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Cysteine-Functionalized Polyaspartic Acid: A Polymer for Coating and Bioconjugation of Nanoparticles and Quantum Dots Nikhil R. Jana,† Nandanan Erathodiyil, Jiang Jiang, and Jackie Y. Ying* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669. Present address: Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata-700032, India
†
Received October 19, 2009. Revised Manuscript Received January 4, 2010 We have synthesized a biocompatible polyaspartic acid-based polymer (molecular weight ∼15 000-25 000) with cysteine on its backbone for use as a capping ligand for functionalized Au, Ag, and CdSe@ZnS nanoparticles. Nearly monodisperse, hydrophobic Au and Ag nanoparticles and CdSe@ZnS quantum dots were first prepared in organic solvents via conventional synthesis and then ligand exchanged to derive polymer-coated water-soluble nanoparticles. Multiple thiol groups in the polymer backbone conferred excellent protection against aggregation of the nanoparticles, and the carboxylic acid groups in the polymer provided the possibility of covalent binding with antibodies. Compared to the conventional thiol-based ligands, this polymer coating led to superior colloidal stability under the experimental conditions involved in the bioconjugation and purification steps. Goat antihuman-IgG (anti-h-IgG) and antimouse epidermal growth factor receptor (anti-m-EGFR) antibodies were conjugated with the polymer-coated nanoparticles and successfully applied to protein detection. This polymer coating exhibited minimal nonspecific interaction with cells and could be broadly applied to cell labeling.
Colloidal nanocrystals such as quantum dots (QDs) and noble metal nanoparticles have great importance in basic and applied research.1-5 They have been functionalized with oligonucleotides, peptides, antibodies, and other molecules and used in biosensing, cellular imaging, and in vivo imaging.2-5 Current research is focused on the synthesis, colloidal stability, biocompatibility, and conjugation chemistry of nanocrystals. Surfactant-mediated nucleation and growth are key in the size control of nanocrystals in the range of 1-10 nm, and methods are well established for the synthesis of nearly monodispersed nanocrystals of QDs,6-10 metals,11-15 and metal oxides.16-19 The nanocrystals were coated with a layer of surfactant molecules that protected them from further growth and the external environment. However, these surfactants also rendered the nanocrystals hydrophobic and *Corresponding author. E-mail:
[email protected]. (1) Murphy, C. J.; Jana, N. R. Adv. Mater. 2002, 14, 80. (2) Eugenii, K.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042. (3) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547. (4) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435. (5) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538. (6) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706. (7) Li, J. J.; Wang, Y. A.; Guo, W.; Keay, J. C.; Mishima, T. D.; Johnson, M. B.; Peng, X. J. Am. Chem. Soc. 2003, 125, 12567. (8) Yin, Y.; Alivisatos, A. P. Nature 2005, 437, 664. (9) Norris, D. J.; Yao, N.; Charnock, F. T.; Kennedy, T. A. Nano Lett. 2001, 1, 3. (10) Pradhan, N.; Goorskey, D.; Thessing, J.; Peng, X. J. Am. Chem. Soc. 2005, 127, 17586. (11) Sun, S.; Murray, C. B.; Weller, D.; Folks, L.; Moser, A. Science 2000, 287, 1989. (12) Jana, N. R.; Peng, X. J. Am. Chem. Soc. 2003, 125, 14280. (13) Shevchenko, E. V.; Talapin, D. V.; Schnablegger, H.; Kornowski, A.; Festin, O.; Svedlindh, P.; Haase, M.; Weller, H. J. Am. Chem. Soc. 2003, 125, 9090. (14) Hiramatsu, H.; Osterloh, F. E. Chem. Mater. 2004, 16, 2509. (15) Zheng, N.; Fan, J.; Stucky, G. D. J. Am. Chem. Soc. 2006, 128, 6550. (16) Hyeon, T.; Lee, S. S.; Park, J.; Chung, Y.; Na, H. B. J. Am. Chem. Soc. 2001, 123, 12798. (17) Sun, S.; Zeng, H. J. Am. Chem. Soc. 2002, 124, 8204. (18) Jana, N. R.; Chen, Y.; Peng, X. Chem. Mater. 2004, 16, 3931. (19) Hou, Y.; Xu, Z.; Sun, S. Angew. Chem., Int. Ed. 2007, 33, 6329.
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prevented further chemical functionalization. Thus, it has been a challenge to synthesize colloidally stable, water-soluble, robust nanocrystals with flexible surface chemistry.2-5 Several schemes have been reported recently for core-shell nanocrystal synthesis20-34 with the shell comprising dendron,23 oligomeric phosphine,27 silica,20-22,25,28,29,32 polymers,24,26,33 and other small molecules.30,34 In those methods, the original surfactants (20) Liz-Marzan, L. M.; Giersig, M.; Mulvaney, P. Langmuir 1996, 12, 4329. (21) Obare, S. O.; Jana, N. R.; Murphy, C. J. Nano Lett. 2001, 1, 601. (22) Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. B 2001, 105, 8861. (23) Guo, W.; Li, J. J.; Wang, Y. A.; Peng, X. J. Am. Chem. Soc. 2003, 125, 3901. (24) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol. 2004, 22, 969. (25) Nann, T.; Mulvaney, P. Angew. Chem., Int. Ed. 2004, 43, 5393. (26) Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktysh, D.; Rogach, A. L.; Keller, S.; Radler, J.; Natile, G.; Parak, W. J. Nano Lett. 2004, 4, 703. (27) Kim, S.-W.; Kim, S.; Tracy, J. B.; Jasanoff, A.; Bawendi, M. G. J. Am. Chem. Soc. 2005, 127, 4556. (28) (a) Selvan, S. T.; Tan, T. T.; Ying, J. Y. Adv. Mater. 2005, 17, 1620. (b) Yi, D. K.; Selvan, S. T.; Lee, S. S.; Papaefthymiou, G. C.; Kundaliya, D.; Ying, J. Y. J. Am. Chem. Soc. 2005, 127, 4990. (c) Selvan, S. T.; Patra, P. K.; Ang, C. Y.; Ying, J. Y. Angew. Chem., Int. Ed. 2007, 46, 2448. (d) Tan, T. T.; Selvan, S. T.; Zhao, L.; Gao, S.; Ying, J. Y. Chem. Mater. 2007, 19, 3112. (29) (a) Jana, N. R.; Earhart, C.; Ying, J. Y. Chem. Mater. 2007, 19, 5074. (b) Jana, N. R.; Ying, J. Y. Adv. Mater. 2008, 20, 430. (c) Earhart, C.; Jana, N. R.; Erathodiyil, N.; Ying, J. Y. Langmuir 2008, 24, 6215. (d) Wei, Y.; Jana, N. R.; Tan, S.; Ying, J. Y. Bioconjugate Chem. 2009, 20, 1752. (30) Smith, A. M.; Duan, H.; Rhyner, M. N.; Ruan, G.; Nie, S. Phys. Chem. Chem. Phys. 2006, 8, 3895. (31) Nikolic, M. S.; Krack, M.; Aleksandrovic, V.; Kornowski, A.; F€orster, S.; Weller, H. Angew. Chem., Int. Ed. 2006, 45, 6577. (32) (a) Zhelev, Z.; Ohba, H.; Bakalova, R. J. Am. Chem. Soc. 2006, 128, 6324. (b) Tu, R.; Liu, B.; Wang, Z.; Gao, D.; Wang, F.; Fang, Q.; Zhang, Z. Anal. Chem. 2008, 80, 3458. (c) Gao, D.; Wang, Z.; Liu, B.; Ni, L.; Wu, M.; Zhang, Z. Anal. Chem. 2008, 80, 8545. (d) van Hal, P. A.; Smits, E. C. P.; Geuns, T. C. T.; Akkerman, H. B.; de Brito, B. C.; Perissinotto, S.; Lanzani, G.; Kronemeijer, A. J.; Geskin, V.; Cornil, J.; Blom, P. W. M.; de Boer, B. D.; de Leeuw, B. M. Nat. Nanotechnol. 2008, 3, 749. (33) (a) Guo, S.-R.; Gong, J.-Y.; Jiang, P.; Wu, M.; Yang, L. Y.; Yu, S.-H. Adv. Funct. Mater. 2008, 18, 872. (b) Yu, W. W.; Chang, E.; Falkner, J. C.; Zhang, J.; Al-Somali, A. M.; Sayes, C. M.; Johns, J.; Drezek, R.; Colvin, V. L. J. Am. Chem. Soc. 2007, 129, 2871. (34) Susumu, K.; Uyeda, H. T.; Medintz, I. L.; Pons, T.; Delehanty, J. B.; Mattoussi, H. J. Am. Chem. Soc. 2007, 129, 13987.
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are exchanged with new ligands or polymer precursors, which are then cross linked or polymerized on the nanocrystal’s surface to produce the shell.20-26,29a,30 However, they often involve complex steps and the associated nanoparticle aggregation problem. The most widely used approach is the small-molecule-based ligandexchange method.35-40 The ligand-exchange schemes offer simplicity, a thin ligand shell, and minimal nanoparticle aggregation, although the colloidal stability may often be affected by the experimental conditions.35-37 Thus, recent efforts have been directed toward synthesizing ligands or polymers with multiple functional groups, which can form direct multiple binding with the nanoparticle surface and improve the colloidal stability.39,41 For example, multiple thiol groups in a single molecule39,40 or a thio-ether/ thiol-containing polymer41 have enhanced the chemisorption and protection of gold nanocrystals and QDs and improved the colloidal stability. These results indicate that a water-soluble polymer with multiple thiol groups can act as a better capping ligand for noble metals and QDs. A block-copolymer-based coating has emerged as an interesting alternative to producing core-shell nanoparticles via polymer self-assembly on the nanoparticle surface.42-44 A simple ligand-exchange method can be used for this polymer coating, and the polymer can be cross linked to enhance the colloidal stability.43 Recently, cysteine-styrene block copolymers were reported as coating materials.44 However, most of these polymers have poor water solubility that adversely impacts the coated particles. Ideally, the polymer coating should have high water solubililty and high binding affinity to nanoparticles so that it can produce colloidally stable, water-soluble nanoparticles. Polyaspartic acid-based polymers have been found to be biocompatible and biodegradable; they have been used as a nanoparticle stabilizer45-47 and drug-delivery agent.48 This report describes the successful synthesis of cysteine-functionalized polyaspartic acid as an effective coating for a wide variety of nanoparticles. Here, the polymer has been designed in such a way that a simple ligand-exchange method can be used to coat this novel polymer on Au, Ag, and ZnS-capped CdSe (CdSe@ZnS coreshell) nanocrystals. The multiple thiol groups on the polymer backbone provide strong chemisorption to the nanoparticle surface, and the carboxyl group imparts water solubility and provides for further functionalization. Polyaspartimide was synthesized according to literature procedures.49 L-Aspartic acid (10 g) was mixed thoroughly with orthophosphoric acid (1 g, 10% by weight of the monomer), and the solid was heated in an oil bath at 180-200 °C for 30 min under argon. The light-yellow solid was ground into a fine powder (35) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27. (36) Aldana, J.; Lavelle, N.; Wang, Y.; Peng, X. J. Am. Chem. Soc. 2005, 127, 2496. (37) Pierrat, S.; Zins, I.; Breivogel, A.; S€oennichsen, C. Nano Lett. 2007, 7, 259. (38) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801. (39) Negishi, Y.; Tsukuda, T. J. Am. Chem. Soc. 2003, 125, 4046. (40) Uyeda, H. T.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 3870. (41) Hussain, I.; Graham, S.; Wang, Z.; Tan, B.; Sherrington, D. C.; Rannard, S. P.; Cooper, A. I.; Brust, M. J. Am. Chem. Soc. 2005, 127, 16398. (42) Spatz, J. P.; Roescher, A.; M€oller, M. Adv. Mater. 1996, 8, 337. (43) Kang, Y.; Taton, T. A. Angew. Chem., Int. Ed. 2005, 44, 409. (44) Abraham, S.; Kim, I.; Batt, C. A. Angew. Chem., Int. Ed. 2007, 46, 5720. (45) Peytcheva, A.; Colfen, H.; Schnablegger, H.; Antonietti, M. Colloid Polym. Sci. 2002, 280, 218. (46) Zhang, Z.; Gao, D.; Zhao, H.; Xie, C.; Guan, G.; Wang, D.; Yu, S. J. Phys. Chem. B 2006, 110, 8613. (47) Sadeghiani, N.; Barbosa, L. S.; Silva, L. P.; Azevedo, R. B.; Morais, P. C.; Lacava, Z. G. M. J. Magn. Magn. Mater. 2005, 289, 466. (48) Nakanishi, T.; Fukushima, S.; Okamoto, K.; Suzuki, M.; Matsumura, Y.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. J. Controlled Release 2001, 74, 295. (49) Xu, W.; Li, L.; Yang, W.; Hu, J.; Wang, C.; Fu, S. J. Macromol. Sci., Pure Appl. Chem. 2003, A40, 511.
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with a mortar and pestle, held at 200 °C for 6 h, and cooled to room temperature. Water was added, and the sample was filtered through a sintered funnel and washed several times with water until the filtrate was neutral. The light-yellow solid obtained was dried under vacuum overnight to obtain polyaspartimide as an off-white powder. For the nucleophilic opening of polyaspartimide, polyaspartimide and methyl-protected L-cysteine/methionine were mixed in a molar ratio of 1:1, and dimethylformamide (DMF) was added. The mixture was held at 50 °C overnight. The thick solution obtained was treated with an aqueous NaOH solution (1 N) and stirred for 1 h at room temperature. The reaction mixture was added to methanol dropwise, and the precipitate that formed was filtered, washed, and dried. Polymer-coated Au, Ag, and QD nanoparticles were synthesized by ligand exchange. First, nearly monodisperse 2-10 nm Au and Ag nanoparticles were derived in toluene in the presence of long-chain fatty acid/fatty amine surfactants as ligands.12 CdSe@ZnS QDs of different colors (corresponding to 2-6 nm in size) were prepared according to literature procedures using octadecene as the high-boiling-point solvent and fatty amines, trioctylphosphine oxide (TOPO), and trioctylphosphine (TOP) as ligands.7 As-synthesized nanoparticles were purified by ethanol precipitation and washed with toluene-ethanol. Next, 10-30 mg of purified nanoparticles was dispersed in 10 mL of a reverse microemulsion, which was prepared by mixing 1 mL of Igepal CO-520 with 9 mL of cyclohexane. A 100 μL polymer solution (100 mg/mL of water) was then added and mixed. In the case of QDs, 100 μL of tetramethylammonium hydroxide (0.1 M solution in methanol) was added to induce ligand exchange. After 5 min of vortex mixing, 1 to 2 mL of ethanol was added to disrupt the reverse microemulsion, and the polymer-coated nanoparticles were collected by centrifugation. The precipitate was washed in ethanol two to three more times and then dissolved in water or a buffer solution. In the ligand-exchange method for aqueous Au and Ag nanoparticles, Au and Ag nanoparticles of 5-100 nm were synthesized by a citrate reduction method or by a seeding growth method in the presence of surfactants.1 After the excess surfactants were removed by centrifugation, the nanoparticles were solubilized in deionized water. Typically, 1.0 mL of a particle solution (with 1 mM of Au or Ag) was prepared, mixed with 100 μL of polymer solution (100 mg/mL of water), and sonicated for 5 min. After 1 h of incubation, the particle solution was centrifuged to remove any free polymers. The precipitated particles were then dissolved in a buffer solution. For the synthesis of polymer-coated Au and Ag nanoparticles in water, 10 mL of an aqueous solution of gold chloride or silver nitrate (1-10 mM) was prepared and mixed with the aqueous polymer solution (1-100 mg/mL). Two to three equivalents of a freshly prepared aqueous sodium borohydride solution were then injected with rapid stirring. After 2 min, stirring was stopped, and the solution was diluted if necessary for spectroscopic and other analyses. For antibody (Ab) conjugation studies, a polymer-functionalized nanoparticle or QD solution was prepared in an aqueous borate buffer (0.02 M) at pH 7.0. The particle concentration was adjusted using a UV-vis spectrophotometer to yield a maximum absorbance of 0.2-0.5 for Au, Ag, and QD solutions. Next, 3 to 4 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride and 5 to 6 mg of N-hydroxy succinimide (NHS), dissolved separately in 1 mL of borate buffer, were added to the particle solution. After 10 min, free reagents were separated using a Sephadex-G25 column, and the particle solution (∼1 mL) was Langmuir 2010, 26(9), 6503–6507
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immediately mixed with 100 μL of Ab solution (1 mg of Ab/mL in borate buffer) and incubated for 2 to 3 h at 4 °C. Next, Ab-bound particles were purified of free Ab and excess reagents by centrifugation at 25 000 rpm for 5 min. Finally, the precipitated particles were dissolved in 500 μL of tris(hydroxymethyl)aminomethane (Tris) buffer (10 mM) at pH 7.0 and held at 4 °C. For protein-detection studies, 1.0 μL of h-IgG solution (1 μg/ mL) was spotted onto a dry nitrocellulose strip. The strip was then incubated in a blocking buffer solution (containing 0.5% bovine serum albumin (BSA), 0.5% Tween 80, and 10 mM Tris-HCl (pH 7.0)) for 1 h. The strips were then incubated with anti-h-IgGconjugated nanoparticle solution for 2 h. Next, the strips were washed with Tris buffer solution (pH 7.0) containing 0.5% Tween 80. For cell-labeling studies, mouse breast cancer cells were subcultured in six-well plates using 500 μL of media, followed by overnight incubation at 40 °C for cell attachment on the well plate surface. Next, 20 μL of anti-m-EGFR-conjugated QD solution was added and mixed with the cell culture medium. After 2 h of incubation at 40 °C, cells were washed with buffer solution, and the cell culture medium was added. Cells were then observed under fluorescence microscopy (Olympus microscope IX71 with DP70 camera) with blue excitation. Confocal fluorescence imaging was performed using an Olympus Fluoview 300 confocal laser scanning system with 488 nm laser excitation. All chemicals were purchased from Aldrich and used as received without further purification. h-IgG, anti-h-IgG produced in goat, and BSA were purchased from Sigma. Anti-m-EGFR produced in goat was purchased from R&D Systems. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400 MHz NMR spectrometer. The purified polymer and Scheme 1. Synthesis of the Cysteine-Functionalized Polymer
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polymer-coated nanoparticle solutions were vacuum dried and used for elemental analysis or dissolved in D2O for NMR study. Absorption spectra were obtained with an Agilent 8453 spectrophotometer using a quartz cell with a 1 cm path length. Fluorescence emission spectra were collected with a Fluorolog FL 3-11 fluorometer using a quartz cell with a 1 cm path length and 390 nm excitation. The quantum yield (QY) was measured using the integrated fluorescence intensity of the CdSe@ZnS QDs and the reference (fluorescein, QY = 97%) under 470 nm excitation. An FEI Technai G2 high-resolution transmission electron microscope was employed for transmission electron microscopy (TEM) studies. Samples were prepared by placing a drop of an aqueous sample on the carbon-coated copper grid, followed by air drying for 24 h. For the staining experiment, the copper grid was dipped into a phosphomolybdic acid solution (1%) for 5-10 s and then dried. Dynamic light scattering (DLS) samples were first filtered through a Millipore syringe filter (0.2 μm pores) and characterized with a model BI-200SM instrument (Brookhaven Instrument Corporation). The synthesis scheme for cysteine-functionalized polyaspartate has been designed such that multiple cysteine/methionine groups could be incorporated during the polymerization steps (Scheme 1). First, polyaspartimide was obtained by heating L-aspartic acid to 200 °C in the presence of orthophosphoric acid.49 Next, the nucleophilic ring opening of polyaspartimide was achieved with methylprotected L-cysteine/methionine. The resulting polymer was watersoluble with an average molecular weight of 15 000-25 000 (Supporting Information (SI)), as determined by gel permeation chromatography (GPC), and was similar to the earlier results on polyaspartimide.49 The proton NMR spectrum of the polymer showed characteristic peaks of cysteine associated with the polymer (SI). Polymer-coated Au, Ag, and QD nanocrystals were prepared by the ligand-exchange method or by the direct reduction of metal salts in the presence of polymer in the case of Au and Ag (Figures 1-3). In the ligand-exchange synthesis, high-quality, nearly monodisperse 1-10 nm Au, Ag, or QD nanocrystals were first prepared in an organic solvent7,12 and then the ligand exchange of polymer was performed in Igepal-cyclohexane reverse micelles. The advantage of using reverse micelles is that both the hydrophobic nanoparticles and the water-soluble polymer could be dissolved in cyclohexane and ligand exchange would occur without any particle aggregation. Particles were precipitated by ethanol
Figure 1. Absorption spectra of solutions of polymer-coated (a, c) Au nanoparticles of three different sizes and (b, d) Ag nanoparticles of two different sizes prepared (a,b) by a ligand-exchange method or (c, d) in the presence of polymer solutions of different concentrations. (a, b) Hydrophobic Au and Ag nanoparticles of different sizes (2-7 nm, ESI Table S1) were prepared first and then used for polymer-based ligand exchange. (c, d) Aqueous polymer solutions of different concentrations: (i) 1.0%, (ii) 0.1%, and (iii) 0.05% were used in the synthesis. Langmuir 2010, 26(9), 6503–6507
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Figure 4. PBS (pH 7.0) solution of polymer-coated nanoparticles prepared by ligand exchange of hydrophobic nanoparticles.
Figure 2. TEM micrographs of polymer-coated (a, b, e) Au, (c, d) Ag, and (f) QD nanoparticles of different sizes prepared by the ligand-exchange method. (e, f) Images are taken after phosphomolybdic acid staining.
Figure 3. Emission spectra of polymer-stabilized CdSe@ZnS QDs of different colors in phosphate-buffered saline (PBS).
addition, and the washed particles were dissolved in aqueous media. NMR spectra of the ligand-exchanged nanoparticles confirmed the presence of the polymer. Broadening of the proton NMR spectra was observed, suggesting polymer adsorption on the nanoparticle surface.35 In the conventional ligand-exchange method, nanoparticles dispersed in one organic solvent are mixed with another miscible organic solvent having the desired ligand.12 Alternatively, in a two-phase extraction method, the hydrophobic nanoparticles are in the organic solvent and the desired ligands are in the aqueous phase; after the solution is shaken, nanoparticles are extracted into the aqueous phase via ligand exchange.12 The first approach was difficult to apply here because the polymer was soluble in water but insoluble in most organic solvents. We have tried the second approach, but the ligand-exchange efficiency was poor, as seen in the significant portion of water-insoluble particles 6506 DOI: 10.1021/la903965t
Figure 5. (a) Schematic and (b) demonstration of h-IgG detection by polymer-stabilized nanocrystals conjugated with goat anti-hIgG. Au and Ag particles of ∼50 nm were used because they have large scattering cross sections. For the controls, protein spots were incubated with polymer-coated nanoparticles without any conjugated antibody, and as expected, protein spots were not observed.
obtained. In contrast, the reverse micelle approach described herein was unique and efficient because it did not produce particle aggregates. Au and Ag nanoparticles could also be synthesized by the direct reduction of the respective metal salts in the presence of polymers. Particles of 2-5 nm could be derived by varying the polymer concentration (Figures 1 and 2). Au and Ag nanoparticles of 10-100 nm, which were prepared in aqueous solution,1 could also be ligand exchanged with polymer by direct incubation with the polymer, followed by the centrifugation-induced separation of free polymer. Spectroscopic studies and TEM were performed before and after ligand exchange. No appreciable changes were observed in the particle size, indicating that the polymer effectively capped the nanoparticles and restricted the particle growth upon ligand exchange. Phosphomolybdic acid was used for polymer staining to visualize the polymer coating. However, the polymer capping was thin and poorly visualized because of poor contrast (Figure 2e,f). Elemental analysis was conducted for a composite of an Au-polymer whereby the Au core was 3 nm in size. It showed that ∼15% of the polymer was bound to the particle, which corresponded to ∼5-10 polymer molecules bound per particle. The DLS study showed that the overall particle diameters were increased by 3-6 nm for Au and Ag and by 15 nm for QD after the polymer coating (SI). The former could be attributed to the nearly monolayer coverage of polymer coating because the polymer has a dimension of ∼4 nm. The larger increase in the DLS particle diameter (i.e., in the case of coated QDs) was possibly due to nonuniform binding of the polymer coating on the particle surface and/or some particle aggregation during the coating steps. The colloidal stability of polyaspartate-functionalized nanoparticles was tested in various buffers, over different ranges of pH, and in the presence of salts (Figure 4). The nanoparticle solutions Langmuir 2010, 26(9), 6503–6507
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Figure 6. Labeling of 4T1 mouse breast cancer cells with polymer-coated CdSe@ZnS conjugated with anti-m-EGFR as studied by (b) fluorescence microscopy and (c) confocal microscopy. (a) No cell labeling was observed with control nanoparticles that were not functionalized with anti-m-EGFR. In the confocal microscopy study, cells were cultured on a circular coverslip placed under the cell culture plate.
were stable for at least several months without any sign of aggregation and precipitation when they were preserved under normal laboratory conditions (i.e., no precautions were taken to protect against sunlight). Particles were stable in the presence of 0.2 M NaCl for more than 1 week. Partial precipitation was observed after 2 to 3 days under a higher salt concentration. We also tested the colloidal stability of QDs exposed to UV light or sunlight. Although the QDs were stable for several hours under UV light or several days under sunlight, they would precipitate under prolonged exposure. Thus, the QD solutions were preferably kept in the dark for long-term stability. The fluorescence stability of the polymer-stabilized QDs was examined at pH’s ranging from 7 to 10. No fluorescence quenching was observed upon ligand exchange and after several months of preservation in buffer solutions. Depending on the size of the QDs, QYs of 10-20% were achieved. These studies illustrated that the multiple thiol and carboxyl groups on the polymer backbone imparted to the nanocrystals excellent colloidal stability and water solubility and that the multiple polar amide bonds and carboxyl groups of the polymer provided water solubility to the polymer-stabilized nanocrystals. We have successfully prepared antibody-conjugated nanoparticles with the novel polymer coating. Remarkably, the polymercoated nanoparticles were robust enough to survive the conditions associated with conjugation chemistry. The common coupling reagent, EDC hydrochloride, was employed to conjugate antibodies to the nanoparticle surface. EDC formed a covalent amide bond between the polymer’s carboxylate group and the antibody’s primary amine group. Anti-h-IgG and anti-m-EGFR were conjugated with the polymer-coated nanoparticles. High-speed centrifugation and size exclusion chromatography (SEC) were used in the purification steps. No particle aggregation or growth was observed during the entire bioconjugation process, indicating that the polymer protection was very effective. However, the use of excessive EDC (>5 mg/mL) would reduce the colloidal stability of the nanoparticles. In such cases, polymer cross linking on the
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particle surface might be useful to stabilize the nanoparticles further. We have examined the application of antibody-functionalized nanoparticles. Goat anti-h-IgG-conjugated nanoparticles could detect membrane-immobilized h-IgG with nanomolar sensitivity (Figure 5). Anti-m-EGFR-functionalized QDs could label the mouse breast cancer cells (Figure 6). Low-resolution fluorescence imaging of different areas of the cells showed that ∼80% of the cells were successfully labeled. Unlike other types of coating that often induced high nonspecific interactions during cell labeling,5 negligible nonspecific interaction was observed with our polymercoated nanoparticles. This could be attributed to the finer overall particle size and the negative surface charge provided by the polymer coating. In conclusion, a new polymer derived from amino acids composed of multiple thiol and carboxylic acid groups has been successfully prepared. This polymer can be used as a coating material for a wide variety of hydrophobic nanocrystals. It provides three combined advantages that are not offered by conventional coating systems. First, the simple ligand-exchange scheme can be used to generate water-soluble nanocrystals. Second, strong binding of the polymer to the nanocrystal’s surface via its multiple thiol groups leads to excellent colloidal stability. Third, the carboxyl groups in the polymer allow for further functionalization with antibodies and other small molecules. Acknowledgment. We thank Dr. Yu Han for TEM characterization. This work was supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). Supporting Information Available: 1H NMR spectra, GPC elution profile, DLS data, and UV-vis spectra. This material is available free of charge via the Internet at http://pubs. acs.org.
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