Gold Nanoparticles with Poly(N-isopropylacrylamide) Formed via

Mar 16, 2010 - Abstract. Abstract Image. Thermoresponsive polymer brushes on 20 nm ... surface of nanoparticles with the help of 2-bromopropionyl brom...
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J. Phys. Chem. C 2010, 114, 5947–5955

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Gold Nanoparticles with Poly(N-isopropylacrylamide) Formed via Surface Initiated Atom Transfer Free Radical Polymerization Exhibit Unusually Slow Aggregation Kinetics Sudipto Chakraborty,† Sandra W. Bishnoi,‡ and Vı´ctor H. Pe´rez-Luna*,† Department of Chemical and Biological Engineering and Biological, Chemical & Physical Sciences, Illinois Institute of Technology, Chicago, Illinois, 60616 ReceiVed: October 31, 2009; ReVised Manuscript ReceiVed: February 8, 2010

Thermoresponsive polymer brushes on 20 nm colloidal gold were formed through atom transfer free radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm) in aqueous media. In this approach, the “graftingfrom” technique was used with atom transfer radical polymerization (ATRP) to grow polymer chains from the surface of gold nanoparticles (∼20 nm). “Grafting from” using the ATRP technique enables dense, uniform, and homogeneous coverage of polymer chains on the surface of gold nanoparticles. Other advantages of ATRP are the growth of polymer chains without appreciable chain termination or chain transfer and that the presence of an active initiator site at the end of the growing polymer chain facilitates synthesis of surface grafted block copolymers. In the present work, pNIPAAm was grown from the surface of nanoparticles with the help of 2-bromopropionyl bromide as the initiator. The polymerization reaction was carried out at room temperature under inert atmosphere and aqueous conditions. The system was found to exhibit thermoresponsive behavior above and below the LCST. This behavior could be exploited to develop aggregation based assays for making drug delivery systems, detection assays, and bioseparations. The hybrid polymer-gold nanoparticle system was characterized using optical absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS). These analytical techniques confirmed the growth of polymer chains in the reaction scheme yielding the final product and a qualitative estimate of polymer chain thickness grafted onto the surface of the nanoparticles. Introduction Nanoparticle-organic material coated hybrid nanostructures can combine the unique optical properties of the metallic core with the properties of a surface tethered organic layer. The ability to tune the organic outer layer renders the potential for various technological applications of these hybrid materials. Recently, metal-polymer hybrid complex uses have been reported in molecular diagnostics1-3 and drug delivery.4,5 In such applications, polymers are chosen based on known bulk properties even though their behavior can be affected by the presence of a surface.6 Organic polymer coated gold nanoparticles can be prepared in a one step synthesis procedure by reduction of gold chlorate (HAuCl4) in a monomer/polymer dispersed solution,7,8 “grafting-to”9-11 and “grafting-from” methods.12,13 The “grafting-to” methods9 are limited by steric interactions of the adsorbing polymer molecules (size exclusion effects) and result in inhomogeneous and limited coverage of the gold surface with the polymer chains. A “grafting-from” technique, on the other hand, offers better control over the polymer layer thickness and surface chain density, and its versatility makes it increasingly attractive. Thus, this method allows better control to prepare well-defined organic-inorganic hybrid nanostructures. The “grafting-from” technique offers better control over surface chain density by introducing initiation sites for chain growth in a controllable manner prior to surface initiated polymerization. Polymerization methods such as cationic,14,15 anionic,16,17 free radical,18-21 reversible addition-fragmentation * Corresponding author. Phone: (312) 567-3963. Fax: (312) 567-8874. E-mail: [email protected]. † Department of Chemical and Biological Engineering. ‡ Biological, Chemical & Physical Sciences.

chain transfer polymerization (RAFT),22,23 and atom transfer radical polymerization (ATRP)24-27 can be used to grow polymer chains on surfaces. Among all these approaches ATRP is attractive because it is a living polymerization process and can give rise to homogeneous polymer layers having a low polydispersity index because polymerization occurs without chain transfer and chain termination steps. Owing to the fact that ATRP is a living polymerization method, initiator sites that are dormant after removal or depletion of monomers can be potentially used to make block copolymers.15,28 Although living free radical polymerization methods provide clear advantages for creating hybrid materials, the high polymer chain density of surface grafted polymers can potentially have different physicochemical properties due to molecular crowding. In the work presented here, ATRP was selected as a “grafting-from” method in order to functionalize gold nanoparticles with a thermoresponsive polymer. As described in this work, the aggregation kinetics of the resulting surface grafted polymer chains were found to significantly differ from those of polymer molecules in solution that were not attached to the nanoparticles. In the present work, the thermoresponsive polymer poly(Nisopropylacrylamide) (pNIPAAm) was grafted to the surface of gold nanoparticles. This polymer is well-known to exhibit a reversible phase transition in water at a point termed the lower critical solution temperature (LCST). Thermoresponsive polymers are of interest owing to the potential applications of these “smart” polymers.29-31 Some of those applications include drug delivery,32,33 nanotechnology,30,34 chromatography,35 biosensing, actuation, and detection.36-38 Below the LCST the pNIPAAm chains adopt an expanded random coil conformation in water. At temperatures above the LCST, these polymers come out of

10.1021/jp910417g  2010 American Chemical Society Published on Web 03/16/2010

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phase and precipitate, changing into a globular compact conformation brought about by the predominance of hydrophobic interactions. The LCST of poly(N-isopropylacrylamide) (pNIPAAm) occurs around 32 °C.26,39,40 The closeness of this value to physiological temperature has led to numerous biomedical applications of this polymer.41-44 In this work, the unique optical properties of colloidal gold and the thermoresponsive behavior of pNIPAAm are combined to create hybrid materials. The materials developed will be useful in applications that demand tuning polymer chains to collapse and expand due to external stimulus such as that occurring by thermal heating when gold nanoparticles absorb visible light near the plasmon resonance frequencies.45,46 Thus, the external stimulus could be provided by taking advantage of the unique optical properties of the metallic core. This could be beneficial for various biosensing, detection, and actuation purposes.47-49 The approach of this work is to graft pNIPAAm on colloidal gold using ATRP, thus taking advantage of this polymerization method to create hybrid nanomaterials. It is expected that molecular crowding of polymers formed by surface initiated ATRP would influence their physicochemical properties when compared to bulk molecules in solution. These differences were investigated by dynamic light scattering (DLS), optical absorption spectroscopy, and Fourier transform infrared spectroscopy (FTIR) and are reported below. Experimental Section Materials. Colloidal gold (monodisperse, 20 nm average particle diameter), N-hydroxysuccinimide (NHS), anhydrous tetrahydrofuran (THF), copper(I) chloride (CuCl), triethylamine (TEA), hydrogen peroxide, hydrochloric acid, and potassium bromide (KBr) were obtained from Sigma. Tween 20 (T20), 16-mercaptohexadecanoic acid (16-MHDA), ethyl alcohol, 2-(2aminoethoxy)ethanol (AEE), N-isopropylacrylamide (NIPAAm), 2-bromopropionyl bromide (2-bpb), copper(II) bromide (CuBr2), 2,2′-dipyridyl (bpy), and nitric acid were purchased from Aldrich. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and sulfuric acid were purchased from Fluka. The monomer NIPAAm was recrystallized three times in hexane to remove the inhibitor and dried under vacuum prior to use. Buffers and Solutions. Phosphate buffer solution (pH 7) at 10 mM concentration was made using sodium phosphate monobasic and sodium phosphate dibasic salts. The exact pH value of the buffer solution was adjusted with HCl and measured with an Accumet AB15 pH meter. Deionized water (electric resisitivity >18 MΩ · cm-1) was obtained from a Barnstead Nanopure Infinity UV/UF water purification system. Tween 20 solutions (1.82 mg/mL) were prepared in degassed sodium phosphate buffer (pH 7), and 0.5 mM 16-MHDA solution was prepared in degassed anhydrous ethanol. All glassware used in the experiments was washed with “piranha solution” (3:7, 30% hydrogen peroxide/concentrated sulfuric acid). (CAUTION: Piranha solution reacts Violently with most organic materials and should be handled carefully.) All glassware was further rinsed with “aqua regia” (1:3, concentrated nitric acid/concentrated hydrochloric acid). Reactions involving initiator immobilization and polymerization of NIPAAm were done in inert nitrogen atmosphere using Schlenk glassware. Instrumentation. Optical Absorption Spectroscopy of Functionalized Gold Colloids (UV-Vis). Optical absorption spectroscopy measurements were performed in a Shimadzu UV-

Chakraborty et al. 2401 PC dual-beam spectrophotometer using 1 cm path length quartz cuvettes. Spectra were collected within the 400-800 nm range . Dynamic Light Scattering (DLS). A Malvern Zetasizer Nano-E system light scattering instrument was used for measuring particle size. Emission wavelength at 633 nm was used for particle measurements. All colloidal samples were filtered using 0.45 µm syringe filters to remove aggregates prior to testing. Fourier Transform Infrared Spectroscopy (FTIR). FTIR was performed on a Bruker Tensor 27 instrument using dry KBr pellets. The data were collected continuously in the 4000-400 cm-1 wavenumber range at a resolution of 4 cm-1. All samples were lyophilized overnight prior to any IR measurement in order to reduce the presence of water in the sample. Surface Modification. A series of surface modifications were performed to functionalize the surface of colloidal gold particles. Characterization of the reaction steps was accomplished using UV-vis, DLS, and FTIR. Each of these steps is described below. Synthesis of Hydroxyl-Terminated Colloidal Gold (Scheme 1A). The surface of colloidal gold particles was modified in the presence of nonionic surfactant (Tween 20) as described before.50 Briefly, gold solutions with a concentration of 0.8 nM were degassed under nitrogen before use. Equal volumes (400 µL) of colloidal gold and Tween 20 in buffer were gently mixed and allowed to stand for a minimum of 30 min at room temperature. This allowed the physisorption of Tween 20 on the colloidal gold surface. Then, 400 µL of 0.5 mM 16-MHDA was added and the final solution was allowed to stand overnight to allow 16-MHDA to be chemisorbed onto the gold surface. Unreacted excess thiol and Tween 20 were removed by repeated centrifugation (three times at 15700g for 15 min) and resuspension in phosphate pH 7 buffer. After a final centrifugation step the 16-MHDA modified gold nanoparticles were reacted with a 1:1 mixture of freshly prepared 50 mM NHS and 200 mM EDC for 10 min. This step produces colloidal gold with NHS active esters on the outermost layer. Unreacted NHS/EDC was removed from the resulting gold dispersions by repeated centrifugation and resuspension in buffer. After this, 22 mM AEE in sodium phosphate buffer (pH 7) was added and allowed to react for 10 min. Excess AEE was removed through microfiltration using Microcon 30 (MW cutoff 30 000) centrifugal filters. The solutions were centrifuged thrice at 6000g for 5 min. The retentate containing hydroxyl-terminated gold particles was resuspended in sodium phosphate buffer containing Tween 20 and centrifuged again. To prevent light-induced flocculation of the colloids and oxidation of the alkanethiols, all colloid solutions were stored in the dark and refrigerated at 4 °C.51 Immobilization of Initiator on Gold Nanoparticles. Scheme 1B gives the overview of this step in the synthesis. In order to attach the initiator, the hydroxyl-terminated gold nanoparticles were first centrifuged to remove excess liquid and concentrate the nanoparticles to a pellet. The retentate was lyophilized to remove any trace amount of water. Anhydrous THF solution containing 2% (v/v) anhydrous TEA by volume was then added to the gold particles. The solution was degassed thoroughly with nitrogen and cooled with ice. A 0.1 M 2-bpb solution was then introduced with vigorous shaking to ensure near-uniform reaction. The reaction time was 2 min and the reaction was carried out at 0 °C. After this reaction step the gold nanoparticles were successively centrifuged and resuspended in THF, ethanol, and deionized water successively.

Aggregation Kinetics of pNIPAAm-Au Nanoparticles

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SCHEME 1: Schematic Illustration of the Surface Modification of Gold Colloida

a (A) 2-(2-Aminoethoxy)ethanol is coupled to carboxyl-functionalized gold nanoparticles using EDC/NHS. The terminal group is the hydroxyl group. (B) Hydroxyl-terminated gold nanoparticles are reacted with initiator 2-bpb in the presence of THF/TEA. (C) Initiator grafted nanoparticles are exposed to NIPAAm in the presence of catalyst (CuCl/CuBr2/bpy) to undergo polymerization forming pNIPAAm grafted nanoparticles.

ATRP on Modified Gold Nanoparticle Surface. The polymerization of NIPAAm on the nanoparticles was carried out by reacting initiator functionalized gold nanoparticles with NIPAAm as shown in Scheme 1C. For the ATRP reaction, 3 mL of initiator coated gold nanoparticles was mixed with an equal volume of 1 M NIPAAm in water. The solution was degassed

for 5 min under nitrogen in the Schlenk tube. The ATRP reaction was initiated by introducing freshly prepared catalyst solution (CuCl/CuBr2/bpy ) 1:0.3:3, CuCl ) 4.3 mM, purged with nitrogen). After 2 h, the reaction was terminated by venting air into the system. After the reaction, the nanoparticles were centrifuged at 15700g and redispersed in buffer (pH 7).

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Figure 1. Normalized optical absorption spectra of gold nanoparticles at each functionalization step.

Results and Discussion Surface Modification of Gold Colloids. The gold nanoparticles having pNIPAAm grafted onto their surface were prepared in three steps as shown in Scheme 1. Synthesis of Hydroxyl-Terminated Colloidal Gold. As described in Scheme 1A, 20 nm gold particles were treated in several steps to bear hydroxyl surface groups. The surface plasmon peak of pure gold colloid solution was at 520 nm (Figure 1), which is consistent with well-dispersed spherical particles in aqueous media. In the next step, when gold solution was treated with 16-MHDA, the plasmon peak shifted to 524 nm. The red shift in plasmon peak value was due to the change in dielectric constant of the media surrounding the gold nanoparticles.52 The absence of significant broadening indicated that there was no evident aggregation of the gold particles on treatment with 16-MHDA.53 When the 16-MHDA modified gold nanoparticles were exposed to freshly prepared NHS/EDC solution, the plasmon peak exhibited a large red shift with the maximum value at 564 nm. The data (Figure 1) also showed broadening of the peak, which reflects the aggregation of gold nanoparticles in solution. This aggregation is most likely due to decreased electrostatic repulsion when the ionizable carboxyl groups are converted to noncharged NHS ester groups, which are also more hydrophobic than carboxyl groups. In the following reaction step with AEE, the surface plasmon peak for the gold nanoparticles shows a blue shift to 538 nm. The broadening of the peak seen in the previous step (formation of NHS ester) was reduced, indicating that aggregation of the particles was reversed and AEE modified nanoparticles spontaneously disperse in buffer (pH 7). The redispersion of gold nanoparticles on treatment with AEE can be explained by the presence of highly hydrated di(ethylene glycol) moieties. Immobilization of Initiator on Gold Nanoparticles. In the next step (Scheme 1B), the hydroxyl-terminated gold nanoparticles were treated with ATRP initiator 2-bpb. Prior to any synthetic step the gold particles having hydroxyl groups attached to them were lyophilized overnight in order to remove water. The moisture-free particles were then suspended in anhydrous THF along with TEA. To achieve initiator immobilization, 2-bpb solution in anhydrous THF was used. Since a thiol selfassembled monolayer can be unstable in acid bromide (which forms upon reaction of 2-bpb with hydroxyl groups or water), the nanoparticles were treated only for a short time (3 min). Special care was taken to keep any moisture from this reaction,

as acyl bromides are very sensitive to moisture, especially in the presence of organic bases such as TEA. The excess initiator was removed by centrifugation and resuspension in THF, ethanol, and finally deionized water. When the initiator modified nanoparticles are resuspended in water, the acyl bromide has already been eliminated by previous THF and ethanol washes. Thus, acid bromides are expected to be absent or at negligible concentrations when the particles are in aqueous medium. In the recent past, surface grafted polymer brushes created using ATRP has been done mostly in organic media and on flat surfaces. However, synthesizing stable gold nanoparticles with ATRP initiator in organic or aqueous media is challenging because of potential aggregation. Hydrophilic nanoparticles in nonpolar media or hydrophobic nanoparticles in aqueous media readily aggregate. Since initiator modified nanoparticles are hydrophobic and aggregate in water, they were synthesized in organic solvent (THF) at the initiator immobilization step. However, the ATRP reaction leading to formation of polymer brushes on the particles was performed in aqueous solution. Figure 1 shows the plasmon resonance curve for initiator capped gold nanoparticles in water. The absence of considerable peak broadening justifies the conclusion that gold particles were not aggregated. Most likely, steric repulsion of di(ethylene glycol) groups contributes significantly to the stability of these initiator functionalized nanoparticles. ATRP on Modified Gold Nanoparticle Surface. In the last step of the reaction (Scheme 1C), initiator immobilized gold nanoparticles were exposed to NIPAAm in aqueous media. CuCl/CuBr2/bpy was used as catalyst. The reaction was carried out in the absence of air, because oxygen can inhibit free radicals and its presence can result in very low yields. At the end of the reaction the excess monomer and catalyst were removed by repeated centrifugation and dispersion in buffer (pH 7). The successful formation of polymer coated gold nanoparticles using ATRP was qualitatively characterized using Fourier transform infrared (FTIR) spectroscopy and dynamic light scattering (DLS) as discussed below. Kinetics of Aggregation of pNIPAAm Molecules. There were significant differences in the kinetics of aggregation of pNIPAAm modified gold nanoparticles and bulk pNIPAAm in solution. Figure 2 shows photographs of both systems at room temperature and different time intervals after being placed in a 40 °C water bath. Clearly, bulk pNIPAAm undergoes precipitation out of solution soon after reaching a temperature above

Aggregation Kinetics of pNIPAAm-Au Nanoparticles

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Figure 2. Photographs of pNIPAAm grafted onto gold nanoparticles by ATRP (top) and bulk polymerized pNIPAAm in aqueous solution (bottom).

the LCST. This is evident in the turbid appearance of the solution occurring in less than 6 min. On the other hand, pNIPAAm functionalized gold nanoparticles exhibit a much slower change in aggregation state. Since the optical properties of gold nanoparticles are strongly dependent on their separation distance, this allows for facile monitoring of aggregation based on optical properties. Figure 2 shows that color changes of pNIPAAm modified Au nanoparticles by the ATRP method do not appear within the first 30 min of bringing these samples to a 40 °C water bath. The changes in color are evident only after 1 h of exposure to the 40 °C environment. This is evident by the color change from reddish to blueish in Figure 2, which indicates aggregation of the gold nanoparticles. It was also noted that no further changes were observed in the appearance of these solutions at longer times (Figure 2). Spectroscopic studies showed differences in ATRP and bulk pNIPAAm, which may explain the different aggregation behaviors. These are discussed further below. Characterization of Particle-Polymer Hybrid Size and Effect of Temperature on Grafted pNIPAAm. The change in size of nanoparticles upon different surface modification steps was characterized using dynamic light scattering (DLS). The change in diameter as a function of temperature for the pNIPAAm grafted nanoparticles was investigated. Figure 3 represents the particle size distribution (diameter) obtained by DLS at various modification steps. As expected, colloidal gold nanoparticles exhibited a mean diameter of 20 nm. After

successive modifications the mean diameter increased and the initiator grafted nanoparticles showed a diameter of 27 nm. After the polymerization step, the diameter of the hybrid pNIPAAm-gold nanoparticle material increased to 46 nm (size determined at room temperature). At this stage, the DLS data show small shoulders around 10 and 200 nm. These are likely due to polymer chains not attached to the nanoparticles and particle aggregates that occurred during the polymerization steps, respectively. It is worth noting that these peaks disappeared from DLS once the particles were cycled through the LCST. The particle size distribution was noticeably increased after polymer growth on the nanoparticles, indicating the formation of grafted pNIPAAm chains with a thickness ca. 10 nm. Since pNIPAAm has an LCST around 32 °C, below which the polymer chains are expected to have a random coil conformation in a highly hydrated state, DLS was also performed above the LCST to confirm the thermoresponsive nature of the chains, where they collapse and attain a globular conformation of more hydrophobic and dehydrated polymer. At 45 °C, the diameter of the particle-polymer hybrid system reduced to 38 nm. This change in average size of the particles by DLS was only observed when polymer was attached to the gold nanoparticles. No such change in diameter size was observed with temperature changes in gold particles in any other modification steps (data not shown). In order to confirm the reversible aggregation behavior of the gold nanoparticle-polymer hybrid systems with respect to

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Figure 4. Reversibility of the particle-polymer hybrid system as it was cycled between temperatures above and below the LCST of the polymer.

Figure 5. Optical absorption spectra of pNIPAAm functionalized gold nanoparticles subjected to heating and cooling.

Figure 3. Size distribution of gold nanoparticles at various stages of modification: (a) gold nanoparticle (with no modification), (b) initiator grafted gold nanoparticlec, (c) gold nanoparticles with pNIPAAm below LCST (25 °C), and (d) gold nanoparticles with polymer above the LCST (45 °C).

temperature, the system was subjected to successive cycles below (25 °C) and above (45 °C) the LCST of pNIPAAm. Figure 4 shows the mean diameter of pNIPAAm modified gold nanoparticles upon cycling above and below the LCST, which clearly indicates that the hydration and collapse behavior of surface grafted pNIPAAm is highly reversible. However, the changes depicted in Figure 4 were observed upon subjecting the particles to temperatures above the LCST for less than 1 min. This is in contrast to Figure 2, which does not show indication of aggregation after 30 min. In fact, color changes indicative of particle aggregation were not observed during the measurements reported in Figure 4. Thus, even though DLS shows changes in particle size above and below the LCST, these changes do not appear to be accompanied by immediate aggregation. In order to obtain a more accurate measurement of optical properties than visual examination can provide, the optical absorption spectra of pNIPAAm modified nanoparticles were obtained at different time intervals when the nanoparticles were held at 40 °C for various amounts of time (Figure 5). The optical

absorption spectrum of pNIPAAm modified gold nanoparticles at room temperature does not seem to exhibit aggregation when the particles are subjected to a temperature of 40 °C for less than 30 min. In fact, it appears that the spectrum becomes narrower initially upon heating, which indicates less aggregation. It is possible that the thermal energy provided can cause some particle separation when the polymer chains have not changed to a hydrophobic state. However, the appearance of a second plasmon peak, indicative of particle aggregation, is evident after 1 h and the changes due to aggregation continue to occur after 2 h. Figure 5 shows that the second peak that appeared upon aggregation begins to decrease after 2 h. This could be occurring because large aggregates begin to sediment in the cuvette and, thus, are not sampled by the spectrophotometer. When these particles are brought to room temperature (below the LCST), the absorption spectrum slowly reverts to that of nonaggregated nanoparticles. These changes are far slower than those observed for bulk pNIPAAm in solution (Figure 2). It is also worth noting that the changes observed in optical absorption spectra are due to aggregation because of the shift and broadening of the plasmon peak. Although local changes in the refractive index can also have an effect on the optical absorption spectra, these changes are very small and occur without only slight changes in the position of the plasmon peak and without broadening.52 The results described above show that DLS indicates changes in the polymer conformation occurring in less than 1 min. Yet, optical absorption data show aggregation kinetics much slower than the time scale for which changes in DLS occur. This appears to indicate that the surface grafted pNIPAAm can undergo a fast change above the transition temperature without

Aggregation Kinetics of pNIPAAm-Au Nanoparticles

Figure 6. FTIR data for gold nanoparticles at each modification step: (a, MHDA) modification with 16-MHDA, (b, AEE) surface reaction with AEE, (c, 2-bpb) 2-bpb initiator on the surface of the nanoparticles, and (d, pNIPAAm) polymer grafted on the nanoparticles.

significant aggregation followed by a much slower change that leads to particle aggregation. Clearly these observations show that pNIPAAm grafted on the gold nanoparticles undergoes a fast transition that does not lead to aggregation and a slower transition resulting in particle aggregation. It is unlikely that the slow aggregation of particles results from a decreased diffusion coefficient of larger particles because salt-induced aggregation of colloidal gold occurs almost instantaneously (not shown). Wu et al.54 reported that pNIPAAm grown at high densities on silica particles by the “grafting-from” technique using ATRP exhibits two transition regions. In that work, it is hypothesized that the first transition occurs near the particle surface due to cooperative effects and molecular crowding and that the second transition occurs on the outer layer of the polymer brush with a more bulk-like behavior. These transitions are not true thermodynamic transitions but result from cooperative conformational transitions in the highly dense environment of polymer brushes near the solid surface.55 The collapse of chains first occurs at the bottom of the brush and progresses toward the external surface.56 The existence of two transitions could help explain the fast change in hydrodynamic radii measured by dynamic light scattering and the anomalously slow aggregation behavior of the gold nanoparticles reported here. As a result, differences in chain conformation between bulk and surface grown pNIPAAm were investigated by FTIR. Such results are presented and discussed below. FTIR Study of Surface Modification of Gold Nanoparticles. Attachment of groups at every modification step was confirmed with the help of FTIR (Figure 6). Samples were completely dried before they were cast to pellets along with KBr and analyzed by FTIR. The presence of 16-MHDA on the nanoparticles was confirmed by the presence of a sharp carbonyl

J. Phys. Chem. C, Vol. 114, No. 13, 2010 5953 peak at 1697 cm-1 (Figure 6b) and the presence of a broad hydroxyl band (2500-3500 cm-1) that overlaps with asymmetric and symmetric C-H stretch bands observed at 2918 and 2850 cm-1, respectively (Figure 6a). Other peaks confirming the presence of MHDA are the O-H out-of-plane vibration observed as a broad peak at 917 cm-1, the C-O stretch at 1215 cm-1, and the in-plane O-H bend vibration at 1410 cm-1 (Figure 6b). These are characteristic features of carboxylic groups and confirm successful modification with MHDA. In the next modification step AEE is attached to the nanoparticles. A peak at 3364 cm-1 due to secondary amine (N-H stretching vibration) and a broad hydroxyl peak around 3000-3700 cm-1 indicate the presence of AEE (Figure 6a). Also, the shift in the carbonyl peak to a lower wavenumber due to the formation of a secondary amide (broader peaks at 1600-1650 cm-1 indicative of amide band) along with absorption bands due to N-H vibration (strong in-plane vibration at 1460 cm-1 and broad out-of-plane N-H bend vibration around 800-500 cm-1) shown in Figure 6b confirm successful coupling of AEE with the carboxyl groups of MHDA through amide bond formation. A saturated, unbranched, asymmetric ether peak at 1124 cm-1 further confirms the presence of AEE. Initiator immobilization was apparent from the presence of a carbonyl peak characteristic of R-bromo-substituted ester at 1743 cm-1 in the absorption FTIR spectrum (Figure 6b). Along with the carbonyl peak, peaks for C-C-O stretch at 1244 cm-1 and O-C-C stretch at 1048 cm-1, all characteristic of ester bond formation, further confirm successful covalent coupling of the initiator (2-bromopropionyl bromide) on the nanoparticles. Other peaks indicative of 2-bpb immobilization were the asymmetric and symmetric C-H stretches of CH3 groups at 2964 and 2877 cm-1 (Figure 6a) and the symmetric C-CH3 bend at 1374 cm-1 (Figure 6b). The broad feature at 3440 cm-1 could indicate partial hydrolysis of the ester bond and/or traces of moisture present in the KBr pellet used to collect the FTIR spectrum. After reaction of the initiator outer shell with NIPAAm, the infrared spectrum of the final product was collected (Figure 6). The presence of a sharp peak at 3300 cm-1 (due to secondary amide N-H stretch in Figure 6a), an amide band around 1650 cm-1 and a broad N-H out-of-plane vibration peak at 713 cm-1 (Figure 6b) confirm the presence of amide group formation. Other peaks indicative of pNIPAAm include the split “umbrella mode” peaks with a 1:1 ratio at 1386 and 1367 cm-1, which are characteristic of isopropyl groups.57 In order to ensure that these data were not from polymer chains formed in bulk instead of being grafted onto gold nanoparticles, the gold nanoparticles were repeatedly centrifuged and resuspended in buffer until the discarded supernatant did not show of the presence of pNIPAAm. The FTIR spectra of ATRP surface polymerized pNIPAAm was compared to that of pNIPAAm prepared in solution by ATRP polymerization. For these purposes, bulk pNIPAAm was cast into a KBr pellet or deposited from solution as a thin film on a KBr window. Both methods, KBr pellet and cast film on KBr window, yielded spectra similar to each other but with features differing from ATRP surface polymerized pNIPAAm on gold nanoparticles. Figure 7 shows the infrared spectra of pNIPPAm cast as a film on a KBr window and Au-pNIPAAm prepared by the ATRP method. The spectral region between 2830 and 3020 cm-1 is shown in Figure 7a. This region contains the C-H stretching vibration regions of methylene and methyl groups. Some differences between ATRP and bulk polymerized pNIPAAm are apparent in this region. The symmetric and

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Figure 7. Comparison of FTIR spectra of pNIPAAm grafted onto gold nanoparticles by ATRP and pNIPAAm prepared in bulk solution by free radical polymerization.

asymmetric C-H stretch vibrations of methyl groups for ATRPprepared pNIPAAm on gold appear slightly shifted to higher frequencies. For ATRP pNIPAAm on gold νCH3a ) 2972 cm-1 and νCH3s ) 2877 cm-1; for bulk pNIPAAm νCH3a ) 2970 cm-1 and νCH3s ) 2876 cm-1. The asymmetric methylene vibration for ATRP pNIPAAm on gold is νCH2a ) 2935 cm-1 and for bulk pNIPAAm is νCH2a ) 2933 cm-1. The symmetric stretches appear as a shoulder at lower frequencies of the symmetric methyl vibrations. The relative intensities of symmetric methyl and methylene vibrations, I(νCH3s)/I(νCH2s), appear to be larger for ATRP pNIPAAm than for bulk pNIPAAm. This could indicate lower mobility of the pNIPAAm chains prepared by the ATRP method.58 Also, the relative ratio of asymmetric methyl vibrations to symmetric methylene vibrations (I2930/I2850) is smaller for ATRP pNIPAAm, also consistent with the picture of a more ordered environment.59 The ATRP pNIPAAm thus appears to have some features indicating that at least a portion of the chains are in a more ordered environment than bulk pNIPAAm. The slight shifts of ATRP pNIPAAm to higher frequencies (∆ν e 2 cm-1), although indicative of less ordered environments (in apparent contradiction to the I2930/I2850 ratios), could be an indication of ATRP pNIPAAm providing two populations of chains: one in an ordered state and another in a disordered state. In fact, the broader shoulder of νCH3as (2972 cm-1) at higher frequencies could be an additional indication of pNIPAAm chains with larger disorder than bulk pNIPAAm. The amide I and II region of the spectra shows even more striking differences between bulk and ATRP-created pNIPAAm (Figure 7b). The carbonyl stretching vibration of bulk pNIPAAm shows only one broad peak centered at 1642 cm-1, whereas ATRP-prepared pNIPAAm on the gold nanoparticles shows two distinctive (and sharper) bands with peak maxima at 1622 and 1659 cm-1. This appears to indicate that ATRP pNIPAAm on gold exists in two clearly distinct environments, whereas the broader peak for bulk pNIPAAm indicates that the carbonyl peaks exist in a continuum of states with different hydrogen bonding levels. The peaks at 1678, 1655, and 1633 cm-1 in amides have been ascribed to dimers, trimers, and higher n-mers, respectively, with free amides assigned to 1690 cm-1.60 This seems to indicate that ATRP pNIPAAm chains exist in one state with higher hydrogen bonding (1659 cm-1, probably composed of trimers) and another state with lower hydrogen bonding (1622 cm-1, probably due to higher n-mers), whereas bulk pNIPAAm consists of one population where the largest component could be higher n-mers. The amide II band (mainly in plane N-H bending and C-N stretching contributions) also indicates larger hydrogen bonding

for the ATRP-prepared pNIPAAm on gold than bulk pNIPAAm (1549 and 1535 cm-1, respectively). In contrast to the amide I region, the amide II region shows only one peak, but this could simply reflect a single interaction of the hydrogen in N-H, whereas the carbonyl group, with the two pairs of unshared electrons, could exhibit different degrees of hydrogen bonding more readily. Other regions of the infrared spectra such as the methylene wagging modes show more intense features for ATRP pNIPAAm on gold than bulk pNIPAAm (not shown), which further supports the existence of pNIPAAm chains in a more ordered state than bulk pNIPAAm.60 This is simply an indication that surface initiated ATRP leads to highly dense polymer chains, which cannot be produced with “grafting-to” methods because of size exclusion limitations for adsorbing random coils. The experimental observations of surface polymerized pNIPAAm versus bulk polymerized pNIPAAm point out the different behaviors in aggregation kinetics and structures of the polymer chains. We hypothesize that these are related and that the slow aggregation kinetics are a result of less mobility of pNIPAAm chains created by surface initiated ATRP on gold. More densely populated chains, being more restricted in their mobility because of molecular crowding, will be slower at changing conformations between hydrated and collapsed states below and above the LCST, respectively. Although it can be argued that the FTIR results were obtained in the dry state and, thus, not directly indicating what is happening in the hydrated state as the polymer chains are cycled above and below the LCST of the polymer, it can be argued that the end state of the pNIPAAm analyzed by FTIR reflects, to some extent, the capability of pNIPAAm chains to rearrange when they are dehydrated. Also, the dehydration of these materials was done at low temperature in a lyophilizer. Thus, we believe the FTIR data reflect, with some degree of accuracy, the fact that pNIPAAm chains prepared by surface initiated ATRP polymerization are more ordered, more crowded, and contain one population that is more extensively hydrogen bonded and another that is less hydrogen bonded than bulk pNIPAAm. The fact that surface polymerized pNIPAAm exhibits fast changes in the light scattering measurements that do not lead to immediate aggregation could be a reflection of the existence of two states of the polymer chains. We hypothesize that the inner segments of the polymer chains of these hybrid materials are able to undergo fast rearrangements that are readily identified by DLS but cannot lead to aggregation.55,56 We also hypothesize that, in order to aggregate, more extensive rearrangement of polymer chains involving the outer segments is necessary in order to create significant aggregation. Because of the progres-

Aggregation Kinetics of pNIPAAm-Au Nanoparticles sion of chain transitions from inner segments first to outer segments later, the slower aggregation kinetics could be a result of the molecular crowding on the dense polymer brushes created by surface initiated ATRP of NIPAAm. Thus, surface initiated polymerization by ATRP could allow tuning of the response kinetics of thermoresponsive polymers by controlling the degree of molecular crowding. Conclusions In summary, the present work describes an approach to prepare gold nanoparticle-pNIPAAm coated hybrid materials using aqueous ATRP. The stepwise functionalization of gold nanoparticles provides a simple route to introduce ATRP initiator on the surface without any significant aggregation of the particles. The immobilized initiator enabled effective growth of dense polymer chains from the particle surface using the “grafting-from” technique. FTIR and optical spectroscopic results confirmed the presence of various layers in successive modification steps. Furthermore, FTIR also confirmed differences in polymer chain structure prepared by surface initiated ATRP and bulk polymerization. Temperature dependence studies using DLS confirm the thermoresponsive behavior of surface grafted pNIPAAm on the gold particles occurring with fast kinetics. Optical absorption spectroscopy showed a much slower process occurring when the polymer chains were cycled above and below the LCST. This latter process was not observed with bulk pNIPAAm in solution. The structural differences in pNIPAAm observed by FTIR could help explain the different aggregation kinetics. One further advantage of these hybrid materials is that the presence of an active ATRP initiator molecule at the end of the polymer chain could also enable further polymerization with other monomers to form block copolymers so that the thermoresponsive behavior and functionality of these nanoparticles could be tailored for applications in drug delivery, medical diagnosis, and nanoactuators. Acknowledgment. The Department of Chemical and Biological Engineering at the Illinois Institute of Technology and the Office of Naval Research are gratefully acknowledged for making this work possible. The authors would like to thank Prof. Georgia Papavasiliou and Michael Turturro for their help and use of the lyophilizer. References and Notes (1) Lee, S.; Pe´rez-Luna, V. H. Anal. Chem. 2005, 77, 7204–7211. (2) Lee, S.; Pe´rez-Luna, V. H. Langmuir 2007, 23, 5097–5099. (3) Thanh, N. T. K.; Vernhet, V.; Rosenzweig, Z. Springer Ser. Chem. Sens. Biosens. 2005, 3, 261–277. (4) Kim, J. H.; Lee, T. R. Drug DeV. Res. 2006, 67, 61–69. (5) van Vlerken, L. E.; Amiji, M. M. Expert Opin. Drug DeliVery 2006, 3, 205–216. (6) Mendez, S.; Curro, J. G.; McCoy, J. D.; Lopez, G. P. Macromolecules 2005, 38, 174–181. (7) Jewrajka, S. K.; Chatterjee, U. J. Polym. Sci. A 2006, 44, 1841–1854. (8) Sarma, T. K.; Chattopadhyay, A. J. Phys. Chem. A 2004, 108, 7837– 7842. (9) Wuelfing, W. P.; Gross, S. M.; Miles, D. T.; Murray, R. W. J. Am. Chem. Soc. 1998, 120 (48), 12696–12697. (10) Lowe, A. B.; Sumerlin, B. S.; Donovan, M. S.; McCormick, C. L. J. Am. Chem. Soc. 2002, 124 (39), 11562–11563. (11) 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 (20), 5811–5821. (12) Mandal, T. K.; Fleming, M. S.; Walt, D. R. Nano Lett. 2002, 2 (1), 3–7. (13) Ohno, K.; Koh, K.-m.; Tsujii, Y.; Fukuda, T. Macromolecules 2002, 35 (24), 8989–8993. (14) Jordan, R.; Ulman, A. J. Am. Chem. Soc. 1998, 120 (2), 243–247. (15) Zhao, B.; Brittain, W. J. J. Am. Chem. Soc. 1999, 121 (14), 3557–3558. (16) Tsubokawa, N.; Ueno, H. J. Appl. Polym. Sci. 1995, 58, 1221–1227.

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