Nanoparticle-assisted tubulin assembly is environment dependent.

Nanomaterials acquire a biomolecular corona upon introduction to biological media, leading to biological transformations such as changes in protein function, unmasking of epitopes, and protein fibrilization. Ex vivo studies to investigate the effect of nanoparticles on protein-protein interactions are typically performed in buffer and are rarely measured quantitatively in live cells. Here, we measure the differential effect of silica nanoparticles on protein association in vitro vs. in mammalian cells. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains that self-assemble like eukaryotic tubulin, first into dimers and then into microtubules in vitro or in vivo. Förster resonance energy transfer labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure their binding interactions in the presence of unfunctionalized silica nanoparticles in buffer and in cells using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in buffer due to protein corona formation. However, these nanoparticles have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment. Thus, the effect of nanomaterials on protein-protein interactions may not be readily translated from the test tube to the cell in the absence of particle surface functionalization that can enable targeted protein-nanoparticle interactions to withstand competitive binding in the nanoparticle corona from other biomolecules.

The Landscape of Gold Nanocrystal Surface Chemistry.

ConspectusGold nanoparticles (AuNPs) exhibit unique size- and shape-dependent properties not obtainable at the macroscale. Gold nanorods (AuNRs), with their morphology-dependent optical properties, ability to convert light to heat, and high surface-to-volume ratios, are of great interest for biosensing, medicine, and catalysis. While the gold core provides many fascinating properties, this Account focuses on AuNP soft surface coatings, which govern the interactions of nanoparticles with the local environments. Postmodification of AuNP surface chemistry can greatly alter NP colloidal stability, nano-bio interactions, and functionality. Polyelectrolyte coatings provide controllable surface-coating thickness and charge, which impact the composition of the acquired corona in biological settings. Covalent modification, in which covalently bound ligands replace the original capping layer, is often performed with thiols and disulfides due to their ability to replace native coatings. N-heterocyclic carbenes and looped peptides expand the possible functionalities of the ligand layer.The characterization of surface ligands bound to AuNPs, in terms of ligand density and dynamics, remains a challenge. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for understanding molecular structures and dynamics. Our recent NMR work on AuNPs demonstrated that NMR data were obtainable for ligands on NPs with diameters up to 25 nm for the first time. This was facilitated by the strong proton NMR signals of the trimethylammonium headgroup, which are present in a distinct regime from other ligand protons' signals. Ligand density analyses showed that the smallest AuNPs (below 4 nm) had the largest ligand densities, yet spin-spin T2 measurements revealed that these smallest NPs also had the most mobile ligand headgroups. Molecular dynamics simulations were able to reconcile these seemingly contradictory results.While NMR spectroscopy provides ligand information averaged over many NPs, the ligand distribution on individual particles' surfaces must also be probed to fully understand the surface coating. Taking advantage of improvements in electron energy loss spectroscopy (EELS) detectors employed with scanning transmission electron microscopy (STEM), a single-layer graphene substrate was used to calibrate the carbon K-edge EELS signal, allowing quantitative imaging of the carbon atom densities on AuNRs with sub-nanometer spatial resolution. In collaboration with others, we revealed that the mean value for surfactant-bilayer-coated AuNRs had 10-30% reduced ligand density at the ends of the rods compared to the sides, confirming prior indirect evidence for spatially distinct ligand densities.Recent work has found that surface ligands on nanoparticles can, somewhat surprisingly, enhance the selectivity and efficiency of the electrocatalytic reduction of CO2 by controlling access to the active site, tuning its electronic and chemical environment, or denying entry to impurities that poison the nanoparticle surface to facilitate reduction. Looking to the future, while NMR and EELS are powerful and complementary techniques for investigating surface coatings on AuNPs, the frontier of this field includes the development of methods to probe the surface ligands of individual NPs in a high-throughput manner, to monitor nano-bio interactions within complex matrices, and to study structure-property relationships of AuNPs in biological systems.

In-Cell Association of a Bioorthogonal Tubulin.

Studies of proteins from one organism in another organism's cells have shown that such exogenous proteins stick more, pointing toward coevolution of the cytoplasm and protein surface to minimize stickiness. Here we flip this question around by asking whether exogenous proteins can assemble efficiently into their target complexes in a non-native cytoplasm. We use as our model system the assembly of BtubA and BtubB from Prosthecobacter hosted in human U-2 OS cells. BtubA and B evolved from eukaryotic tubulins after horizontal gene transfer, but they have low surface sequence identity with the homologous human tubulins and do not respond to tubulin drugs such as nocodazole. In U-2 OS cells, BtubA and B assemble efficiently into dimers compared to in vitro, and the wild-type BtubA and B proteins subsequently are able to form microtubules as well. We find that generic crowding effects (Ficoll 70 in vitro) contribute significantly to efficient dimer assembly when compared to sticking interactions (U-2 OS cell lysate in vitro), consistent with the notion that a generic mechanism such as crowding can be effective at driving assembly of exogenous proteins, even when protein-cytoplasm quinary structure and sticking have been modified in a non-native cytoplasm. A simple Monte Carlo model of in vitro and in-cell interactions, treating BtubA and B as sticky dipoles in a matrix of sticky or nonsticky crowders, rationalizes all the experimental trends with two adjustable parameters and reveals nucleation as the likely mechanism for the time-scale separation between dimer- and tubule formation in-cell and in vitro.

Adsorption and Molecular Display of a Redox-Active Protein on Gold Nanoparticle Surfaces.

Engineered gold nanoparticles (AuNPs) have great potential in many applications due to their tunable optical properties, facile synthesis, and surface functionalization via thiol chemistry. When exposed to a biological environment, NPs are coated with a protein corona that can alter the NPs' biological identity but can also affect the proteins' structures and functions. Protein disulfide isomerase (PDI) is an abundant protein responsible for the disulfide formation and isomerization that contribute to overall cell redox homeostasis and signaling. Given that AuNPs are widely employed in nanomedicine and PDI plays a functional role in various diseases, the interactions between oxidized (oPDI) and reduced (rPDI) with 50 nm citrate-coated AuNPs (AuNPs) are examined in this study using various techniques. Upon incubation, PDI adsorbs to the AuNP surface, which leads to a reduction in its enzymatic activity despite limited changes in secondary structures. Partial enzymatic digestion followed by mass spectrometry analysis shows that orientation of PDI on the NP surface is dependent on both its oxidation state and the PDI:AuNP incubation ratios.

Anionic nanoparticle-induced perturbation to phospholipid membranes affects ion channel function.

Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties.

Regulating and Directionally Controlling Electron Emission from Gold Nanorods with Silica Coatings.

Dielectric coatings offer a versatile means of manipulating hot carrier emission from nanoplasmonic systems for emerging nanocatalysis and photocathode applications, with uniform coatings acting as regulators and nonuniform coatings providing directional photocurrent control. However, the mechanisms for electron emission through dense and mesoporous silica (SiO2) coatings require further examination. Here, we present a systematic investigation of photoemission from single gold nanorods as a function of dense versus mesoporous silica coating thicknesses. Studies with dense coatings on gold nanostructures clarify the short (∼1 nm) attenuation length responsible for severely reduced transmission through the silica conduction band. By contrast, mesoporous silica is much more transmissive, and a simple geometric model quantitatively recapitulates the electron escape probability through nanoscopic porous channels. Finally, photoelectron velocity map imaging (VMI) studies of nanorods with coating defects verify that photoemission occurs preferentially through the thinner regions, illustrating new opportunities for designing photocurrent distributions on the nanoscale.

Opportunities for Electrocatalytic CO2 Reduction Enabled by Surface Ligands.

To achieve high selectivity in enzyme catalysis, nature carefully controls both the catalyst active site and the pocket or environment that mediates access and the geometry of a reactant. Despite the many advantages of heterogeneous catalysis, active sites on a surface are rarely defined with atomic precision, making it difficult to control reaction selectivity with the molecular precision of homogeneous systems. In colloidal nanoparticle synthesis, structural control is accomplished using a surface ligand or capping layer that stabilizes a specific particle morphology and prevents nanoparticle aggregation. Usually, these surface ligands are considered detrimental for catalysis because they occupy otherwise active surface sites. However, a number of examples have shown that surface ligands can play a beneficial role in defining the catalytic environment and enhancing performance by a variety of mechanisms. This perspective summarizes recent advances and opportunities using surface ligands to enhance the performance of nanocatalysts for electrochemical CO2 reduction. Several mechanisms are discussed, including selective permeability, modulating interfacial solvation structure and electric fields, chemical activation, and templating active site selection. These examples inform strategies and point to emerging opportunities to design nanocatalysts toward molecular level control of electrochemical CO2 conversion.

Isolation Methods Influence the Protein Corona Composition on Gold-Coated Iron Oxide Nanoparticles.

Upon exposure to a biological environment, nanoparticles (NPs) acquire biomolecular coatings, the most studied of which is the protein corona. This protein corona gives NPs a new biological identity that will determine various biological responses including cellular uptake, biodistribution, and toxicity. The standard method to isolate NPs from a biological matrix in order to study their coronas is centrifugation, but more gentle means of retrieval may enable deeper understanding of both irreversibly bound hard coronas and more loosely bound soft coronas. In this study, magnetic gold-coated iron oxide NPs were incubated with rainbow trout gill cell total protein extracts and mass spectrometric proteomic analysis was conducted to determine the composition of the protein coronas isolated by either centrifugation or magnetic retrieval. The number of washes were varied to strip away the soft coronas and isolate the hard corona. Hundreds of proteins were adsorbed to the NPs. Some proteins were common to all isolation methods and many others were particular to the isolation method. Some qualitative trends in protein character were discerned from quantitative proteomic analyses, but more importantly, a new kind of protein corona was identified, mixed corona, in which the labile or inert nature of the protein-NP interaction is dependent upon sample history.

Surface-Enhanced Raman Spectroscopy-Scanning Electrochemical Microscopy: Observation of Real-Time Surface pH Perturbations.

Understanding and controlling chemical dynamics at electrode interfaces is key to electrochemical applications in sensing, electrocatalysis, and energy storage. Here, we introduce colocalized surface-enhanced Raman scattering-scanning electrochemical microscopy (SERS-SECM) as a multimodal tool able to simultaneously probe and affect electrochemical interfaces in real time. As a model system to demonstrate SERS-SECM, we used a self-assembled monolayer of 4-mercaptopyridine (4MPy), a pH sensitive Raman indicator, anchored to silver nanoparticles as a substrate. We modulated the local pH at the surface with chronoamperometry, inducing the hydrogen evolution reaction (HER) at the SECM tip and observed subsequent Raman peak height changes in the 4MPy. We then performed cyclic voltammetry of HER at the SECM tip while measuring SERS spectra every 200 ms to highlight the technique's real-time capabilities. Our results show the capability to sensitively interrogate and trigger chemical/electrochemical dynamic surface phenomena. We hope SERS-SECM will provide insight on the link between heterogeneous and homogeneous reactivity at electrochemical interfaces.

How Do Proteins Associate with Nanoscale Metal-Organic Framework Surfaces?

It is well known that colloidal nanomaterials, upon exposure to a complex biological medium, acquire biomolecules on their surface to form coronas. Porous nanomaterials present an opportunity to sequester biomolecules and/or control their orientation at the surface. In this report, a metal-organic framework (MOF) shell around gold nanorods was compared to MOF nanocrystals as potential protein sponges to adsorb several common proteins (lysozyme, beta-lactoglobulin-A, and bovine serum albumin) and potentially control their orientation at the surface. Even after correction for surface area, MOF shell/gold nanorod materials adsorbed more protein than the analogous nanoMOFs. For the set of proteins and nanomaterials in this study, all protein-surface interactions were exothermic, as judged by isothermal titration calorimetry. Protein display at the surfaces was determined from limited proteolysis experiments, and it was found that protein orientation was dependent both on the nature of the nanoparticle surface and on the nature of the protein, with lysozyme and beta-lactoglobulin-A showing distinct molecular positioning.

Virus-Sized Gold Nanorods: Plasmonic Particles for Biology.

Plasmons, collective oscillations of conduction-band electrons in nanoscale metals, are well-known phenomena in colloidal gold and silver nanocrystals that produce brilliant visible colors in these materials that depend on the nanocrystal size and shape. Under illumination at or near the plasmon bands, gold and silver nanocrystals exhibit properties that enable fascinating biological applications: (i) the nanocrystals elastically scatter light, providing a straightforward way to image them in complex aqueous environments; (ii) the nanocrystals produce local electric fields that enable various surface-enhanced spectroscopies for sensing, molecular diagnostics, and boosting of bound fluorophore performance; (iii) the nanocrystals produce heat, which can lead to chemical transformations at or near the nanocrystal surface and can photothermally destroy nearby cells. While all the above-mentioned applications have already been well-demonstrated in the literature, this Account focuses on several other aspects of these nanomaterials, in particular gold nanorods that are approximately the size of viruses (diameters of ∼10 nm, lengths up to 100 nm). Absolute extinction, scattering, and absorption properties are compared for gold nanorods of various absolute dimensions, and references for how to synthesize gold nanorods with four different absolute dimensions are provided. Surface chemistry strategies for coating nanocrystals with smooth or rough shells are detailed; specific examples include mesoporous silica and metal-organic framework shells for porous (rough) coatings and polyelectrolyte layer-by-layer wrapping for "smooth" shells. For self-assembled-monolayer molecular coating ligands, the smoothest shells of all, a wide range of ligand densities have been reported from many experiments, yielding values from less than 1 to nearly 10 molecules/nm2 depending on the nanocrystal size and the nature of the ligand. Systematic studies of ligand density for one particular ligand with a bulky headgroup are highlighted, showing that the highest ligand density occurs for the smallest nanocrystals, even though these ligand headgroups are the most mobile as judged by NMR relaxation studies. Biomolecular coronas form around spherical and rod-shaped nanocrystals upon immersion into biological fluids; these proteins and lipids can be quantified, and their degree of adsorption depends on the nanocrystal surface chemistry as well as the biophysical characteristics of the adsorbing biomolecule. Photothermal adsorption and desorption of proteins on nanocrystals depend on the enthalpy of protein-nanocrystal surface interactions, leading to light-triggered alteration in protein concentrations near the nanocrystals. At the cellular scale, gold nanocrystals exert genetic changes at the mRNA level, with a variety of likely mechanisms that include alteration of local biomolecular concentration gradients, changes in mechanical properties of the extracellular matrix, and physical interruption of key cellular processes-even without plasmonic effects. Microbiomes, both organismal and environmental, are the likely first point of contact of nanomaterials with natural living systems; we see a major scientific frontier in understanding, predicting, and controlling microbe-nanocrystal interactions, which may be augmented by plasmonic effects.

Surface Coating Structure and Its Interaction with Cytochrome c in EG6-Coated Nanoparticles Varies with Surface Curvature.

The composition, orientation, and conformation of proteins in biomolecular coronas acquired by nanoparticles in biological media contribute to how they are identified by a cell. While numerous studies have investigated protein composition in biomolecular coronas, relatively little detail is known about how the nanoparticle surface influences the orientation and conformation of the proteins associated with them. We previously showed that the peripheral membrane protein cytochrome c adopts preferred poses relative to negatively charged 3-mercaptopropionic acid (MPA)-gold nanoparticles (AuNPs). Here, we employ molecular dynamics simulations and complementary experiments to establish that cytochrome c also assumes preferred poses upon association with nanoparticles functionalized with an uncharged ligand, specifically ω-(1-mercaptounde-11-cyl)hexa(ethylene glycol) (EG6). We find that the display of the EG6 ligands is sensitive to the curvature of the surface-and, consequently, the effective diameter of the nearly spherical nanoparticle core-which in turn affects the preferred poses of cytochrome c.

Gold nanorod impact on mechanical properties of stretchable hydrogels.

Double-network hydrogels have attracted much attention because of their superior mechanical properties, which are more similar to rubbers and soft tissues than classic hydrogels. In this report, plasmonic gold nanorods (AuNRs) were incorporated into a stretchable double-network hydrogel, composed of alginate and acrylamide. The impact of gold nanorod concentration and surface chemistry on bulk mechanical properties such as Young's modulus and elongation at break was investigated. AuNRs with three different surface chemistries, cetyltrimethylammonium bromide, thiolated poly(ethylene glycol), and 11-mercaptoundecanoic acid were successfully dispersed into alginate/polyacrylamide hydrogels. The AuNR-loaded hydrogels could be reversibly stretched, leading to AuNR reversible alignment along the stretch direction as judged by polarized optical spectroscopy. With the proper surface chemistry, hydrogel nanorod composites were able to be stretched to more than 3000% their initial length without fracturing. These results show that plasmonic gold nanorods can be well dispersed in multi-component polymer systems, certain surface chemistries can enhance the bulk mechanical properties, and AuNR orientation can be controlled through varying strains on the matrix.

Ultrasonic Nebulization for TEM Sample Preparation on Single-Layer Graphene Grids.

Spray-coating using ultrasonic nebulization is reported for depositing nanoparticles on a TEM grid without many of the drying artifacts that are often associated with drop-casting. Spray-coating is suitable for preparing TEM samples on fragile support materials, such as suspended single-layer graphene, that rupture when samples are prepared by drop-casting. Additionally, because ultrasonic nebulization produces uniform droplets, nanoparticles deposited by spray-coating occur on the TEM grid in clusters, whose size is dependent on the concentration of the nanoparticle dispersion, which may allow the concentration of nanoparticle dispersions to be estimated using TEM.

Quantitative Imaging of Organic Ligand Density on Anisotropic Inorganic Nanocrystals.

A longstanding challenge in nanoparticle characterization is to understand anisotropic distributions of organic ligands at the surface of inorganic nanoparticles. Here, we show that using electron energy loss spectroscopy in an aberration-corrected scanning transmission electron microscope we can directly visualize and quantify ligand distributions on gold nanorods (AuNRs). These experiments analyze dozens of particles on graphene substrates, providing insight into how ligand binding densities vary within and between individual nanoparticles. We demonstrate that the distribution of cetyltrimethylammonium bromide (CTAB) on AuNRs is anisotropic, with a 30% decrease in ligand density at the poles of the nanoparticles. In contrast, the distribution of (16-mercaptohexadecyl)trimethylammonium bromide (MTAB) is more uniform. These results are consistent with literature reported higher reactivity at the ends of CTAB-coated AuNRs. Our results demonstrate the impact of electron spectroscopy to probe molecular distributions at soft-hard interfaces and how they produce spatially heterogeneous properties in colloidal nanoparticles.

Solution NMR Analysis of Ligand Environment in Quaternary Ammonium-Terminated Self-Assembled Monolayers on Gold Nanoparticles: The Effect of Surface Curvature and Ligand Structure.

We report a solution NMR-based analysis of (16-mercaptohexadecyl)trimethylammonium bromide (MTAB) self-assembled monolayers on colloidal gold nanospheres (AuNSs) with diameters from 1.2 to 25 nm and gold nanorods (AuNRs) with aspect ratios from 1.4 to 3.9. The chemical shift analysis of the proton signals from the solvent-exposed headgroups of bound ligands suggests that the headgroups are saturated on the ligand shell as the sizes of the nanoparticles increase beyond ∼10 nm. Quantitative NMR shows that the ligand density of MTAB-AuNSs is size-dependent. Ligand density ranges from ∼3 molecules per nm2 for 25 nm particles to up to 5-6 molecules per nm2 in ∼10 nm and smaller particles for in situ measurements of bound ligands; after I2/I- treatment to etch away the gold cores, ligand density ranges from ∼2 molecules per nm2 for 25 nm particles to up to 4-5 molecules per nm2 in ∼10 nm and smaller particles. T2 relaxation analysis shows greater hydrocarbon chain ordering and less headgroup motion as the diameter of the particles increases from 1.2 nm to ∼13 nm. Molecular dynamics simulations of 4, 6, and 8 nm (11-mercaptoundecyl)trimethylammonium bromide-capped AuNSs confirm greater hydrophobic chain packing order and saturation of charged headgroups within the same spherical ligand shell at larger nanoparticle sizes and higher ligand densities. Combining the NMR studies and MD simulations, we suggest that the headgroup packing limits the ligand density, rather than the sulfur packing on the nanoparticle surface, for ∼10 nm and larger particles. For MTAB-AuNRs, no chemical shift data nor ligand density data suggest that two populations of ligands that might correspond to side-ligands and end-ligands exist; yet T2 relaxation dynamics data suggest that headgroup mobility depends on aspect ratio and absolute nanoparticle dimensions.

One low-dose exposure of gold nanoparticles induces long-term changes in human cells.

We report the in vitro long-term (20 wk) changes in cells exposed to well-characterized gold nanoparticles (Au NPs) with varying shapes and surface coatings under both chronic (exposure to Au NPs continuously over 20 wk) and nonchronic (initial acute cell exposure to Au NPs, followed by 20 wk in NP-free cell media) conditions. Both chronic and nonchronic Au NPs exposures at low dose induce modifications at the gene level after long periods. In attempt to overcome from the injuries caused by nanoparticle exposure, genes related to oxidative stress, cell cycle regulation, and inflammation are among those presenting differential expression levels. Surprisingly, the nonchronic exposure induced more gene expression changes than its chronic counterpart and the stress effects caused by this type of exposure were sustained even after 20 wk without any additional NP exposure. NP surface chemistry played an important role in the alteration of gene regulation. Overall, our data suggest that (i) cells can adaptively respond to chronic, low-level NP insults; (ii) the cell stress response is not reversible over time upon removal of NPs upon acute, nonchronic exposure; and (iii) polyethylene glycol is not as benign a surface chemistry as is generally supposed.

Quantification of Lipid Corona Formation on Colloidal Nanoparticles from Lipid Vesicles.

Formation of a protein corona around nanoparticles when immersed into biological fluids is well-known; less studied is the formation of lipid coronas around nanoparticles. In many cases, the identity of a nanoparticle-acquired corona determines nanoparticle fate within a biological system and its interactions with cells and organisms. This work systematically explores the impact of nanoparticle surface chemistry and lipid character on the formation of lipid coronas for 3 different nanoparticle surface chemistries (2 cationic, 1 anionic) on 14 nm gold nanoparticles exposed to a series of lipid vesicles of 4 different compositions. Qualitative (plasmon band shifting, ζ-potential analysis, dynamic light scattering on the part of the nanoparticles) and quantitative (lipid liquid chromatography/mass spectrometry) methods are developed with a "pull-down" scheme to assess the degree of lipid corona formation in these systems. In general, cationic nanoparticles extract 60-95% of the lipids available in vesicles under the described experimental conditions, while anionic nanoparticles extract almost none. While electrostatics apparently dominate the lipid-nanoparticle interactions, primary amine polymer surfaces extract more lipids than quaternary ammonium surfaces. Free cationic species can act as lipid-binding competitors in solution.

Peripheral Membrane Proteins Facilitate Nanoparticle Binding at Lipid Bilayer Interfaces.

Molecular understanding of the impact of nanomaterials on cell membranes is critical for the prediction of effects that span environmental exposures to nanoenabled therapies. Experimental and computational studies employing phospholipid bilayers as model systems for membranes have yielded important insights but lack the biomolecular complexity of actual membranes. Here, we increase model membrane complexity by incorporating the peripheral membrane protein cytochrome c and studying the interactions of the resulting membrane systems with two types of anionic nanoparticles. Experimental and computational studies reveal that the extent of cytochrome c binding to supported lipid bilayers depends on anionic phospholipid number density and headgroup chemistry. Gold nanoparticles functionalized with short, anionic ligands or wrapped with an anionic polymer do not interact with silica-supported bilayers composed solely of phospholipids. Strikingly, when cytochrome c was bound to these bilayers, nanoparticles functionalized with short anionic ligands attached to model biomembranes in amounts proportional to the number of bound cytochrome c molecules. In contrast, anionic polymer-wrapped gold nanoparticles appeared to remove cytochrome c from supported lipid bilayers in a manner inversely proportional to the strength of cytochrome c binding to the bilayer; this reflects the removal of a weakly bound pool of cytochrome c, as suggested by molecular dynamics simulations. These results highlight the importance of the surface chemistry of both the nanoparticle and the membrane in predicting nano-bio interactions.

Oxidation State of Capping Agent Affects Spatial Reactivity on Gold Nanorods.

Despite enormous progress toward controlling the shapes and surface chemistry of colloidal nanoparticles, spatial control of nanoparticle surface chemistry remains a major challenge. In recent years, there have been tantalizing reports demonstrating anisotropic silica coating of gold nanorods in which silica is deposited only on the sides by functionalizing the nanorods with poly(ethylene glycol) methyl ether thiol (PEG-thiol) prior to silica coating, but such results have been difficult to reproduce. We report that the oxidation state of PEG-thiol is key to anisotropic silica coating, with the disulfide, not the thiol, leading to side silica coating. PEG-disulfide appears to selectively functionalize the ends of gold nanorods, and robust methods are developed to reliably deposit side silica shells on PEG-disulfide functionalized gold nanorods.