Langmuir 2005, 21, 11495-11499
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pH-Responsive Capsules Derived from Nanocrystal Templating Hongwei Duan, Min Kuang, Gang Zhang, Dayang Wang,* Dirk G. Kurth, and Helmuth Mo¨hwald Max Planck Institute of Colloids and Interfaces, D-14424, Germany Received June 17, 2005. In Final Form: August 18, 2005 In the current work we demonstrate a facile and versatile way to create hydrophilic polymeric capsules by integration of Au nanocrystal templating, surface-initiated atom-transfer radical polymerization, and selective chemical cross-linking of polymer shells. Capsules of the homopolymer of 2-(dimethylamino)ethyl methacrylate and its copolymers with 2-(diethylamino)ethyl methacrylate and poly(ethylene glycol) methyl ether methacrylate were constructed. They swell at low pH and shrink at high pH. On the basis of the pH sensitivity of the resulting capsules, encapsulation and release of a drug model, rhodamine 6G, were realized. Furthermore, by cleaving Au-S bonds between Au cores and polymer shells, capsules containing free Au cores were generated, paving a simple pathway to introduce more functionality to the polymeric capsules.
Introduction To date, enormous efforts have been devoted to the fabrication of hydrophilic polymeric capsules with sizes ranging from several nanometers to a few micrometers in view of their diverse applications, particularly serving as drug delivery vehicles and protection shells of active species.1 Among the existing strategies of synthesis of capsules, a simple, versatile, and efficient way is to use colloidal particles as sacrificial templates.2 On the basis of in situ polymerization of hydrophobic monomers absorbed within the walls of lipid vesicles, polymeric capsules can be constructed which have a broad size distribution according to the size polydispersity of vesicle templates.3 Jiang and co-workers generated micrometersized capsules using noncovalently connected micelles.4 On the basis of the electrostatic interaction of oppositely charged polyions, a layer-by-layer approach was developed to coat various colloidal particles with hybrid multilayers of polymers, nanoparticles, and/or biomolecules.2b,5 Subsequent removal of colloidal cores may leave behind composite capsules. In extending this layer-by-layer method to less than 200 nm colloidal spheres one encounters difficulties in avoiding aggregation of spheres for practical concentrations. It is well known that capsules less than 200 nm in size are needed to suppress clearance of reticuloendothelial systems for applications in drug delivery.6 Polymeric capsules of sizes around 100 nm have been formed using block copolymer micelles as templates.7 This method usually requires deliberate design of block copolymers that should be composed of both cross-linkable * To whom correspondence should be addressed. Fax:+49 331 567 9202. E-mail:
[email protected]. (1) Hollow and Solid Spheres and Microspheres: Science and Technology Associated with Their Fabrication and Application. Mater. Res. Soc. Proc. Vol. 372 (Ed: Wilcox, D. L.; Berg, M.; Bernat, T.; Kellerman, D.; Cochran, J. K.), MRS Pittsburgh, PA 1995. (2) (a) Bergbreiter, D. E. Angew. Chem., Int. Ed. 1999, 38, 2870. (b) Caruso, F. Chem. Eur. J. 2000, 6, 413. (3) Sauer, M.; Streich, D.; Meier, W. Adv. Mater. 2001, 13, 1649. (4) Liu, X.; Jiang, M.; Yang, S.; Chen, M.; Chen, D.; Yang, C.; Wu, K. Angew. Chem., Int. Ed. 2002, 41, 2950. (5) (a) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202. (b) Gittins, D. I.; Caruso, F. J. Phys. Chem. 2001, 105, 6846. (6) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. Adv. Drug Delivery Rev. 2002, 54, 135.
and degradable blocks. Feldheim and co-workers produced capsules 5-200 nm in size by trapping and aligning Au nanocrystals in channels of membranes, followed by polymerization of pyrrole-based monomers.8 Prolonging the polymerization time results in aggregation within channels. Herein we present an alternative way to produce pHsensitive polymer capsules of sizes ranging from tens to hundreds of nanometers. The current work relies on the success of surface-initiated atom-transfer radical polymerization (ATRP) on colloidal particles.9 The groups of Walt and Xia succeeded in constructing capsules from silica particles coated with hydrophobic polymer brushes obtained by means of surface-initiated ATRP.9a,f Since their hydrophobic shells are not chemically cross-linked, the resulting capsules are expected to decompose in organic solvents. They are therefore not capable of loading drugs, restricting their usage as drug delivery vehicles. In the current work we fabricated cross-linkable (co)polymer brushes on Au nanocrystals by means of surface-initiated ATRP. After cross-linking the polymer brushes, followed by etching out the Au cores, hydrophilic polymer capsules were formed. Scheme 1 depicts our protocol. Our study should represent the first report on preparation of hydrophilic, colloidally stable, and pH-sensitive polymer capsules based on surface-initiated ATRP on nanocrystals. The potential of the resulting capsules in drug delivery applications is also demonstrated. Our protocol has several advantages over the existing methods to create polymer capsules. First, Au nanocrystals provide excellent tem(7) (a) Ma, Q.; Remsen, E. E.; Kowalewski, T.; Schaefer, J.; Wooley, K. L. Nano Lett. 2001, 1, 651. (b) Huang, H. Y.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1999, 121, 3805. (c) Clark, C. G, Jr.; Wooley, K. L., Curr. Opin. Colloid Interface Sci. 1999, 4, 122. (8) Marinakos, S. M.; Novak, J. P.; Brousseau, L. C.; House, A. B.; Edei, E. M.; Feldhaus, J. C.; Feldheim, D. L. J. Am. Chem. Soc. 1999, 121, 8518. (9) (a) Mandal, T. K.; Fleming, M. S.; Walt, D. R. Chem. Mater. 2000, 12, 3481.(b) Pyun, J.; Matyjaszewski, K.; Kowalewski, T.; Savin, D.; Patterson, G.; Kickelbick, G.; Huesing, N. J. Am. Chem. Soc. 2001, 123, 9445. (c) Nuss, S.; Bo¨ttcher, H.; Wurm, H.; Hallensleben, M. L. Angew. Chem. Int. Ed. 2001, 40, 4016. (d) Ohno, K.; Koh, K.; Tsujii, Y.; Fukuda, T. Angew. Chem. Int. Ed. 2003, 42, 2751. (e) Chen, X.; Randall, D. P.; Perruchot, C.; Watts, J. F.; Patten, T. E.; von Werne, T.; Armes, S. P. J. Colloid Interface Sci. 2003, 257, 56. (f) Kamata, K.; Lu, Y.; Xia, Y. J. Am. Chem. Soc. 2003, 125, 2384.
10.1021/la051638x CCC: $30.25 © 2005 American Chemical Society Published on Web 10/14/2005
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Scheme 1. Schematic Illustration of the Procedure of Fabrication of pH-Sensitive Polymer Capsules Utilizing Au Nanocrystals as Templates, Surface-Initiated ATRP, and Selectively Cross-Linking of PDMA
plates since they can be simply and reproducibly prepared with defined dimension and surface chemistry.10 Second, surface-initiated ATRP is a “grafting from” coating strategy, which can efficiently prevent aggregation of nanoparticles.9 Third, ATRP allows synthesis of a large number of hydrophilic (co)polymers, especially stimuliresponse ones.11 Fourth, the controlled cleavage of bonds between Au cores and cross-linked polymer shells allows inclusion of free Au nanocrystals inside polymer capsules, which can be further employed to probe the local environment.9f,12 Experimental Section Materials. Hydrogen tetrachloroaurate trihydrate, sodium citrate dihydrate, 2-hydroxyethyl disulfide, 11-mercapto-1-undecanol, bromine, 2-bromoisobutyryl bromide, 2-(dimethylamino)ethyl methacrylate (DMA), 2-(diethylamino)ethyl methacrylate (DEA), poly(ethylene glycol) methyl ether methacrylate (PEGMA), copper(I) bromide, 2,2′-bipyridine (BPy), (-)-sparteine (SP), 1,2-bis(2-iodoethoxy) ethane (BIE), potassium cyanide (KCN), rhodamine 6G (R6G), and 6-carboxyfluoresceine (6-CF) were all purchased from Aldrich and used as received. Synthesis of ATRP Initiators. ATRP initiators, 2,2′-dithiobis[1-(2-bromo-2-methylpropionyloxy)ethane] (DTBE) and 11,11′-dithiobis[1-(2-bromo-2-methylpropionyloxy)undecane] (DTBU), were synthesized based on the method reported by Hawker and co-workers.13 Briefly, 2-bromo-2-methylpropionyl bromide (14.8 mmol) was added dropwise to a mixture of disulfide (6.17 mmol) and triethylamine (31.5 mmol) in 150 mL of dichloromethane at 0 °C under argon atmosphere. The solution was stirred at 0 °C for 1 h and then for another 2 h at room temperature. After filtering off the precipitates, the organic phase was extracted with a 2 N Na2CO3 solution saturated with NH4Cl to remove the excess bromides. Subsequent removal of dichloromethane yielded DTBE and DTBU. Capping ATRP Initiators on Au Nanocrystals. Twelve nanometer Au nanocrystals were prepared by citrate reduction of chloroauric acid.10 Au nanocrystals capped with DTBE and DTBU were synthesized as described elsewhere.14 Typically, 4.6 mg of DTBE was dissolved in 10 mL of THF, which was added dropwise into 40 mL of an aqueous solution of Au nanocrystals. After incubation for 6-12 h, followed by removal of excess (10) Daniel, M.; Astruc, D. Chem. Rev. 2004, 104, 293. (11) (a) F. Zeng, Y. Shen, S. Zhu, R. Pelton, J. Polym. Sci. Polym. Chem. 2000, 38, 3821. (b) Liu, S.; Weaver, J. V. M.; Tang, Y.; Billingham, N. C.; Armes, S. P.; Tribe, K. Macromolecules 2002, 35, 6121. (12) Kim, M.; Sohn, K.; Na, H. B.; Hyeon T. Nano Lett. 2002, 2, 1383. (13) Shah, R. R.; Merreceyes, D.; Husemann, M.; Rees, I.; Abbott, N. L.; Hawker, C. J.; Hedrick, J. L. Macromolecules 2000, 33, 597. (14) Duan, H.; Wang, D.; Kurth, D. G.; Mo¨hwald, H. Angew. Chem. Int. Ed. 2004, 43, 5639.
Duan et al. initiators by centrifugation, DTBE-capped Au nanocrystals were obtained and suspended in water for further use. After incubating 10 mL of a THF solution of 7.2 mg of DTBU with 40 mL of an Au nanocrystal aqueous dispersion for 16 h, followed by centrifugation/THF wash, DTBU-capped Au nanocrystals were obtained and dispersed in dimethylformamide (DMF). To minimize the deterioration of the colloidal stability of the resulting capped nanoparticles, the capping density of DTBE and DTBU was optimized as about 800 ligands per particle, determined by thermogravimetric analysis (TGA). ATRP on Au Nanocrystals. Growth of polymer brushes on Au nanocrystals was performed based on a modified method described in our previous study.15 Aqueous ATRP of DMA on Au nanocrystals was carried out in aqueous media using DTBEcapped Au nanocrystals as initiators and CuBr/BPy complexes as catalysts at room temperature. The DMA concentration was 50 vol %. The molar ratio of DTBE/CuBr/BPy was 1/1/2. After 2 h ATRP under Ar, Au nanocrystals coated by poly[(2dimethylamino)ethyl methacrylate], marked as Au@pDMA, were collected by repeating the centrifugation/THF wash/redispersion cycle three times. ATRP of DMA and DEA was carried out in DMF, initiated by DTBU-capped Au nanocrystals. The monomer concentration was 50 vol %, and the volume ratio of DMA and DEA is 1:3. Polymerization was conducted for 8 h under Ar at 40 °C, catalyzed by CuBr/SP. The molar ratio of DTBU/CuBr/SP was 1/1/2. The product was denoted as Au@pDME. After 8 h of copolymerization of DMA and DEA, PEGMA (volume ratio of DMA/DEA/PEGMA ) 1:3:0.5) was subsequently added to the reaction mixture. After further polymerization for 6 h, the statistical copolymer brushes of DMA, DEA, and PEGMA were generated on Au cores, whose outmost parts are expected to contain more poly(ethylene glycol) (PEG) chains. The polymercapped Au nanoparticles, denoted as Au@pMEPEG, were collected by centrifugation. Preparation of Polymer Capsules. Armes and co-workers demonstrated that BIE may selectively quaternize more reactive amine groups of pDMA in the presence of both pDMA and pDEA.16 In the present work, therefore, we employed BIE to selectively cross-link pDMA moieties of shells on Au cores. The molar ratios of BIE to DMA were chosen as 1:2 and 1:6, corresponding to 100% and 30% target cross-linking degree of pDMA moieties on Au cores, as suggested by Armes and co-workers.16a KCN aqueous solutions (15 and 0.15 mg/mL) were used to etch Au cores to yield the hollow capsules. Encapsulation and Release of R6G. To encapsulate R6G we incubated 1 mL of an aqueous dispersion of the pMEPEG capsules (1013 capsules) in 1 mL of an aqueous solution of 10 mg of R6G of pH 6 for 10 min and then adjusted the pH to 12 by dropping 1 M NaOH aqueous solution. After removal of R6G precipitates by centrifugation at 10 000g for 30 min and washing with water of pH 12, orange-yellow dispersions of R6G-loaded capsules were obtained. Release of R6G was realized by dialyzing the R6G-loaded capsules against water of pH 6 overnight. Characterization. Dynamic light scattering (DLS) measurements were performed by a Malvern HPPS 500. Transmission electron microscope (TEM) images were obtained by a Zeiss EM 912 Omega microscope at an acceleration voltage of 120 kV. Atomic force microscopy (AFM) images were obtained by a Dimension 3100 AFM (Digital Instruments, CA). Luminescence spectra were obtained with a Spex Fluorolog 1680 spectrophotometer (the excitation wavelength is 524 nm). 1H NMR spectra were recorded with a Bruker DMX 400 spectrometer. TGA was implemented by a Netzsch 209. The molecular weight of polymer brushes, cleaved from Au cores, was determined by gel permeation chromatography (Polymer Standards Service GmbH, Mainz, Germany); the eluent used is N,N-dimethylformamide.
Results and Discussion pMEPEG Capsules. Recently, we succeed in growing hydrophilic polymer brushes on both aqueous and organic (15) Duan, H.; Kuang, M.; Wang, D.; Kurth, D. G.; Mo¨hwald, H. Angew. Chem. Int. Ed. 2005, 44, 1717. (16) (a)Bu¨tu¨n, V.; Wang, S.; de Paz Benez, M.; Robinson, K.; Billingham N. C.; Armes, S. P.; Tuzer, Z. Macromolecules 2000, 33, 1. (b)Bu¨tu¨n, V.; Armes, S. P.; Billingham N. C. Polymer 2001, 42, 5993.
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Table 1. Morphological Parameters of the Polymer-Coated Au Nanoparticles samples
〈Dh〉a (nm)
polymer shell thickness (nm)
Au@pDMA Au@pDME Au@pMEPEG
32 24 54
10 6 21
〈Dh〉 of corresponding capsulesb (nm) 64 42 210 (or 266)c
a DLS measurements were carried out in water at pH 6. b The capsules were obtained by using a BIE/DMA molar ratio of 1/2, corresponding to 100% target cross-linking degree of pDMA moieties. DLS measurements were conducted in water at pH 6. c The capsules were obtained using a BIE/DMA molar ratio of 1/6, corresponding to 30% target cross-linking degree of pDMA moieties. DLS measurements were conducted in water at pH 6.
nanocrystals via surface-initiated ATRP; their chain length may efficiently be tuned by the polymerization period.15 Herein we extended this study to form capsules using Au nanocrystals as templates and BIE to selectively cross-link pDMA moieties in the polymer shells.11b,16 The growth of polymer brushes on Au nanocrystals was demonstrated by 1H NMR spectroscopy (Figure S1, Supporting Information). Table 1 and Table S1 (Supporting Information) summarize the characterization data of the Au@pDMA, Au@pDME, and Au@pMEPEG nanoparticles obtained here. On the basis of the TGA data the density of polymer brushes on the nanocrystals is calculated as about 0.5 chain/nm2. After consecutively crosslinking the pDMA moieties with BIE and removing the Au cores by etching with 15 mg/mL KCN aqueous solution, we obtained hollow capsules from these nanoparticles. Upon addition of etching solution, the reddish transparent dispersions of the coated nanoparticles became opalescent, visually indicating removal of Au nanoparticles. The capsules derived from Au@pDMA and Au@pDME nanoparticles are 65 and 40 nm in mean sizes. Their TEM images indicate the presence of numbers of aggregates, which is due to interparticle cross-linking. In the case of using Au@pMEPEG nanoparticles as templates, their outer shells are rich in PEGMA chains, whose PEG chains may suppress the interparticle cross-linking.16a Figure 1a shows a typical TEM picture of the capsules derived from Au@pMEPEG nanoparticles in which one can see uniform 135 nm capsules with a dark outer shell and a light center. Figure 1b shows the hydrodynamic radius distribution of pMEPEG capsules, revealing that no aggregates are formed during the cross-linking reaction. The polydispersity index is as low as 0.08,17 indicating the relatively narrow size distribution of the capsules. In comparison with the original Au@pMEPEG nanoparticles, the pMEPEG capsules are much larger, 210 nm at pH 6 (Figure 1b and Table 1). This should be due to the swelling of hydrophilic moieties, PEG, and quaternized pDMA chains of the as-made capsules after removal of the spatial restriction imposed by Au cores. This observation is similar to previous reports on polymer capsules derived from shell cross-linked micelles.6 AFM imaging of the thin film cast from pMEPEG capsule dispersion reveals that closely packed capsules remain spherical and no collapsed capsules are observed (Figure 2). This suggests a robust mechanical stability of the pMEPEG capsules obtained. Figure 3a shows that the hydrodynamic radius (〈Rh〉) of pMEPEG capsules gradually increases as the environmental pH is reduced from 12 to 2, demonstrating the pH sensitivity of the resulting capsules. It is worth noting (17) On the basis of the cumulant analysis of DLS, the polydispersity index is derived from µ2/Γ2, where µ2 is the second cumulant of the decay function and Γ the average characteristic line width (Chu, B. Laser Light Scattering, 2nd ed: Academic Press: New York, 1991)
Figure 1. (a) TEM image of pMEPEG capsules with 100% target cross-linking degree of pDMA moieties. (b) Hydrodynamic radius distribution of pMEPEG capsules in pH 6 water.
Figure 2. AFM image of the thin film obtained by casting pMEPEG capsules with 100% target cross-linking degree of pDMA moieties.
that this pH-dependent size variation is reversible; its repetition does not cause flocculation. Reduction of the cross-linking degree may increase the size of pMEPEG capsules but has little influence on the profile of their pH-dependent size variation (Figure 3a). Although both pDMA and pDEA are pH sensitive, the pH response of pMEPEG capsules may mainly arise from pDEA moieties as the pDMA moieties were partially or fully quanternized by BIE. Since the pKa of pDEA is about 7.3,16 it can be
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Figure 4. Photoluminescence spectra of R6G at pH 6 (dotted line) and the R6G-loaded pMEPEG capsules at pH 12 (solid line). The target cross-linking degree of pDMA moieties of pMEPEG capsules is 30%. The insets in Figure 3 are photographs of R6G-loaded pMEPEG capsules (left) and pMEPEG capsules after release of R6G by dialyzing against water of pH 6 overnight (right) under a UV lamp.
Figure 3. (a) Plot of the hydrodynamic diameter of pMEPEG capsules with different target cross-linking degrees of pDMA moieties: 30% (0) and 100% (9), versus pH. (b) Plots of hydrodynamic diameter of pDMA (9) and pDME capsules (0) versus pH. Their target cross-linking degrees of pDMA moieties are 100%.
protonated at low pH. The electrostatic repulsion between the protonated amine groups of the pDEA moieties of the pMEPEG leads to swelling. Figure 3b shows that highly quaternized pDMA ones are not very pH sensitive, while pDME capsules still remain sensitive to the environmental pH due to protonation of the pDEA moieties of the capsules. This creates an opportunity to tune the permeability of the pDME and pMEPEG capsules by the environmental pH. Encapsulation and Release of R6G. Encouraged by this pH sensitivity, we encapsulated a drug model, R6G, using pMEPEG capsules. The solubility of R6G in water depends on the pH, being soluble at low pH and precipitating at high pH. Note that pMEPEG capsules may shrink with increasing environmental pH, suggesting that their pore sizes are reduced at high pH (Figure 3a). After incubating pMEPEG capsules with 30% target crosslinking degree of pDMA moieties in R6G aqueous solution of pH 6, followed by adjusting the pH gradually to 12, R6G precipitates were removed by centrifugation, leading to R6G-containing capsules, as shown in the inset of Figure 4 (left). As determined by absorption spectroscopy, the R6G loading amount per capsule is on the order of 10-13 mg. Compared with that of R6G in aqueous solution at pH 6, the emission peak of the R6G-loaded capsules is blue shifted by 10 nm (Figure 4). This may be due to the formation of R6G aggregates, suggesting that not R6G molecules but their aggregates are encapsulated inside pMEPEG capsules. These capsules are rather stable at pH 12; few precipitates are visible within at least 3 months. When the R6G-loaded pMEPEG capsules were dialyzed against water of pH 6 we found that the dialyzing water immediately turned fluorescent, exhibiting the same emission band as that of R6G molecules. This suggests that R6G molecules were released out of pMEPEG capsules. After dialyzing overnight, all dyes encapsulated were completely released from pMEPEG capsules, as
Figure 5. TEM image of pMEPEG capsules with free Au nanocrystals. Their target cross-linking degree of pDMA moieties is 100%.
shown in the inset of Figure 4 (right). The encapsulation and release of R6G were also realized by using pMEPEG capsules with 100% target cross-linking degree of pDMA moieties. On the other hand, we failed to use pMEPEG capsules to confine 6-CF, which is soluble at high pH and precipitates at low pH. This may be expected due to the fact that the pMEPEG capsules swell at low pH; the pores in the capsule walls are too large to confine 6-CF aggregates. pMEPEG Capsules with Au Cores Inside. The kinetic process of etching Au by KCN is well established.18 Use of dilute KCN solution, 0.15 mg/mL in our work, does not etch out the entire Au cores but only cleaves the Au-S bond between Au cores and the polymer shells, leaving behind capsules containing free Au nanocores. Figure 5 shows a typical TEM image of pMEPEG capsules with detached 12 nm Au cores. Using silica-coated Au coreshell spheres as templates, polymer capsules with movable Au cores have recently been created.9f,12 Compared with the existing technique, our process should be more simple and easier to extend to other inorganic nanocores. Since their plasmon resonance is exceedingly sensitive to the environmental variation,10,19 usually associated with (18) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498.
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apparent color change, the free Au nanocores are expected to serve as inner sensors to detect changes of the local environment,9f which is the current topic in our laboratory. Conclusion In summary, we succeeded in creating hydrophilic polymeric capsules of sizes in the 40-300 nm range by integration of Au nanocrystal templating, surface-initiated ATRP, and selective chemical cross-linking of pDMA. The capsules obtained are colloidally stable, mechanically robust, and pH sensitive. It is also possible to use these capsules for controlled encapsulation and release of a drug model, R6G. The PEG-rich outer shells of the as-made pMEPEG capsules are biologically inert and capable of minimizing nonspecific interaction, so the pMEPEG (19) Mulvaney, P. Langumir 1996, 12, 788.
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capsules should be good candidates for serving as drug delivery vehicles. By controlling the core etching process, our method also allows incorporation of functional nanoparticles within polymer capsules, providing an opportunity to introduce additional functionality to the capsules. Acknowledgment. We thank J. Hartmann and R. Pitschke for help with TEM. This work is financially supported by the Max Planck Society. Supporting Information Available: 1H NMR spectra of Au@pDME and Au@pMEPEG nanoparticles, and a summary of characterization data of the resulting pDMA, pDME, and pMEPEG brushes on Au nanocrystals. This material is available free of charge via the Internet at http://pubs.acs.org. LA051638X
