ACKNOWLEDGEMENTS

This invention was made with government support under Grant Nos.

RO1CA108468, U54A119338 and U01HL080711 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Number 61/478,078 filed April 22, 2011, hereby incorporated by reference in its entirety.

BACKGROUND

Many human cancers are treated by surgical resection, chemotherapy, and/or radiation. A complete surgical resection is an important predictor of patient survival. Preoperative imaging improves tumor detection but provides limited guidance during surgery. Thus, there is a need to develop strategies that help the surgeon to delineate tumor margins and to determine if the tumor has been completely removed. Nanometer- sized particles such as quantum dots (QDs) have been conjugated to monoclonal antibodies, peptides, or small molecules. These nanoparticles can be used to target malignant tumor cells and visualize a tumor during surgery. See Singhal et al., Annu Rev Med. 2010; 61 : 359-373.

Size tuned quantum dots may be prepared with amphiphilic multidentate ligands and modified with desired molecular entities. See Kairdolf et al, J Am Chem Soc, 2008, 130, 12866-12867 and US Published Patent Application Number 2011/0260111.

Quantum dots typically contain cadmium or other potentially toxic elements. Thus, there is a need to develop nanometer-sized particles that do not pose safety issues. To overcome the potential toxicity concerns of QDs, nanoparticles for in vivo tumor targeting and spectroscopic detection has moved to pegylated colloidal gold and iron oxide

nanoparticles. See Qian et al, Nature Biotechnology, 2008, 26, 83 - 90, Hadjipanayis et al, Cancer Research, 2010, 70(15):6303-6312, and Peng et al, Int J Nanomedicine. 2008 September; 3(3): 311-321

A couple approaches are used for the chemical synthesis of gold nanoparticles. One approach uses sodium citrate as a reducing agent and stabilizing ligand. Turkevich et al, Discuss Faraday Soc, 1951 , 11, 55. While the particle size can be controlled by the gold precursor/citrate molar ratio, the resulting particles are not well protected and are prone to aggregation upon storage or exposure to salts. In another approach, alkanethiols are used to stabilize gold particles in a two-phase emulsion process. This method yields monolayer-protected gold clusters that are highly stable, but the available range of nanoparticle sizes is limited. See, e.g., Brust et al, J Chem Soc, Chem Commun, 1994, 801-02 and Templeton et al, Acc Chem Res, 2000, 33, 27. Thus, there is a need to identify improved methods of producing stable gold particles. SUMMARY

Disclosed herein are polymer ligands used to synthesize metal particles such as colloidal gold nanocrystals. In certain embodiments, the metal particles are protected by an inner coordinating layer and an outer polymer layer and are broadly soluble in water and polar solvents. In other embodiments, the particles can further shed their outer polymer layer and become soluble in nonpolar organic solvents. In certain embodiments, the polymer coatings are permeable to organic molecules.

In certain embodiments, the disclosure relates to metal particles, such as gold metal particles, coated with a first polymer comprising or consisting essentially of monomers with a chemical group with two or more heteroatoms, e.g., carboxylic acid groups, carboxamide groups, thioacid groups, urea groups, quinidine groups, and monomers with a hydrophobic group, e.g., an alkyl chain, mono or polycyclic carbocycle or aromatic or combinations of hydrophobic groups. In certain embodiments, the alkyl groups may be saturated or unsaturated. In certain embodiments, the metal is cobalt, nickel, palladium, platinum, copper, silver, gold, or aluminum. In certain embodiments, the first polymer non-covalently binds to a surface coating polymer comprising hydrophobic groups forming a hydrophobic layer. The surface coating optionally contains additional hydrophilic groups. Typically, the hydrophilic groups are carboxylic acid or amine groups and the hydrophobic groups are alkyl chains. Typically, the alkyl chains may contain from 4 to 22 carbons.

In certain embodiment, the disclosure relates to compositions comprising metal particles wherein the diameter of the metal particles, not including the polymer coating, are between about or greater than 5 nanometers (nm) to about 200 nanometers (nm).

In certain embodiments, the invention relates to compositions wherein the particles are a size of about 8 to 150 nm in diameter and vary in size of less than about 10%, 20%, or 30%. In certain embodiments, greater than 80%, 90%, or 95% of the particles are between about 50 to 80 nm or between about 55 to 70 nm. In certain embodiments, greater than 80%), 90%>, or 95% of the particles are between about 20 to 50 nm or between about 10 to 20 nm. In certain embodiments, greater than 80%, 90%, or 95% of the particles are between about 6 to 16 nm.

In certain embodiments, the first polymer is polyacrylic acid wherein 30 % to 50 % of the carboxylic acid groups are alkyl amides. In certain embodiments, the hydrophobic layer comprises non-covalently bound hydrophobic molecule such as a dye or therapeutic agent.

In certain embodiments, a polypeptide, polynucleotide, polyethylene glycol, therapeutic agent or other molecule comprising a primary amine group is covalently conjugated to the surface coating polymer. The polypeptide may be a protein, receptor, ligand, antibody, antibody mimic, aptamer, nucleic acid, or fragment. The therapeutic agent may be an anti-cancer agent or anti-inflammatory agent.

In certain embodiments, the disclosure relates to methods of producing a particle comprising mixing a metal complex with a polymer solution comprising monomers with carboxylic acid groups and monomer with alkyl chains under conditions such that a particle comprising a first polymer layer is formed. Typically the particles comprise a second surface coating polymer. In certain embodiments, the method further comprises the step of removing the second surface coating polymer by mixing the solution with an acidic media exposing a hydrophobic layer on the exterior of the particle.

In certain embodiments, the metal complex comprises gold. In certain

embodiments, the metal complex comprises a halogen ligand. In certain embodiments, the metal complex is selected from a metal oxide, gold salt, gold(III) salt, hydrogen tetrachloroaurate, sodium tetrachloroaurate, gold monochloride, gold dichloride, tetragold octachloride, gold trichloride, digold hexachloride, gold trifloride, gold tribromide, sodium aurate, gold(III) oxide, digold hexabromide, silver salt, silver halide, silver chloride, sliver bromide, silver monofluoride, sliver(II) fluoride, disilver fluoride, copper salt, copper halide, copper (I) chloride, and copper (II) chloride, copper (I) fluoride, copper (II) fluoride, tetrachloroaluminate, aluminum salt, aluminum hydroxide, aluminum oxide, and aluminum chloride, cobalt salt, cobalt chloride, hexamminecobalt(III) chloride, nickel salt, nickel chloride, palladium salt, palladium chloride, platinum salt, potassium

hexachloroplatinate, hexachloroplatinic acid, platinum (II) chloride, and platinum (IV) bromide. In certain embodiment, the polymer solution comprising monomers with carboxylic acid groups and monomers with alkyl chains is polyacrylic acid wherein greater than 10 % of the carboxylic acid groups are alkyl amides. Typically, about 30 % to 50 % of the carboxylic acid groups are alkyl amides.

In certain embodiments, the ratio of carboxylic acid groups to metal atoms is between about 15: 1 to about 1 : 10 respectively.

In certain embodiments, the disclosure relates to compositions comprising particles made by methods disclosed herein.

In certain embodiments, the disclosure relates to uses of compositions disclosed herein for applications in diagnostics, surface-enhanced Raman scattering (SERS), photothermal therapy, and in vivo delivery of therapeutic and imaging agents.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 schematically illustrates certain embodiments of the disclosure, (a) Schematic illustration of multidentate-protected AuNCs with pH-sensitive properties, including solubility control by surface charge modification (1→ 2) and outer layer shedding (2→ 3). (b) Dynamic light scattering data showing a dramatic change in the hydrodynamic size of the AuNCs after shedding of the surface layer, (c) TEM images showing (top) a thick polymer coating (white) surrounding water-soluble AuNCs and (bottom) a much thinner polymer shell after outer layer shedding in chloroform. The chain-shaped arrangement of nanoparticles was an artifact caused by drying on the TEM grid.

Figure 2 shows data on the comparison of AuNC nucleation and growth kinetics observed using traditional monovalent or multidentate polymer ligands. (a) Color photograph (top) and absorption spectra (bottom) at different reaction times during the citrate reduction procedure, (b) Color photograph (top) and absorption spectra (bottom) at different reaction times during AuNC synthesis in the presence of multidentate ligands. The labels 2 and 8 in the absorption spectra correspond to vial numbers in the photos. The reaction time for completion was -9 h for the multidentate procedure, which is >50-fold longer than that (10 min) of the citrate procedure.

Figure 3 shows experiments indicating broad solubility and stability of

multidentate-polymer-coated AuNCs. (a) Color photograph showing stable AuNCs dissolved in a broad range of solvents, (b) Color photograph showing extraction of AuNCs into the aqueous phase at basic pH and into the organic phase (chloroform) at acidic pH. (c) Absorption spectra in the presence of 0.1-2.5 M NaCl.

Figure 4 illustrates pH-triggered cooperative transition in solubility as measured by precipitation of multidentate-protected AuNCs. (a) Plots of AuNC solubility (black) and the degree of carboxylic acid deprotonation (red) as a function of pH. The solubility curve closely mirrors the titration curve of acetic acid but is much sharper because of a cooperative effect (see the text), (b) Hydrodynamic diameter as a function of pH, showing the formation of nanoparticle aggregates at lower pH. (c) Schematic illustration of pH- triggered precipitation of nanoparticles. Surface carboxylate ions are protonated as the pH decreases, resulting in a decrease in electrostatic repulsion and eventual precipitation from solution.

Figure 5 shows data from SERS studies of 60 nm multidentate-protected AuNCs demonstrating that small reporter molecules are able to move across the multidentate polymer coating and adsorb on the gold surface: (red) negative control (multidentate- coated AuNCs without the reporter dye); (black) SERS spectra of multidentate-protected AuNCs after the addition of MGITC; (green) standard SERS nanoparticles encoded with MGITC. = 633 nm; laser power = 3 mW; integration time = 1 s.

Figure 6 shows transmission electron micrographs of gold nanocrystals

(unstained). Samples in water (top) and chloroform (bottom) were imaged without counterstaining, showing uniform and well dispersed particles (scale bar: 30 nm).

Figure 7 is a graph showing particle size as a function of polymer concentration. Gold nanocrystals were synthesized as described above, varying the concentration of the polymer (measured as the ratio of carboxylic acid functional groups to gold atoms or COOH:Au). Transmission electron micrographs were taken of the samples and analyzed to determine size of the nanocrystals.

DETAILED DESCRIPTION

Disclosed herein are polymer ligands used to synthesize metal particles such as colloidal gold nanocrystals. In certain embodiments, the metal particles are protected by an inner coordinating layer and an outer polymer layer and are broadly soluble in water and polar solvents. In other embodiments, the particles can further shed their outer polymer layer and become soluble in nonpolar organic solvents. In certain embodiments, the polymer coatings are permeable to organic molecules. Polymer synthesis

In certain embodiments, the disclosure relates to particles made in the presence of polymers disclosed herein. In one example, an amphiphilic polymer (~ 3500 Daltons) was synthesized using carbodiimide chemistry. Poly(acrylic) acid (Sigma Aldrich, MW = 1800 Daltons) (518 mg) and 533 mg of dodecylamine were dissolved in 10 mL DMF. Dicyclohexylcarbodiimide (DCC) (609 mg) dissolved in a minimum amount of DMF was added dropwise and the solution was mixed vigorously for 24 hours to give an amphiphilic polymer with 40% of the carboxylic acid functional groups modified with a 12-carbon aliphatic chain. Insoluble dicyclohexylurea (DCU) byproduct was removed using vacuum filtration. The product was diluted into basic water (pH=10) and vacuum filtered to remove hydrophobic impurities. The filtered solution was acidified with HC1 to precipitate the polymer, which was collected using filtration and lyophilized to yield a white powder.

Instead of polyacrylic acid a number of other polymers with monomers comprising carboxylic acids could be utilized to provide a polymer with carboxylic acid groups and alkyl amides. Hydro lyzed polymaleic acid anhydride, copolymers of maleic and acrylic acid, and polyaspartic acid polypeptide are contemplated to be coupled to a variety of alkyl amines and used as the amphiphilic polymer. Other copolymers could be used such as acrylic acid-hydroxymethylacrylate copolymers. In another example, the amphiphilic polymer may be prepared by the reaction of the poly(isobutylene-alt-maleic anhydride) backbone with n-octylamine. See Janczewski et al, Nature Protocols, 2011 , 6, 1546-1553, hereby incorporated by reference in its entirety.

Synthesis of nanocrystals using polymer ligands

In certain embodiments, the disclosure relates to nanometer sized metal particle made in the presence of polymers disclosed herein. In one example, a hydrogen tetrachloroaurate (HAuCU) solution (50 of a 50 mM) and 9.8 mL of deionized water was added to a 50 mL flask and brought to a boil under vigorous stirring. Separately, an aqueous polymer solution was prepared to give a stock solution of 0.15 M carboxylic acid (polymer concentration about 40 mg/mL). For a ratio of 10: 1 carboxylic acid: Au, 167 of the polymer stock solution was added to the gold precursor solution and the reaction was allowed to progress for 10 hours under reflux. The reaction was then cooled to room temperature and the nanocrystals were purified using dialysis (MWCO = 100 kDa) against 50 mM borate buffer or repeated pelleting/redispersion using ultracentrifugation (x3 at 80k g for 45 min). Methods of use

Uses of compositions disclosed herein include applications in diagnostics, surface- enhanced Raman scattering (SERS), photothermal therapy, and in vivo delivery of therapeutic and imaging agents. In diagnostic applications, the particles may be conjugate to targeting moieties and/or dyes. These conjugates can be used in assays. In certain embodiments, the disclosure relates to methods of detecting an analyte by conjugating, covalently or noncovalently, a moiety with affinity to the analyte to the surface of particles disclosed herein, and mixing the conjugate with a sample suspected of containing the analyte, and determining whether the moiety binds the analyte by evaluating a physical property of the particle and/or dye conjugated or incorporated into the particle, e.g., fluorescence and/or surface-enhanced Raman scattering. In certain embodiments, the moiety is antibody, fragment, or antibody mimic, and the analyte is a molecule comprising an epitope or ligand. An optical encoding approach jointly using surface enhanced Raman scattering (SERS) and fluorescence for spectral encoding is disclosed using

organic-metal-quantum dot (QD) hybrid nanoparticles (OMQ NPs) with a nanolayered structure. See Wang et al, JACS, 2012, 134:2993-3000, hereby incorporated by reference in its entirety. In certain embodiments, the disclosure contemplates a substrate such as a glass slide or silicon wafer, e.g., microarray, containing or coated with a composition comprising particles disclosed herein, e.g., gold particles with an average size of between about 50 to 80 nm or about 60 to 70 nm.

In certain embodiments, particles comprise or are conjugated to nucleic acids, e.g., single stranded DNA or RNA. The particles are mixed with a test sample suspected of containing a complementary nucleic acid, and hybridization with the complementary nucleic acid is determined by evaluation of a physical property of the particle or dye. See Lim et al., Nature Nanotechnology, 2011, 6:452-460, hereby incorporated by reference in its entirety.

Conjugating molecules or polypeptides to the polymers can be accomplished using a variety of methods. Typically, primary amine containing compounds and proteins may be conjugated to the carboxylic acid groups on the polymer mediated by a coupling reagent such as ED AC. See Yang et al, Small, 2009, 5(2):235-43, hereby incorporated by reference in its entirety. The outside of particles disclosed herein may contain carboxylic acid or amine groups that may be coupled to any molecule containing a free amine/thiol or carboxylic acid group respectively to form an amide or thioester conjugate. This allows one to conjugate nucleic acids and polypeptides such as antibodies or fragments to the exterior of the particle. Other coupling methods are contemplated, e.g., poly-histidine sequence may be recombinantly incorporated into a polypeptide sequence of the targeting moiety. A poly-histidine chelating agent may be coupled to the polymer surface, e.g., NTA-Ni. Mixing the histidine tagged polypeptide sequence attaches it to the polymer surface linked through the chelating agent. The avidin/streptavidin-biotin interactions may be used, e.g., biotin may be coupled to the polymer surface and streptavidin may be expressed as a chimera.

In the case where the particles disclosed herein are exposed to solution at a pH low enough to shed their outer polymer layer, the exposing hydrophobic alkyl chains are exposed. Mixing these single layered particles with the same or modified polymers with hydrophobic groups allows one to coat the polymer with desired molecules. For example, one can conjugate hydrophilic molecules, e.g., polypeptide or drug, to hydrophobic moieties, e.g., fatty acids or steroids, or polymers with hydrophobic groups. Mixing the conjugates with the single layered particles will cause the hydrophobic groups to incorporate into the hydrophobic alkyl chains exposing the hydrophilic molecule on the surface of the particles, e.g., amine groups or protected amine groups. With a protected amine group an amine group is exposed by mixing the particle with a deprotecting agent.

In certain embodiments, the disclosure contemplates uses of particles disclosed herein for photothermal therapy (PTT). Photothermal agents may be used to achieve the selective heating of a target area. When the PTT agents absorb light, overheating of area around the light absorbing species occurs. The heat produced can destroy local cells or tissues. Gold and silver nanoparticles have enhanced absorption in the visible and NIR regions on account of surface plasmon resonance (SPR) oscillations. See Huang et al, Lasers Med Sci, 2008, 23 :217-228, hereby incorporated by reference in its entirety. In certain embodiments, the disclosure contemplates methods comprising administering compositions of particles disclosed herein to a subject diagnosed with cancer, optionally the particles comprise cancer targeting moiety, e.g., antibodies specific to biomarkers on cancer cells, and exposing the subject to light sufficient to produce heat and kill cells or destroy tissue where the particles, e.g., gold or silver particles, accumulated in the subject, e.g., the area of a tumor. In certain embodiments, the particles comprise drugs, e.g., anticancer agents, incorporated into the hydrophobic area of the polymer of conjugated to the exterior of the particle optionally through a biodegradable linker. In certain embodiments, the targeting moiety is a monoclonal antibody-610 that targets a surface antigen for use in treating colon carcinoma. See Cerdan et al., Magn Reson Med, 1989, 12: 151-63 1989, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is an antibody to carcinoembryonic antigen (CEA) that targets CEA for use in treating colon tumors. See Tiefenauer et al., Magn Reson Imaging, 1996, 14:391-402, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is a monoclonal antibody L6 that targets a surface antigen for use in treating intracranial tumor. See Remsen et al., Am J Neuroradiol, 1996, 17:411-18, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is transferrin that targets transferrin receptor for use in treating carcinoma. See Kresse et al, Magn Reson Med, 1998, 40:236- 42, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is a monoclonal antibody to Her-2, e.g., herceptin, for use in treating breast cancer. See Lee et al, Nat Med, 2007, 13:95-9; Artemov et al, Magn Reson Med, 2003, 49:403-8; and Huh et al, J Am Chem Soc, 2005, 127: 12387-91, all hereby incorporated by reference in their entirety.

In certain embodiments, the targeting moiety is the EPPT peptide that targets underglycosylated mucin- 1 antigen (uMUC-1) for use in treating breast, colon, pancreas and lung cancer. See Moore et al, Cancer Res, 2004, 64: 1821-7, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is folic acid that targets folate receptor for use in treating mouth carcinoma and cervical cancer. See Chen et al., PDA J Pharm Sci Technol, 2007, 61 :303-13; Sun et al, Small, 2006, 4:372-9; and Sonvico et al, Bioconjug Chem, 2005, 16: 1181-8, all hereby incorporated by reference in their entirety.

In certain embodiments, the targeting moiety is methotrexate that targets folate receptor for use in treating cervical cancer. See Kohler et al, Langmuir, 2005, 21 :8858- 64, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is a monoclonal antibody A7 that targets colorectal tumor antigen for use in treating colorectal carcinoma. See Toma et al., Br J Cancer, 2005, 93: 131-6, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is chlorotoxin peptide that targets membrane-bound matrixmetalloproteinase-2 (MMP-2) for use in treating glioma. See Veiseh et al, Nano Lett, 2005, 5: 1003-8, hereby incorporated by reference in its entirety. In certain embodiments, the targeting moiety is F3 peptide that targets surface- localized tumor vasculature for use in treating glioma. See Reddy et al, Clin Cancer Res, 2006, 12:6677-86, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is RGD or RGD4C that targets integrins for use in treating melanoma and epidermoid carcinoma. See Zhang et al,

Cancer Res, 2007, 67: 1555-62 and Uchida et al, J Am Chem Soc, 2006, 128: 16626-33, both hereby incorporated by reference in their entirety.

In certain embodiments, the targeting moiety is luteinizing hormone releasing hormone (LHRH) that targets LHRH receptor for use in treating breast cancer. See Leuschner et al, Breast Cancer Res Treat, 2006, 99:163-76, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is CREKA peptide that targets clotted plasma proteins for use in treating breast cancer. See Simberg et al., Proc Natl Acad Sci U S A, 2007, 104:932-6, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is an antibody to prostate specific membrane antigen (PSMA) that targets PSMA for use in treating prostate cancer. See Serda et al, Mol Imaging, 2007, 6:277-88, hereby incorporated by reference in its entirety. EXAMPLES

Use of multidentate polymer ligands to synthesize protected colloidal gold

nanocrystals (AuNCs) with broad size tunability and pH-sensitive surface properties

The carboxylic acid functional groups of the polymer ligands are able to coordinate with the surface atoms of the NCs and behave in the same manner as capping ligands. However, the large number of functional groups in the polymer considerably alters the nucleation and growth kinetics of the NCs relative to that for the monovalent ligands. Typically, a polymer molecule has about 14 carboxylic groups available for binding to the NC surface. This multidentate effect significantly increases the binding affinity of the ligand and results in an extremely stable surface coating. The surface coating also contains a large number of extended alkyl chains that are able to interact with the hydrophobic side chains of the free polymer, leading to a thick hydrophobic middle layer and a hydrophilic outer layer (exposed carboxylic acid groups). This allows highly stable AuNCs with tunable sizes of 5-150 nm to be prepared. As discussed in more detail below, the multidentate-protected NCs can be isolated, purified, and redispersed, practically behaving as chemical reagents.

In one example, an amphiphilic polymer (- 3500 Daltons) was synthesized by coupling dodecylamine to poly acrylic acid using carbodiimide chemistry. A hydrogen tetrachloroaurate (HAuCW) solution boiled in the presence of the amphiphilic polymer resulted in the colloidal gold nanocrystals with multidentate ligands. As shown in Figure la, immediately after synthesis, the AuNCs are protected by an inner coordinating layer and an outer polymer layer and are soluble in water and polar solvents. Transmission electron microscopy (TEM) revealed a thick polymer coating around the AuNCs (Figure lc). When a portion of the surface carboxylate groups are neutralized at a lower pH, the NCs irreversibly shed their outer polymer layer and become soluble in nonpolar organic solvents such as chloroform. After the loss of this outer polymer layer, the NCs are encapsulated by a single layer of the multidentate ligands, and their hydrodynamic size decreases by 15-20 nm (Figure lb). This large size decrease indicates that the inner coordinating layer (formed by multidentate binding) is much tighter than the outer polymer layer (consisting of a hydrophobic layer and exposed carboxylic acid groups).

Although it is not intended that any of the embodiments disclosed herein be limited by any particular mechanism, two conditions are believed to cause shedding of the outer layer: (i) the surface charges are neutralized at acidic pH (below the pKa value of acetic acid) and (ii) the charge-neutralized NCs are exposed to a nonpolar solvent such as chloroform. Once the outer layer is lost, the NCs are soluble in nonpolar solvents and cannot be redissolved in water or polar solvents unless excess polymer ligands are added.

As shown in Figure 2, the use of multidentate polymer ligands gives rise to different nucleation and growth mechanisms in comparison with those for monovalent ligands. With the traditional citrate method, the AuNC nucleation and growth process undergoes a transient stage in which the solution initially becomes clear (reduction to atomic Au clusters) and subsequently progresses to a gray intermediate before developing the characteristic ruby-red color of gold colloids (Figure 2a). This was confirmed by monitoring the absorbance, which first shows a broad absorption spectrum (black curve) that rapidly becomes blue-shifted and narrows to form the surface plasmon resonance (SPR) peak typical of monodisperse AuNCs (red curve). The transient intermediate is noticeably absent when the NCs are prepared using the multidentate polymer ligand (Figure 2b), indicating that the polymer-induced NC formation proceeds through different nucleation and growth mechanisms: the AuNCs slowly appear and increase in concentration as the synthesis progresses. Absorption spectra measured during NC growth illustrate the appearance and gradual red- shifting of the SPR peak (Figure 2b).

The NC growth rate is reduced by nearly 2 orders of magnitude relative to that reported for monovalent ligands. Slower growth kinetics allows better control of the AuNC size. The size can also be tuned by changing the ratio of polymer ligands to Au atoms, as in the synthesis by citrate reduction. Using these strategies, multidentate- protected AuNCs with a wide range of sizes (5-150 nm with about 10% variation) were prepared.

The multidentate-protected AuNCs are soluble in water and polar solvents such as dimethylformamide and acetone (Figure 3a). However, adding an aqueous solution of AuNCs to a nonpolar solvent such as chloroform results in two separate phases, with the negatively charged AuNCs remaining in the aqueous phase (Figure 3b). When the surface charge is reduced at lower pH, the AuNCs rapidly shed their outer polymer layer, exposing hydrophobic alkyl chains. This process can be used to transfer AuNCs with a wide range of sizes into a variety of nonpolar solvents. Furthermore, in contrast to citrate- stabilized particles that aggregate at salt concentrations of 10 mM or higher, the polymer- coated AuNCs are highly resistant to salt-induced aggregation at concentrations as high as 2.5 M (shown by the SPR peak in Figure 3c). The particles also exhibit high stability in serum solutions, suggesting that the polymer-coated AuNCs are likely to be well-suited for diagnostic applications.

As shown in Figure 4a, the protected NCs are sensitive to pH and can be precipitated and isolated under acidic conditions. The precipitated NCs can subsequently be purified and redissolved by raising the solution pH to 6-7. Significantly, nanoparticle precipitation takes place in a pH range that is much narrower than the titration curve of carboxylic acid (Figure 4b). On the basis of the fitted experimental data, 95% of the particles are estimated to be soluble at pH 4.85, while only 5% are soluble at pH 4.25. This sharp transition in solubility is triggered by a pH change of only 0.6 units. If it is assumed that the acid-base equilibrium closely follows the titration curve, this solubility transition corresponds to a 40% change in carboxyl neutralization [from 35% neutral carboxyl groups (-COOH) at pH 4.85 to 75% neutral groups at pH 4.25].

The multidentate polymer coatings were found to be permeable to small molecules such as organic dyes. This is surprising because TEM showed that the polymer coatings are fairly thick with few or no defects (Figure lc). Experimental evidence comes from SERS studies in which the reporter molecules had to move across the polymer coating and then adsorb on the AuNC. AuNCs with a diameter of 60-70 nm are optimal for surface Raman enhancement (at 633-647 nm excitation). The polymer concentration and reaction time of the synthesis procedure was optimized to prepare AuNCs with an average diameter of 60 nm. After the addition of a Raman reporter dye [malachite green isothiocyanate (MGITC)], the AuNCs exhibited strong SERS signals (Figure 5).

Synthesis of SERS-active gold nanocrystals

Gold nanocrystals were prepared as described above except a carboxylic acid/Au ratio of 1 : 1 was used to yield particles with a diameter of about 60 nm. After cooling to room temperature, 10 of a 1 mM aqueous solution of malachite green was added to the nanocrystal solution and was stirred for 1 hour. The particles were pelleted using centrifugation at 1000 g and were washed to remove excess dye and polymer. The pellet was then redispersed in a 50 mM borate buffer to yield purified SERS-active particles. Transfer of gold nanocrystals to hydrophobic solvents

Gold nanocrystals were immediately transferred to hydrophobic solvents such as chloroform using liquid- liquid extraction. Briefly, gold nanocrystals were diluted 1 : 10 into 50 mM aqueous HC1. The nanocrystal solution and an equal volume of chloroform were added to a vial and gently shaken for 10 seconds to extract the nanocrystals into the hydrophobic phase.