Facile Synthesis of Highly Biocompatible Poly(2-(methacryloyloxy

Biocompatibles UK Ltd., Chapman House, Farnham Business Park, Weydon ..... as doubly functional polymer support for immobilization of enzyme oxidase...
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Langmuir 2006, 22, 11022-11027

Facile Synthesis of Highly Biocompatible Poly(2-(methacryloyloxy)ethyl phosphorylcholine)-Coated Gold Nanoparticles in Aqueous Solution Jian-Jun Yuan, Andreas Schmid, and Steven P. Armes* Department of Chemistry, The UniVersity of Sheffield, Brook Hill, Sheffield S3 7HF, United Kingdom

Andrew L. Lewis Biocompatibles UK Ltd., Chapman House, Farnham Business Park, Weydon Lane, Farnham, Surrey, GU9 8QL, United Kingdom ReceiVed June 7, 2006. In Final Form: September 20, 2006 Diblock copolymers comprising a highly biocompatible poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) block and a poly(2-(dimethylamino)ethyl methacrylate) (PDMA) block were evaluated for the synthesis of sterically stabilized gold nanoparticles in aqueous solution. The PDMA block becomes partially protonated on addition of HAuCl4, and the remaining nonprotonated tertiary amine groups reduce the AuCl4- counterion to zerovalent gold in situ. This approach results in the adsorption of the PDMA block onto the gold nanoparticle surface while the PMPC chains serve as a stabilizing block, producing highly biocompatible gold sols in aqueous solution at ambient temperature without any external reducing agent. The size and shape of gold nanoparticles could be readily controlled by tuning synthesis parameters such as the block composition and the relative and absolute concentrations of the PMPC-PDMA diblock copolymer and HAuCl4. These highly biocompatible gold sols have potential biomedical applications.

Introduction Gold nanoparticles have been of particular interest over the past decade because of their unique applications in many areas such as biomedical materials, optics, and electronics.1 The surface engineering of gold nanoparticles plays an essential role in improving their biocompatibility and colloidal stability and hence imparting desirable optical properties, as well as enabling conjugation of bioactive functional groups.1,2 Thiol chemistry has been widely used for the surface modification of gold nanoparticles with chemical species ranging from small molecules2 to synthetic polymers3-16 to biomacromolecules being * Author to whom correspondence should be addressed. E-mail: [email protected]. (1) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293-346. (2) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (3) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802. (4) Wuelfing, W. P.; Gross, S. M.; Miles, D. T.; Murray, R. W. J. Am. Chem. Soc. 1998, 120, 12696-12697. (5) Nuss, S.; Bottcher, H.; Wurm, H.; Hallensleben, M. L. Angew. Chem., Int. Ed. 2001, 40, 4016-4018. (6) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226-8230. (7) Corbierre, M. K.; Cameron, N. S.; Sutton, M.; Mochrie, S. G. J.; Lurio, L. B.; Ruhm, A.; Lennox, R. B. J. Am. Chem. Soc. 2001, 123, 10411-110412. Corbierre, M. K.; Cameron, N. S.; Lennox, R. B. Langmuir 2004, 20, 28672873. (8) Lowe, A. B.; Sumerlin, B. S.; Donovan, M. S.; McCormick, C. L. J. Am. Chem. Soc. 2002, 124, 11562-11563. (9) Mangeney, C.; Ferrage, F.; Aujard, I.; Marchi-Artzner, V.; Jullien, L.; Ouari, O.; Rekai, E. D.; Laschewsky, A.; Vikholm, I.; Sadowski, J. W. J. Am. Chem. Soc. 2002, 124, 5811-5821. (10) Mandal, T. K.; Fleming, M. S.; Walt, D. R. Nano Lett. 2002, 2, 3-7. (11) Ohno, K.; Koh, K.-M.; Tsujii, Y.; Fukuda, T. Macromolecules 2002, 35, 8989-8993. (12) Shan, J.; Nuopponen, M.; Jiang, H.; Kauppinen, E.; Tenhu, H. Macromolecules 2003, 36, 4526-4533. Raula, J.; Shan, J.; Nuopponen, M.; Niskanen, A.; Jiang, H.; Kauppinen, E.; Tenhu, H. Langmuir 2003, 19, 34993504. Shan, J.; Nuopponen, M.; Jiang, H.; Viitala, T.; Kauppinen, E.; Kontturi, K.; Tenhu, H. Macromolecules 2005, 38, 2918-2926. (13) Zhu, M.; Wang, L.; Exarhos, G. J.; Li, A. D. Q. J. Am. Chem. Soc. 2004, 126, 2656-2657. (14) Shimmin, R. G.; Schoch, A. B.; Braun, P. V. Langmuir 2004, 20, 56135620.

reported.17-19 These gold nanoparticles were typically produced using conventional reducing agents such as NaBH4 in the presence of thiol-functional molecules as stabilizers. In some cases, this approach has certain obvious disadvantages, such as the use of organic solvents, the generation of small molecule byproducts from the reducing agents, and the relatively poor long-term stability of Au-S bonds, and multiple steps are required for the final functionalized gold nanoparticles. On the other hand, it has been recently demonstrated that gold nanoparticles can be synthesized by employing certain copolymers that can act as both a reducing agent and a stabilizer simultaneously.20-24 Thus, colloidally stable gold sols can be conveniently prepared in a single step at ambient conditions without any external reducing agents, which aids purification of these nanoparticles. However, reports of tailoring the surface chemistry of gold nanoparticles by this facile approach remain rare. Recently, Ishii et al.24 reported the synthesis of PEGylated gold nanoparticles with biotin moieties by using R-biotinylPEG-block-poly[2-(dimethylamino)ethyl methacrylate)] (PDMA). The tertiary amine methacrylate-based PDMA block reduces (15) 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, 16398-16399. (16) Luo, S.; Xu, J.; Zhang, Y.; Liu, S.; Wu, C. J. Phys. Chem. B 2005, 109, 22159-22166. (17) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. Li, Z.; Jin, R.; Mirkin, C. A.; Letsinger, R. L. Nucleic Acids Res. 2002, 30, 1558-1562. (18) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P. J.; Schultz, P. G. Nature 1996, 382, 609-611. (19) de la Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo, J.; Can˜ada, J.; Ferna´ndez, A.; Penade´s, S. Angrew. Chem., Int. Ed. 2001, 40, 2257-2261. (20) Bronstein, L. M.; Sidorov, S. N.; Gourkova, A. Y.; Valetsky, P. M.; Hartmann, J.; Breulmann, M.; Co¨lfen, H.; Antonietti, M. Inorg. Chim. Acta 1998, 280, 348-354. (21) Yu, S. H.; Co¨lfen, H.; Mastai, Y. J. Nanosci. Nanotechnol. 2004, 4, 291-298. (22) Sakai, T.; Alexandridis, P. Langmuir 2004, 20, 8426-8430. Sakai, T.; Alexandridis, P. J. Phys. Chem. B 2005, 109, 7766-7777. (23) Sun, X.; Dong, S.; Wang, E. Mater. Chem. Phys. 2006, 96, 29-33. Chen, H.; Wang, Y.; Wang, Y.; Dong, S.; Wang, E. Polymer 2006, 47, 763-766. (24) Ishii, T.; Otsuka, H.; Kataoka, K.; Nagasaki, Y. Langmuir 2004, 20, 561-564.

10.1021/la0616350 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/10/2006

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Table 1. Molecular Weight Data of the Various Copolymer Stabilizers Used for the Synthesis of Gold Nanoparticles copolymera PDMA102 PMPC30-PDMA10 PMPC30-PDMA30 PMPC30-PDMA60 PMPC30-PDMA100 PEO45-PDMA30

Mn (GPC, g/mol)b Mw/Mnb method of synthesis 16 100 11 000 22 000 34 000 46 000 9000

1.09 1.28 1.27 1.29 1.32 1.13

GTP ATRP ATRP ATRP ATRP ATRP

a The subscripts indicate the mean degrees of polymerization (DP) of each block. b As determined by aqueous GPC using poly(2vinylpyridine) calibration standards.

AuCl4- to zerovalent gold, while the PEG (aka PEO) block serves as a stabilizer. The biotin-functionalized gold nanoparticles can be aggregated in the presence of streptavidin. This reduction in the degree of dispersion of the sol has an associated color change, suggesting its possible application in immunodiagnostic assays. The 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) monomer possesses phosphorylcholine groups, which are also found on the outside of cell membranes.25-28 This biomimetic monomer has been used to produce highly biocompatible copolymer surface coatings that are remarkably resistant to protein adsorption and bacterial/cellular adhesion.25-28 Furthermore, controlled-structure poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC)based hydrogels,29 micelles,30 and vesicles31 have been prepared in our laboratory using atom transfer radical polymerization (ATRP).32 Recently, we have become interested in conjugating PMPC chains to the surface of inorganic nanoparticles to combine the high biocompatibility of PMPC with interesting optical, electronic, and magnetic properties.33-35 For example, ultrafine silica and tin(IV) oxide nanoparticles have been coated with PMPC chains using an electrostatically adsorbed polyelectrolytic ATRP macroinitiator.33,34 More recently, colloidably stable suspensions of superparamagnetic magnetite sols have been prepared using PMPC-block-poly(glycerol monomethacrylate) copolymers.35 Such biocompatible sols are expected to have potential applications as high-performance contrast agents in magnetic resonance imaging. Herein, we report the synthesis of colloidally stable gold nanoparticles coated with PMPC chains using a series of PMPC-PDMA diblock copolymers (see Table 1 for details of the copolymers used in this work). The synthesis protocol for the preparation of PMPC-coated gold nanoparticles using these copolymers is summarized in Figure 1. Experimental Section Materials. HAuCl4 was purchased from Aldrich and was used as received. Deionized water was used in all experiments. The PMPC-PDMA diblock copolymers and PMPC homopolymer were all synthesized using ATRP as described previously.36 The PDMA102 (25) Hayward, J. A.; Chapman, D. Biomaterials 1984, 5, 135-142. (26) Ishihara, K. Trends Polym. Sci. 1997, 5, 401-407. (27) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323-330. (28) Nakaya, T.; Li, Y. J. Prog. Polym. Sci. 1999, 24, 143-181. (29) Li, C.; Tang, Y.; Armes, S. P.; Morris, C. J.; Rose, S. F.; Lloyd, A. W.; Lewis, A. L. Biomacromolecules 2005, 6, 994-999. (30) Giacomelli, C.; Le Men, L.; Borsali, R.; Lai-Kee-Him, J.; Brisson, A.; Armes, S. P.; Lewis, A. L. Biomacromolecules 2006, 7, 817-828. (31) Du, J.; Tang, Y.; Lewis, A. L.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 17982-17983. (32) Lobb, E. J.; Ma, I.; Billingham, N. C.; Armes, S. P.; Lewis, A. L. J. Am. Chem. Soc. 2001, 123, 7913-7914. (33) Chen, X.; Armes, S. P. AdV. Mater. 2003, 15, 1558-1562. (34) Chen, X. Y.; Armes, S. P.; Greaves, S. J.; Watts, J. F. Langmuir 2004, 20, 587-595. (35) Yuan, J. J.; Armes, S. P.; Takabayashi, Y.; Prassides, K. P.; Leite, C. A. P.; Galembeck, F.; Lewis, A. L. Langmuir 2006, in press. (36) Ma, Y.; Tang, Y.; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salvage, J. P. Macromolecules 2003, 36, 3475-3484.

Figure 1. Schematic representation of the synthesis of biocompatible, sterically stabilized gold nanoparticles using a PMPC30-PDMA30 diblock copolymer stabilizer. homopolymer was synthesized by group transfer polymerization. Molecular weight and polydispersity data for each polymeric stabilizer are summarized in Table 1. Gold Nanoparticle Synthesis. The following protocol is typical. Copolymer-stabilized gold nanoparticles were simply synthesized by adding an aqueous solution of copolymer at various concentrations (see later) into an aqueous solution of HAuCl4. This reaction mixture was stirred at room temperature for 24 h. The final sol had good colloidal stability and a red color, indicating the formation of gold nanoparticles. Transmission Electron Microscopy (TEM). Samples were prepared by drying a drop of a dilute dispersion of gold nanoparticles onto a carbon-coated copper grid and were analyzed using a Phillips CM100 electron microscope operating at 100 kV. Dynamic Light Scattering (DLS). A Malvern Nanosizer instrument operating at a laser wavelength of 633 nm and a fixed detector angle of 173° was used for DLS measurements on highly dilute aqueous gold dispersions. Aqueous Electrophoresis. Measurements were carried out using a Malvern Zetasizer NanoZS instrument. The zeta potential (ζ) was calculated from the electrophoretic mobility (u) using the Smoluchowsky relationship, ζ ) ηu/, assuming that κa . 1 (where η is the solution viscosity,  is the dielectric constant of the medium, and κ and a are the Debye-Hu¨ckel parameters and the particle radius, respectively). The solution pH was adjusted by the addition of HCl or NaOH. UV-Vis Absorption Spectroscopy. Spectra were recorded for dilute aqueous dispersions of gold nanoparticles in a 1-cm quartz cuvette using a Perkin-Elmer Lambda 2 spectrophotometer. FT-IR Spectroscopy. Copolymer-stabilized gold sols were centrifuged at 20 000 rpm for 1 h, and the sedimented nanoparticles (or platelike particles) were dried in an oven overnight at 80 °C. KBr disks were prepared using 1-2 mg of each sample. Spectra were recorded using a Series II Nicolet Magna 550 spectrometer. All spectra were recorded at 4 cm-1 resolution, and typically 64 scans were averaged per spectrum. Background spectra were recorded using a blank KBr disk. Thermogravimetry (TGA). TGA runs were conducted using a Perkin-Elmer Pyris 1 instrument. Dried gold nanoparticles were obtained using the same purification procedure as that described for the FT-IR studies. TGA measurements were carried out by heating samples in air to 800 °C at a heating rate of 20 °C min-1. The observed mass loss was attributed to the quantitative degradation of the copolymer stabilizer, with the remaining incombustible residues being assumed to be pure gold.

Results and Discussion PDMA was selected as the anchor block since, according to Ishii et al., this should also act as a reducing agent when generating the gold sol.24 Moreover, the basic nature of PDMA means that this block will be protonated by the acidic gold precursor (HAuCl4), leading to the intimate association of the AuCl4counterions with the cationic PDMA chains just prior to the in-situ reduction to zerovalent gold. This should ensure efficient

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Figure 2. Gold nanoparticles synthesized using a PDMA102 homopolymer and a PMPC30-PDMA30 diblock copolymer: (A) UV-vis absorption spectra; (B) a representative TEM image of gold nanoparticles synthesized using PDMA102 homopolymer; (C) a representative TEM image of gold nanoparticles prepared using PMPC30-PDMA30 diblock copolymer; and (D) the particle size distribution of gold nanoparticles determined from C. The conditions were as follows: [DMA]/[HAuCl4] molar ratio ) 9.25; [HAuCl4] ) 1.2 × 10-6 moles; the solution pH is 6.5; synthesis was conducted for 24 h at 20 °C.

adsorption of the PDMA chains onto the gold sol surface. In contrast, the PMPC chains were expected to provide a highly hydrated, adsorbed layer. As expected, stable aqueous sols of gold nanoparticles were readily prepared by mixing aqueous stock solutions of HAuCl4 with various PMPC-PDMA diblock copolymers. Each reaction solution was stirred at room temperature for typically 24 h. The final dispersion had a characteristic red color, indicating the formation of colloidal gold nanoparticles. In our preliminary control experiments, we examined gold nanoparticle formation in the presence of PMPC30 and PDMA102 homopolymers. A solution containing 3.0 mL of a 0.4 mM solution of aqueous HAuCl4 and 0.50 mL of an aqueous solution containing 0.35 wt % PMPC30 did not show any color change after stirring for 24 h, indicating that, unlike PEO homopolymer,22 the PMPC chains alone do not reduce AuCl4- to zerovalent gold. In contrast, on mixing 3.0 mL of an aqueous solution of 0.4 mM HAuCl4 with 0.50 mL of an aqueous solution containing 0.35 wt % PDMA102 homopolymer (the [DMA]/[HAuCl4] molar ratio ) 9.25), the solution gradually turned red after stirring at 20 °C for 24 h. This color change indicated the formation of ultrafine gold nanoparticles.37 UV-vis absorption spectroscopy studies indicated a maximum absorption at 525 nm, which corresponds to the surface plasmon absorption of gold nanoparticles (see Figure 2A). The size and shape of the gold nanoparticles were examined by transmission electron microscopy (TEM). As shown in Figure 2B, an approximately spherical morphology was obtained, with particle diameters ranging from 2 to 14 nm, and the numberaverage diameter was estimated to be 8.7 ( 4.2 nm (as shown in Figure S1). Dynamic light scattering (DLS) studies on the same gold sol indicated an intensity-average diameter of 41 nm. This apparent discrepancy is partly because DLS is sensitive to (37) Wilcoxon, J. P.; Williamson, R. L.; Baughman, R. J. Chem. Phys. 1993, 98, 9933-9950.

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the larger particles in the size distribution and also because DLS “sees” the hydrated layer of PDMA chains that is adsorbed onto the surface of the gold nanoparticles. Clearly, the PDMA block acts as an effective reducing agent and promotes the formation of gold nanoparticles at room temperature. The block copolymer-stabilized gold nanoparticles were synthesized as follows. First, 0.50 mL of a 1.0 wt % aqueous solution of PMPC30-PDMA30 diblock copolymer ([DMA] ) 11.1 × 10-6 moles) was added to 3.0 mL of an aqueous solution of 0.4 mM HAuCl4 aqueous solution (i.e., 1.2 × 10-6 moles of HAuCl4). This reaction solution initially had a solution pH of 6.5. The mixture was stirred for 24 h at room temperature, yielding an aqueous red sol with a final solution pH of 6.3. UV-vis absorption spectroscopy studies indicated a maximum absorption at 530 nm for the surface plasmon absorption of gold (see Figure 2A). TEM studies indicated well-defined, spherical gold nanoparticles (see Figure 2C) and a number-average diameter of 10 ( 1.4 nm, with diameters ranging from 7 to 14 nm (see Figure 2D). DLS studies indicated an intensity-average diameter of 45 nm for this gold sol. Allowing for polydispersity effects, this value is in reasonable agreement with 10-nm gold cores being coated with a highly hydrated layer of the PMPC30-PDMA30 diblock copolymer. Compared to the nanoparticles prepared using the PDMA102 homopolymer, this gold sol is more spherical, has a narrower size distribution, and has a slightly larger diameter. The diblock copolymer architecture obviously improves the polymeric stabilizer efficiency, as well as conferring a highly biocompatible surface coating of PMPC chains. The 5-nm red shift in the absorption maximum is presumably due to the slightly larger nanoparticle diameter.37-40 To further address the role played by the PMPC block in producing these gold nanoparticles, a PEO45-PDMA30 diblock copolymer was evaluated as a stabilizer. The gold nanoparticles were prepared under the same conditions used for the gold sol synthesized with the PMPC30PDMA30 copolymer. TEM studies indicated the formation of ill-defined gold particles consisting of gold plates (mainly hexagons and triangles) with diameters of several hundred nanometers and also smaller particles of tens of nanometers (Figure S2A). UV-vis absorption spectroscopy studies indicated a maximum absorption at 539 nm due to the smaller population and a broader absorption envelope because of the larger population (Figure S2B). PEO alone was reported to be active for the reduction of AuCl4- at ambient temperature, which possibly contributes to the ill-defined gold particle morphology.22 In contrast, PMPC does not play an active role for the roomtemperature reduction of AuCl4-; instead, it acts solely as a steric stabilizer. Aqueous electrophoresis measurements were conducted to assess the zeta potentials of the gold nanoparticles prepared using both the PDMA102 homopolymer and the PMPC30-PDMA30 block copolymer (see Figure 3). Both sols exhibited an isoelectric point at around pH 8.6. The block copolymer-coated sol showed good long-term colloidal stability for at least 2 months and also on varying the solution pH from 2 to 12. No flocculation was observed at around its IEP. Remarkably, such PMPC30-PDMA30 stabilized gold sols can be readily redispersed in aqueous solution to form stable colloidal dispersions even after drying at 80 °C overnight. In contrast, the gold sol prepared using the PDMA102 homopolymer stabilizer exhibited inferior pH-dependent colloidal stability. When the solution pH was adjusted to near the isoelectric (38) Templeton, A. C.; Pietron, J. J.; Murray, R. W.; Mulvaney, P. J. Phys. Chem. B 2000, 104, 564-570. (39) Lu, Y.; Yin, Y.; Li, Z.-Y.; Xia, Y. Nano Lett. 2002, 2, 785-788. (40) Kang, Y.; Taton, T. A. Angew. Chem., Int. Ed. 2005, 44, 409-411. Kang, Y.; Taton, T. A. J. Am. Chem. Soc. 2003, 125, 5650-5651.

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Figure 3. Zeta potential vs pH curves obtained for gold sols prepared using the PDMA102 homopolymer and the PMPC30-PDMA30 diblock copolymer. The synthesis conditions were the same as those described in the caption for Figure 2.

Figure 5. Gold nanoparticles prepared at 20 °C using a PMPC30PDMA60 block copolymer by varying the [DMA]/[HAuCl4] molar ratio. (A), (B), and (C) are TEM images of gold nanoparticles prepared using PMPC30-PDMA60 at [DMA]/[HAuCl4] molar ratios of 20.8, 13.8, and 6.9, respectively; (D) UV-vis absorption spectra recorded for these three gold sols. The reaction time in each case was 24 h.

Figure 4. Gold nanoparticles synthesized using three PMPCPDMA diblock copolymers (each with a fixed PMPC block length and a variable PDMA block length). Representative TEM images of gold nanoparticles prepared using (A) PMPC30-PDMA60, (B) PMPC30-PDMA100, and (C) PMPC30-PDMA10, respectively; (D) UV-vis absorption spectra recorded for these three gold sols. The [DMA]/[HAuCl4] molar ratio was 9.25; [HAuCl4] ) 1.2 × 10-6 moles; syntheses were conducted for 24 h at 20 °C.

point of this latter sol, the color of the solution changed from red to violet, indicating aggregation of the primary nanoparticles (see Figure S3A).17,18 On standing, the aggregated particles sedimented to the bottom of the vial. UV-vis absorption spectroscopy studies indicated that the maximum absorption shifted from 525 nm at either pH 7 or pH 10 to 560 nm at pH 8.3 (Figure S3A). DLS measurements indicated an intensityaverage sol diameter of approximately 1120 nm at pH 8.3, which is much larger than the 41-nm diameter observed at pH 7.0 and indicates substantial flocculation. TEM studies also indicated aggregation of the primary gold nanoparticles (Figure S3B). The surface charge of the PDMA102-derived sol was strongly dependent on the degree of protonation of the weakly basic adsorbed PDMA chains. In contrast, the diblock copolymerderived sol remained colloidally stable even at its isoelectric point, since the highly hydrated zwitterionic PMPC chains on the particle surface provide very effective steric stabilization. The PMPC30-PDMA30 block copolymer-stabilized gold nanoparticles were further characterized using FT-IR spectroscopy

and thermogravimetric analyses. The sol was centrifuged at 20 000 rpm for 1 h, and the sedimented nanoparticles were dried at 80 °C overnight for FT-IR and thermogravimetry studies. FT-IR spectroscopy provided reasonable qualitative evidence that the PMPC30-PDMA30 diblock copolymer was adsorbed onto the gold nanoparticles, since the band observed at 1720 cm-1 (see Figure S4) is assigned to the ester carbonyl stretch of the copolymer. Thermogravimetric studies indicated that the average copolymer content of the gold nanoparticles was about 45% by mass. The size of the gold nanoparticles could be readily controlled by varying the block composition, the relative block copolymer/ HAuCl4 molar ratio, and the HAuCl4 concentration. Three PMPC-PDMA copolymers, namely, PMPC30-PDMA60, PMPC30-PDMA100, and PMPC30-PDMA10, were evaluated to examine the effect of block composition on gold nanoparticle formation. Typically, 3.0 mL of an aqueous solution of 0.4 mM HAuCl4 was mixed with 0.50 mL aqueous copolymer solution ([DMA] ) 11.1 × 10-6 moles), giving a [DMA]:[HAuCl4] molar ratio of 9.25. The reactions were conducted in stirred solutions for 24 h at 20 °C. As shown in Figure 4A, the gold nanoparticles prepared using PMPC30-PDMA60 have a spherical morphology with diameters ranging from 3 to 12 nm and a number-average diameter of 6.6 ( 1.9 nm (see Figure S5B). Such nanoparticles have a smaller mean diameter and a slightly broader size range compared to the sols synthesized using PMPC30-PDMA30. Clearly, increasing the PDMA block length can produce smaller nanoparticles. PMPC30-PDMA100 was also used for the synthesis of gold nanoparticles. TEM studies indicated a spherical particle morphology (see Figure 4B). These particles have a numberaverage diameter of 8.0 ( 5.5 nm, but a much broader size distribution, with diameters ranging from 3 to 35 nm (see Figure S5C). Thus, if the PDMA block is too long, this type of stabilizer is not well-suited for the synthesis of high-quality gold nanoparticles. On the other hand, we also examined the synthesis of gold nanoparticles using PMPC30-PDMA10. As shown in

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Figure 6. Gold nanoparticles prepared at 20 °C using the PMPC30PDMA30 diblock copolymer at various copolymer and HAuCl4 concentrations. (A), (B), and (C) are typical TEM images obtained for gold nanoparticles prepared at PMPC30-PDMA30 concentrations of 0.45, 3.6, and 10.5%, respectively; (D) UV-vis absorption spectra obtained for three gold sols prepared using (a) 0.45, (b) 3.6, and (c) 10.5% copolymer, respectively. The [DMA]/[HAuCl4] molar ratio was held constant at 9.25, and each synthesis was conducted for 24 h.

Figure 4C, a mixture of gold hexagons and triangles, as well as relatively large spherical sols, was observed. These particles have a number-average diameter of 75 ( 25 nm, with diameters ranging from 34 to 168 nm (see Figure S5D). Obviously, a relatively short PDMA block is not sufficient for efficient gold sol stabilization. DLS studies indicated intensity-average diameters of 42, 50, and 130 nm for gold sols synthesized using the PMPC30-PDMA60, PMPC30-PDMA100, and PMPC30-PDMA10 copolymers, respectively. These data are in reasonable agreement with those obtained from TEM measurements, allowing for the highly hydrated copolymer layer adsorbed onto the gold sol and polydispersity effects. Figure 4D shows the UV-vis absorption spectra of gold sols synthesized using three copolymers. Like the gold sols prepared using PMPC30-PDMA30 (see Figure 2A), those synthesized using PMPC30-PDMA60 and PMPC30PDMA100 exhibited absorption maxima at 530 nm, indicating gold nanoparticles of 6-10 nm diameter. However, the absorption maximum obtained for a gold sol prepared using PMPC30PDMA10 exhibited a significant red shift to 562 nm, indicating much larger gold nanoparticles in this dispersion. This is consistent with the results from DLS (130 nm) and TEM studies (Figure 4C). The [copolymer]:[HAuCl4] molar ratio could be also exploited to control the size of the gold nanoparticles. For example, 0.50 mL of an aqueous solution of PMPC30-PDMA60 ([DMA] ) 11.1 × 10-6 moles) was mixed with 2.0, 3.0, or 6.0 mL of an aqueous solution of 0.4 mM HAuCl4, producing [DMA]: [HAuCl4] molar ratios of 20.8, 13.8, and 6.9. As shown in Figure 5A, the highest [DMA]:[HAuCl4] molar ratio resulted in a 50nm-diameter gold sol. At a [DMA]:[HAuCl4] molar ratio of 13.8, somewhat smaller gold nanoparticles of about 10 nm were obtained (see Figure 5B), while the lowest molar ratio gave a 7-nm sol (see Figure 5C). DLS studies indicated intensity-average diameters of 180, 56, and 40 for the gold sols synthesized using 2.0, 3.0, and 6.0 mL of the 0.4 mM aqueous HAuCl4 solution, respectively. The latter

Figure 7. Gold nanoparticles prepared at 20 °C using various concentrations of PDMA102 homopolymer and HAuCl4: (A) UVvis absorption spectra obtained for gold sols prepared using 0.29, 0.43, 1.3, and 3.9% PDMA102; (B) is a representative TEM image of gold nanoparticles synthesized at 3.9% PDMA102. The [DMA]/ [HAuCl4] molar ratio was held constant at 9.25, and each synthesis was conducted for 24 h.

two diameters are in reasonable agreement with the TEM observations allowing for the additional hydrodynamic thickness of the adsorbed copolymer overlayer and polydispersity effects. However, the DLS diameter of 180 nm is much larger than that obtained from TEM, indicating some degree of flocculation of the primary particles. The UV-vis absorption spectra of these three sols are shown in Figure 5D. The absorption maxima are blue-shifted from 545 to 526 nm, indicating a reduction in the mean diameter of the gold nanoparticles. In contrast, this variation in particle size was not observed for the corresponding sols synthesized using PDMA102 homopolymer, where the absorption maximum remained almost constant at around 523 nm, regardless of the stabilizer concentration (see Figure S6). DLS studies also indicated that there were no significant size differences, with intensity-average diameters of around 30-40 nm being obtained for each sol. TEM studies on selected samples also suggested similar results (data not shown). Therefore, using PMPC-PDMA block copolymer stabilizers offers convenient control over the mean gold sol diameter by simply varying the [DMA]:[HAuCl4] molar ratio. (41) Licciardi, M.; Tang, Y.; Billingham, N. C.; Armes, S. P.; Lewis, A. L. Biomacromolecules 2005, 6, 1085-1096.

Coated Gold Nanoparticles in Aqueous Solution

Finally, we attempted to synthesize gold sols at higher concentrations of both polymer and HAuCl4. The [DMA]: [HAuCl4] molar ratio was held constant at 9.25. Aqueous solutions (3.50-mL aliquots) containing PMPC30-PDMA30 and HAuCl4 were prepared, and the block copolymer concentrations were selected to be 0.45, 3.6, and 10.5 wt %, respectively. The reactions were conducted at room temperature for 24 h with continuous stirring. As shown in Figure 6A, the gold nanoparticles synthesized at a block copolymer concentration of 0.45 wt % have a mean diameter of around 23 nm. This value is much larger than that obtained for the sols synthesized using a block copolymer concentration of 0.045 wt % (see Figure 1C). DLS studies indicated an intensity-average diameter of 43 nm. When the concentration was increased to 3.6 wt % for PMPC30-PDMA30, gold nanoparticles with a mean diameter of around 100 nm were obtained. In addition, gold platelets of up to 500 nm were observed as byproducts. DLS studies indicated intensity-average diameters of around 183 nm. The gold particles synthesized at a PMPC30PDMA30 concentration of 10.5 wt % had a typical TEM diameter of around 200 nm, but some platelets of up to 500 nm were also observed. DLS studies indicated intensity-average diameters of around 350 nm. UV-vis absorption spectroscopy studies were consistent with larger gold nanoparticles being formed at higher block copolymer concentration. As shown in Figure 6D, the absorption maxima were red-shifted with increasing PMPC30PDMA30 concentration. Again, the mean size of gold nanoparticles synthesized using the PDMA homopolymer did not appear to depend on the PDMA (or HAuCl4) concentration under similar synthesis conditions. As shown in Figure 7A, gold sols prepared at 0.29, 0.43, 1.3, and 3.9% PDMA each exhibit absorption maxima at around 521 nm, indicating similar mean sol diameters. TEM studies confirmed these observations. Figure 7B shows a representative image of a gold sol synthesized at 3.9 wt % PDMA. The nanoparticles have a well-defined spherical morphology with a mean diameter of about 10 nm. DLS studies indicated similar intensity-average diameters of 38-50 nm for the four PDMA homopolymer-stabilized sols. (42) Li, C.; Madsen, J.; Armes, S. P.; Lewis, A. L. Angew. Chem., Int. Ed. 2006, 45, 3510-3513.

Langmuir, Vol. 22, No. 26, 2006 11027

Conclusions In summary, PMPC-PDMA diblock copolymers can be used to prepare high-quality gold nanoparticles in aqueous solution at room temperature without any external reducing agent. The PDMA block promotes the in-situ reduction of AuCl4- to zerovalent gold and subsequently binds onto the surface of gold nanoparticles, while the PMPC acts as a stabilizing block, producing highly biocompatible gold sols. The size and shape of gold nanoparticles can be controlled by varying the block composition of the PMPC-PDMA stabilizer, the relative proportions of copolymer and HAuCl4, and the absolute concentrations of copolymer and HAuCl4. Moreover, it should be relatively easy to design “next-generation” gold nanoparticles with surface functional groups such as fluorescent labels, folic acid,41 -SH,42 or -NH2 located at the end of the PMPC blocks. These surface-functionalized biocompatible gold nanoparticles should allow cell imaging and other biomedical applications. Acknowledgment. We thank Dr. Y. Ma, Dr. V. Bu¨tu¨n, and Ms. E. S. Read for the synthesis of the various copolymers. EPSRC is acknowledged for a postdoctoral fellowship for J.J.Y. (Platform grant, GR/S25845). S.P.A. is the recipient of a 5-year Royal Society/Wolfson Research Merit Award. Supporting Information Available: Particle size distribution of gold nanoparticles synthesized using the PDMA102 homopolymer; TEM image and UV-vis spectrum of gold nanoparticles synthesized using the PEO45-PDMA30 block copolymer; UV-vis spectra and TEM images recorded for PDMA102-stabilized gold nanoparticles at different solution pH showing the aggregation of nanoparticles at around their isoelectric point; FT-IR spectra of PMPC30-PDMA30 and PMPC30PDMA30-stabilized gold nanoparticles; size distribution of gold nanoparticles prepared using PMPC30-PDMA30, PMPC30-PDMA60, PMPC30-PDMA100, and PMPC30-PDMA10; UV-vis spectra of gold nanoparticles synthesized using PDMA102 homopolymer at various [DMA]:[HAuCl4] molar ratios. This material is available free of charge via the Internet at http://pubs.ac.org. LA0616350