Modulation of the Surface Charge on Polymer-Stabilized Gold

Nov 6, 2009 - ... Design (CAMD), School of Chemical Sciences and Engineering, The University of New South Wales, Sydney, NSW 2052, Australia...
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Modulation of the Surface Charge on Polymer-Stabilized Gold Nanoparticles by the Application of an External Stimulus Cyrille Boyer, Michael R. Whittaker, Kyloon Chuah, Jingquan Liu, and Thomas P. Davis* Centre for Advanced Macromolecular Design (CAMD), School of Chemical Sciences and Engineering, The University of New South Wales, Sydney, NSW 2052, Australia Received July 27, 2009. Revised Manuscript Received September 24, 2009 A new approach to controlling the charge on gold nanoparticle surfaces is described. The method exploits the simultaneous coattachment of both charged and neutral polymers onto gold surfaces. The charged and neutral polymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, and the RAFT end-group functionality was used as the anchor for attachment to gold. The approach described is general and can be applied to a wide range of monomers; those exemplified in the paper are poly(2-aminoethyl methacrylamide) (P(AEA)), poly(acrylic acid) (PAA), and poly(N,N-diemthylaminoethyl acrylate) (P(DMAEA)) together with neutral polymers based on poly(oligoethylene oxide) acrylate (P(OEG-A)). The hybrid polymer-stabilized GNPs thus formed were characterized in solution using dynamic light scattering and zeta potential measurements, transmission electron microscopy, UV-visible spectroscopy, X-ray photoelectron spectroscopy, and attenuated total reflection-Fourier-transform IR spectroscopy. The grafting densities of the polymers on GNPs were measured using thermal gravimetric analyses (TGA), as 0.4 chains/nm2 (for PAA), 0.9 chains/nm2 (for neutral polymers, such as P(NIPAAm), and 0.6 chain/nm2 for the positive charged polymers P(AEA) and P(DMAEA). The directed coassembly of two different polymers (one charged and one noncharged) on the gold nanoparticle surfaces provided a mechanism (dependent on molecular weight) for shielding the surface charge imparted by the charged polymer component, allowing for a range of surface charges on the GNPs from -30 to þ39 mV. In further work, the surface-charges were modulated by an external stimulus (temperature). The charge-modulation was controlled by the use of thermosensitive neutral polymers coassembled with charged polymers. The thermosensitive polymers exemplified in this paper are P(oligoethylene oxide acrylate-co-diethylene oxide acrylate) (P(OEG-A-coDEG-A)) and P(N-isopropyl acrylamide) (P(NIPAAm). The temperature of the aqueous phase (from 15 to 70 °C) was then adjusted to tune the zeta potentials of the hybrid GNPs from þ39 or -30 to ∼0 mV. Finally, by manipulating the solution pH, reversible aggregation behavior of the hybrid GNPs could be induced.

Introduction 1

Functional gold nanoparticles (GNPs) are emerging as a promising class of material for nanotechnology and biotechnology applications.2-9 The attachment of well-defined and responsive polymer layers onto the GNP surface confers not only stability in aqueous solution but can also impart additional properties. Two different strategies have been widely employed for the grafting of polymers layers to GNP surfaces: “grafting from”10-13 or “grafting to”.14,15 *Address correspondence to [email protected]. (1) Chen, P. C.; Mwakwari, S. C.; Oyelere, A. K. Nanotechnol., Sci. Appl. 2008, 1, 45–66. (2) Weissleder, R. Science 2006, 312(5777), 1168–1171. (3) De, M.; Ghosh, P. S.; Rotello, V. M. Adv. Mater. 2008, 20(22), 4225–4241. (4) Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M. Adv. Drug Delivery Rev. 2008, 60(11), 1307–1315. (5) Giljohann, D. A.; Seferos, D. S.; Prigodich, A. E.; Patel, P. C.; Mirkin, C. A. J. Am. Chem. Soc. 2009, 130(6), 2073–2074. (6) Zhao, W.; Chiuman, W.; Lam, J. C. F.; McManus, S. A.; Chen, W.; Cui, Y.; Pelton, R.; Brook, M. A.; Li, Y. J. Am. Chem. Soc. 2008, 130(11), 3610–3618. (7) Storhoff, J. J.; Lazarides, A. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L.; Schatz, G. C. J. Am. Chem. Soc. 2000, 122(19), 4640–4650. (8) Chen, L.; Hu, J.; Richards, R. J. Am. Chem. Soc. 2009, 131(3), 914–915. (9) Han, J.; Liu, Y.; Guo, R. J. Am. Chem. Soc. 2009, 131(6), 2060–2061. (10) Dong, H.; Zhu, M.; Yoon, J. A.; Gao, H.; Jin, R.; Matyjaszewski, K. J. Am. Chem. Soc. 2008, 130(39), 12852–12853. (11) Li, Y.; Smith, A. E.; Lokitz, B. S.; McCormick, C. L. Macromolecules 2007, 40(24), 8524–8526. (12) Raula, J.; Shan, J.; Nuopponen, M.; Niskanen, A.; Jiang, H.; Kauppinen, E. I.; Tenhu, H. Langmuir 2003, 19(8), 3499–3504. (13) Wei, Q.; Ji, J.; Shen, J. Macromol. Rapid Commun. 2008, 29(8), 645–650. (14) Lowe, A. B.; Sumerlin, B. S.; Donovan, M. S.; McCormick, C. L. J. Am. Chem. Soc. 2002, 124(39), 11562–11563. (15) Zhang, T.; Zheng, Z.; Ding, X.; Peng, Y. Macromol. Rapid Commun. 2008, 29(21), 1716–1720.

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Recently, reversible addition-fragmentation transfer polymerization (RAFT) has received a lot of attention for a large range of applications, such as in bio- and nanotechnology16-24 owing to the versatility of this method and the large range of functional polymers that can be synthesized. In addition, welldefined polymers synthesized via RAFT polymerization can also bind directly to GNPs through the RAFT end-group (typically di- and tri-thio compounds)25-27 making it ideal (16) (a) Boyer, C.; Liu, J.; Bulmus, V.; Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H. Macromolecules 2008, 41(15), 5641–5650. (b) Boyer, C.; Bulmus, V.; Priyanto, P.; Teoh, M. H.; Amal, R.; Davis, T. P. J. Mater. Chem. 2009, 19(1), 111–123. (c) Boyer, C.; Priyanto, P.; Davis, T. P.; Pissuwan, D.; Bulmus, V.; Kavallaris, M.; Teoh, W. Y.; Amal, R.; Carroll, M.; Woodward, R.; St. Pierre, T. J. Mater. Chem. 2009, DOI: 10.1039/b914063h. (d) Zarei, H.; Boyer, C.; Bulmus, V.; Nateghi, E.; Davis, T. P. ACSNano 2008, 2, 757–765. (17) Boyer, C.; Bulmus, V.; Liu, J.; Davis, T. P.; Stenzel, M. H.; BarnerKowollik, C. J. Am. Chem. Soc. 2007, 129(22), 7145–7154. (18) Boyer, C.; Bulmus, V.; Davis, T. P.; Ladmiral, V.; Liu, J.; Perrier, S., Chem. Rev. Published online September 18, 2009. http://pubs.acs.org/doi/full/10.1021/ cr9001403. (19) McCormick, C. L.; Sumerlin, B. S.; Lokitz, B. S.; Stempka, J. E. Soft Matter 2008, 4(9), 1760–1773. (20) York, A. W.; Zhang, Y.; Holley, A. C.; Guo, Y.; Huang, F.; McCormick, C. L. Biomacromolecules 2009, 10, 936–943. (21) Kakwere, H.; Perrier, S. J. Am. Chem. Soc. 2009, 131(5), 1889–1895. (22) Strandwitz, N. C.; Khan, A.; Boettcher, S. W.; Mikhailovsky, A. A.; Hawker, C. J.; Nguyen, T.-Q.; Stucky, G. D. J. Am. Chem. Soc. 2008, 130(26), 8280–8288. (23) De, P.; Li, M.; Gondi, S. R.; Sumerlin, B. S. J. Am. Chem. Soc. 2008, 130 (34), 11288–11289. (24) Heredia, K. L.; Nguyen, T. H.; Chang, C.-W.; Bulmus, V.; Davis, T. P.; Maynard, H. D. Chem. Commun. 2008, 28, 3245–3247. (25) Fustin, C.-A.; Colard, C.; Filali, M.; Guillet, P.; Duwez, A.-S.; Meier, M. A. R.; Schubert, U. S.; Gohy, J.-F. Langmuir 2006, 22(15), 6690–6695. (26) Hotchkiss, J. W.; Lowe, A. B.; Boyes, S. G. Chem. Mater. 2007, 19(1), 6–13. (27) Roth, P. J.; Theato, P. Chem. Mater. 2008, 20(4), 1614–1621.

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for the “grafting to” modification of GNP. Conveniently, both thiol and disulfide groups are easily obtained after the facile cleavage of the RAFT end-group either by aminolysis or reduction to thiols.28-31 This approach has led to many GNP/polymer hybrid materials.11,14,32-36 Recently, many research teams have developed predesigned polymers coatings for GNPs which are able to respond to different external stimuli such as temperature37-39 and pH36,35,40 and specific compounds41 such as polysaccharides42,43 and polynucleotides.5,6,44,45 There are numerous studies reporting the coating of GNPs with thermosensitive polymers, such as P(N-isopropylacrylamide), P(NIPAAm).46-48 The presence of only one critical solution temperature (LCST) for PNIPAAm at 31-32 °C in water limits the flexibility of this approach.49,50 To increase the flexibility of response, more complex synthetic methodologies have been described including copolymerization with pH-sensitive monomers51,52 or by the addition of hydrophilic/hydrophobic monomers, such as acrylamide10 during the polymerization. PEG blocks have also been reported in an attempt to improve the antifouling properties of GNPs.53,54 The antifouling properties of (28) Scales, C. W.; Convertine, A. J.; McCormick, C. L. Biomacromolecules 2006, 7(5), 1389–1392. (29) York, A. W.; Scales, C. W.; Huang, F.; McCormick, C. L. Biomacromolecules 2007, 8(8), 2337–2341. (30) Li, M.; De, P.; Gondi, S. R.; Sumerlin, B. S. J. Polym. Sci., Part A: Polym. Chem. 2008, 46(15), 5093–5100. (31) (a) Boyer, C.; Bulmus, V.; Davis, T. P. Macromol. Rapid Commun. 2009, 30 (7), 493–497. (b) Boyer, C.; Granville, A.; Davis, T. P.; Bulmus, V. J. Polym. Sci.; Part A: Polym. Chem. 2009, 47, 3773–3794. (32) Sumerlin, B. S.; Lowe, A. B.; Stroud, P. A.; Zhang, P.; Urban, M. W.; McCormick, C. L. Langmuir 2003, 19(14), 5559–5562. (33) McCormick, C. L.; Lowe, A. B. Acc. Chem. Res. 2004, 37(5), 312–325. (34) Smith, A. E.; Xu, X.; Abell, T. U.; Kirkland, S. E.; Hensarling, R. M.; McCormick, C. L. Macromolecules 2009, 42(8), 2958–2964. (35) Kim, B. J.; Bang, J.; Hawker, C. J.; Chiu, J. J.; Pine, D. J.; Jang, S. G.; Yang, S.-M.; Kramer, E. J. Langmuir 2007, 23(25), 12693–12703. (36) Nuopponen, M.; Tenhu, H. Langmuir 2007, 23(10), 5352–5357. (37) Shan, J.; Zhao, Y.; Granqvist, N.; Tenhu, H. Macromolecules 2009, 42(7), 2696–2701. (38) Bhattacharjee, R. R.; Chakraborty, M.; Mandal, T. K. J. Phys. Chem. B 2006, 110(13), 6768–6775. (39) Boyer, C.; Whittaker, M. R.; Lutzon, M.; Davis Thomas, P. Macromolecules 2009, 42(18), 6917–6926. (40) Feng, C.; Shen, Z.; Li, Y.; Gu, L.; Zhang, Y.; Lu, G.; Huang, X. J. Polym. Sci., Part A: Polym. Chem. 2009, 47(7), 1811–1824. (41) Pasquato, L.; Pengo, P.; Scrimin, P. J. Mater. Chem. 2004, 14(24), 3481– 3487. (42) Toyoshima, M.; Miura, Y. J. Polym. Sci., Part A: Polym. Chem. 2009, 47 (5), 1412–1421. (43) Guo, C.; Boullanger, P.; Jiang, L.; Liu, T. Biosens. Bioelectron. 2007, 22(8), 1830–1834. (44) Elbakry, A.; Zaky, A.; Liebl, R.; Rachel, R.; Goepferich, A.; Breunig, M. Nano Lett. 2009, 9(5), 2059–2064. (45) Ghosh, P. S.; Kim, C.-K.; Han, G.; Forbes, N. S.; Rotello, V. M. ACS Nano 2008, 2(11), 2213–2218. (46) Yusa, S.-i.; Fukuda, K.; Yamamoto, T.; Iwasaki, Y.; Watanabe, A.; Akiyoshi, K.; Morishima, Y. Langmuir 2007, 23(26), 12842–8. (47) Ishii, T.; Otsuka, H.; Kataoka, K.; Nagasaki, Y. Langmuir 2004, 20(3), 561– 564. (48) Zhu, M.-Q.; Wang, L.-Q.; Exarhos, G. J.; Li, A. D. Q. J. Am. Chem. Soc. 2004, 126(9), 2656–2657. (49) Kujawa, P.; Segui, F.; Shaban, S.; Diab, C.; Okada, Y.; Tanaka, F.; Winnik, F. M. Macromolecules 2006, 39(1), 341–348. (50) Van Durme, K.; Rahier, H.; Van Mele, B. Macromolecules 2005, 38(24), 10155–10163. (51) Zhou, Y.; Jiang, K.; Chen, Y.; Liu, S. J. Polym. Sci., Part A: Polym. Chem. 2008, 46(19), 6518–6531. (52) Zheng, P.; Jiang, X.; Zhang, X.; Zhang, W.; Shi, L. Langmuir 2006, 22(22), 9393–9396. (53) Jeon, H. J.; Go, D. H.; Choi, S.-y.; Kim, K. M.; Lee, J. Y.; Choo, D. J.; Yoo, H.-O.; Kim, J. M.; Kim, J. Colloids Surf., A 2008, 317(1-3), 496–503. (54) Tang, T.; Krysmann, M. J.; Hamley, I. W. Colloids Surf., A 2008, 317(1-3), 764–767. (55) Salmaso, S.; Caliceti, P.; Amendola, V.; Meneghetti, M.; Magnusson, J. P.; Pasparakis, G.; Alexander, C. J. Mater. Chem. 2009, 19(11), 1608–1615. (56) Ernst, O.; Lieske, A.; Hollaender, A.; Lankenau, A.; Duschl, C. Langmuir 2008, 24(18), 10259–10264.

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GNPs can also be controlled using the LCST of PNIPAAm blocks.55,56 Shen et al.57 grafted a thermosensitive hyperbranched electrolyte onto GNPs extending the LCST temperature range of the hybrid/GNPs in solution, from 25 to 50 °C. Moreover, the presence of tertiary amine in the hyperbranched structure, allowed further manipulation of the hydrophilic/hydrophobic balance by modifying the pH of the aqueous solution. Our group reported a new class of thermoresponsive gold nanoparticles obtained by grafting “onto” of thermoresponsive PEG derivatives polymers creating nanoparticles with antifouling surfaces39 that were stable in fetal bovine serum for several days. Control of the GNP surface charge is critically important in many applications, for example, for gene delivery, biological recognition, and biosensors. One example, the in-vivo and invitro delivery of polynucleotides requires vectors with cationic surface charges for complexation and then delivery.44 Herein, we demonstrate a novel strategy to manipulate the zeta potential or surface charge of polymer-coated hybrid polymer/GNPs using two different stimuli, that is, pH and temperature. The work described herein focuses on GNPs but could be easily applied to any polymer brush on a wide range of surfaces.

Results and Discussion The synthesis of a series of functional polymers was achieved using LFRP-RAFT18,58,59 polymerization, as described in Scheme 1. A summary of the copolymer/polymer characteristics is given in Table 1. The synthesized polymers are classified according to their structure, that is, neutral/uncharged polymers (noted as Px(0) in the text, where x corresponds to the sample number) based on (P(oligoethylene glycol) acrylate) (P(OEG-A)); thermoresponsive polymers based on either P(oligoethylene acrylate-co-diethylene glycol acrylate) (P(OEG-A-co-DEG-A)) or P(N-isopropyl acrylamide), P(NIPAAm); anionic polymers obtained from the polymerization of tert-butyl acrylate (P(t-Bu A)) followed by the cleavage of the tert-butyl group in the presence of trifluoroacetic acid yielding P(acrylic acid), P(AA) (designated as Px(-) in the text); and finally, cationic polymers obtained from the polymerization of 2-dimethylaminoethyl acrylate or 2-aminoethyl methacrylamide (Table 1), P(DMAEA) and P(AEA) (designated as Px(þ)), respectively. The polymerization of 2-AEA was carried out in an acetic buffer (pH = 5.5) to avoid any aminolysis of the RAFT functionality at 40 °C. All polymers were characterized by 1H NMR (Figures S1-S3 in the Supporting Information), gel permeation chromatography (GPC), and UV-visible spectroscopy. The data (as shown in Table 1) indicates accord between the targeted and experimental molecular weights together with narrow polydispersities, below 1.2-1.3, (obtained from GPC using either P(styrene) or PEG calibrations), consistent with effective RAFT control over the polymerizations. The maintenance of RAFT end-groups (after purification), an essential requirement for the subsequent “grafting-to” modification of the GNPs, was confirmed by UV-vis spectroscopy using the characteristic absorbance peak at 305 nm from the trithiocarbonate end-group and the following equation: fRAFT = 100  [abs305nm/εRAFT]/[polymer]0, where abs305nm, εRAFT, and [polymer]0 corresponding to the absorbance of RAFT agent, extinction coefficient of RAFT agent,60 and polymer concentration, (57) Shen, Y.; Kuang, M.; Shen, Z.; Nieberle, J.; Duan, H.; Frey, H. Angew. Chem., Int. Ed. 2008, 47(12), 2227–2230. (58) Benaglia, M.; Chiefari, J.; Chong, Y. K.; Moad, G.; Rizzardo, E.; Thang, S. H. J. Am. Chem. Soc. 2009, 131(20), 6914–6915. (59) Moad, G.; Rizzardo, E.; Thang, S. H. Polymer 2008, 49(5), 1079–1131. (60) Boyer, C.; Liu, J.; Long, W.; Tipett, M.; Bulmus, V.; Davis, T. P. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 7207–7224.

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Article Scheme 1. Schematic Representation of Polymers Synthesized in This Study

Table 1. A Summary of Polymers Used in This Study entry

polymers

a

Mn (g/mol)

R (%) M

b

c

Mn (g/mol)

Mn (g/mol)b (1H NMR)

Mn (g/mol)d (SEC)

PDI d (SEC)

fRAFT (%)e

T (LCST)f (°C)

P1(þ) P(DMAEA) 10 200 65 6 650 6 200 10 500 1.15 85 1.26g 90 P2(þ) P(AEA) 10 000 60 6 000 7 500 14 500g g g 1.34 90 P3(-) P(AA) 7 400 12 600 P4(0) P(OEG-A) 20 000 70 14 000 14 100 12 300 1.18 90 94 P5(0) P(OEG-A) 45 000 65 27 000 28 500 24 400 1.25 90 92 P6(0) P(NIPAAm) 30 000 65 28 500 18 500 28 700 1.24 95 32 P7(0) P(DEG-A) 30 000 65 19 500 24 400 21 500 1.23 95 15 P8(0) P(OEG-A-co-DEG-A) 32 000 65 20 800 26 100 22 200 1.26 90 22 a Theoretical molecular weight calculated by the following equation: Mn = [M]0/[CTA]0  MWmonomer þ MWCTA. b Determined by 1H NMR. c Molecular M monomer CTA d e þ MW . Measured by DMAc GPC (P(styrene) calibration). RAFT weight calculated by the following equation: Mn = [M]0/[CTA]0  R  MW functionality quantified by UV-visible spectroscopy using the following equation: fRAFT = 100  [abs305nm/εRAFT]/[polymer]0, where abs305nm, εRAFT, and [polymer]0 correspond to the absorbance of RAFT agent, extinction coefficient, and polymer concentration, respectively. f LCST determined by turbidity measurement at 500 nm assessed by UV-vis spectroscopy. g Determined by water GPC (PEO calibration).

respectively. In all cases the RAFT end-group functionalization was greater than 85%. The UV spectroscopy results are in good accord with structural data derived from 1H NMR (see Supporting Information). It is noteworthy that the tert-butyl group cleavage, under acid conditions, occurs without any significant loss of the RAFT end-groups, a finding consistent with an earlier study.61 The LCST behavior of P(NIPAAm) and P(OEG-co-DEG-A) (1 mg/mL) in water was measured by collecting UV-visible turbidity data (at λ = 500 nm) as a function of temperature. P(DEG-A) was found to have a LCST of 15 °C, while P(OEG-A) has a LCST close to 92 °C (Figures S4 and S5 in the Supporting Information). By adjusting the proportion of these two monomers in the copolymers it was possible to tune the LCST from 15 to 92 °C (Table S1 in the Supporting Information). A similar result was reported by Lutz for methacrylic monomers.62 As expected the P(NIPAAm) exhibits a characteristic LCST close to (61) Whittaker, M. R.; Monteiro, M. J. Langmuir 2006, 22(23), 9746–9752. (62) Lutz, J.-F. J. Polym. Sci., Part A: Polym. Chem. 2008, 46(11), 3459–3470.

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31-32 °C (Table 1). It is noteworthy that the LCST of P(NIPAAm) can be tuned using molecular weight control at low molecular weights. However, in this work the molecular weight was maintained close to 20 000 g/mol. Highly uniform gold nanoparticles (GNP) were synthesized via the established citrate reduction method63 (reduction of HAuCl4 by boiling with sodium citrate) to yield spherical gold nanoparicles with a highly uniform diameter close to 20 nm as observed by transmission electron microscopy (TEM) (average diameter assessed by TEM was 18 nm) and by dynamic light scattering (DLS) (Figure S6 in the Supporting Information). The “grafting-to” modification of the GNPs was achieved by addition of the RAFT-functionalized polymers to the GNPs in aqueous solution. The strong affinity of the trithiocarbonate functionality for gold provides an anchoring point and the polymer chains assemble onto the gold surface.26 After polymer attachment, the optical properties (i.e., the color of the nanoparticle solution, see Figure 1) of the GNPs change with a small red-shift (63) Frens, G. Nat. Phys. Sci. 1973, 241(105), 20–2.

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Figure 1. Typical TEM images of hybrid GNPs: (A) GNPs-P4(0), (B) GNPs-P3(-), (C) GNPs-P1(þ), high concentration of P1(þ), i.e., 10 mg/mL, (D) GNPs-P2(þ), high concentration of P2(þ), i.e., 10 mg/mL, (E) GNPs-P1(þ) (obtained with low concentration of P1(þ), i.e. 1 m/mL), F- GNPs-P2(þ) (obtained with low concentration of P2(þ) (1 mg/mL). Insets: pictures of GNP/polymers dispersed in distilled water and TEM images of GNPs using a contrast agent (phosphonic acid tungsten).

caused by modification of the characteristic plasmon resonance absorption (Figure S7 in the Supporting Information). The modification of GNPs using either P(DMAEA) or P(AEA) requires special care to avoid formation of large aggregates (Figure 1E,F). Successful cationic polymer attachment was ensured by the prior removal of excess sodium citrate (from the GNP synthesis) as described by Decher.64,65 The polymer concentrations and molecular weights are important design criteria in maintaining welldispersed and nonaggregated nanoparticles during the grafting-to process. A low polymer concentration yields nanoparticles with limited stability.66 If inadequate stabilization occurs then a blue solution results (see insets of Figure 1E,F). The redispersal (in water) of these GNPs becomes impossible after centrifugation. Destabilization of GNPs occurs because of electrostatic interactions between amine functionality and the negatively charged gold surface, resulting in the bridging flocculation of the nanoparticles by the polymer chains (Figure 1E,F). However, using carefully optimized grafting conditions, flocculation can be avoided resulting in the formation of stabilized hybrid organic/inorganic nanoparticles that can be purified easily by repeated washing/centrifugation cycles (using ultrapure water to remove extraneous polymers). Successful polymer coating was confirmed by a slight shift (ca. 5-10 nm) in the visible absorbance caused by the surface plasmon resonance (SPR effect) assessed by UV-vis. spectro(64) (65) (66) (67) 3736.

Schneider, G.; Decher, G. Nano Lett. 2004, 4(10), 1833–1839. Schneider, G. F.; Decher, G. Nano Lett. 2008, 8(11), 3598–3604. Schneider, G.; Decher, G. Langmuir 2008, 24(5), 1778–1789. Coffer, J. L.; Shapley, J. R.; Drickamer, H. G. J. Am. Chem. Soc. 1990, 112,

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scopy.67 The SPR effect arises from the collective oscillation of conduction electrons of the gold upon irradiation with visible light. The characteristic wavelength of maximum absorption and its width depends on the size, shape, and dielectric environment of the GNPs. For spherical GNPs dispersed in water only a single absorption maximum is evident (∼520 nm for 20 nm particles). The SPR wavelength exhibits a red-shift with any increase in nanoparticle size, along with an overall decrease in the maximum absorbance intensity. A stable, well dispersed solution of GNPs appears red, as the average interparticle distance is greater than the particle diameter. When particle aggregation occurs, with the average interparticle distance becoming smaller than the particle diameter, the solution turns blue.68 The SPR effect is also a function of the effective refractive index of the metal surface that is determined by the mass of the substances present at the interface.69 Consequently in a well-dispersed and stable hybrid GNP solution, the changes in the SPR wavelength (red shift) that occur as a result of the polymer coating can be related to changes in the refractive index at the GNP surface, which can be directly attributed to the mass of the polymer coating. The hybrid particles could be redispersed easily following centrifugation. Moreover, zeta-potential measurements yielded data that confirmed a change of charge on the hybrid GNP surfaces (Table 2). GNPs coated by P(OEG-A):GNPs-P4(0) or P5(0), P(OEG-A-co-DEG-A):GNPs-P8(0), and P(NIPAAm): GNPs-P6(0) yielded neutral nanoparticles. In contrast, P(DMAEA) and P(AEA) grafts yielded positively charged nanoparticles: þ29 mV (GNPs-P1(þ)) and þ39 mV (GNPs-P2(þ)), respectively. Finally, gold nanoparticles coated with P(AA), that is, GNPs-P3(-), demonstrated a slight charge change (-40 to -30 mV) indicating that the negatively charged P(AA) polymer had replaced the citrate ions on the GNP surface. DLS results confirmed the formation of well-dispersed GNPs in water for P(OEG-A), P(NIPAAm), and P(AA) stabilization, and with these polymers it was possible to obtain GNPs with zeta-potentials over the range -30 and þ39 mV with relatively narrow sizes (10), destabilized (the solution turned blue consistent with the formation of large aggregates, as shown by DLS) and precipitation was observed. In contrast, GNPs-[P1(þ): P5(0)] or GNPs-[P2(þ):P5(0)] remained stable (no significant λmax shift was observed by UV-vis spectroscopy ca. 525535 nm) and confirmed by DLS (data not shown). Finally, the coassembly of thermoresponsive polymers, that is, P(NIPAAm) (P6(0)), or P(OEG-A-co-DEG-A) (P8(0)), was used to modulate the surface charges. The LCST of these polymers was used to reversibly screen and reveal the charged groups of the coassembled polymer, as shown in Scheme 2. The coassembly process (as described previously) was adopted using thermoresponsive polymers to yield GNPs-[P8(0): P(cationic or anionic)] or GNPs-[P6(0):P(cationic or anionic)]. Langmuir 2010, 26(4), 2721–2730

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Figure 5. (A) UV-visible spectra of GNPs/[P8(0):P2(þ)], comFigure 4. (A) Zeta-potential (mV) and (B) number size (nm) assessed by DLS of (blue bullet, b) GNPs-[P8(0): P2(þ)], (black square, 9) GNPs-[P8(0):P1(þ)], (red triangle, 2) GNPs-[P8(0):P3(-)], surface composition: 50:50 wt %) and (pink diamond, () GNPs-[P8(0)] vs temperatures (°C); (C and D) Typical TEM pictures of GNPs-[P8(0):P3(-)], composition 50/50 wt %, below LCST (10 °C) and above (LCST) (40 °C), respectively. Inset: pictures of the GNP solutions. Note: zeta-potential was measured in the presence of 1 mM KCl at pH = 6.5, temperature ramp: 0.5 °C/min. Scheme 2. Illustration of Switchable Surface Charge on Gold Nanoparticles

The thermoresponsive polymers were engineered to have a greater chain length than the charged polymers, thus ensuring a neutral GNP surface below the LCST of the neutral polymer. Both XPS and ATR-FTIR yielded results confirming the presence of both polymers on the GNP surfaces after purification. The surface composition of the GNPs was determined via XPS analysis (as before). A packing density close to 0.7-0.8 chain/nm2 was found by TGA analysis. As expected, at low temperature the GNP hybrid particles exhibited a neutral surface (∼0 mV) and sizes in the range of 25-45 nm. The evolution of the charge (zetapotential) with temperature is shown in Figure 4A. As the Langmuir 2010, 26(4), 2721–2730

position 50/50 wt % and (B) UV-visible spectra of GNPs-P8(0) vs temperature from 18 to 25 °C. Insets show pictures of the GNP solutions at 18 and 25 °C. (C) Evolution of absorption maximum (nm) vs temperature for different GNPs: (red bullet) GNPs/[P8(0): P3(-)] and (blue triangle) GNPs/[P8(0):P1(þ)], surface composition 50/50 wt %; (black square) GNPs/[P8(0)]. Note: temperature increases 0.5 °C/ min, GNP concentration, 5 mg/mL. Note: GNPs was dispersed in solution with 1 mM KCl at pH = 6.5.

temperature increased the zeta potential progressively changed from neutral to negative for GNP/[P3(-):P8(0)] and neutral to positive for GNP/[P1(þ) or P2(þ):P8(0)]. This change occurred over a narrow temperature range (∼5 °C in the case of P8(0) and a slightly broader temperature range, i.e., ca. 5-10 °C in the case of P5(0), data not shown) corresponding to the LCST of the polymers. The size of the particles as assessed by DLS was found to be largely unaffected by the LCST transition (Figure 4B). TEM confirmed the presence of monodisperse GNP particles in solutions above the LCST (Figure 4C,D). The zeta-potential changes did not influence the particle sizes or distribution (PDI stays close to 0.1-0.2) as determined by DLS. Analysis using UV-visible spectroscopy confirmed that the GNP absorbance remains unaffected (i.e., ca. 525-540 nm, Figure 5) during the LCST transition. In contrast, in the absence of cationic/anionic polymers, the particles tend to aggregate above the LCST resulting in a shift of the plasmon resonance from 525 to 540 to 600 nm (a color shift from red to purple/blue is observed, Figure 5). Below the LCST the neutral polymer chains mask the charge from the coassembled polymer, that is, the charged one. At temperatures above the LCST, the thermosensitive polymer chains undergo a transition into a more compact form, exposing the charged polymers at the surface. P(OEGA-co-DEG-A) copolymers present several advantages over DOI: 10.1021/la902746v

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Figure 6. (A and B) Typical TEM pictures of GNPs-[P8(0):P1(þ)] above LCST at pH = 6.5 and pH = 10, respectively. (C and D) Typical TEM pictures of GNPs-[P8(0):P3(-)] above LCST at pH = 6.5 and pH = 3.0, respectively (inset: zoom of aggregates particles). (E) Evolution of GNPs number sizes distribution (nm) assessed by DLS vs temperature for (red bullet) GNPs-[P8(0): P3(-)] at pH = 3.0 and (blue triangle) GNPs/[P8(0):P2(þ)] at pH = 10, (black square) GNPs-[P8(0)] at pH = 6.5. Note: temperature ramp, 0.5 oC/min; GNP concentration, 5 mg/mL.

P(NIPAAm) in the synthesis of these hybrid materials. These PEG-related copolymers are known to confer antifouling properties,16d,39 and tunable LCST behavior (via the DEG-A component). Moreover, when P(OEG-A-co-DEG-A) is used as the neutral polymer component, complete reversibility in zeta potential variability is observed, that is, a reduction of temperature back through the LCST results in a remasking of surface charge. The hybrid GNPs can be cycled several times (at least five times) through the LCST with minimal change in behavior. The influence of pH was also investigated as an experimental variable. For GNPs/[P1(þ) or P2(þ) with thermosensitive polymers], at low pH (regardless of temperature) the GNP/polymers yielded stable dispersions. As pH was increased from 6.5 to pH > 10 for GNPs/[P1(þ):P8(0)] and GNPs/[P2(þ):P8(0)] the particles aggregated at temperatures above the LCST of the neutral polymers (Figure 6A and B). This aggregation can be observed directly by a color change in the nanoparticle solution, that is, the GNP solution changes from red to blue-purple at T > TLCST, and an increase in particle size measured by DLS (Figure 6E). The plasmon band of the hybrid GNPs became broader and a decrease in the intensity of the absorption peak was observed (data no shown). DLS also confirms the formation of continually growing large aggregates (>500 nm, DLS data not shown in Figure 6E because of the fast precipitation of nanoparticles) of the GNPs-[P6(0):P1(þ)] hybrid nanoparticles, while a smaller size 2728 DOI: 10.1021/la902746v

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was observed (around 200-300 nm) for GNPs-P8(0) particles. TEM analysis also confirms the systematic formation of aggregates comprising 20-30 particles each in the P(OEG-A-co-DEGA) (P8(0)) system, while in the P(NIPAAm) (P6(0)) stabilized GNPs, the aggregates consisted of a larger number of individual particles. The destabilization of these GNPs at high temperature can be explained by the absence of positive charges (confirmed by zeta potential) to stabilize the GNPs. Indeed, P1(þ) and P2(þ) show pKa values around 8 and 9, respectively. In contrast, the GNPs-[P3(-):P8(0)] are stable at the pH of the ultrapure water (pH = 6.5, Figure 6C) but are unstable at pH values lower than 4.0 and for T > TLCST. The observation of large aggregates by both DLS (∼200-300 nm: Figure 6E) and TEM (20-30 particles, Figure 6D) is attributed to neutralization of the COO- anion at low pH. The formation of smaller aggregates when using P(OEGA-co-DEG-A), (P8(0)) instead of P(NIPAAm) (P6(0)) in these systems is not fully understood, but probably reflects differences in polymer-polymer and polymer-solvent interactions both above and below the LCST. In addition, when P8(0) was used as the neutral polymer, reversibility of behavior around the LCST seemed more effective, in contrast to P(NIPAAm) stabilization, where recovery of a well-dispersed nanoparticle solution required vigorous stirring. Protein Adsorption on Hybrid Gold Nanoparticles. In this part, we describe an investigation into the protein adsorption behavior of the hybrid GNPs obtained by the self-assembly of both polymers, that is, noncharged and thermosensitive polymers and charged polymers, such as P1(þ) and P2(-), using bovine serum albumin (BSA) solution and fetal bovine serum (FBS). GNPs coated by P8(0) presented excellent resistance to protein adsorption below and above the LCST, as we have demonstrated elsewhere.16b,39 As expected GNPs-P1(þ) showed a strong and rapid adsorption of protein onto their surfaces resulting in a rapid aggregation as observed by DLS (size increases from 40 nm to > 1 μm, in less 10 min, ∼5.5 mg/m2 of BSA). This aggregation results in a color change from red to blue and a rapid precipitation caused by the strong electrostatic interactions between the P1(þ) polymer layer and BSA. It is known that BSA presents different regions: neutral and positively and negatively charged domains in its tertiary structure.71,72 A rapid precipitation was also observed in fetal bovine serum (FBS), used here as a model biological fluid. In contrast, GNPs-P2(-) presented improved stability in FBS than P1(þ). The potential to tune the nanoparticle surface charge using external stimuli may have applications in biosensor research or drug delivery. It has been established that uncharged nanoparticles display a longer circulation half-life time in the body than charged ones. However, uncharged particles present a lower cell uptake than charged ones.45 We investigated the protein adsorption of these GNPs at different temperatures to study how the LCST behavior affected their “stealth” characteristics. BSA was introduced into a solution of GNPs/[P1(þ): P8(0)], with MnP(OEG-A-co-DEG-A) > MnP(DMAEA). Below the LCST, the solution does not change color (red-pink), this demonstrates that there is no (or very low) absorption of protein (∼0.1 mg/m2 of BSA determined by Braford’s assay for GNPs-[P1(þ):P8(0)], composition 50/50 wt %)) onto the GNPs surfaces as demonstrated by a Bradford assay. This result reflects the antifouling properties of the P(OEG-A-co-DEG-A) material and confirmed previous results.39 Even if the concentration of BSA (71) Rezwan, K.; Meier, L. P.; Rezwan, M.; Voeroes, J.; Textor, M.; Gauckler, L. J. Langmuir 2004, 20(23), 10055–10061. (72) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Langmuir 2005, 21(20), 9303–9307.

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was increased to 10 mg/mL, there was no change to the stability of these GNPs after several hours (14 h) as observed by both UV-visible spectroscopy and DLS. This result indicates that the charges of the coassembled polyelectrolyte are totally screened by the PEG-analogue polymers. When the temperature was increased above the LCST, the solution turned blue as previously observed with the GNPs-P1(þ) hybrid nanoparticles. DLS measurements showed a rapid aggregation, and a quick precipitation of the hybrid particles. At the LCST the charges are revealed and the adsorption of protein onto the GNP surfaces takes place, as confirmed by a Bradford assay (∼5.5 mg/m2 of BSA). As a control experiment, GNPs-P5(0) was subjected to a similar procedure, and in this case, no change was observed. The change in protein adsorption is therefore not attributable to a change in the LCST of the polymers, but results from electrostatic interactions between P1(þ) and BSA. In the case of GNPs-[P3(-):P6(0)], there was no change

Conclusion This paper describes a new and facile strategy to control the zeta-potential of GNPs using the coassembly of two different polymers, that is, noncharged and charged (cationic or anionic) polymers, onto GNP surfaces. By judicious selection of molecular weights, the charge can be totally screened to yield neutral GNPs. When the chain-lengths of the charged and noncharged polymers are similar, the concentration ratio of the two polymers can be used to tune the surface charges of hybrid GNPs with zeta potentials ranging from -30 to þ30 mV. The coassembly of a thermo-responsive and noncharged polymer, that is, P(NIPAAm) or P(OEG)-with a charged polymer allows for temperature modulation of the zeta-potential. At temperatures above the LCST, the thermo-responsive polymers are in a collapsed form revealing the charged polymers at the surface, while below the LCST, P(OEG-A-co-DEG-A) or P(NIPAAm) effectively screen the charged polymers. It was found that during the LCST transition that there was a progressive change in the surface charge between the two extremes with intermediate surface charges presented. In addition, the GNPs present an antifouling surface below the LCST, while above the LCST, these particles aggregates rapidly. The control of surface charge may be a significant factor in designing particles for effective cell uptake45 and for gene delivery.16c Materials and Methods Materials. Oligoethylene glycol acrylate (OEG-A) (number average molecular weight Mn = 450 g/mol, PDI = 1.02), di(ethylene glycol) ethyl ether acrylate, and dimethylaminoethyl acrylate (DMAEA, Aldrich) were purified via an alumina column to remove the inhibitor prior to use. N-Isopropylacrylamide (NIPAAm) (Aldrich, 99%) was crystallized twice from hexane prior to use. 2-Aminoethyl methacrylamide hydrochloride (AEA/ HCl, Polymer Sciences) was used as received. 2,20 -Azobisisobutyronitrile (AIBN) was purchased from Wako Chemicals and was crystallized twice from methanol prior to use. Hydrogenotetrachloroaurate (III) hydrate (HAuCl4, 99.9%, Aldrich) and trisodium citrate dehydrate (99%, Aldrich) were used as received. Deionized water used for these experiments was purified by Milli-Q system with a resitivity of 17.9 mΩ/cm. Methods. LCST Measurement. The lower critical solution temperature, LCST,was determined by a UV-vis spectroscopy at 500 nm. The polymer concentrations were 0.2 mg/mL (0.2% wt) in water with a heating rate of 1 °C/min. The temperature at which 10% of the maximum absorbance of the solution was observed was defined as the LCST. Langmuir 2010, 26(4), 2721–2730

Determination of Particle Size. GNPs (or GNPs/polymer) solutions were prepared in distilled water with GNPs concentration of 1 mg/mL (or 5 mg/mL). The solution was filtered trough Millipore nylon filters (pore size 0.45 μm) to eliminate dust and large contaminants. The size measurements were carried out in quartz cuvette and the temperature was allowed to equilibrate for 5 min. For the determination of size versus temperature the heating rate was 0.5 °C/min. Determination of zeta-potential. The zeta potential was measured at neutral pH (6.5) in the presence of a low concentration of KCl solution (1 mM) as the medium. Syntheses. Synthesis of Gold Nanoparticles. Citrate-stabilized gold nanoparticles (20 nm) were prepared using published procedures.63 Briefly, all glassware was first washed with an aquaregia solution (25 v % nitric acid and 75 v % of concentrated hydrochloric acid), then rinsed with Milli-Q water several times and dried. Milli-Q water (100 mL) and 1% solution trisodium citrtate dehydrate (5 mL, 1.053 g of trisodium citrtate) were mixed. The solution was heated up to boiling point with vigorous stirring and then hydrogenotetrachloroaurate (III) hydrate stock solution (0.01 M) (2.54 mL) was introduced rapidly using a syringe. The solution was boiled for a further 30 min with vigorous stirring. A progressive change of color was observed from yellow to wine-red. The solution was cooled down and stored in a refrigerator at 5 °C until required. The particles were characterized by TEM and DLS analyses. Synthesis of RAFT Agents. The synthesis of 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (BSTP, 1 of Scheme 1) and 4-cyanopentanoic acid dithiobenzoate (CDTB, 2 of Scheme 1) and are described in Supporting Information. RAFT Polymerizations. All of the polymers synthesized were characterized by 1H NMR, gel permeation chromatography (GPC) and UV-visible spectroscopy. RAFT Polymerization of Dimethyl Aminoethyl Acrylate (DMAEA). In the presence of 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (BSTP), this was carried out as per P(NIPAAm) above. An example of polymerization of DMAEA is given for [DMAEA]0/[BSTP]0/[AIBN]0 = 100/1/0.2 (P1(þ) in Table 1). DMAEA (0.52 g, 3.64 mmol), 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (10 mg, 3.64  10-5 mol), AIBN (1.2 mg. 7.3  10-6 mol), and dry and distilled acetonitrile (obtained by distillation) (10 mL) were mixed. The solution was cooled in an ice bath and purged with nitrogen for 30 min before heating to 60 °C. After 6 h, the solution was partially evaporated under vacuum, and the polymer was precipitated in hexane (at 0 °C) to yield a viscous yellow product. The precipitation was repeated twice more to ensure the purity of the polymer. The product was dried in vacuo to yield a yellow viscous product. The viscous product (P(DMAEA), 1 g) was dissolved in 15 mL of cold acid water ([HCl] = 0.5 M) to quaternize tertiary amine. The pH was checked and acidified with a few drops of concentrated HCl (12 M) if required. The solution was rapidly dialyzed using distilled water to remove the unreacted HCl for 3 h after which the product recovered by freeze-drying.

RAFT Polymerization of 2-Aminoethyl Methacrylamide Hydrochloride (AEA, HCl). In the presence of 4-cyanopentanoic acid dithiobenzoate (CDTB) as shown in Scheme 1C, polymerizations were conducted at 45 °C employing VA-044 as the primary radical source and CDTB as the RAFT chain transfer agent. All polymerizations with this monomer were performed directly in an acetic acid buffer at pH 5.2, (0.27 mol/L acetic acid and 0.73 mol/L sodium acetate) with an initial monomer concentration ([M]0) of 1 M. An example of AEA polymerization follows: for a feed ratio of [AEA]0/[CDTB]0/[AIBN]0 = 80.0/1.0/ 0.2 (P2(þ) in Table 1). AEA (1.6 g (0.01 mol), CDTB (34.5 mg, 1.23  10-4 mol), ACVA (6.9 mg, 2.46  10-5 mol), and acetic buffer (10 mL) were mixed. The reaction mixture was purged with nitrogen in an ice bath for 30 min and then allowed to polymerize in a temperature-controlled oil bath held at 70 °C for 6 h. The polymerization solution was dialyzed against water for 48 h to DOI: 10.1021/la902746v

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Article remove monomers (at pH 5.2 at 5 °C, water was changed hourly), and then freeze-dried. The solid was dissolved in ethanol and the polymer was precipitated in diethyl ether. The precipitation was repeated twice more to ensure the purity of the polymer. The product was dried in vacuo to yield a pink powder. RAFT Polymerization of tert-Butyl Acrylate (t-Bu A). This was carried out in the presence of 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (BSTP) as shown in Scheme 1D. An example of polymerization of t-Bu A is given for [t-Bu A]0/[BSTP]0/[AIBN]0 = 120/1/0.2. t-Bu A (0.65 g, 0.005 mol), 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (10 mg, 3.64  10-5 mol), AIBN (1.2 mg. 7.3  10-6 mol), and acetonitrile (10 mL) were mixed. The solution was cooled in an ice bath and purged with nitrogen for 30 min before heating to 60 °C. After 6 h, the solution was partially evaporated under vacuum, and the polymer was precipitated in pentane (at 0 °C) to yield a viscous yellow product at the bottom of the Erlenmeyer. The precipitation was repeated twice more to ensure the purity of the polymer. The product was dried in vacuo to yield a yellow viscous product.

Synthesis of P(AA) by Cleavage of tert-Butyl Group of P(t-Bu A). An example of cleavage of the tert-butyl acrylate groups is given (P3(-) in Table 1): 1.28 g of P(t-Bu A) was dissolved in 30 mL of chloroform; ca. 10 equiv of trifluoroacetic acid (TFA) compared to tert-butyl group (i.e., 10 g of TFA) was added to the chloroform solution. The solution was stirred for 24 h at room temperature. A yellow precipitate appears during the reaction. The solvent and TFA were removed by distillation to yield a yellow powder. The yellow powder was dissolved in methanol and precipitated in acetone. The product was dissolved in water and rapidly dialyzed against water for 6 h. Finally, the solution was freeze-dried to yield yellow powder.

RAFT Polymerization of N-Isopropyl Acrylamide (NIPAAm). This was performed in the presence of 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (BSTP) as shown in Scheme 1A. An example of polymerization of NIPAAm is given for [NIPAAm]0/[BSTP]0/[AIBN]0 = 250/1/0.2 (P6(0) in Table 1). NIPAAm (1.13 g, 0.01 mol), 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (10 mg, 3.64  10-5 mol), AIBN (1.2 mg. 7.3  10-6 mol) and acetonitrile (10 mL) were mixed. The solution was cooled in an ice bath and purged with nitrogen for 30 min before heating to 65 °C. After 5 h, the solution was partially evaporated under vacuum, and the polymer was precipitated in cold diethyl ether (at 0 °C). The precipitation was repeated twice more to ensure the purity of the polymer. The product was dried in vacuo to yield a yellow powder.

RAFT Polymerization of Di(ethylene glycol) Ethyl Ether (DEG-A) . This was performed in the presence of 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (BSTP) as shown in Scheme 1B. An example of polymerization of DEG-A is given for [DEG-A]0/[BSTP]0/[AIBN]0 = 250.0/1.0/0.2 (P8(0) in Table 1). DEG-A (3.10 g, 16.6 mmol), 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (30 mg, 3.64  10-5 mol), AIBN (2.0 mg. 1.22  10-5 mol), and acetonitrile (10 mL) were mixed. The solution was cooled in an ice bath and purged with nitrogen for 30 min before heating to 60 °C. After 5 h, the solution was

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Boyer et al. partially evaporated under vacuum, and the polymer was precipitated in cold diethyl ether (at 0 °C) yielding a viscous yellow product. The precipitation was repeated twice more to ensure the purity of the polymer. The product was dried in vacuo to yield a yellow viscous product. A similar process was repeated for all the polymerizations with OEG-A and the copolymerization with OEG-A and DEG-A. Protein Adsorption. GNP/polymer nanoparticles (1.0 mg/ mL) were suspended in aqueous solution with bovine serum albumin (BSA) at an initial concentration of 1.0 mg/mL (pH = 6.5). The samples were then shaken at room temperature for 3 h to reach adsorption equilibrium. GNPs were removed by centrifugation and the supernatant was analyzed for BSA. The supernatant BSA concentrations were determined via the Bradford method73 using a UV-visible spectrometer (Varian Cary 300 scan). An aliquot of the supernatant (1 μL) was added to Bradford’s reactant (3 mL), and mixed for 2 min at room temperature (the solution becomes blue). The concentration of BSA was measured by absorption at 595 nm. The test was repeated three times for each sample and the average was taken as the BSA concentration at equilibrium. The amount of BSA adsorbed was calculated by difference from a reference solution (solution treated using the same method but without gold nanoparticles). BSA adsorbed per m2 was calculated using the following equation: BSA adsorbed = nBSA/GNPSurface = [([BSA]0 [BSA]eq)V]/SurfaceGNP, where [BSA]0, [BSA]eq, V and SurfaceGNP correspond to BSA concentrations measured by Bradford’s assay without GNPs and BSA concentrations in the presence of GNPs after 14 h, volume of the solution, and surface of gold nanoparticles used for the assay, respectively. The surface of gold nanoparticles was estimated using the following equation: SurfaceGNP = 1.5/(dF), where F and d are the mass volume and diameter, respectively. Grafting of Polymer “to” GNPs. A stirred GNP solution (10 mL of 1 mg/mL previously obtained) was placed in an ice bath for 30 min. Cooled polymer solution (1 mL concentration: 30 mg/L) was added to the GNP solution, followed by stirring for 180 min. Then, the GNPs were purified by centrifugation at 20 000 rpm, for 30 min at 5 °C followed by resuspension in cooled water. This process was repeated three times. GNPs/polymer nanoparticles were stored in solution (10 mg/mL) or freeze-dried. After freezedrying the hybrid GNP/polymer nanoparticles could be redispersed easily in water (in contrast to GNPs with no polymer coating).

Acknowledgment. T.P.D. acknowledges the Australian Research Council for the award of Discovery Grants and a Federation Fellowship. Supporting Information Available: NMR spectra of polymers, DLS of “naked” GNPs and GNPs-coated polymers, TEM picture of “naked” GNPs, UV-visible spectra of GNPs/polymers. This material is available free of charge via the Internet at http://pubs.acs.org. (73) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.

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