidfunctionalized gold nanoparticles. J. Am. Chem. Soc. 2005, 127, 12873-12881. 135. You, C.-C.; De, M.; Rotello, V. M., Contrasting effects of exterior and interior hydrophobic moieties in the complexation of amino acid functionalized gold clusters with αchymotrypsin. Org. lett. 2005, 7, 5685-5688. 136. Hamad, I.; Al-Hanbali, O.; Hunter, A. C.; Rutt, K. J.; Andresen, T. L.; Moghimi, S. M., Distinct polymer architecture mediates switching of complement activation pathways at the nanosphere− serum interface: implications for stealth nanoparticle engineering. ACS Nano 2010, 4, 6629-6638. 137. Barlow, P. G.; Donaldson, K.; MacCallum, J.; Clouter, A.; Stone, V., Serum exposed to nanoparticle carbon black displays increased potential to induce macrophage migration. Toxicol. Lett. 2005, 155, 397-401. 138. Zhu, M.; Li, Y.; Shi, J.; Feng, W.; Nie, G.; Zhao, Y., Exosomes as Extrapulmonary Signaling Conveyors for Nanoparticle-Induced Systemic Immune Activation. Small 2012, 8, 404-412. 139. Dobrovolskaia, M. A.; Aggarwal, P.; Hall, J. B.; McNeil, S. E., Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution. Mol. Pharm. 2008, 5, 487-495. 140. Peek, L. J.; Middaugh, C. R.; Berkland, C., Nanotechnology in vaccine delivery. Adv. Drug Delivery Rev. 2008, 60, 915-928. 141. Fang, C.; Shi, B.; Pei, Y.-Y.; Hong, M.-H.; Wu, J.; Chen, H.-Z., In vivo tumor targeting of tumor necrosis factor-α-loaded stealth nanoparticles: effect of MePEG molecular weight and particle size. Eur. J. Pharm. Sci. 2006, 27, 27-36. 142. Manolova, V.; Flace, A.; Bauer, M.; Schwarz, K.; Saudan, P.; Bachmann, M. F., Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 2008, 38, 1404-1413. 143. Zahr, A. S.; Davis, C. A.; Pishko, M. V., Macrophage uptake of core-shell nanoparticles surface modified with poly (ethylene glycol). Langmuir 2006, 22, 8178-8185. 144. Demento, S. L.; Eisenbarth, S. C.; Foellmer, H. G.; Platt, C.; Caplan, M. J.; Saltzman, W. M.; Mellman, I.; Ledizet, M.; Fikrig, E.; Flavell, R. A., Inflammasome-activating nanoparticles


Page 19 of 21

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as modular systems for optimizing vaccine efficacy. Vaccine 2009, 27, 3013-3021. 145. Gal, N.; Lassenberger, A.; Herrero-Nogareda, L.; Scheberl, A.; Charwat, V.; Kasper, C.; Reimhult, E., Interaction of Size-Tailored PEGylated Iron Oxide Nanoparticles with Lipid Membranes and Cells. ACS Biomater. Sci. Eng. 2017, 3, 249-259. 146. MacLeod, M. J.; Johnson, J. A., PEGylated NHeterocyclic Carbene Anchors Designed To Stabilize Gold Nanoparticles in Biologically Relevant Media. J. Am. Chem. Soc. 2015, 137, 7974-7977. 147. Barenholz, Y. C., Doxil®—the first FDA-approved nano-drug: lessons learned. J. Controlled Release 2012, 160, 117134. 148. Ishida, T.; Wang, X.; Shimizu, T.; Nawata, K.; Kiwada, H., PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner. J. Controlled Release 2007, 122, 349355. 149. Wang, X.; Ishida, T.; Kiwada, H., Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J. Controlled Release 2007, 119, 236-244. 150. Tomasetti, L.; Liebl, R.; Wastl, D. S.; Breunig, M., Influence of PEGylation on nanoparticle mobility in different models of the extracellular matrix. Eur. J. Pharm. Biopharm. 2016, 108, 145-155. 151. Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M., PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv. Drug Delivery Rev. 2016, 99, 28-51. 152. Uz, M.; Bulmus, V.; Alsoy Altinkaya, S., Effect of PEG Grafting Density and Hydrodynamic Volume on Gold Nanoparticle–Cell Interactions: An Investigation on Cell Cycle, Apoptosis, and DNA Damage. Langmuir 2016, 32, 5997-6009. 153. Gal, N.; Lassenberger, A.; Herrero-Nogareda, L.; Scheberl, A.; Charwat, V.; Kasper, C.; Reimhult, E., Interaction of size-tailored PEGylated iron oxide nanoparticles with lipid membranes and cells. ACS Biomat. Sci. Eng. 2017, 3, 249-259. 154. Jenkins, S. I.; Weinberg, D.; al-Shakli, A. F.; Fernandes, A. R.; Yiu, H. H.; Telling, N. D.; Roach, P.; Chari, D. M., ‘Stealth’nanoparticles evade neural immune cells but also evade major brain cell populations: Implications for PEG-based neurotherapeutics. J. Controlled Release 2016, 224, 136-145. 155. Rodriguez, P. L.; Harada, T.; Christian, D. A.; Pantano, D. A.; Tsai, R. K.; Discher, D. E., Minimal "Self" Peptides That Inhibit Phagocytic Clearance and Enhance Delivery of Nanoparticles. Science 2013, 339, 971-975. 156. Parodi, A.; Quattrocchi, N.; van de Ven, A. L.; Chiappini, C.; Evangelopoulos, M.; Martinez, J. O.; Brown, B. S.; Khaled, S. Z.; Yazdi, I. K.; Enzo, M. V.; Isenhart, L.; Ferrari, M.; Tasciotti, E., Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions. Nat. Nanotechnol. 2013, 8, 61-68. 157. Hu, C.-M. J.; Zhang, L.; Aryal, S.; Cheung, C.; Fang, R. H.; Zhang, L., Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 10980-10985. 158. Rao, L.; Meng, Q.-F.; Bu, L.-L.; Cai, B.; Huang, Q.; Sun, Z.-J.; Zhang, W.-F.; Li, A.; Guo, S.-S.; Liu, W.; Wang, T.-H.; Zhao, X.-Z., Erythrocyte Membrane-Coated Upconversion Nanoparticles with Minimal Protein Adsorption for Enhanced Tumor Imaging. ACS Appl. Mater. Interfaces 2017, 9, 2159-2168. 159. Sun, H.; Su, J.; Meng, Q.; Yin, Q.; Chen, L.; Gu, W.; Zhang, Z.; Yu, H.; Zhang, P.; Wang, S.; Li, Y., Cancer Cell Membrane-Coated Gold Nanocages with HyperthermiaTriggered Drug Release and Homotypic Target Inhibit Growth and Metastasis of Breast Cancer. Adv. Funct. Mater. 2017, 27, 1604300.

160. Rao, L.; He, Z.; Meng, Q.-F.; Zhou, Z.; Bu, L.-L.; Guo, S.-S.; Liu, W.; Zhao, X.-Z., Effective cancer targeting and imaging using macrophage membrane-camouflaged upconversion nanoparticles. J. Biomed. Mater. Res. A 2017, 105, 521-530. 161. Kreuter, J.; Shamenkov, D.; Petrov, V.; Ramge, P.; Cychutek, K.; Koch-Brandt, C.; Alyautdin, R., Apolipoproteinmediated Transport of Nanoparticle-bound Drugs Across the Blood-Brain Barrier. J. Drug Target. 2002, 10, 317-325. 162. Choi, K.; Riviere, J. E.; Monteiro-Riviere, N. A., Protein corona modulation of hepatocyte uptake and molecular mechanisms of gold nanoparticle toxicity. Nanotoxicology 2017, 11, 64-75. 163. Oh, W.-K.; Kim, S.; Choi, M.; Kim, C.; Jeong, Y. S.; Cho, B.-R.; Hahn, J.-S.; Jang, J., Cellular uptake, cytotoxicity, and innate immune response of silica− titania hollow nanoparticles based on size and surface functionality. ACS Nano 2010, 4, 53015313. 164. Kedmi, R.; Ben-Arie, N.; Peer, D., The systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation. Biomaterials 2010, 31, 68676875. 165. Tarasova, N. K.; Gallud, A.; Ytterberg, A. J.; Chernobrovkin, A. L.; Ruiz, J.; Astruc, D.; Antipov, A.; Fedutik, Y.; Fadeel, B.; Zubarev, R. A., Cytotoxic and Pro-inflammatory Effects of Metal-based Nanoparticles on THP-1 Monocytes Characterized by Combined Proteomics Approaches. J. Proteome Res. 2016, 16, 689–697. 166. Savitski, M. M.; Reinhard, F. B.; Franken, H.; Werner, T.; Savitski, M. F.; Eberhard, D.; Molina, D. M.; Jafari, R.; Dovega, R. B.; Klaeger, S., Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 2014, 346, 1255784. 167. Sun, H.; Gong, S.; Carmody, R. J.; Hilliard, A.; Li, L.; Sun, J.; Kong, L.; Xu, L.; Hilliard, B.; Hu, S., TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis. Cell 2008, 133, 415-426. 168. Oslakovic, C.; Cedervall, T.; Linse, S.; Dahlbäck, B., Polystyrene nanoparticles affecting blood coagulation. Nanomed. Nanotechnol. Biol. Med. 2012, 8, 981-986. 169. Salvador-Morales, C.; Zhang, L.; Langer, R.; Farokhzad, O. C., Immunocompatibility properties of lipid–polymer hybrid nanoparticles with heterogeneous surface functional groups. Biomaterials 2009, 30, 2231-2240. 170. Fuentes, E.; Yameen, B.; Bong, S.-J.; Salvador-Morales, C.; Palomo, I.; Vilos, C., Antiplatelet effect of differentially charged PEGylated lipid-polymer nanoparticles. Nanomedicine 2017, 13, 1089-1094. 171. Moyano, D. F.; Goldsmith, M.; Solfiell, D. J.; Landesman-Milo, D.; Miranda, O. R.; Peer, D.; Rotello, V. M., Nanoparticle hydrophobicity dictates immune response. J. Am. Chem. Soc. 2012, 134, 3965-3967. 172. Flemming, A., Autoimmunity: Nanoparticles engineered for antigen-specific immunotherapy. Nat. Rev. Immunol. 2016, 16, 204-205. 173. Fiering, S., Cancer immunotherapy: Making allies of phagocytes. Nat. Nanotechnol. 2017, advance online publication. 174. Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O. C., Factors Affecting the Clearance and Biodistribution of Polymeric Nanoparticles. Mol. Pharm. 2008, 5, 505-515. 175. Arvizo, R. R.; Miranda, O. R.; Moyano, D. F.; Walden, C. A.; Giri, K.; Bhattacharya, R.; Robertson, J. D.; Rotello, V. M.; Reid, J. M.; Mukherjee, P., Modulating pharmacokinetics, tumor uptake and biodistribution by engineered nanoparticles. PLoS One 2011, 6, e24374. 176. Kim, B.; Han, G.; Toley, B. J.; Kim, C.-k.; Rotello, V. M.; Forbes, N. S., Tuning payload delivery in tumour cylindroids using gold nanoparticles. Nat. Nanotechnol. 2010, 5, 465-472.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

177. Elci, S. G.; Jiang, Y.; Yan, B.; Kim, S. T.; Saha, K.; Moyano, D. F.; Yesilbag Tonga, G.; Jackson, L. C.; Rotello, V. M.; Vachet, R. W., Surface Charge Controls the Sub-Organ Biodistributions of Gold Nanoparticles. ACS Nano 2016, 10, 5536– 5542. 178. Zhu, Z. J.; Carboni, R.; Quercio, M. J.; Yan, B.; Miranda, O. R.; Anderton, D. L.; Arcaro, K. F.; Rotello, V. M.; Vachet, R. W., Surface properties dictate uptake, distribution, excretion, and toxicity of nanoparticles in fish. Small 2010, 6, 2261-2265. 179. Giret, S.; Wong Chi Man, M.; Carcel, C., MesoporousSilica-Functionalized Nanoparticles for Drug Delivery. Chem. Eur. J. 2015, 21, 13850-13865. 180. Ilinskaya, A. N.; Dobrovolskaia, M. A., Interaction Between Nanoparticles and Plasma Proteins: Effects on Nanoparticle Biodistribution and Toxicity. Pol. Nanopart.Nanomed., Springer: 2016; 505-520. 181. Bae, Y. H.; Park, K., Targeted drug delivery to tumors: Myths, reality and possibility. J. Controlled Release 2011, 153, 198205. 182. Nagayama, S.; Ogawara, K.-i.; Fukuoka, Y.; Higaki, K.; Kimura, T., Time-dependent changes in opsonin amount associated on nanoparticles alter their hepatic uptake characteristics. Int. J. Pharm. 2007, 342, 215-221. 183. Mizuhara, T.; Saha, K.; Moyano, D. F.; Kim, C. S.; Yan, B.; Kim, Y. K.; Rotello, V. M., Acylsulfonamide-Functionalized Zwitterionic Gold Nanoparticles for Enhanced Cellular Uptake at Tumor pH. Angew. Chem., Int. Ed. 2015, 54, 6567–6570. 184. Li, Y.; Zhang, X.; Cao, D., Nanoparticle hardness controls the internalization pathway for drug delivery. Nanoscale 2015, 7, 2758-2769. 185. Anselmo, A. C.; Mitragotri, S., Impact of particle elasticity on particle-based drug delivery systems. Adv. Drug Delivery Reviews 2017, 108, 51-67. 186. Anselmo, A. C.; Zhang, M.; Kumar, S.; Vogus, D. R.; Menegatti, S.; Helgeson, M. E.; Mitragotri, S., Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting. ACS Nano 2015, 9, 3169-3177. 187. Tao, S. L.; Desai, T. A., Micromachined devices: the impact of controlled geometry from cell-targeting to bioavailability. J. Controlled Release 2005, 109, 127-138. 188. Beningo, K. A.; Lo, C.-M.; Wang, Y.-L., Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions. Methods Cell Biol. 2002, 69, 325-339. 189. Sun, J.; Zhang, L.; Wang, J.; Feng, Q.; Liu, D.; Yin, Q.; Xu, D.; Wei, Y.; Ding, B.; Shi, X., Tunable rigidity of (polymeric core)–(lipid shell) nanoparticles for regulated cellular uptake. Adv. Mater. 2015, 27, 1402-1407.

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190. Ding, H.-m.; Ma, Y.-q., Role of physicochemical properties of coating ligands in receptor-mediated endocytosis of nanoparticles. Biomaterials 2012, 33, 5798-5802. 191. Yi, X.; Shi, X.; Gao, H., Cellular uptake of elastic nanoparticles. Phys. Rev. Lett. 2011, 107, 098101. 192. Yi, X.; Gao, H., Kinetics of receptor-mediated endocytosis of elastic nanoparticles. Nanoscale 2017, 9, 454-463. 193. Banquy, X.; Suarez, F.; Argaw, A.; Rabanel, J.-M.; Grutter, P.; Bouchard, J.-F.; Hildgen, P.; Giasson, S., Effect of mechanical properties of hydrogel nanoparticles on macrophage cell uptake. Soft Matter 2009, 5, 3984-3991. 194. Del Pino, P.; Yang, F.; Pelaz, B.; Zhang, Q.; Kantner, K.; Hartmann, R.; Martinez de Baroja, N.; Gallego, M.; Möller, M.; Manshian, B. B., Basic physicochemical properties of polyethylene glycol coated gold nanoparticles that determine their interaction with cells. Angew. Chem., Int. Ed. 2016, 55, 5483-5487. 195. Zhang, L.; Cao, Z.; Li, Y.; Ella-Menye, J.-R.; Bai, T.; Jiang, S., Softer zwitterionic nanogels for longer circulation and lower splenic accumulation. ACS Nano 2012, 6, 6681-6686. 196. Zhang, L.; Feng, Q.; Wang, J.; Zhang, S.; Ding, B.; Wei, Y.; Dong, M.; Ryu, J.-Y.; Yoon, T.-Y.; Shi, X., Microfluidic synthesis of hybrid nanoparticles with controlled lipid layers: understanding flexibility-regulated cell–nanoparticle interaction. ACS Nano 2015, 9, 9912-9921. 197. Hajipour, M. J.; Laurent, S.; Aghaie, A.; Rezaee, F.; Mahmoudi, M., Personalized protein coronas: a “key” factor at the nanobiointerface. Biomater. Sci. 2014, 2, 1210-1221.


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