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Jul 21, 2005 - Cross-linked poly(4-vinylbenzyl chloride) (PVBC) microspheres were first synthesized by ... Furthermore, polymer microspheres of fairly...
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Polymer Microspheres with Permanent Antibacterial Surface from Surface-Initiated Atom Transfer Radical Polymerization Zhenping Cheng,† Xinlin Zhu,† Z. L. Shi,‡ K. G. Neoh,‡ and E. T. Kang*,

‡

School of Chemistry and Chemical Engineering, Suzhou University, Suzhou, 215006, People’s Republic of China, and Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 119260

Cross-linked poly(4-vinylbenzyl chloride) (PVBC) microspheres were first synthesized by suspension copolymerization of 4-vinylbenzyl chloride (VBC) in the presence of a cross-linking agent, ethylene glycol dimethacrylate (EGDMA). Subsequent modification of the microsphere surfaces via surface-initiated atom transfer radical polymerization (ATRP) of 2-(dimethylamino)ethyl methacrylate (DMAEMA), using the VBC units of PVBC on the microsphere surface as the macroinitiators, gave rise to well-defined (nearly monodisperse) and covalently tethered poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) brushes. Quaternization of the tertiary amine groups of the PDMAEMA brushes with alkyl bromides (1-bromododecane or 1-bromohexane) gave rise to a high concentration of quaternary ammonium salt (QAS) on the microsphere surfaces. The chemical composition of the microsphere surfaces at various stages of surface modification was characterized by X-ray photoelectron spectroscopy (XPS). The bactericidal effect of the QAS-functionalized microspheres on Escherichia coli and Staphylococcus aureus was demonstrated. The permanence of the bactericidal activity was also demonstrated through the repeated applications of the surface-modified PVBC microspheres without any significant loss of their surface activity or functionality. 1. Introduction The antimicrobial agent in liquid form has the inherent problem of residual toxicity. This problem could be resolved if the antimicrobial agent could be immobilized on a substrate surface. Methods of immobilizing antimicrobial agents on various substrate surfaces have been widely studied.1 Among the surface functionalization techniques, polymer brushes from surface-initiated polymerizations have been widely used to tailor the surface properties of substrates, such as wettability,2-4 biocompatibility,5,6 corrosion resistance,7 and antibacterial effect.8,9 The advantage of covalently tethered polymer brushes over other surface modification methods (e.g., self-assembled monolayers) is their mechanical and chemical robustness, coupled with a high degree of synthetic flexibility toward the introduction of a wide variety of functional groups. A large number of quaternary ammonium salts (QAS) exhibit good bactericidal properties.9-21 Quaternized cationic polymers can exhibit higher antimicrobial activities than the corresponding low molecular weight model compounds.22-24 For ease of recovery, it will also be advantageous to immobilize antimicrobial macromolecules on solid substrates. Methods for functionalizing planar surfaces, such as glass, high-density polyethylene (HDPE), low-density polyethylene (LDPE), polyprolylene (PP), nylon, poly(ethylene terephthalate), and filter paper surfaces, with quaternized vinylpyridine * To whom correspondence should be addressed. Tel.: +656874-2189. Fax: +65-6779-1936. E-mail: [email protected]. † School of Chemistry and Chemical Engineering, Suzhou University, Suzhou, 215006, People’s Republic of China. ‡ Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 119260.

polymers have been reported.9,15,25,26 For introducing functional moieties to substrate surfaces via grafting or graft polymerization, good control over the brush density, polydispersity, and composition will be highly desirable. Among the major controlled polymerization strategies developed, living radical polymerizations, atom transfer radical polymerization (ATRP) in particular,27-29 have become the most popular techniques. The ATRP process has good tolerance for a wide range of functional monomers and requires less stringent experimental conditions. ATRP has been successfully applied to the preparation of well-defined polymer brushes on different substrates, such as silicon,29-36 SiO2,37,38 gold,39 and polymers.40-47 Recently, antimicrobial polymers have been attached directly on glass and filter paper surfaces via surface-initiated ATRP.8 The microspheres have the advantages of large specific surface area, ease of dispersion, ease of packing (in pack column applications), and ease of recovery and handling (in comparison to nano- or submicron spheres). Furthermore, polymer microspheres of fairly uniform sizes can be readily prepared via suspension or emulsion polymerization. They have been widely used as absorbents, affinity bioseparators, and drug and enzyme carriers.48 In the present work, we report on a simple process for the preparation of polymer microspheres with permanent antimicrobial surfaces. The process involved (i) synthesis of cross-linked poly(4-vinylbenzyl chloride) (PVBC) microspheres via suspension polymerization, (ii) modification of the microsphere surfaces with covalently grafted and well-defined (nearly monodisperse) poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) brushes from surface-initiated ATRP of DMAEMA, and (iii) quaternization of PDMAEMA brushes by alkyl bromides (1-bromododecane or 1-bromohexane). The 4-vinylbenzyl chloride (VBC) units of

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PVBC chains on the microsphere surfaces were used conveniently as macroinitiators for the surface-initiated ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA). 2. Experimental Section 2.1. Materials. The monomer, 4-vinylbenzyl chloride (VBC, 90+%), the cross-linking agent, ethylene glycol dimethacrylate (EGDMA, 98%), and the stabilizer, poly(vinyl alcohol) (PVA, 87-89% hydrolyzed, Mw ) 125 000), were obtained from Aldrich Chemical Co. (Milwaukee, WI). 1-Bromohexane (98%) was obtained from Avocado Research Chemical Ltd. (Lancashire, UK), 1-bromododecane (97%) was obtained from Fluka Chemical Co. (St. Louis, MO), and N,N-dimethylformamide (DMF, analytical reagent) and tetrahydrofuran (THF, analytical reagent) were obtained from Fisher Scientific Co. (Leics, UK). 2-(Dimethylamino)ethyl methacrylate (DMAEMA, 98%) was obtained from Aldrich Chemical Co., passed through an inhibitor-removing column (Aldrich Chemical Co.), and then stored under an argon atmosphere at -10 °C. The initiator R,R′-azobisisobutyronitrile (AIBN, 97%) was obtained from Kanto Chemical Co. (Tokyo, Japan) and was recrystallized in anhydrous ethanol. Copper(II) bromide (CuBr2, 99+%) from Aldrich Chemical Co. was dissolved in the deionized water, filtered, condensed in vacuo at 30 °C, crystallized in a vacuum oven with P2O5, filtered, and dried. 2,2′-Bipyridyl (bpy, 99+%) was supplied by Aldrich Chemical Co. and was recrystallized in acetone. Copper(I) chloride (CuCl, 98+%) was supplied by Aldrich Chemical Co. and was dissolved in hydrochloric acid, precipitated into a large amount of deionized water, filtered, washed with anhydrous ethanol, and then dried in vacuo. Escherichia coli (ATCC DH5R), or E. coli, and Staphylococcus aureus (Newman), or S. aureus, were obtained from American Type Culture Collection. All other solvents (reagent or HPLC grade) were obtained from Fisher Scientific Co. 2.2. Preparation of PVBC Microspheres. The cross-linked PVBC microspheres were prepared by suspension polymerization.49 The details are as follows: VBC (5.0 mL, 31.9 mmol), EGDMA (1.5 mL, 7.8 mmol), and AIBN (0.12 g, 0.71 mmol) were dissolved in heptane (7.2 mL). The resulting solution was dispersed in an aqueous medium, prepared by dissolution of PVA (0.25 g) in water (80 mL). The polymerization was carried out in a magnetically stirred glass flask (100 mL) at 78 °C for 8 h. The stirring rate was kept constant at 700 rpm during the polymerization process. After polymerization, the PVBC microspheres were washed exhaustively with ethanol, and then with water, to remove the diluent and unreacted monomer. They were subsequently dried in vacuo at 50 °C. The microspheres were sieved and a proper size fraction (500-600 µm in diameter) was isolated. 2.3. Surface-Initiated ATRP of DMAEMA on PVBC Microspheres and Quaternization by Alkyl Bromide. Surface-initiated ATRP of DMAEMA on PVBC microspheres was accomplished in a magnetically stirred glass flask (100 mL) by immersing the microspheres (1.2 g) into a reaction mixture containing 6.0 mL (34.9 mmol) of DMAEMA, 9.8 mg (0.1 mmol) of CuCl, 2.2 mg (0.01 mmol) of CuBr2, 46 mg (0.29 mmol) of bpy, and 10 mL of DMF. The suspension was purged with argon for approximately 20 min to remove the dissolved oxygen. The flask was then sealed. Polymer-

ization was carried out at 100 °C for 36 h. The stirring rate was kept constant at 100 rpm during polymerization. At the end of the polymerization reaction, the microspheres were subjected to exhaustive washing, first with THF, then with THF:water (1:1, v/v) mixture, and finally with water. They were dried in vacuo at 50 °C prior to being exposed to 6 mL of 1-bromododecane or 1-bromohexane for the quaternization reaction. After stirring in the alkyl halides at 70 °C for 48 h, the microspheres were rinsed with THF, water, and methanol, in that order, and dried in vacuo at 50 °C for 48 h. 2.4. Characterizations. Surface compositions were measured by X-ray photoelectron spectroscopy (XPS) on an AXIS HSi spectrometer (Kratos Analytical Ltd., Manchester, Lancashire, UK) with a monchormatized Al KR X-ray source (1486.6 eV photons) at a constant dwell time of 100 ms and a pass energy of 40 eV. The anode voltage was 15 kV, and the anode current was 10 mA. The pressure in the analysis chamber was maintained at 7.0 × 10-6 Pa or lower during each measurement. The substrates were mounted on the standard sample studs by means of double-sided adhesive tapes. The core-level signals were obtained at a photoelectron takeoff angle of 90° (with respect to the sample surface). All binding energies (BEs) were referenced to the C 1s hydrocarbon peak at 284.6 eV. In peak synthesis, the line width (full-width at half-maximum) of the Gaussian peaks was maintained constant for all components in a particular spectrum. Surface elemental stoichiometries were determined from XPS spectral area ratios and were reliable to within (5%. The elemental sensitivity factors were calibrated using stable binary compounds of well-established stoichiometries. 2.5. Determination of the Antibacterial Effect of PVBC Microspheres. E. coli, a Gram-negative bacteria, was cultivated in 50 mL of a 3.1% yeast-dextrose broth (containing 10 g/L peptone, 8 g/L beef extract, 5 g/L sodium chloride, 5 g/L glucose, and 3 g/L yeast extract at a pH of 6.8)50 at 37 °C. All glassware and polymer samples were sterilized in an autoclave at 120 °C for 20 min or with UV irradiation before the experiments. The E. coli containing broth was centrifuged at 2700 rpm for 10 min. After removal of the supernatant, the cells were washed twice with a sterile phosphate buffer solution, PBS (containing 5.4 g of sodium dihydrogen phosphate monohydrate and 8.66 g of anhydrous disodium hydrogen phosphate in 1 L of distilled water, adjusted to pH 7.0), and resuspended in PBS at a concentration of 105 cells/mL. Thirteen millgrams of the PVBC microspheres were introduced into 20 mL of this suspension in an Erlenmeyer flask. The flask was shaken at 200 rpm at 37 °C. The viable cell counts of E. coli were carried out using a surface spread-plate method. After a predetermined period of time, 1 mL of the bacteria culture was taken from the flask, and decimal serial dilutions with PBS were repeated with each initial sample. A drop of 0.1 mL of the diluted sample was then spread onto a triplicate solid growth agar plate. After incubation of the plates at 37 °C for 24 h, the number of viable cells (colonies) was counted manually and the results, after multiplication with the dilution factor, were expressed as mean colony forming units per milliliter. For the Gram-positive bacteria, S. aureus, the same assay procedures were used as those described above for E. coli.

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Figure 1. Schematic diagram illustrating the process for preparing microspheres with permanent bactericidal surfaces.

3. Results and Discussion 3.1. Surface Modification. Figure 1 outlines the synthetic pathway for the preparation of PVBC microspheres with antibacterial surfaces. The process involved (i) synthesis of cross-linked PVBC microspheres via suspension polymerization (PVBC-1), (ii) modification of the microsphere surfaces via covalent grafting of well-defined PDMAEMA brushes from surface-initiated ATRP of DMAEMA (PVBC-2), and (iii) quaternization of the PDMAEMA brushes with 1-bromododecane and 1-bromohexane (PVBC-3 (R ) C12H25) and PVBC-3 (R ) C6H13), respectively). It is noted that the benzyl chloride group serves as the active ATRP initiator on the microsphere surface to produce the well-defined (nearly monodisperse)34 DMAEMA polymer brushes. Thus, ATRP of DMAEMA was carried out directly on the microsphere surface of PVBC-1. CuCl/CuBr2 and the bpy ligand served as the catalyst system in the ATRP reaction, and DMF was used as the solvent. After exhaustive washing, the PVBC-2 microspheres were quaternized with alkyl bromides (1-bromododecane or 1-bromohexane) to obtain the antibacterial PVBC-3 microspheres. The morphology of the PVBC microspheres prior to and after surface functionalization, as revealed by optical microscopy, is shown in Figure 2. Thus, the surface functionalization process did not result in any significant changes in the morphology of microspheres. The chemical composition of the microsphere surfaces at various stages of surface modification was determined by XPS. Figure 3 shows the XPS wide scan spectra of the PVBC-1, PVBC-2, PVBC-3 (R ) C12H25), and PVBC-3 (R ) C6H13) microspheres. The O ls, C ls, and Cl 2p peak components are discernible in the wide scan spectrum of PVBC-1 in Figure 3a. The appearance of a strong N 1s signal at the binding energy (BE) of about 399.1 eV is consistent with the presence of tertiary amines associated with the grafted DMAEMA polymer (PDMAEMA) on the PVBC-2 microspheres (Figure 3b). The appearance of the Br 3d spectrum after quaternization of the grafted PDMAEMA brushes is consistent with

Figure 2. Optical microscopy images of (a) PVBC-1 and (b) PVBC-3 (R ) C12H25).

the incorporation of the alkyl bromide chain on the PDMAEMA brushes (Figure 3c,d). Figure 4a shows the C 1s core-level spectrum of the PVBC-1 surface. The peak components at the BEs of about 284.6, 286.3, and 288.6 eV are attributable to the

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Figure 3. XPS wide scan spectra of (a) PVBC-1, (b) PBVC-2, (c) PVBC-3 (R ) C12H25), and (d) PVBC-3 (R ) C6H13). The main peak components observed are Cl 2p (BE ∼200 eV), C 1s (BE ∼285 eV), O 1s (BE ∼532 eV), Br 3d (BE ∼68 eV), and N 1s (BE ∼399 eV).

Figure 4. (a, b) C 1s and Cl 2p core-level spectra of the PVBC-1 surface and (c, d) C 1s and N 1s core-level spectra of the PVBC-2 surface.

C-H/C-C, C-O/C-Cl, and OdC-O species,51,52 respectively. Figure 4b shows the Cl 2p core-level spectrum of the PVBC-1 surface. The Cl 2p core-level spectrum consists of the Cl 2p3/2 and Cl 2p1/2 doublet at the BEs of about 200.0 and 201.8 eV, respectively,51 attributable to the covalently bonded chlorine (C-Cl) species. The presence of a weak OdC-O component in the curve-fitted C 1s core-level spectrum is consistent with the presence of EGDMA cross-linking agent in the microsphere. A [Cl]:[OdC-O] ratio of about 1.6:1 suggests a [VBC]:[EGDMA] ratio of about 3.2:1 on the microsphere surface. The VBC units on the cross-linked PVBC-1 microsphere surface can serve as initiators for the surfaceinitiated ATRP (Figure 1). ATRP of DMAEMA on PVBC-1 surfaceswas carried out in DMF at 100 °C, using a [DMAEMA]:[CuCl]:[CuBr2]:[bpy] ratio of 200: 1:0.1:3 to obtain the microspheres with well-defined PDMAEMA brushes on the surfaces (PVBC-2 microspheres). Figure 4 shows the C 1s (part c) and N 1s (part d) core-level spectra of the PVBC-2 surface. The five peak components at the BEs of about 284.6, 285.6, 286.0, 286.3, and 288.5 eV are attributable to the C-H/ C-C, C-N, C-N+, C-O/C-Cl, and OdC-O species,51,52 respectively. The [C-H/C-C]/[C-N/C-N+]/[C-O/CCl]/[OdC-O] ratio, as determined from the curve-fitted C 1s core-level spectrum, is about 3.6:2.6:1.0:0.9, in

Figure 5. C 1s, Br 3d, and N 1s core-level spectra of (a-c) PVBC-3 (R ) C12H25) surface and (d-f) PVBC-3 (R ) C6H13) surface.

reasonable agreement with the theoretical ratio of 3:3: 1:1 for PDMAEMA. However, the slightly higher than expected [C-H/C-C] surface concentration suggests a contribution of the underlying PVBC to the concentration of these carbon species. Thus, the thickness of the PDMAEMA layer grown on the PVBC-1 microsphere surface is on the order of the probing depth of the XPS technique (about 8 nm in an organic matrix53). The N 1s core-level spectrum (Figure 4d) shows two peak components at the BEs of about 399.1 and 401.6 eV, attributed to the C-N and C-N+ species, respectively. It is noted that neutral PDMAEMA should exhibit only one N 1s peak component at the BE of 399.1 eV. The appearance of the C-N+ species suggests (i) partial quaternization of PDMAEMA by the ω-chloride of the main chain of PDMAEMA from the ATRP process54-56 and (ii) amination of the PVBC-1 microspheres with the DMF solvent.57 The [C-N]/[C-N+] ratio of 3.8:1, as determined from the curve-fitted N 1s core-level spectrum (Figure 4d), is consistent with the [C-N]/[C-N+] ratio of 4:1, as determined from the curve-fitted C 1s core-level spectrum (Figure 4c). The partial quaternization process is also consistent with the appearance of an anionic chloride species in the Cl 2p core-level spectrum (inset of Figure 4d) of the PVBC-2 surface. Thus, the ATRP of DMAEMA has been successfully accomplished on the microsphere surfaces, using the VBC units of PVBC as the macroinitiators. Subsequently, the PVBC-2 surfaces were quaternized with 1-bromododecane or 1-bromohexane to obtain the surface-functionalized antibacterial microspheres (PVBC3). Parts a, b, and c of Figure 5 show the respective C 1s, Br 3d, and N 1s core-level spectra of the PVBC-3 (R ) C12H25) surface. The corresponding C 1s, Br 3d, and N 1s core-level spectra of the PVBC-3 (R ) C6H13) microspheres are shown in Figure 5d-f. The four C 1s peak components in Figure 5a at the BEs of about 284.6, 286.0, 286.3, and 288.6 eV are attributable to the C-H/ C-C, C-N+, C-O/C-Cl, and OdC-O species, respectively. The [C-H/C-C]/[C-N+]/[C-O/C-Cl]/[OdC-O] ratio, as determined from the curve-fitted C 1s corelevel spectrum, is about 13.6:4.2:1.1:1.0, in fairly good agreement with the theoretical ratio of 14:4:1:1 for PDMAEMA quaternized with 1-bromododecane (PVBC-3 (R ) C12H25)). Similarly, the BEs at about 284.6, 286.0, 286.3, and 288.6 eV in Figure 5d are attributed, respectively, to the C-H/C-C, C-N+, C-O/C-Cl, and OdC-O species of PDMAEMA quaternized with 1-bromohexane (PVBC-3 (R ) C6H13)). The [C-H/C-C]/

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Figure 6. Viable E. coli cell number as a function of time in contact with different microspheres: PVBC-1, PVBC-3 (R ) C6H13), and PVBC-3 (R ) C12H25). The cell number was determined by the surface spread method.

Figure 7. Viable S. aureus cell number as a function of time in contact with different microspheres: PVBC-1 and PVBC-3 (R ) C12H25). The cell number was determined by the surface spread method.

[C-N+]/[C-O/C-Cl]/[OdC-O] ratio of 8.4:4.2:1.2:1.0 again is in fairly good agreement with the theoretical ratio of 8:4:1:1. The Br 3d core-level spectra consist of the Br 3d5/2 and Br 3d3/2 doublet at the respective BEs of about 67.4 and 68.5 eV for both the PVBC-3 (R ) C12H25) and PVBC-3 (R ) C6H13) surfaces, attributable to the ionic Br (Br-) species.9,58 On the other hand, the N 1s core-level spectra of the two quaternization surfaces show predominantly a single peak component at the BE of about 401.9 eV (Figure 5c,f), attributable to the C-N+ species. It is noted that the neutral C-N peak component of PVBC-2 at the BE of 399.1 eV has disappeared almost completely after quaternization with the alkyl bromides (1-bromododecane or 1-bromohexane), indicating complete quaternization of the PDMAEMA brushes on the PVBC-2 microsphere surface. 3.2. Antibacterial Effect of Functionalized PVBC Microspheres. The antibacterial properties of 1-bromododecane and 1-bromohexane quaternized PDMAEMA brushes on the PVBC-3 microspheres were evaluated. The antibacterial effect of the functionalized microspheres was investigated by contacting the microspheres with viable cells in suspension. In each antibacterial experiment, 150 microspheres with an average diameter of 550 µm (∼1.4 cm2 in total surface area) were used and were incubated with 20 mL of the bacterial suspension. The antimicrobial activity experiments were first conducted with suspensions of Gram-negative bacteria (E. coli), containing 1 × 105 cells/mL. The results are shown in Figure 6. For the pristine PVBC microspheres (PVBC-1), the number of viable cells in the suspension decreases by 40% or less after 120 min. This relatively low reduction may have resulted from natural apoptosis. However, in the presence of PVBC-3 (R ) C6H13) and PVBC-3 (R ) C12H25) microspheres, the viable cell number decreases by 1 and 3 orders of magnitude, respectively, after 15 min. Thus, bactericidal efficiencies of 90% and 99.9% have been achieved, respectively, with the two types of microspheres. All bacteria have been eliminated by PVBC-3 (R ) C12H25) and PVBC-3 (R ) C6H13) microspheres after 30 and 60 min, respectively. The potency of the hydrophobic chain containing the quaternary group has been most frequently studied in antibacterial effect. It has been found that C10 and C12 chains were most effective for S. aureus and P. aeruginosa.59 A similar relationship between the alkyl chain length and the antibacterial activity against E. coli and B. subtilis has also been reported.60 Thus, the hydrophobic portion of the molecule plays a significant role in determining the antibacterial effect of the polycation. For the present microspheres, the bactericidal efficiency

of PVBC-3 (R ) C12H25) is significantly higher than that of PVBC-3 (R ) C6H13). The results are thus consistent with those reported earlier.59,60 The antimicrobial action probably arises from the ability of the hydrophobic chains to increase cell permeability and to disrupt the cell membranes.13,15,25,26,61 The antimicrobial activity experiments were also carried out on suspensions of Gram-positive bacteria, S. aureus, in cultures containing 1 × 105 cells/mL. Figure 7 shows the results on viable S. aureus cell number vs contact time with PVBC-1 and PVBC-3 (R ) C12H25) microspheres. It can be seen that the PVBC-3 (R ) C12H25) microspheres also have a higher bactericidal efficiency for S. aureus, reaching 99.0% and 99.99% after 15 and 30 min, respectively. However, by comparison of the data in Figures 6 and 7, the bactericidal efficiency of the PVBC-3 (R ) C12H25) microspheres for S. aureus is lower than that of the corresponding microspheres for E. coli. Based on the fact that the functionalized microspheres terminate both Gram-negative and Gram-positive bacteria, it is reasonable to expect that many bacterial species will be susceptible.8 The permanence of the antimicrobial activity was investigated by repeated washing of the used microspheres with alcohol to recover the original surface, followed by renewed exposure of the PVBC-3 microspheres to a new bacteria (E. coli) culture. The antimicrobial capability has been restored almost to its original level. It is likely that materials from the dead cells accumulate on the surface of the microsphere via hydrophobic interactions. The materials were removed by the solvent, with the concomitant restoration of the antimicrobial activity of the microsphere surface. 4. Conclusions A simple three-step process for synthesizing microspheres with well-structured and permanent bactericidal surfaces was demonstrated. The process involved (i) synthesis of cross-linked PVBC microspheres via suspension polymerization, (ii) modification of the microsphere surfaces via covalent bonding of well-defined PDMAEMA brushes from surface-initiated ATRP of DMAEMA, and (iii) quaternization of PDMAEMA brushes by alkyl bromides (1-bromododecane or 1-bromohexane). The assays with E. coli and S. aureus showed that the polycationic chains introduced on the microsphere surface via quaternization of the tertiary amine groups with 1-bromodedocane and 1-bromohexane possessed desirable bactericidal activity. It was found that about 1.4 cm2 of the functionalized microsphere surfaces was sufficient to terminate 105 bacteria

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within a few minutes. The surface functionality of the microspheres can be regenerated via a simple solvent washing process. The present microspheres with robust and well-defined surface structure and functionality via controlled suspension polymerization and surface-initiated ATRP can serve as prototypes in industrial process applications, for example, as effective packing in antiseptic columns and devices. Literature Cited (1) Vigo, T. L. Antimicrobial Polymers and Fibers: Retrospective and Prospective. ACS Symp. Ser. 2001, 792 (Bioactive Fibers and Polymers), 175. (2) Uyama, Y.; Kato, K.; Ikada, Y. Surface Modification of Polymers by Grafting. Adv. Polym. Sci. 1998, 137, 1. (3) Motornov, M.; Minko, S.; Eichhorn, K. J.; Nitschke, M.; Simon, F.; Stamm, M. Reversible Tuning of Wetting Behavior of Polymer Surface with Responsive Polymer Brushes. Langmuir 2003, 19 (19), 8077. (4) Minko, S.; Mu¨ller, M.; Motornov, M.; Nitschke, M.; Grundke, K.; Stamm, M. Two-Level Structured Self-Adaptive Surfaces with Reversibly Tunable Properties. J. Am. Chem. Soc. 2003, 125 (13), 3896. (5) Groll, J.; Amirgoulova, E. V.; Ameringer, T.; Heyes, C. D.; Ro¨cker, C.; Nienhaus, G. U.; Mo¨ller, M. Biofunctionalized, Ultrathin Coatings of Cross-Linked Star-Shaped Poly(ethylene oxide) Allow Reversible Folding of Immobilized Proteins. J. Am. Chem. Soc. 2004, 126 (13), 4234. (6) McGurk, S. L.; Green, R. J.; Sanders, G. H. W.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Molecular Interactions of Biomolecules with Surface-Engineered Interfaces Using Atomic Force Microscopy and Surface Plasmon Resonance. Langmuir 1999, 15 (15), 5136. (7) Bergbreiter, D. E.; Crooks, R. M.; Bruening, M. L.; Zhou, Y.; Liu, Y.; Aguilar, G.; Zhao, M. Thin Hyperbranched Films Grafted to Gold, Silicon, and Aluminum. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1997, 38 (1), 943. (8) Lee, S. B.; Koepsel, R. R.; Morley, S. W.; Matyjaszewski, K.; Sun, Y.; Russell, A. J. Permanent, Nonleaching Antibacterial Surfaces. 1. Synthesis by Atom Transfer Radical Polymerization. Biomacromolecules 2004, 5 (3), 877. (9) Cen, L.; Neoh, K. G.; Kang, E. T. Surface Functionalization Technique for Conferring Antibacterial Properties to Polymeric and Cellulosic Surfaces. Langmuir 2003, 19 (24), 10295. (10) Kanazawa, A.; Ikeda, T.; Endo, T. Novel Polycationic Biocides: Synthesis and Antibacterial Activity of Polymeric Phosphonium Salts. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 335. (11) Nakagawa, Y.; Tawaratani, T.; Kourai, H.; Horie, T.; Shibasaki, I. Adsorption of Escherichia coli onto Insolubilized Lauryl Pyridinium Iodide and its Bacteriostatic Action. Appl. Environ. Microbiol. 1984, 47 (1), 88. (12) Kawabata, N.; Hayashi, T.; Matsumoto, T. Removal of Bacteria from Water by Adhesion to Cross-Linked Poly(vinylpyridinium halide). Appl. Environ. Microbiol. 1983, 46, 203. (13) Chen, C. Z.; Beck-Tan, N. C.; Dhurjati, P.; Dyk, T. K. v.; LaRossa, R. A.; Cooper, S. L. Quaternary Ammonium Functionalized Poly(propylene imine) Dendrimers as Effective Antimicrobials: Structure-Activity Studies. Biomacromolecules 2000, 1, 473. (14) Chen, C. Z.; Cooper, S. L. Interactions Between Dendrimer Biocides and Bacterial Membranes. Biomaterials 2002, 23, 3359. (15) Lin, J.; Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Insights into Bactericidal Action of Surface-Attached Poly(vinylN-hexylpyridinium) Chains. Biotechnol. Lett. 2002, 24, 801. (16) Lin, J.; Murthy, S. K.; Olsen, B. D.; Gleason, K. K.; Klibanov, A. M. Making Thin Polymeric Materials, Including Fabrics, Microbicidal and also Water-Repellent. Biotechnol. Lett. 2003, 25, 1661. (17) Kim, J. Y.; Lee, J. K.; Lee, T. S.; Park, W. H. Synthesis of Chitooligosaccharide Derivative with Quaternary Ammonium Group and its Antimicrobial Activity against Streptococcus Mutans. Int. J. Biol. Macromol. 2003, 32, 23. (18) Thorsteinsson, T.; Ma´sson, M.; Kristinsson, K. G.; Hja´lmarsdo´ttir, M. A.; Hilmarsson, H.; Loftsson, T. Soft Antimicrobial Agents: Synthesis and Activity of Labile Environmentally Friendly

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Received for review February 22, 2005 Revised manuscript received June 11, 2005 Accepted June 18, 2005 IE050225O