WO2014046807A1

WO2014046807A1 - Hydrophobic shielding for enhanced nanoparticle stability

Info

Publication number: WO2014046807A1
Application number: PCT/US2013/054628
Authority: WO
Inventors: Konstantin Sokolov; Timothy Larson; Andrew Ellington
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 2012-09-20
Filing date: 2013-08-13
Publication date: 2014-03-27

Abstract

Nanoparicles comprising hydrophobic shielding layers.

Description

HYDROPHOBIC SHIELDING FOR ENHANCED NANOPARTICLE STABILITY

Statement of Government Interest

[01 ] This invention was made with support under Grant Number RO 1 EB008101 and R21 CA 135315 awarded by National Institute of Health. The U.S. government has certain rights in the invention.

Cross-Reference to Related Applications

[02] This Application claims the benefit of U.S. Provisional Application Serial No. 61/703,629 filed on September 20, 2012, which is incorporated by reference.

BACKGROUND

[03] Gold nanoparticles (GNPs) have been proposed as optical contrast agents and drug carriers for a variety of biochemical and biomedical applications. Their strong and tunable optical properties, in combination with an easily modified surface chemistry, have spurred this interest. These applications generally require modifying the surface chemistry in order to render the nanoparticles biocompatible, to provide functionality, and to stabilize the nanoparticle suspension against unwanted aggregation in biological media. A variety of modifications have been investigated for this purpose, including combinations of proteins, self-assembled monolayers of small molecules, and natural or synthetic polymers. The polymer polyethylene glycol (PEG), modified to have a thiol group on one end (methoxy-PEG-thiol or mPEG-SH), has found wide use in surface modification due to its lack of toxicity, resistance to protein adsorption, and ease with which it forms monolayers on gold surfaces. PEG has been used in drug formulations for decades, and has been shown to increase the blood half-life of a variety of pharmaceutically active compounds without adverse toxic effects.

[04] There have been a number of studies investigating the stability, toxicity, protein and cellular interactions, and in vivo biodistribution of PEGylated gold nanoparticles. Multiple studies have shown that PEGylation reduces protein adsorption and can reduce cellular uptake of nanoparticles. PEGylated gold spheres have been used as negative controls for cellular interaction, demonstrating that PEGylated spheres generally do not bind to a variety of cancer cell lines in the absence of a specific targeting ligand. [05] It has been widely reported that the liver and spleen accumulate the majority of systemically injected nanoparticles, and that nanoparticle clearance is dependent on both size and surface chemistry. PEGylated gold nanoshells were demonstrated as the first plasmonic nanoparticle based agents for photothermal therapy of a cancer in vivo, with their tumor accumulation attributed to the enhanced permeability and retention (EP ) effect.

[06] It has been shown that gold nanoparticle conjugates can be destabilized by thiol- containing small molecules. A PEG layer on nanoparticles can reduce adsorption of serum proteins and can diminish non-specific cellular interactions; it also increases the blood half-life that can improve the tumor targeting efficiency of a nanoparticle formulation. However, surface coatings formed using thiol conjugation chemistry can be displaced by thiol-containing small molecules, and the essential amino acid cysteine is present in the blood.

DRAWINGS

[07] A more complete understanding of this disclosure may be acquired by referring to the following description taken in combination with the accompanying figures in which:

[08] FIGURE 1 shows, top left: TEM image of GNP. The average size by TEM was 17.1 +/- 1.33; Top right: Three reconstructed size distributions of stock particles via DLS. The cumulants analysis of DLS for the same sample shows a diameter of 19.7 +/- 0.15 and a polydispersity of .068 +/- .003; Bottom: UV-Vis spectra of the stock GNP, GNP-PEG, and GNP- alkyl-PEG.

[09] FIGURE 2 shows displacement of FITC-PEG-SH from GNP surface by solutions with different concentrations of cysteine in PBS as a function of time. Schematic illustrates cysteine displacing FITC-PEG-SH, which leads to increased fluorescence, as indicated by the DTT control. Note that 200 and 400 μΜ cysteine lines overlap.

[ 10] FIGURE 3 shows DLS size distribution of freshly prepared GNP-PEG (black, solid line) and GNP-alkyl-PEG (grey, solid line) in complete media.

[11] FIGURE 4 shows hydrodynamic diameter of GNP-PEG (black) and GNP-alkyl- PEG (grey) as a function of time over a 5 days period in complete media (DMEM) with 5% FBS. The observed standard deviation was less than 1% of the measured diameters. [12] FIGURE 5 shows the results of a viability assay conducted using MTS reagent. No significant difference was found between any of the samples. Each sample is normalized relative to the cell only control

[13] FIGURE 6 shows Protein adsorption to GNP-PEG (dotted white) and GNP-alkyl- PEG (grey) over a period of 5 days in complete media with 5% FBS. The negative controls contained particles that had not been exposed to serum.

[14] FIGURE 7 shows brightfield (a, b) and darkfield (c, d) images of cells incubated with either freshly prepared GNP-PEG (a, c) or GNP-PEG pre-incubated in media for 2 days (b, d). Nanoparticles appear red in brightfield transmittance mode and green to orange in darkfield reflectance mode. Scale bar is ca. 20 μιη.

[15] FIGURE 8 shows transmitted brightfield images of cells incubated for 24 hours with either GNP-PEG (top row) or GNP-alkyl-PEG (bottom row)). The nanoparticles were pre- incubated in complete media for 1 day, 3 days and 5 days. The presence of nanoparticles is readily apparent in cells treated with GNP-PEG as a dark contrast due to light absorption by the particles, while there is no detectable particles visible in the GNP-alkyl-PEG samples. Scale bar is ca. 20 um.

[16] FIGURE 9 shows raw absorbance spectra of GNP-PEG and GNP-alkyl-PEG after incubation with cells. The time indicates how long the particles were pre-incubated in media before adding particles to cells. The aggregation of the 1 day GNP-PEG sample is noticeable, while the 3 day GNP-PEG sample indicates significantly higher uptake than all other samples.

[17] FIGURE 10 shows spectra of nanoparticles after incubation with cells normalized to one at the absorption maximum. The time indicates how long the particles were pre-incubated in media before adding particles to cells. The normalization makes the aggregation of the GNP- PEG particles at 1 day and 3 days more noticeable. There is no noticeable aggregation in GNP- alkyl-PEG

[18] FIGURE 11 shows a schematic illustration of the displacement of mPEG-SH molecules in GNP-PEG by small thiolated molecules, e.g. cysteine and cysteine, leading to significant adsorption of proteins (upper row). The interactions between nanoparticles and serum proteins is altered when a hydrophobic shield is inserted between the thiol moiety and the outer mPEG layer in GNP-alkyl-PEG (bottom row). The hydrophobic shield drastically reduces non-specific interactions of the GNP-alkyl-PEG with macrophages. [19] FIGURE 12 shows UV-Vis spectra of gold nanoparticle solutions in complete media after 1, 3, and 5 days. The spectra demonstrate that there is no significant aggregation of the particles in the media.

[20] FIGURE 13 shows top: Sample spectral curves from known quantities of serum albumin mixed with Coomassie Blue. The amount of protein is determined from an increase in the absorption of the dye at 595 nm. Bottom: A calibration curve of the OD at 595 nm with a linear fit to the curve over the protein concentration range of interest. A calibration curve was generated for every experiment.

[21 ] FIGURE 14 shows hydrodynamic diameter of GNP-PEG (black) and GNP-alkyl- PEG (grey) as a function of time over 5 days period in complete media (DMEM) with 5% FBS. The error bars are visible at this scale, and the GNP-PEG data is outside of the displayed size range on days 4 and 5.

[22] While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as defined by the appended claims.

DESCRIPTION

[23] The present disclosure generally relates to hydrophobic shielding of surfaces, and in particular surfaces with a hydrophobic shielding layer that may increase stability and reduce opsonization in biological fluids, leading to enhanced efficacy and reduced toxicity.

[24] While the present disclosure is exemplified with reference to nanoparticle surfaces, the present disclosure applies to any surface that may be implanted in a living body. Surfaces implanted or indwelling in a living body (e.g., gold nanoparticles) may opsonize quickly and, depending on the surface, may have short half-lives. This limits their suitability for use in a living body, such as, for example, their ability to be used as diagnostic and therapeutic agents. Additionally, the usage of gold particles in vitro may also be limited by their stability over a period of days. The present disclosure provides surfaces and treatments for surfaces that address these issues. [25] The present disclosure is based, in part, on the observation that the mPEG-thiol layer on GNPs can be displaced by cysteine molecules, and that mPEG-thiol modified GNPs (GNP-PEG) adsorb proteins within 24 hours of being placed in cell culture media supplemented with fetal bovine serum (FBS). The present disclosure is also based, in part, on the observation that protein adsorption to nanoparticles may be reduced by including a hydrophobic shielding layer having a small alkyl chain as a hydrophobic shield between the nanoparticle surface and the outer hydrophilic PEG layer.

[26] The hydrophobic shielding layer includes an outer layer comprising synthetic polymers that do not generally opsonize (e.g., PEG) and an inner layer formed from a small hydrophobic molecule (e.g., alkyl chain). The inner layer is proximate to the surface. The inner layer may be covalently bound to the surface or associated to the surface. In operation, the hydrophobic shielding layer increases the surface's compatibility with biological fluids and reduces opsonization in biological fluids, which may lead to enhanced efficacy and reduced toxicity.

[27] One aspect of the present invention is directed to a composition comprising a nanoparticle having a surface and a hydrophobic shielding layer disposed on the surface, wherein the hydrophobic shielding layer comprises a hydrophilic outer layer and a hydrophobic inner layer.

[28] In one embodiment, the nanoparticle is a metal nanoparticle.

[29] In another embodiment, the metal nanoparticle is a gold nanoparticle.

[30] In another embodiment, the nanoparticle is a magnetic or semiconducting nanoparticle.

[31] Examples of suitable inner layers include, but are not limited to alkyl chains. The size of the layer can vary from few nanometers (typically in the range of 1-20 nm) for nanoparticles to microns and tens of microns for other material surfaces including, but not limited to, implantable devices and sensors.

[32] For purposes of this patent application, the term "alkyl" or "small alkyl" means a saturated or unsaturated aliphatic hydrocarbon group which may be straight or branched having 1 to 20 carbon atoms in the chain. "Branched" means that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkyl chain. The alkyl may be substituted with one or more alkyl group substituants, which may be the same or different, and include for instance halo, cycloalkyl, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy.

[33] Examples of suitable outer layer polymers include, but are not limited to, polymers comprising poly ethylene glycol (e.g., PEG and derivatives of PEG), poly(ethylene oxide), polyvinyl alcohol, poly lactic acid, poly(oxazoline), polyglycerol, poly(N-(2- hydroxypropyl)methacrylamide)), and poly-N-vinylpyrrolidone.

[34] The outer layer also may comprise one or more targeting moieties, diagnostic moieties (e.g., fluorescence dyes), or therapeutic agents embedded or associated with the outerlayer polymers. For example, the outer layer may comprise targeting moieties such as, for example, peptides, antibodies or antibody fragments, small molecules (e.g., folic acid (folate)), aptamers, targeting ligands (e.g., transferring), macromolecules (e.g., hyaluronic acid, which bind to cluster determinant 44 overexpressed in various tumors). The outer layer may comprises therapeutic agents or therapeutic moieties such as, for example, genes (or gene fragments) siPv A, one or more drugs.

[35] In one embodiment, the hydrophilic outer layer comprises a synthetic polymer.

[36] In another embodiment, the hydrophilic outer layer comprises a biological polymer such as an antibody, a nucleic acid, a peptide, or an aptamer.

[37] In another embodiment, the hydrophilic outer layer comprises a mixture of synthetic and biological polymers as described above.

[38] In another embodiment, the hydrophobic inner layer comprises a saturated or unsaturated alkyl chain.

[39] In another embodiment, the hydrophobic inner layer is covalently bound to the surface.

[40] In certain embodiments, the surface may be the surface of an implantable material or device exposed to bodily fluids. For example, the surface may be a surface of a particle (e.g., nanoparticle), an implantable sensor or monitor, a stent (e.g., a cardiac stent), a catheter, or any other at least partially implantable or at least partially indwelling biomaterial or medical device.

[41] In certain embodiments, the present disclosure provides compositions comprising a nanoparticle with a hydrophobic shielding layer. The nanoparticle is formed at least in part by one or more metals, such as gold, although other materials are also suitable. Examples of suitable metals include, but are not limited to, Au, Ag, Pt, Ti, Al, Si, Ge, Cu, Cr, W, Fe, or a corresponding oxide. Suitable nanoparticles may have any desired geometry. For example, suitable nanoparticles may be spherical, or may have other shapes such as rods, ellipsoids, triangle, and stars.

[42] Another aspect of the present invention is directed to a method comprising providing a surface and adding to the surface a hydrophobic shielding layer comprising a hydrophilic outer layer and a hydrophobic inner layer.

[43] Another aspect of the present invention is directed to a method comprising providing a surface and preventing or reducing interactions between nanoparticles as described above and an immune system by providing on the surface of the nanoparticles a hydrophobic shielding layer comprising a hydrophobic inner layer and a hydrophilic outer layer.

[44] In certain embodiments, the hydrophobic inner layer comprises either saturated or unsaturated alkyl chains.

[45] In another embodiment, the hydrophilic outer layer comprises mPEG.

[46] In other embodiments, the hydrophilic outer layer comprises a mixture of mPEG molecules and one or a composition of antibodies, nucleic acids, peptides, and aptamers.

[47] In another embodiment, the surface is a surface of a nanoparticle, a biomedical material, or an at least partially indwelling medical device.

[48] Another aspect of the present invention is directed to a method of improving stability of a metal nanoparticle comprising applying a hydrophobic shielding layer to the surface of the metal nanoparticle. The metal nanoparticle may be a gold nanoparticle. The hydrophobic shielding layer may comprise a hydrophobic inner layer and a hydrophilic outer layer.

[49] In one embodiment, the metal nanoparticle is a magnetic or semiconducting nanoparticle.

[50] Another aspect of the present invention is directed to a method of preventing or reducing protein adsorption on a metal nanoparticle comprising applying a hydrophobic shielding layer to the surface of the metal nanoparticle. The metal nanoparticle may be a gold nanoparticle. The hydrophobic shielding layer may comprise a hydrophobic inner layer and a hydrophilic outer layer.

[51] In one embodiment, the metal nanoparticle is a magnetic or semiconducting nanoparticle. [52] To facilitate a better understanding of the present invention, the following examples of certain aspects of some embodiments are given. In no way should the following examples be read to limit, or define, the entire scope of the invention.

EXAMPLES

[53] Materials and Methods

[54] Materials. All reagents were purchased from Sigma-Aldrich unless mentioned otherwise, and used without further purification. Purified 18.2 ΜΩ water was obtained using a Millipore Direct-Q 3 system. Methoxy-PEG-thiol, 5 kDa and 10 kDa, was obtained from Creative PEGworks. Coomassie Plus Protein Assay Reagent for use in the Bradford assay was purchased from Pierce.

[55] Instrumentation. Dynamic light scattering measurements were carried out using disposable cuvettes in a DelsaNano (Beckman Coulter). Size distribution reconstructions were obtained using the supplied NNLS algorithm. The cumulants analysis in the DelsaNano software package was used to obtain the average sizes in the week long study. Each size measurement was done using 300 acquisitions and 3 repetitions to ensure that the DLS measurements were reproducible. UV-Vis measurements and fluorescence kinetic data were acquired using an HT Synergy Plate Reader. Microscopy images were collected on a Leica 6000DM upright microscope using a 20x objective and a SPOT color imaging camera. A halogen and a Xe lamps were used to collect bright-field transmittance and dark-field reflectance images, respectively, from samples on covers lips. Twenty foure well plates were imaged directly on an inverted Leica DM3000 B microscope with a Leica DFC290 camera, a 20x objective and a halogen lamp.

[56] Synthesis of mPEG-alkyl-SH and PEGylation of GNPs. We used a reaction protocol that was based on standard NHS cross-linking reactions available from Pierce.

Methoxy-PEG-thiol (mPEG-SH) was dissolved in water at 500 μΜ and stored at -20°C. A mPEG-alkyl-SH was synthesized using a mercaptododecanoic acid n-hydroxysuccinimidyl ester (MDDA-NHS, Sigma) and a 10 kDa methoxy-PEG-amine. The MDDA-NHS was dissolved in dimethyl formamide at 100 mM concentration. Then, 200 μί of MDDA-NHS was added to 2 mL of an aqueous solution of methoxy-PEG-amine at 1 mM. A significant amount of precipitate formed upon the addition of MDDA-NHS to the aqueous solution, which is commonly observed when hydrophobic NHS molecules are added to aqueous solutions. The reaction was left overnight, and then the 2 mL solution was dialyzed twice against 1.5 L of water to remove excess MDDA-NHS. The remaining precipitated MUDA-NHS was removed via centrifugation at 18,000 g for 30 minutes, and the supernatant containing methoxy-PEG-alkyl-thiol was aliquoted and stored at -20 °C.

[57] Modification of GNPs with either mPEG-SH or mPEG-alkyl-SH was

accomplished by adding either one of the PEG stock solution to GNP solution (1.64 nM) to reach the final PEG concentration of 24 uM. The reaction mixture was incubated at room temperature for 30 minutes, and then GNP-PEGs or GNP-alkyl-PEGs were centrifuged at 14,000 g for 60 minutes at 4°C. The pellets containing nanoparticles were redispersed in either cell culture media (DMEM supplemented with 5% fetal bovine serum) or phosphate buffered saline (PBS) for future studies.

[58] The density of mPEG-SH on GNPs was assayed using reduction of 5,5'- dithiobis(2-nitrobenzoic acid (Ellman's reagent, Pierce). Briefly, gold nanoparticles were pelleted by centrifugation after incubation with mPEG-SH and the concentration of free mPEG- SH molecules in the supernatant was compared to a control solution of mPEG-SH that was not incubated with nanoparticles. The total amount of PEG molecules adsorbed on nanoparticles was obtained by subtracting the amount of mPEG-SH in the supernatant solution from the control mPEG-SH solution. Then the PEG density was calculated by dividing the total amount of adsorbed PEG molecules by the number of nanoparticles and an average surface area of a single nanoparticle.

[59] Fluorescence kinetics assay. GNP-PEG-FITC were synthesized by mixing GNP with a mixture of mPEG-SH (5 kDa, Creative PEGworks) and FITC-PEG-SH (5 kDa, Nanocs) at a ratio of 4: 1 mPEG:FITC-PEG, and left to react for 30 minutes. The mixture was used to prevent aggregation during the centrifugation washing step (12,000 g, 60 minutes), after which nanoparticles were resuspended in PBS. Small aliquots of either cysteine, DTT, or PBS were placed in wells of a black fluorescence 96 well plate, and then the same amount of GNP-PEG- FITC solution in PBS was rapidly added to each well and a fluorescence kinetics assay was begun on the HT Synergy plate reader. The 475-495nm excitation and the 518-538nm emission filters were used for measurements of FITC fluorescence.

[60] Protein Adsorption Assays. All GNP samples were sterile-filtered through a 0.2 micron filter. Particles for DLS measurements were placed in a sterilized plastic cuvette with the top covered in foil then wrapped in parafilm to prevent contamination, then measured daily. As shown in Figure 12, there was no observed precipitation or aggregation of nanoparticles.

[61] Particles used in the Bradford assay were split into three separate 400 aliquots prior to incubation in complete media. At each time point (1, 3 or 5 days) the particles were washed in PBS three times, with dilution of the pellet out to 5 mL during each step. This washing was required to sufficiently remove all media proteins. After the final washing step the particles were resuspended in 200 μΐ, PBS, and then three separate 150 μΐ, aliquots were mixed with 150 μΐ. of Coomassie Plus reagent as received from Pierce. Particle only controls were prepared by mixing 50 μΐ, of each GNP sample with 250 μΐ, of PBS to account for potential losses during centrifugation. The spectra of the particle controls were subtracted from the spectra of the particles mixed with Coomassie reagent. Calibration curves were generated using a series dilution of the serum albumin provided with the Coomassie Plus kit from Pierce

[62] Cell Culture and Media experiments. Cells were grown and passaged in DMEM media supplemented with 5% FBS. All experiments with nanoparticles were conducted in phenol-free media. The initial assays with GNP-PEG were carried out using coverslips.

Coverslips were sterilized in 70% ethanol, rinsed in a sterile PBS, and then placed in 6 well plates. Each well was seeded at 200,000 cells/well and left to grow overnight. The old media was removed and then 1 mL of either fresh or pre -incubated GNP-PEGs at 1.4 nM were added to the cells for 4 hours.

[63] Twenty four hours assays with GNP-PEGs and GNP-alkyl-PEGs were conducted in triplicate in 24 well plates with 3 samples per particle per time point. For this assay, cells were seeded at 50,000 cells/well. 400 μί of each particle sample at 1.4 nM was incubated with cells for 24 hours prior to optical imaging.

[64] The viability assay was conducted on a 96 well plate. Each sample was run in six replicates.. Cells were seeded at 5000 cells/well, left to grow overnight, and then incubated with either media, GNP-PEG, GNP-alkyl-PEG, or the mPEG-SH and mPEG-alkyl-SH molecules. The nanoparticles were used at stock concentration of ca. 1.4 nM and PEG molecules at

concentrations of 10 nM and 1 μΜ; the upper concentration of PEG molecules was chosen to approximately match concentration of PEG molecules conjugated to 1.4 nM solution of 18 nm diameter particles according to a published data on PEG density on GNP.²⁹ Cells were washed in media, 100 μΕ of fresh media was added to each well, then a background absorbance was acquired at 490 nm. MTS reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium) was prepared at 2 mg/mL in PBS and phenazine methosulfate (PMS) was prepared at 0.92 mg/mL in PBS. The MTS and PMS solutions were mixed in a 20: 1 ratio, then 20 μΐ, of this mixture was added to each well and allowed to react for 3 hours before measurements were taken at 490 nm. The background absorbance was subtracted and the optical density of each sample was normalized relative to the cell only control.

[65] Results.

[66] Effect of cysteine on mPEG-SH gold nanoparticle coating. Citrate-capped gold spheres were synthesized following the Turkevich method by adding sodium citrate to a refluxed solution of chlorauric acid. Turkevich, J.; Stevenson, P. C; Hillier, J. The Formation of Colloidal Gold. The Journal of Physical Chemistry 1953, 57, 670-673. Recent literature results demonstrate that citrate plays a role both as a precursor to the nucleating reagent and as a buffer to control the pH. The reactivity of gold ions increases with decreasing pH, and this effect is responsible for the ellipticity observed with the synthesis of larger particles using the Frens method. Frens, G. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Colloid Suspensions. Nature 1973, 241, 20-22. Our reaction ratios were controlled such that the synthesis proceeded slowly, requiring approximately 10 minutes to complete. The resulting gold colloid was characterized by transmission electron microscopy (TEM), optical spectroscopy, and dynamic light scattering (DLS) (Figure 1). The DLS and TEM measurements were in good agreement, with a typical gold core size of 18 nm. The DLS measurements were consistently found to be 1-2 nm larger than the sizes derived from TEM images, which is consistent with a citrate layer causing an increase in the hydrodynamic radius. We assume that the gold nanoparticle synthesis goes to completion with the reaction resulting in a gold particle concentration of 1.64 nM giving the size of ca. 17 nm as determined by TEM (Figure 1).

[67] In order to test the impact of cysteine on PEGylated GNPs, the particles were coated with a mixture of 5 kDa mPEG-SH and FITC-PEG-SH at a 4: 1 ratio. The mixture was required to avoid aggregation, as a coating of 100% FITC-PEG-SH resulted in aggregation of GNPs upon centrifugation. This is not unexpected because even though FITC molecule does not have a strong binding energy with itself, the effect of small binding energies across multiple moieties has been shown to induce aggregation of nanoparticles in other situations. The

PEGylated GNPs were resuspended in PBS, and varying amounts of cysteine and a DTT control were added to aliquots of the GNP-PEG-FITC solution, then the fluorescence intensity was recorded over time. DTT was used as a control because of its ability to efficiently displaced thiolated compounds from gold surfaces. Incubation of GNP-PEG-FITC with DTT at a high concentration resulted in an increase in fluorescence intensity of the suspension that indicates release of the FITC-PEG-SH molecules from gold nanoparticles. The observed increase in fluorescence signal is due to fluorescence quenching near gold surfaces that is well documented. The experiment demonstrated that increasing concentrations of cysteine led to increasing amounts of displaced PEG molecules, with a pronounced effect even at concentration of cysteine as low as 25 μΜ (Figure 2). Based on previously published literature, we assumed that the PEG was completely displaced after 1 hour exposure to 47 mM DTT. The fluorescence data in Figure 1 are linearly rescaled to show the percentage of displaced FITC-PEG-SH molecules from 0% displacement for the GNP control sample to 100% displacement for GNPs treated with DTT.

[68] The fact that cysteine readily displaces densely packed mPEG-SH layers on GNP is somewhat surprising given the resistance of PEG coatings to adsorption of large molecules. We determined the density of mPEG-SH molecules on the gold particle surface, using a previously published procedure. Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C. W. Nanoparticle Size and Surface Chemistry Determine Serum Protein Adsorption and Macrophage Uptake. Journal of the American Chemical Society 2011, 134, 2139-2147. Our protocol led to a density of 2.6 PEG/nm², which corresponds to the high PEG density particle group (density more than 1 PEG/nm ) tested by Walkey et ai; this density is also consistent with the highest PEG density of 3.5 PEG/nm² for 15 nm diameter gold spheres that was reported in the previous study. Some insight into this process can be gained from a review of the literature on ligand exchange mechanisms of the gold-sulfur bond. As noted by Caragheorgheopol and Chechik, gold ligand exchange mechanisms showed a "very rich and diverse chemistry". Thiols, thiolates, and disulfides can be involved in ligand exchange reactions with the specific mechanism depending on the ligand pair involved. The final equilibrium point of a ligand exchange reactions depends on both the concentration and the nature of the ligands involved, and it can take a long time before equilibrium is achieved.

[69] Conjugates of GNPs with PEG-alkyl-thiols. A recent publication by Maus et al. showed that cysteine-terminal peptides can bind directly to the surface of gold nanoparticles pre- coated with 3 kDa mPEG-SH. Maus, L.; Dick, O.; Bading, H.; Spatz, J. P.; Fiammengo, R. Conjugation of Peptides to the Passivation Shell of Gold Nanoparticles for Targeting of Cell- Surface Receptors. ACSNano 2010, 4, 6617-6628. This result shows that cysteine containing peptides are capable of penetrating through a PEG layer and interacting directly with the gold surface of PEG-coated GNPs. This observation is similar to our results with cysteine, which are described above. Maus et al. also demonstrated that the penetration effect could be avoided when an alkyl group is inserted between the PEG and the thiol moieties. Here, we assess the impact of a hydrophobic alkyl chain, which we call a hydrophobic shield, between the thiol group and the PEG portion of the polymer molecule on stabilization of gold nanoparticles in biological environment that contains small thiolated compounds such as cysteine and cystine. Methoxy-PEG-alkyl-thiol (mPEG-alkyl-SH) was synthesized by reacting a 20-fold molar excess of mercaptododecanoic acid NHS ester (MDDA-NHS) with methoxy-PEG-amine (see Methods for details). Gold nanoparticles were PEGylated by adding either mPEG-SH or mPEG-alkyl-SH molecules to a water suspension of GNPs at room temperature for 30 minutes followed by purification via centrifugation. DLS measurements of the resulting GNP-PEG and GNP-alkyl- PEG in media supplemented with 5% fetal bovine serum (complete media) are shown in Figure 2. The addition of mPEG-SH and mPEG-alkyl-SH coatings caused a slight red-shift in the plasmon peak position of ca. 2 nm and 4 nm and an increase of ca. 1.5 % and 7.6% in the peak extinction coefficient of coated nanoparticles, respectively (Figure 1). Larger optical changes associated with the mPEG-alkyl-SH layer are attributed to a higher refractive index change on the nanoparticle surface due to the alkyl moiety.

[70] Protein Adsorption on GNP-PEG and GNP-alkyl-PEG. To explore the stability of different PEG coatings in biological media, GNP-PEG and GNP-alkyl-PEG with 10 kDa mPEG moiety were synthesized, washed via centrifugation, resuspended in complete media with 5% fetal bovine serum (FBS), and sterile filtered into a clean, sterile cuvette. For these experiments 10 kDa PEG was chosen to match the particle formulation with the longest blood half-life from the data presented by Perrault et al. Perrault, S. D.; Walkey, C; Jennings, T.; Fischer, H. C; Chan, W. C. W. Mediating Tumor Targeting Efficiency of Nanoparticles through Design. Nano Lett. 2009, 9, 1909-1915. The size of the particles was measured by DLS over a period of 5 days (Figure 3). Cell culture media contains PBS, a number of divalent cations in the mM range, sulfur containing cystine and methionine, and a few other small molecules that do not have sulfur. FBS adds a much more complicated composition that includes various types of proteins and all of the small molecules found in the non-clotting potion of blood plasma.

[71 ] The GNP-PEG and GNP-alkyl-PEG showed very different behavior in complete media over the period of 5 days (Figure 4). There was no observable aggregation of either particle type as determined by UV-Vis spectra of gold nanoparticle solutions in complete media after 1, 3, and 5 days. (Figure 12). However, the size of the GNP-PEG began to gradually increase after two days (Figure 4). These changes are likely due to the formation of a protein corona around the particles, a process that was found to proceed gradually over a period of hours and days. By contrast, the GNP-alkyl-PEG maintained a constant size during the observation time, indicating that there is far less interactions between the GNP-alkyl-PEG and serum proteins. This result is consistent with our hypothesis that the alkyl group would provide a protective effect in biological media. The effectiveness of the alkyl group is not unexpected, as Maus et al. showed that the alkyl group can eliminate the binding of cysteine-terminal peptides to the gold surface. Additionally, mPEG-alkyl-SH was shown to greatly enhance the stability of silver nanoparticles in the presence of high ionic strength solvents.

[72] To further investigate interactions between PEG-coated GNPs and serum proteins we carried out an assay to directly detect protein adsorption on nanoparticle surface using a Coomassie blue reagent for a Bradford protein assay. GNP-PEG and GNP-alkyl-PEG were suspended in compete media with 5% FBS and were kept in an incubator at 37 °C and 5% C0₂ for 1 day, 3 days, and 5 days. The particles were then washed three times in PBS via centrifugation and added to either PBS or the Coomassie Plus reagent from Pierce. The PBS control was used to calculate the concentration of nanoparticles and to obtain gold nanoparticle baseline spectra. The control was also used to monitor potential aggregation of nanoparticles. Calibration curves showed that the Coomassie Plus assay is sensitive to serum albumin concentrations down to 0.5 μg mL, which is equivalent to a detection limit of approximately 15 albumin proteins per particle in our experiments (Figure 13). It is important to note that the protein corona formed on particles in serum contains many different proteins, which can lead to some variability in the response of Coomassie Plus. Control measurements carried out using GNP-PEG and GNP-alkyl-PEG that were not exposed to serum proteins confirmed that the particles alone had a significantly lower effect on the Coomassie Plus reagent when compared to particles incubated in complete media. The supernatant of the final wash step after incubation of nanoparticles with serum also did not show any presence of proteins. Each sample was split into three separate aliquots prior to the initial wash for the Bradford assay. Triplicate measurements on the same particles showed that the Bradford assay had a standard deviation less than 1%, indicating that the majority of the variation in this assay stems from particle losses during centrifugation. As can be seen in Figure 4, both GNP-PEG and GNP-alkyl-PEG have a significant amount of adsorbed proteins at 24 hours, and there is no statistically significant difference in protein adsorption between GNP-PEG and GNP-alkyl-PEG at this early time point. At later time points GNP-PEG has significantly more adsorbed protein than GNP-alkyl-PEG, which agrees with the DLS data shown in Figure 4. It is also interesting to compare the protein adsorption data with the hydrodynamic size radius measurements taken at 24 hours. While there is a significant degree of protein adsorption at this time points, it is not immediately obvious in the DLS data. However, a slight increase in the hydrodynamic diameter of the GNP-alkyl-PEG particles of about 3 nm was observed during the first few days (Figure 14). This result suggests that significant adsorption of proteins to GNP-PEG particles may not be easily detectable using DLS and that more direct protein detection methods might be required. Alternately, as the GNP- PEG particles increase in hydrodynamic size between three and five days, there is no observable increase in adsorbed proteins using the Coomassie assay. This is possibly due to the fact that this assay only detects proteins adsorbed with a relatively slow K_0ff, as the washing steps require approximately 3 hours in PBS with increasingly dilute serum concentrations. Previously, Casals et al. noted the presence of both a hard protein corona and a soft transient protein corona that is detectable by DLS measurements but detaches upon washing. Our DLS measurements and the results of Coomassie assay are consistent with the formation of a similar soft protein corona on top of a harder protein corona on GNP-PEG in media with serum proteins.

[73] Effect of pre-incubation in media on uptake by macrophages. After we had shown that cysteine disrupts the mPEG-SH layer on GNP-PEG and that GNP-PEG slowly adsorb proteins in biological media containing cystine, we carried out experiments to test the effect of the observed protein adsorption on interactions with macrophages. First, we conducted an MTS cell viability assay to determine whether PEGylated GNP or the PEG molecules themselves would have an impact on cell viability at the concentrations used in our experiments. Using pairwise t tests, we found that there was no significant difference in cell viability between control and cells incubated with either PEG-coated nanoparticles or PEG molecules (Figure 5). [74] Initially, we looked at how pre-incubation of GNP-PEG in media with serum influences their uptake by macrophages (Figure 7). There were no detectable particles in the cells incubated with freshly prepared GNP-PEG particles (Figure 7, a and c), in agreement with previous literature reporting no non-specific interactions at short time points for GNPs with high- density PEG layers. However, significant uptake of nanoparticles was observed after GNP-PEG were first pre-incubated in media for 2 days and then added to macrophage cells for a period of 4 hours (Figure 7, b and d). Our results summarized above (see Figures 2, 4 and 6) indicate that pre-incubation of GNP-PEG in media with serum leads to disruption of the PEG layer by small thiol-containing molecules that results in protein adsorption on nanoparticle surface. The adsorbed proteins are recognized by macrophage receptors leading to rapid cell uptake. Particles appear red or blue in the brightfield transmitted images depending on their aggregation state, and as various shades of green to orange in the darkfield images (Figure 7). The difference in color between the two imaging modes is caused by the fact that absorption dominates nanoparticle contrast in brightfield images while only scattering is visible in darkfield images of optically thin samples.

[75] Next, a comparison study was conducted using GNP-PEG and GNP-alkyl-PEG pre-incubated in media for 1 day, 3 days, or 5 days, and then added to cells for 24 hours. As seen in Figure 8, there is a very strong uptake in macrophages treated with the GNP-PEG sample while no detectable uptake was observed in cells treated with GNP-alkyl-PEG. The differences in cellular uptake correlate well with the assays comparing both hydrodynamic radius and protein adsorption on GNP-PEG and GNP-alkyl-PEG in complete media (see Figures 4 and 6). It should be noted that there were some degree of protein adsorption to the GNP-alkyl-PEG (Figure 6); this result and the distinct difference in cellular uptake of the two nanoparticles (see Figure 8) suggest that the nature of the protein corona could be more important than the amount of adsorbed proteins in mediating cellular interactions. Additionally, we acquired optical spectra of the particle supernatants after they had been incubated with cells for 1 day (Figure 9 and 10). The supernatant showed that the 1 day pre-incubated GNP-PEGs underwent some aggregation but the particles became increasingly stable in the complete media over time with very little aggregation by the 5 day time point (Figure 10). The observed trend can be explained by the evolution of the protein corona over time. It is known that RAW macrophages excrete lysozyme, and that lysozyme can induce aggregation of gold nanoparticles. It is possible that as the protein coating develops around the GNP-PEG during pre-incubation in complete media they become more resistant to aggregation induced by macrophage secretions. The GNP-alkyl-PEG spectra were very stable and showed no aggregation at any time point, as were the particle solutions prior to addition to cells (data not shown). The optical microscopy data agrees well with the UV- Vis spectroscopy of nanoparticle suspensions.

[76] Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as defined by the appended claims. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part, by the appended claims.

Claims

What is claimed is:

1. A composition comprising a nanoparticle having a surface and a hydrophobic shielding layer disposed on the surface, wherein the hydrophobic shielding layer comprises a hydrophilic outer layer and a hydrophobic inner layer.

2. The composition of claim 1 , wherein the nanoparticle is a metal nanoparticle.

3. The composition of claim 1 , wherein the metal nanoparticle is a gold nanoparticle.

4. The composition of claim 1 , wherein the nanoparticle is a magnetic or a

semiconducting nanoparticle.

5. The composition of claim 1, wherein the hydrophilic outer layer comprises a synthetic polymer.

6. The composition of claim 1, wherein the hydrophilic outer layer comprises a biological polymer such as an antibody, a nucleic acid, a peptide, an aptamer.

7. The composition of claim 1, wherein the hydrophilic outer layer comprises a mixture of synthetic and biological polymers.

8. The composition of claim 1, wherein the hydrophobic inner layer comprises a saturated or unsaturated alkyl chain.

9. The composition of claim 1, wherein the hydrophobic inner layer is covalently bound to the surface.

10. A method comprising providing a surface and adding to the surface a hydrophobic shielding layer comprising a hydrophilic outer layer and a hydrophobic inner layer.

11. A method comprising providing a surface and preventing or reducing interactions between nanoparticles as described in claims 1 through 10 and an immune system by providing on the surface of the nanoparticles a hydrophobic shielding layer comprising a hydrophobic inner layer and a hydrophilic outer layer.

12. The method of claim 10 or 11, wherein the hydrophobic inner layer comprises either saturated or unsaturated alkyl chains.

13. The method of claim 10 or 11, wherein the hydrophilic outer layer comprises mPEG.

14. The method of claim 10 or 11, wherein the hydrophilic outer layer comprises a mixture of mPEG molecules and one or a composition of antibodies, nucleic acids, peptides, aptamers.

15. The method of claim 10 or 11, wherein the surface is a surface of a nanoparticle, a biomedical material, or an at least partially indwelling medical device.

16. A method of improving stability of a metal nanoparticle comprising applying a hydrophobic shielding layer to the surface of the metal nanoparticle.

17. The method of claim 16, wherein the metal nanoparticle is a gold nanoparticle.

18. The method of claim 16, wherein the metal nanoparticle is a magnetic or

semiconducting nanoparticle.

19. The method of claim 16, wherein the shielding layer comprising a hydrophobic inner layer and a hydrophilic outer layer.

20. A method of preventing or reducing protein adsorption on a metal nanoparticle comprising applying a hydrophobic shielding layer to the surface of the metal nanoparticle.

21. The method of claim 20, wherein the metal nanoparticle is a magnetic or

semiconducting nanoparticle.

22. The method of claim 20, wherein the metal nanoparticle is a gold nanoparticle.

23. The method of claim 20, wherein the shielding layer comprising a hydrophobic inner layer and a hydrophilic outer layer.