Biomacromolecules 2004, 5, 877-882
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Permanent, Nonleaching Antibacterial Surfaces. 1. Synthesis by Atom Transfer Radical Polymerization Sang Beom Lee,† Richard R. Koepsel,‡ Scott W. Morley,† Krzysztof Matyjaszewski,| Yujie Sun,§ and Alan J. Russell*,†,‡,⊥ Department of Bioengineering, Department of Chemical and Petroleum Engineering, and Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, Department of Surgery, McGowan Institute for Regenerative Medicine, Suite 200, 100 Technology Drive, Pittsburgh, Pennsylvania 15219, and Center for Macromolecular Engineering, Department of Chemistry, 4400 Fifth Avenue, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 Received September 12, 2003; Revised Manuscript Received January 23, 2004
We have grown an antimicrobial polymer directly on the surfaces of glass and paper using atom transfer radical polymerization (ATRP). The method described here results in potentially permanent nonleaching antibacterial surfaces without the need to chemically graft the antimicrobial material to the substratum. The tertiary amine 2-(dimethylamino)ethyl methacrylate was polymerized directly onto Whatman #1 filter paper or glass slides via atom transfer radical polymerization. Following the polymerization, the tertiary amino groups were quaternized using an alkyl halide to produce a large concentration of quaternary ammonium groups on the polymer-modified surfaces. Incubating the modified materials with either Escherichia coli or Bacillus subtilis demonstrated that the modified surfaces had substantial antimicrobial capacity. The permanence of the antimicrobial activity was demonstrated through repeated use of a modified glass without significant loss of activity. Quaternary amines are believed to cause cell death by disrupting cell membranes allowing release of the intracellular contents. Atomic force microscopic imaging of cells on modified glass surfaces supports this hypothesis. Introduction A large number of applications can be found for selfsterilizing surfaces. The facile production of such surface materials has been a goal of a number of research groups for many decades. A variety of techniques have resulted in surface treatments that are more or less effective. The vast majority of the previous methods for production of antimicrobial surfaces have either applied antimicrobial compounds to surfaces or mixed antimicrobials with coating materials.1-13 In this report, we demonstrate a new method for the generation of antimicrobial surfaces in which an antimicrobial material is polymerized directly on the target surface using atom transfer radical polymerization (ATRP). The mechanism by which an antimicrobial acts determines how it can be used in surface treatments. Most conventional antimicrobials act by diffusing into the cell and disrupting essential cell functions. To use this type of compound for surface treatment, the antimicrobial must be released from the surface matrix. These leachable biocides are typically compositions containing antibiotics, phenols, iodine, quaternary ammonium compounds, or heavy metals such as silver, tin, and mercury.1-4 The fact that the antimicrobial is * To whom correspondence should be addressed. † Department of Bioengineering, University of Pittsburgh. ‡ Department of Chemical and Petroleum Engineering, University of Pittsburgh. § Department of Chemistry, University of Pittsburgh. | Carnegie Mellon University. ⊥ McGowan Institute for Regenerative Medicine.
free to leave the surface has serious adverse effects on the durability and useful life of the treated material, though it does not appear to impact marketability. Another potentially serious problem with leaching-based technologies is that the compounds released into the environment at sub lethal concentrations have the effect of increasing drug resistance throughout the microbial realm. A type of renewable releasing surface coating for fabrics and other surfaces has been described that releases halide ions that act as antimicrobial agents.5 The antimicrobial capacity of these compounds can be renewed through a bleach treatment. These materials are effective at killing bacteria but need to be regenerated in order to maintain activity. The more unconventional route to the production of surface-active antimicrobial materials is where the bound antimicrobial stays affixed to the surface through covalent interactions.6-11 A number of surface active compounds have been tested in attempts to address the deficiencies inherent in leaching type antimicrobial surfaces. Predominant among these is the chemical bonding of active antimicrobial substances to various surfaces. Tiller et al. have described methods for treating flat surfaces such as glass, high-density polyethylene (HDPE), low-density polyethylene (LDPE), polyprolylene (PP), and nylon, poly(ethylene terephthalate) with poly(4-vinyl-pyridine) modified with pendant quaternary ammonium salts.9-11 The antibacterial properties of these materials were assessed by spraying aqueous suspensions of bacterial cells on the surfaces and counting the survivors.
10.1021/bm034352k CCC: $27.50 © 2004 American Chemical Society Published on Web 02/27/2004
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Interestingly, when the applied polymer was a polyquaternary amine, it was able to kill bacteria that were resistant to other types of cationic antimicrobials.11 This same group has also demonstrated the utility of immobilizing antimicrobial polymers on fabrics12,13 and polyolefins.13 The active material for all of the above biocidal surfaces was synthesized by either classical free radical polymerization or simple coupling reactions and then applied to an activated surface. These types of reactions fail to strictly control the monomer distribution, polydispersity, molecular weight, polymer topology, and density of functional groups in a way that will allow rational modification of the polymer for increased anti-microbial activity. The control of polymer compositions, architectures, and functionalities for the development of materials with biological properties has long been of interest in polymer chemistry. ATRP is one kind of controlled/living radical polymerization process (CRP) that provides a new and versatile method for the synthesis of polymers with controlled molecular weights and low polydispersities.14-17 In general, CRP processes provide compositionally homogeneous well-defined polymers (with predictable molecular weights, narrow molecular weight distribution, and high degree of end-functionalization) and have been the subject of recent review articles.15,16 Our laboratories have begun an exhaustive study of how ATRP can be used to make bioactive polymers.18 Polymeric materials made from vinyl monomers containing primary, secondary, or tertiary amino groups including monomers such as 2-(dimethylaminoethyl methacrylate) (DMAEMA), 4-vinyl pyridine (4-VP), N-substituted acrylamides, N-acryloyl pyrrolidine, N-acryloyl piperidine, and acryl-L-amino acid amides are useful for various applications such as water-soluble polymers and coordination reagents for transition metals.19-24 Most of these vinyl monomers can be used as substrates for ATRP reactions to make either homopolymers or a variety mixed copolymers. Some monomeric compounds can easily be converted to chemical forms with known antimicrobial activity. An example of this is the facile conversion of DMAEMA to a corresponding series of quaternary amines. We predicted that combining the controlled size of long polymer chains produced by ATRP with an effective quaternary ammonium group would result in a highly effective biocidal polymer. Further, this polymer can be produced directly on a solid surface which would provide that surface with permanent antimicrobial activity. In this study, we have used ATRP as a robust mechanism for growing long chain, low polydispersity polymers on Whatman #1 filter paper and on amino glass slides using DMAEMA as a monomer. The tertiary amino group of the DMAEMA, which is pendant to the main chain of the polymer, is then easily quaternized by ethyl bromide to provide an effective biocidal functionality. Experimental Section Materials. Whatman #1 filter papers and NH2 glass slides (aminopropyltrimethoxysilane-coated microscope slides) were purchased from Sigma-Aldrich Chemical Co. DMAEMA
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was obtained from Aldrich and was passed through a column of basic alumina and then distilled prior to polymerization. CuBr, 2,2′-bipyridine (bpy), 2-bromoisobutyryl bromide, propionyl bromide, and all other reagents were commercial products and used without further purification. Immobilization of ATRP Initiator on Whatman #1 Filter Paper. A paper with an immobilized ATRP initiator was prepared by reacting 5 mL of 2-bromoisobutyryl bromide with the hydroxyl groups present on the filter paper (25 mm × 25 mm) for 24 h at room temperature. The filter paper was thereafter thoroughly washed with dichloromethane, then acetone, then water. The number of initiator sites on a given paper sample was controlled by reacting the hydroxyl groups present on the filter paper with a mixture of 2-bromoisobutyryl bromide and an increasing amount of propionyl bromide; the propionyl bromide acts as blocking agent since it does not include a functionality for ATRP initiation. In this manner one can obtain papers or NH2 glass slides with 0% to 100% ATRP initiator functionalities. ATRP of DMAEMA on Paper Modified with ATRP Initiator and Its Quaternization by Ethyl Bromide. ATRP of DMAEMA on the paper modified with ATRP initiator was accomplished by immersing the initiator-modified paper into a reaction mixture containing 5 g of DMAEMA, 0.035 g of CuBr, 0.070 g of 2,2′-bipyridine (bpy), and 5 g of 1,2dichlorobenzene. The polymerization was carried out at 80 °C for 48 h. After completion of the polymerization, the paper was subjected to intense washing, first with THF, then THF:water, and finally water, and then air-dried before being placed in a solution of 5 mL of methyl bromide in 15 mL of nitromethane. After stirring the reaction mixture at 30 °C for 24 h, the paper was rinsed with THF, methanol, and water and dried under a vacuum for 24 h. To control the polydispersity of the polymer on paper, ATRP of DMAEMA was also carried out in the presence of the sacrificial initiator, ethyl 2-bromoisobutyrate containing Cu(I)Br, bpy. Surface Analysis. Polymers, cleaved from filter papers by acid hydrolysis, were analyzed by gel permeation chromatography at 30 °C in 0.1% tetrabutylammonium bromide solution in DMF using a Waters 717 column calibrated against PMMA standards. Atomic force microscope (AFM) measurements were obtained on polymers on flat glass, using a Digital Instruments (Santa Barbara, CA) Nanoscope III in tapping mode with a 2 Hz scan rate. Polymers were grown via surface initiated polymerization on filter paper and removed by acid hydrolysis in a 1 mM HCl aqueous solution overnight. The HCl aqueous solution was removed by evaporation, and the remaining polymer was dissolved in 100 µL of DMF and analyzed by GPC. Antimicrobial Activity Determination. Antimicrobial testing was performed using a modified ASTM standard: E 2149-01 Standard Test Method for Determining the Antimicrobial ActiVity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions.25 A colony of Escherichia coli K12 or Bacillus subtilis 168 grown on a Luria agar (L-agar) plate was used to inoculate 5 mL of Luria broth in a sterile 50 mL conical tube. The culture was incubated at 37 °C while being shaken at 300 rpm (G24 Environmental
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Permanent, Nonleaching Antibacterial Surfaces Scheme 1
Scheme 2
Incubator Shaker, New Brunswick Scientific) for 18-20 h. The cells were diluted with Sorensen’s Phosphate Buffer (pH 6.8, 0.3 mM KH2PO4) to the desired concentration. The actual number of cells used for a given experiment was determined by standard serial dilution. Bacillus spores were prepared by growing a culture in M9 medium containing 1% Brain Heart Infusion overnight at 37 °C. The culture was centrifuged and the pellet resuspended in distilled water. The suspension was heated at 85 °C for 20 min, pelleted, resuspended, heated at 80 °C for 20 min, and stored at 4 °C. The preparation was about 95% spores by phase contrast microscopy. Spore samples were boiled for 2 min immediately prior to use in experiments. Polymer modified samples were weighed (paper samples) or measured for area (glass samples) and incubated with 5 mL of cell suspension in a 50 mL conical tube (Falcon) at 37 °C and 300 rpm. Samples were taken after 1 h, diluted appropriately, plated on L-agar plates and the number of viable cells was determined as colony forming units (CFU) after overnight incubation at 37 °C. Atomic Force Microscopy. AFM images were obtained using a DI Dimension 3100 AFM operating in the tapping mode. Tips were NSC15, noncontact Silicon cantilevers, and observations were made in air. Data were recorded in both the height and phase lag modes.
Table 1. Results from GPC after Hydrolysis
Results and Discussions Surface Modification. Scheme 1 outlines the synthetic pathway for the ATRP polymerzation and subsequent quaternization of DMAEMA on solid surfaces. 2-Bromoisobutyryl bromide was reacted with the hydroxyl groups of the cellulose in filter paper and the free amine groups on amino glass slides via esterification or amidation, respectively, to produce the active ATRP initiator on the surfaces. ATRP was then used to polymerize DMAEMA to the initiated surfaces. Cu(I)Br and the ligand bpy served as catalysts in the ATRP reaction, and 1,2-dichlorobenzene was used as the solvent. After washing, the materials were quaternized with ethyl bromide using nitromethane as a solvent. In some experiments, a “blocking agent” was used to synthesize filter papers with varying surfaces densities of the polymer as shown in Scheme 2. Propionyl bromide was mixed with stoichiometrically varying amounts of 2-bro-
a
percent of initiator on surface
Mn/g mol-1
PDI
100 100a 50 10
21 100 21 900 23 000 19 300
2.22 1.62 2.21 2.14
In the presence of sacrificial initiator.
moisobutyryl bromide to vary the density of active ATRP initiation sites on the paper. The propionyl bromide reacts with the hydroxyl groups found on the filter paper to produce a nonpolymerizable site. To determine the molecular weight of the polymers synthesized by this method, papers were prepared with different initiator densities, and the completed polymer chains were cleaved from the surface by HCl hydrolysis. The molecular weights and polydispersities of the cleaved polymers were determined using gel permeation chromatography (GPC). The GPC data for these experiments, presented in Table 1, show that the extent of the polymerization, and thus the length of the polymer chains, and the polydispersity index (PDI) are not greatly influenced by the number of initiation sites. Sacrificial initiators or Cu(II)Br deactivators are thought to limit the polydispersitiy of polymer grown on paper. When the grafted polymer chains, obtained in the presence of a sacrificial initiator, were cleaved from the surface by HCl hydrolysis, the PDI of polymer obtained was 1.62. This is in agreement with the prediction and indicates less termination of the chain during polymerization in the presence of the sacrificial initiator. FT-IR analysis (Figure 1) was used to demonstrate the presence of polymer grafted onto a paper surface. A carbonyl peak at 1720 cm-1 is a positive indicator for the poly(2(dimethylamino)ethyl methacrylate) (PDMAEMA). This carbonyl peak is clearly seen on the grafted paper (Figure 1D) but not on pure paper (Figure 1A) or paper modified with initiator but not subjected to ATRP (Figure 1C). However, as seen in Figure 1B, paper that was not modified with initiator but still subjected to ATRP conditions in the presence of a sacrificial initiator revealed a small carbonyl peak. This peak can be attributed to PDMAEMA that is physiosorbed to the paper. This implies that the washing procedure was not able to completely remove the polymer. Antimicrobial Activity. The ability of the modified surfaces to kill bacteria was tested for both the paper and
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Lee et al. Table 2. Antimicrobial Activity of Modified Paper and Glassa treated sampleb paper paper paper paper glass glass
organism
number of bacteria added to sample
number of bacteria remaining
E. coli E. coli B. subtilis B. subtilis spores E. coli B. subtilis
2.6 × 108 1.6 × 109 6.2 × 106 2.5 × 106 9.4 × 106 1.0 × 104
0 4.9 × 105 6.0 × 102 1.3 × 106 1.4 × 104 0
a Samples were incubated with the number of bacteria as indicated. Following incubation the number of viable cells was determined by serial dilution. b All samples tested were compared to an untreated control of the same material.
Figure 1. FT-IR of the grafted and ungrafted papers: pure paper (a); pure paper in the presence of sacrificial initiator (b); initiatormodified paper (c); and PDMAEMA-modified paper (d).
glass preparations. We used a dynamic testing protocol for these experiments because we wished to avoid issues of accessibility of the cells to the surface and the adverse effects drying has on some cells. For the paper experiments, 2.5 × 2.5 cm pieces of modified paper were shaken with 5 mL of a bacterial suspension for 1 h at 37 °C. The number of viable cells in the suspension was determined before and after incubation by dilution of the samples followed by overnight incubation on agar plates. The presumption that the antimicrobial activity was not diffusible was tested by punching paper dots from treated and untreated papers with a hole punch and placing them on the surface of an agar plate which was previously inoculated with approximately 1 × 105 E. coli. The lack of a zone of growth inhibition around any of the paper dots indicated that the material was not diffusing out of the paper. Further experiments were performed with the paper under the conditions of the shaking experiments except that there were no cells present. In these experiments some antimicrobial material was released. After repeated washing the paper no longer released measurable amounts of antimicrobial activity. The paper itself retained activity. Further experiments were done with papers that were washed sufficiently to remove this loosely bound material. Supernatants from these experiments were passed through a series of filters with defined molecular weight cutoffs. The activity was partially retained by a 100 000 mw cutoff filter and completely retained by a 50 000 mw cutoff filter. This implies that the activity removed from the paper was associated with a molecule greater than 50 000 Da. Since the material hydrolyzed from the paper (see Table 1) was no larger that 23 000 Da we believe that we are not washing loose polymer from the paper but are, rather, generating microfibers and small paper pieces with polymer attached to them. Treated glass slides did not release antimicrobial activity into the buffer when shaken under experimental conditions. The nature of the material removed from the paper is the subject of ongoing investigation, as is the effect of surface density of polymer on effectiveness.
Figure 2. Live/dead analysis of E. coli incubated with papers. Panel A. cells incubated with nonmodified paper for 15 min. Panel B. Cells incubated with modified paper for 15 min.
The initial antimicrobial activity experiments used suspensions of E. coli that contained 5 × 106 cells. In these cases, no viable cells were recovered. The concentration of cells was incrementally increased to try and determine the upper limit of the biocidal activity. As seen in Table 2, when the number of E. coli was increased to 1.6 × 109, some cells survived the treatment. Even though some cells survived, it should be noted that the paper was still extremely effective, killing a total of more than 109 cells within 1 h. Treated paper was also tested for its ability to kill spores. A suspension of B. subtilis spores was mixed with paper samples and incubated as for E. coli cells. The treatment reduced the number of viable spores by 52% in the 1 h
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Figure 3. AFM images of glass surfaces. The pictures are: E. coli cells on plain glass A and B; E. coli cells on quaternized glass C and D; and quaternized glass without added cells E and F. Two data modes are shown for each location, height mode (A, C, and E) and the phase lag mode (B, D, and F). For the height mode, orange is the base color and represents the substrate surface. Relative height progresses through the spectrum with purple indicating the tallest structure. For the phase lag mode, orange represents the largest attraction between the tip and the substrate and purple is the weakest interaction
incubation. Additional experiments using this material to kill bacterial spores is currently underway. Treated glass samples were 1.25 × 2.5 cm and were incubated with 2 mL of bacterial suspension. It should be noted that because paper is a fibrous material it has a much larger surface area than a piece of glass with the same dimensions. Because of this, a lower number of cells was incubated with the samples (Table 2). Glass samples were tested for their ability to kill E. coli and B. subtilis vegetative cells. The treated glass killed >9 × 106 E. coli and > 1 × 104 B. subtilis. While it is beyond the scope of this study, it will be interesting to determine the range of organisms that can be killed by these surfaces. Based on the fact that the treated materials killed both gram negative and gram positive bacteria, it is reasonable to expect that many bacterial species will be susceptible. To confirm that the bacteria were indeed killed and not just bound to the surface, we used the LIVE/DEAD BacLight kit from Molecular Probes to differentiate living versus killed bacteria. E. coli cells were incubated with modified and nonmodified papers as described above except that samples were taken at various times and the cells were stained and viewed with a fluorescence microscope. Figure 2 shows the
results of this experiment after 15 min incubation. The kit contains two fluorescent dyes that stain the bacteria such that live cells are stained green and dead cells are stained red. The figure shows that after 15 min the majority of cells are dead when incubated with the modified paper but are still alive with the nonmodified paper. This result fits well with time course experiments that show the majority of cells are dead within 15 min of incubation with the treated paper (data not shown). The permanence of the antimicrobial activity was tested by repeated washing followed by a challenge with bacteria. We used glass slides to test the reusability of the modified surfaces because, as noted above, the paper began to fragment after a single use. Glass slides were prepared and incubated with approximately 1 × 106 CFU of E. coli. Two sets of slides were prepared. Following incubation with E. coli, the glasses were washed with either distilled water or with a detergent solution (0.1% SDS in distilled water), dried, and retested. The intervals between test challenges varied with the entire series completed over the course of 4 weeks. Interestingly, when the glass was washed with pure water, the antimicrobial activity disappears after two rounds of exposure to bacteria. Glass washed with SDS retains its antimicrobial activity. Washing the inactivated glass with
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detergent resulted in the antimicrobial capacity returning to its original level. It is likely that material from the dead cells accumulates on the surface of the glass through a hydrophobic interaction. That material is then removed by the detergent with the concomitant restoration of the antimicrobial activity of the surface. This result agrees with the notion that the mechanism of action of quaternary ammonium involves disruption of the plasma membrane causing the release of intracellular material. To further assess the mechanism of cell death, atomic force microscopy (AFM) was used to probe treated and untreated samples before and after exposure to bacteria (Figure 3). E. coli was imaged on unmodified glass (Figure 3A,B) and quaternized glass (Figure 3C,D). These images were then compared to a sample of quaternized glass that had not been exposed to any bacteria (Figure 3E,F). When the height mode images are compared, it is clear that some material has accumulated on the quaternized glass incubated with E. coli (compare E with A and C). The predominant background color of the image is yellow with green spots indicating material is covering the surface. The phase lag mode images indicate that plain glass and quaternized glass have a similar strong attraction to the tip (yellows and greens as predominant background colors in B and F). Bacteria have a smaller attraction to the tip than the glass surfaces showing up as blue to purple spots (compare B and D with their corresponding height mode images A and C). Perhaps the most interesting result comes from comparison of C and D. The green spots in height mode that indicate the presence of material on the surface have become purple spots in phase lag mode. The fact that the material deposited on the surface has the same lack of attraction to the tip as the intact bacteria suggests that this material is of bacterial origin. We believe that the interaction of the bacteria with the quaternized surface has an adverse effect on the integrity of the bacteria causing components of the cell to spread out onto the surface. It is not yet established whether the polymer must pierce the membrane to elicit its effect, but with ATRP, we will be able to probe whether these quaternary ammoniums function by disrupting membranes through association or through direct penetration. A number of natural polycationic compounds such as the antibiotic polymyxin and several small antimicrobial peptides are thought to work by displacing the divalent cations that are thought crucial to the organization of the lipopolysaccharide of bacterial cell walls.26 This type of activity would hold true for the compounds described here and by others.5-13 Additionally, these amphiphilic oligo-cations are often described as penetrating the plasma membrane thereby causing leakage of the intracellular contents. The action of polycationic compounds in Gram-positive bacteria, which have a thick cell wall, is thought to be similar in that the oligo-cation first penetrates the outer cell wall and finally reaches and disrupts the plasma membrane causing leakage.9 We argue that the polymers synthesized in this study exhibit this type of membrane-penetrating mechanism and that the debris seen on the quaternized glass slides (Figure 3D) is intracellular components which leaked out of the cell during its death.
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Conclusion In conclusion, we have successfully developed a method for producing an antimicrobial polymer using ATRP which can be synthesized on a number of common materials including glass and paper. Using ATRP to perform a living radical polymerization of DMAEMA gives polymer chains of controlled molecular weights and low polydispersities. Subsequent quaternization of the amino group provides the biocidal functionality using the polymer chain as a delivery mechanism. When the substratum was paper, a 6.25 cm2 piece was sufficient to kill 109 bacteria in a few minutes. Furthermore, progress has been made in understanding the mechanism of action of this polymer and similar cationic substances using atomic force microscopy. The possible uses of a permanent, nonleaching biocidal surface treatment such as the one described here would include treatment of food packaging, everyday household items, as well as military applications. Acknowledgment. This work was supported by the DoD Multidisciplinary University Research Initiative (MURI) (DAAD19-01-1-0619) program administered by the Army Research Office. References and Notes (1) Golubovich, V. N.; Rabotnova, I. L. Microbiology 1974, 43, 948. (2) Nohr, R. S.; Macdonald, G. J. J. Biomater., Sci. Polym. Ed. 1994, 5, 607. (3) Shearer, A. E.; Paik, J. S.; Hoover, D. G.; Haynie, S. L.; Kelley, M. J. Biotechnol. Bioeng. 2000, 67, 141. (4) Klueh, U.; Wagner, V.; Kelly, S.; Johnson, A.; Bryers, J. D. J. Biomed. Mater. Res., Appl. Biomater. 2000, 53, 621. (5) Sun, G.; Xu, X.; Bickett, J. R.; Williams, J. F. Ind. Eng. Chem. Res. 2001, 40, 1016. (6) Lin, J.; Qiu, S.; Lewis, K.; Klibanov, A. M. Biotechnol. Prog. 2002, 18, 1082-1086. (7) Abel, T.; Cohen, J. I.; Engel, R.; Filshtinskaya, M.; Melkonian, A.; Melkonian, K. Carbohydr. Res. 2002, 337, 2495. (8) Chen, Y.; Worley, S. D.; Kim, J.; Wei, C.-I.; Chen, T.-Y.; Santiago, J. I.; Williams, J. F.; Sun, G. Ind. Eng. Chem. Res. 2003, 42, 280. (9) Tiller, J. C.; Liao, C.-J.; Lewis, K.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5981. (10) Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2002, 79, 465. (11) Lin, J.; Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Biotechnol. Lett. 2002, 24, 801. (12) Lin, J.; Qiu, S.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2003, 83, 168. (13) Lin, J.; Murthy, S. K.; Olsen, B. D.; Gleason, K. K.; Klibanov, A. M. Biotechnol. Lett. 2003, 25, 1661. (14) Wang, J.-S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614. (15) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921. (16) Kamigaito, M.; Ando, T.; Sawamoto, M. Chem. ReV. 2001, 101, 3689. (17) Carlmark, A.; Malmstro¨m, E. J. Am. Chem. Soc. 2002, 124, 900. (18) Lee, S. B.; Russell, A. J.; Matyjaszewski, K. Biomacromolecules 2003, 4, 1386. (19) Matyjaszewski, K. Chem. Eur. J. 1999, 5, 3095. (20) Patten, T. E.; Matyjaszewski, K. AdV. Mater. 1998, 10, 901. (21) Coessens, V.; Pintauer, T.; Matyjaszewski, K. Prog. Polym. Sci. 2001, 26, 337. (22) Teodorescu, M.; Matyjaszewski, K. Macromol. Rapid Comm. 2000, 21, 190. (23) Teodorescu, M.; Matyjaszewski, K. Macromolecules 1999, 32, 4826. (24) Zhang, X.; Matyjaszewski, K. Macromolecules 1999, 32, 1763. (25) E2149-01: Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions, in Annual Book of ASTM Standard 2002; International, A., Ed.; West Conshohocken, PA, 2002; pp 15971600. (26) Varra, M. Microbiol. ReV. 1992, 56, 395.
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