Langmuir 2008, 24, 2051-2056
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Antibacterial Properties of Novel Poly(methyl methacrylate) Nanofiber Containing Silver Nanoparticles Hyeyoung Kong and Jyongsik Jang* Hyperstructured Organic Materials Research Center, School of Chemical and Biological Engineering, College of Engineering, Seoul National UniVersity, 56-1 Shinlim Kwanak, Seoul 151-742, Korea ReceiVed June 8, 2007 Poly(methyl methacrylate) (PMMA) nanofiber containing silver nanoparticles was synthesized by radical-mediated dispersion polymerization and applied to an antibacterial agent. UV-vis spectroscopic analysis indicated that the silver nanoparticles were continually released from the polymer nanofiber in aqueous solution. The antibacterial properties of silver/PMMA nanofiber against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria were evaluated using minimum inhibitory concentration (MIC), the modified Kirby-Bauer method, and a kinetic test. The MIC test demonstrated that the silver/PMMA nanofiber had enhanced antimicrobial efficacy compared to that of silver sulfadiazine and silver nitrate at the same silver concentration.
Introduction Various antimicrobial agents have been developed for curing and preventing diseases in public health hygiene and antifouling in biomedical industry.1-6 Among them, silver ions and silver nanoparticles have been recognized as an excellent antimicrobial agents because of their effective biocidal ability and nontoxicity to human cells.7-11 The possible mechanism of killing microorganisms by silver ions may be explained as follows: (1) silver ion inhibits ATP synthesis via binding to the ATP synthesis enzyme molecules in the cell wall, (2) silver ion enters the cell and binds with DNA, leading to the DNA denaturation, (3) silver ion blocks the respiratory chain of microorganisms in the cytochrome oxidase and NADH-succinate-dehydrogenase region.12,13 It has been reported that the mode of antibacterial action of silver nanoparticles is similar to that of silver ion.14 However, the effective biocidal concentration of silver nanoparticles is at a nanomolar level in contrast to a micromolar level of silver ions.15 * Author to whom correspondence should be addressed. Tel: (+82) 2880-7069; fax: (+82) 2-888-1604; e-mail:
[email protected]. (1) Vigo, T. L. Antimicrobial polymers and fibers: retrospective and prospective. In BioactiVe Fibers and Polymers; Edwards, J. V., Vigo, T. L., Eds.; American Chemical Society: Washington, D.C., 2001; pp 175-182, and references therein. (2) Lu, L.; Rininsland, F. H.; Wittenburg, S. K.; Achyuthan, K. E.; McBranch, D. W.; Whitten, D. G. Langmuir 2005, 21, 10154-10159. (3) Krishnan, S.; Wang, N.; Ober, C. K.; Finlay, J. A.; Callow, M. E.; Callow, J. A.; Hexemer, A.; Sohn, K. E.; Kramer, E. J.; Fischer, D. A. Biomacromolecules 2006, 7, 1449-1462. (4) Kurt, P.; Wood, L.; Ohman, D. E.; Wynne, K. J. Langmuir 2007, 23, 4719-4723. (5) Fuchs, A. D.; Tiller, J. C. Angew. Chem., Int. Ed. 2006, 45, 6759-6762. (6) Rosemary, M. J.; MacLaren, I.; Pradeep, T. Langmuir 2006, 22, 1012510129. (7) Dias, H. V. R.; Batdorf, K. H.; Fianchini, M.; Diyabalanage, H. V. K.; Carnahan, S.; Mulcahy, R.; Rabiee, A.; Nelson, K.; Waasbergen, L. G. J. Inorg. Biochem. 2006, 100, 158-160. (8) Balogh, L.; Swanson, D. R.; Tomalia, D. A.; Hagnauer, G. L.; McManus, A. T. Nano Lett. 2001, 1, 18-21. (9) Ramstedt, M.; Cheng, N.; Azzaroni, O.; Mossialos, D.; Mathieu, H. J.; Huck, W. T. S. Langmuir 2007, 23, 3314-3321. (10) Sambhy, V.; MacBride, M. M.; Peterson, B. R.; Sen, A. J. Am. Chem. Soc. 2006, 128, 9798-9808. (11) Shi, Z.; Neoh, K. G.; Kang, E. T. Langmuir 2004, 20, 6847-6852. (12) Klueh, U.; Wagner, V.; Kelly, S.; Johnson, A.; Bryers, J. D. J. Biomed. Mater. Res. 2000, 53, 621-631. (13) Kumar, R.; Howdle, S.; Mu¨nstedt, H. J. Biomed. Mater. Res. 2005, 75B, 311-319. (14) Dibrov, P.; Dzioba, J.; Gosink, K. K.; Ha¨se, C. C. Antimicrob. Agents Chemother. 2002, 46, 2668-2670.
Silver-impregnated polymer matrix provides antimicrobial efficacy with a sustained release of silver.16-18 The silver/polymer fabric minimizes the transmission of infective agents and enhances patient comfort as well as facile application for health care.19 Youngs et al. reported that the synthesized fiber encapsulating silver(I) N-heterocyclic carbene complexes facilitated the release of silver ions with maximum bactericidal activity over a longer period of time than that of aqueous silver.19 Kluech and coworkers demonstrated that silver-coated poly(ethylene terephthalate) fabric was fabricated by the deposition of silver onto the polymeric substrate, and the resultant fabric effectively prevented the attachment of microorganisms on the silver active surface.12 In general, template synthesis20-23 and electrospinning techniques have been widely used for the fabrication of polymer nanofibers. However, these methods require complicated multistep procedures such as hard template removal, or application of a supplementary electric potential to create a jet as the origin of fiber formation.24 Therefore, it is desirable to develop a simple and reliable approach to synthesize polymer nanofibers with enhanced antimicrobial activities. We previously reported that the silver nanoparticle-embedded polymer nanofiber was fabricated by radical-mediated dispersion polymerization, and this novel polymerization technique was found to be a facile onestep method for polymer nanofiber synthesis.25 In the present work, antibacterial properties of novel silver/polymer nanofibers were evaluated using antibacterial kinetics, minimum inhibitory concentration, and the modified Kirby-Bauer test. Escherichia coli and Staphylococcus aureus were selected as Gram-negative and Gram-positive bacteria. The silver/polymer nanofiber had (15) Lok, C.-N.; Ho, C.-M.; Chen, R.; He, Q.-Y.; Yu, W.-Y.; Sun, H.; Tam, P. K.-H.; Chiu, J.-F.; Che, C.-M. J. Proteome Res. 2006, 5, 916-924. (16) Silver, S. FEMS Microbiol. ReV. 2003, 27, 341-353. (17) Voccia, S.; Ignatova, M.; Je´roˆme, R.; Je´roˆme, C. Langmuir 2006, 22, 8607-8613. (18) Li, Z.; Lee, D.; Sheng, X.; Cohen, R. E.; Rubner, M. F. Langmuir 2006, 22, 9820-9823. (19) Melaiye, A.; Sun, Z.; Hindi, K.; Milsted, A.; Ely, D.; Reneker, D. H.; Tessier, C. A.; Youngs, W. J. J. Am. Chem. Soc. 2005, 127, 2285-2291. (20) Lee, K. J.; Oh, J. H.; Kim, Y.; Jang, J. AdV. Mater. 2006, 18, 2216-2219. (21) Jang, J.; Bae, J. Angew. Chem., Int. Ed. 2004, 43, 3803-3806. (22) Jang, J. AdV. Polym. Sci. 2006, 199, 189-259. (23) Jang, J.; Bae, J.; Park, E. AdV. Funct. Mater. 2006, 16, 1400-1406. (24) Czaplewski, D. A.; Verbridge, S. S.; Kameoka, J.; Craighead, H. G. Nano Lett. 2004, 4, 437-439. (25) Kong, H.; Jang, J. Chem. Commun. 2006, 3010-3012.
10.1021/la703085e CCC: $40.75 © 2008 American Chemical Society Published on Web 01/29/2008
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Figure 1. A schematic illustration of the fabrication of silver nanoparticles/polymer nanofiber using the radical-mediated dispersion polymerization. (a) Photograph image of Ag+/PVA aqueous solution. (b) TEM image of Ag/PVA linear assembly. (c) TEM image of Ag/PMMA nanofiber.
enhanced antibacterial efficacy compared to that of silver sulfadiazine and silver nitrate at the same silver concentration. Experimental Section Materials. Poly(vinyl alcohol) (PVA), silver nitrate (AgNO3), and silver sulfadiazine were purchased from Aldrich (Milwaukee, WI) and used without further purification. Methyl methacrylate (MMA) was also provided from Aldrich and applied after purification. 2,2-Azobis(isobutyronitrile) (AIBN) as a radical initiator was supplied from Junsei (Japan) and used after purification. For the bacterial test, Escherichia coli (ATCC 8739) and Staphylococcus aureus (ATCC 6538) were provided from Fisher Company. Fabrication of Polymer Nanofiber Containing Silver Nanoparticles. In a typical procedure, 0.05 wt % of PVA was dissolved in distilled water (100 mL), and silver precursor, AgNO3 (0.29 µM), was added to the polymeric aqueous solution. After the solution was mixed, 2,2-azobis(isobutyronitrile) (AIBN, 0.29 µM) and methyl methacrylate (MMA, 0.02 M) were sequentially injected into the aqueous silver solution. MMA monomer was polymerized with vigorous stirring at 60 °C for 24 h. After polymerization, the synthesized silver/PMMA nanofiber was precipitated for 24 h and washed by ethanol to remove the residual reagents. Organic Reagent Test. The silver/polymer nanofibers were introduced to distilled water (20 mL) and incubated at 25 °C for 24 h. A 1 mL amount of aqueous solution containing the released silver nanoparticle was then placed in test tube. To compare the reaction of the released silver nanoparticle with that of silver salt, silver nitrate (0.017 mg) was dissolved in distilled water (20 mL) and 1 mL of aqueous solution was prepared in another test tube. Consecutively, 0.5 mL of rhodanine solution (20 mM) was added to silver nitrate aqueous solution and the released silver nanoparticle solution. The color changes were monitored by digital camera. Antimicrobial Tests. E. coli and S. aureus were cultivated in sterilized LB broth and then incubated overnight at 37 °C with a shaking incubator. The bacterial suspensions employed for the tests contained from 106 to 107 colony forming units (CFU). For the kinetic test, 1 µg of solid-state silver compound (silver/polymer
nanofiber, silver nitrate, and silver sulfadiazine) was prepared in each sterilized test tube and inoculated with an equivalent volume of E. coli suspension. In each tube, 50 µL volumes were removed as a function of contact time (min) and cultured in LB agar plates. The LB agar plates were kept at 37 °C for 24 h, and the number of survival colonies was counted. The minimum inhibitory concentration (MIC) test was performed according to a reported procedure.19 Sterilized LB solutions were prepared (2 mL) in five test tubes, and the first tube was mixed with 1 mL of silver compounds solution (with the same amount of silver). Consecutively, a 1 mL aliquot of the mixed solution of first tube was transferred to second test tube. In the same manner, five serial dilutions were made. The resultant solutions were inoculated with 1 mL of E. coli or S. aureus suspension. After incubation at 37 °C for 24 h, fresh organisms were reinoculated and incubated for an additional 24 h. The survival of organisms was observed by visual inspection. The concentration of Ag in the silver nitrate and silver sulfadiazine was calculated as the mass ratio of Ag to silver compounds. In the case of Ag/PMMA nanofiber, the Ag concentration was obtained using an ICP-atomic emission spectrometer. For the modified Kirby-Bauer method, an equal amount of solidstate silver sulfadiazine and silver/PMMA nanofiber were pelletized by a hydraulic press. Sample pellets were placed on the E. coli growth LB agar plate and incubated overnight at 37 °C. The zone of inhibition was measured. Characterization. Photographs of transmission electron microscopy (TEM) were obtained with a JEOL JEM-200CX. Acceleration voltage for TEM was 200 kV. In the sample preparation, Ag/polymer nanofibers were diluted in distilled water and cast onto a copper grid. Fourier transform infrared (FT-IR) spectra were recorded on a Bomem MB 100 spectrometer (Quebec, Canada) in the absorption mode at a resolution of 4 cm-1 and 32 scans. The UV-vis spectra were taken at 25 °C with a Perkin-Elmer Lambda-20 spectrometer. Energy dispersive X-ray (EDX) analysis was performed with a Philips CM-20 microscope coupled to an EDX facility. Elemental analysis was conducted with an EA1110 apparatus (CE Instruments). Thermogravimetric analysis (TGA) was carried out in air using a
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Langmuir, Vol. 24, No. 5, 2008 2053
TGA 2050 analyzer (TA Instruments). The weight loss of the Ag/ PMMA nanofibers was measured from ambient temperature up to 800 °C, at a rate of 10 °C min-1 to determine the silver content.
Results and Discussion The overall synthetic procedure of silver nanoparticles/polymer nanofibers using radical-mediated dispersion polymerization is illustrated in Figure 1. Silver ions were dissolved in aqueous PVA solution (Figure 1a), and the hydroxyl group of PVA was coordinated with silver ion. High shear flow was induced by vigorous stirring, and AIBN, as reductant and radical initiator, was added into the Ag+/PVA aqueous solution. Subsequently, Ag/PVA complexes were linearly assembled in the shear flow and more tightly assembled because of the dipole-dipole interaction of silver nanoparticle surface (Figure 1b). The carboxyl group of MMA can form a hydrogen bond with the hydroxyl group of PVA coated on silver nanoparticles. When MMA monomer was injected into the reaction medium and then polymerized for 24 h, the silver nanoparticles/PMMA nanofiber could be obtained. The TEM image of silver/PMMA nanofibers reveals that silver nanoparticles are embedded in the polymer nanofiber (Figure 1c). The silver/polymer nanofiber was characterized to obtain detailed information on its composition. First of all, the FT-IR spectrum of the silver/polymer nanofiber had both characteristic peaks of PVA and PMMA. For PVA, the peaks at 1150 and 1195 cm-1 were attributed to C-O stretching and the broad peak near 3400 cm-1 was designated as an O-H stretching vibration. The bands at 1738 cm-1 and 1220 cm-1 resulted from the CdO stretching vibration of PMMA. Elemental analysis was performed to provide quantitative information on the carbon and hydrogen atoms contained in the nanofiber. The result provided the composition of 39.5 wt % carbon and 5.5 wt % hydrogen. Since the repeating unit of PMMA has five carbon and eight hydrogen atoms, the carbon-to-hydrogen (C/H) weight ratio of PMMA is considered to be approximately 7.5. The estimated C/H weight ratio of the nanofiber is about 7.2, which is similar to that of PMMA. The slight deviation may be attributed to the PVA stabilizer because its repeating unit includes two carbon and four hydrogen atoms. EDX analysis was conducted to examine the presence of silver in the nanofiber and displayed the distinct peaks of carbon, oxygen, and silver, demonstrating the presence of silver embedded in the PMMA nanofibers. The silver/polymer nanofiber was further examined by thermogravimetric analysis (TGA) in order to obtain quantitative information on the silver content. From TGA measurement, it was confirmed that the silver/PMMA nanofibers were composed of about 29 wt % of silver. According from these data, the silver/polymer nanofiber was successfully fabricated by radical-mediated dispersion polymerization. Figure 2a shows the photograph image of the silver/polymer nanofiber in aqueous solution at different nanofiber contents. When the silver/PMMA nanofiber was dispersed in aqueous solution, the color of the solution changed from pale brown to darker brown with increasing nanofiber content. The brownish color is associated with the silver nanoparticles which are released from the polymer nanofiber.26 UV-visible spectroscopic analysis verifies the releasability of active silver nanoparticles more qualitatively. In the Figure 2b UV-vis spectra, the maximum absorbance peak appears at ca. 400 nm, which is consistent with the absorption of silver nanoparticles.27 As a consequence of (26) Yin, B.; Ma, H.; Wang, S.; Chen, S. J. Phys. Chem. B 2003, 107, 88988904. (27) Yonezawa, T.; Onoue, S.-Y.; Kimizuka, N. AdV. Mater. 2001, 13, 140142.
Figure 2. (a) Photograph image of the silver/PMMA nanofiber aqueous solution with different nanofiber contents and (b) UV-vis spectra of released silver nanoparticles from the silver/PMMA nanofiber as a function of immersion time. For photograph and UV-vis spectra, the Ag/PMMA nanofiber powder was dispersed in distilled water. In the photograph image, the concentration of the nanofibers increased from the left: 1, 1 mg; 2, 2 mg; 3, 3 mg; 4, 4 mg; 5, 5 mg.
continuous release of the silver nanoparticles, the intensity at 400 nm increases gradually in proportion to the immersion time. The silver concentrations in the aqueous solution were analyzed by an ICP-atomic emission spectrometer. The silver concentration was 6.14 ppm after 12 h release time. As the release time increased from 24 to 48 h, the released silver concentration was measured to be 13.31 and 20.79 ppm, respectively. In addition, the rate of releasability of silver nanoparticles was also calculated to be approximately 0.43 µg/(mL‚h). Long-term releasability performance can be considered an important factor from the viewpoint of practical applications. The silver/PMMA nanofiber was immersed in aqueous solution for 6 months and characterized by TEM. In the pristine silver/ polymer nanofiber, silver nanoparticles are embedded in the polymer nanofiber (Figure 3a). After 6 months, the silver nanoparticles are removed from the polymer nanofiber (Figure 3b) and exist in the supernatant (Figure 3c). The diffusion of silver nanoparticles from the silver/PMMA nanofiber could be explained as follows. The glass transition temperature (Tg) of bulk PMMA is ca. 125 °C, but Tg of polymer decreases with decreasing polymer film thickness. It has been reported that Tg of PMMA is reduced to lower temperature (Tg of thin film PMMA: 75.3 °C) resulting in more flexible PMMA.28,29 (28) Jang, J.; Oh, J. H. AdV. Funct. Mater. 2005, 15, 494-502.
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Figure 4. Photograph images of (a) silver nitrate solution and (b) released silver nanoparticle solution as a function of rhodanine addition times.
Figure 3. TEM images of the silver embedded polymer nanofiber: (a) as prepared and (b) after the 6 month immersion experiment. (c) TEM image of the silver nanoparticles which diffused into the supernatant.
Furthermore, PMMA is sufficiently porous to allow water to pass through the polymer nanofiber.29 As a result, the silver nanoparticles can diffuse out of the PMMA nanofiber. It has been reported that metallic silver can be oxidized to silver ion.30 To investigate the oxidation of released silver nanoparticles, an organic reagent test was performed as an analytical technique. In general, rhodanine is a selective and sensitive reagent for the detection of silver salt. When silver salt coexists with rhodanine in aqueous solution, the precipitates are formed involving the replacement of acidic imino-hydrogen group of rhodanine by silver ion.31 Figure 4a represents the images of aqueous silver nitrate solution as a function of rhodanine addition times. After rhodanine addition, red-yellow precipitates were immediately formed and gradually changed to black-brown (29) Rege, K.; Raravikar, N. R.; Kim, D.-Y.; Schadler, L. S.; Ajayan, P. M.; Dordick, J. S. Nano Lett. 2003, 3, 829-832. (30) Kumar, R.; Mu¨nstedt, H. Biomaterials 2005, 26, 2081-2088. (31) Stephen, W. I.; Townshend, A. J. Chem. Soc. 1965, 3738-3746.
because of the formation of Ag-rhodanine complexes. In contrast, no precipitates were observed in the released silver solution, and the color slowly faded after 48 h (Figure 4b). The faint color change without precipitation is thought to be related to the formation of a few Ag-rhodanine complexes, originating from rarely formed silver ions. This result means that only small amounts of silver nanoparticles can be oxidized to silver ion under our experimental conditions and that most of them exist as silver nanoparticles. Silver nanoparticles/polymer nanofiber was evaluated for antibacterial properties using various methods such as antibacterial kinetics, minimum inhibitory concentration (MIC), and the modified Kirby-Bauer test. Gram-negative bacteria, E. coli, and Gram-positive bacteria, S. aureus, were chosen for the tests. For the comparison of activity, silver nitrate and silver sulfadiazine were also examined as model compounds. Antibacterial kinetics can be analyzed by a plot of % reduction32 versus contact time, which provides the effectiveness for releasing the silver nanoparticle in aqueous solution. An equivalent amount of silver compound was placed in small volumes of aqueous E. coli solution and observed for periods of contact time (Figure 5). As the contact time increases, the % reduction increases and approaches an asymptotic value. The result shows that the silver/ PMMA nanofiber has a faster kill rate than silver sulfadiazine and silver nitrate. This enhanced antimicrobial activity of the silver/PMMA nanofiber could be explained as follows. Silver nitrate and silver sulfadiazine have an antimicrobial property by releasing silver ions. When the silver ions contact bacteria, black (32) Yeo, S. Y.; Jeong, S. H. Polym. Int. 2003, 52, 1053-1057.
Antibacterial Properties of Ag/Polymer Nanofiber
Langmuir, Vol. 24, No. 5, 2008 2055 Table 1. Minimum Inhibitory Concentration (MIC) Results of Various Silver Compoundsa E. coli concn of Ag (ng/mL)b
1 day
2 day
1 day
2 day
Ag/polymer
6933 2311 770 257 86 7056 2352 784 261 87 7047 2349 783 261 87
+ + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + +
+ + + + + + + + + + + +
AgNO3
SSDc
Figure 5. The plot of % reduction versus contact time (min) of different silver compounds on E. coli. The % reduction was calculated as % reduction ) (A - B)/A × 100 (where A is the number of surviving E. coli colonies in the blank solution and B is the number of surviving E. coli colonies in silver compound solution).
Figure 6. Photograph images of the zone of inhibition of (a) silver sulfadiazine and (b) silver/PMMA nanofiber by the modified KirbyBauer test. Two silver compounds were pelletized with a hydraulic press and placed on the lawn of E. coli. After 24 h incubation, the zone of inhibition was measured. Both pellet sizes are ca. 13 mm in diameter.
precipitates are formed via ion reduction or salt formation,16,33 and those precipitates deteriorate the antimicrobial ability of (33) Joerger, R.; Klaus, T.; Granqvist, C. G. AdV. Mater. 2000, 12, 407-409.
S. aureus
silver compounds
+: growth, -: no growth. b The concentration of Ag in the Ag/ polymer was measured by ICP-atomic emission spectrometer. c SSD: silver sulfadiazine. a
silver nitrate and silver sulfadiazine. On the other hand, the silver/ PMMA nanofiber releases silver nanoparticles of ca. 7 nm in diameter and contacts bacteria without direct precipitation. As mentioned above, the effective concentration of silver nanoparticles is much lower than that of silver ions,15 and it can be represented that silver nanoparticles have an enhanced biocidal ability than that of silver ions at the same concentration. Moreover, the negative charge of silver nanoparticles can induce metal depletion of Gram-negative bacterial cell.34,35 Metal ions undergo complex formation with adjacent negatively charged barrier molecules in the Gram-negative cell, and a loose construction of barrier molecules is induced by the metal depletion. Therefore, the metal depletion results in cell death because of the disorganization of the outer membrane.36 An MIC test was also performed to study the quantitative antimicrobial activity of the silver/polymer nanofiber. Serial diluted silver solutions were each incubated with equal volumes of E. coli and S. aureus solution, and growth or no-growth was determined by visual inspection. The MIC of silver/PMMA nanofiber is 3-fold lower than that of silver nitrate and 9-fold lower than that of silver sulfadiazine for 24 h incubation as shown in Table 1. After 24 h incubation, fresh organisms were reinoculated and incubated for additional 24 h. Consequently, the MIC values of the 48 h incubation are higher than those of 24 h incubation because of the addition of new organisms. Nevertheless, the silver/polymer nanofiber has better sustained activity than silver nitrate and silver sulfadiazine in the 48 h incubation test. From this result, it can be concluded that the silver/polymer nanofiber has superior antibacterial activity to that of AgNO3 and silver sulfadiazine, and the enhanced antibacterial performance is preserved. The Ag/PMMA nanofiber also shows the excellent biocidal effect on Gram-positive bacteria S. aureus. In the case of Gram-positive bacteria, the enhanced antimicrobial activity of the nanofiber can be described by the cell structure. Gram-positive S. aureus has a loose cell wall, which can be readily attacked by nanosize particles.37 As a result, (34) Sondi, I.; Salopek-Sondi, B. J. Colloid Inerface Sci. 2004, 275, 177-182. (35) Amro, N. A.; Kotra, L. P.; Wadu-Mesthrige, K.; Bulychev, A.; Mobashery, S.; Liu, G.-Y. Langmuir 2000, 16, 2789-2796. (36) Lenoir, S.; Pagnoulle, C.; Galleni, M.; Compe`re, P.; Je´roˆme, R.; Detrembleur, C. Biomacromolecules 2006, 7, 2291-2296. (37) Chen, C. Z.; Beck-Tan, N. C.; Dhurjati, P.; Dyk, T. K.; LaRossa, R. A.; Cooper, S. L. Biomacromolecules 2000, 1, 473-480.
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the released silver nanoparticles could pass through the S. aureus cell wall and bind with DNA, causing DNA denaturation. The contact biocidal property of silver sulfadiazine and silver/ polymer nanofiber was also investigated by using a modified Kirby-Bauer technique. Silver compound was made into a pellet and placed on a lawn of E. coli in an agar plate. The contact antibacterial property can be measured by the clear zone of inhibition around the pellet after 24 h incubation (Figure 6). The diameter of the zone of inhibition for the silver/PMMA nanofiber is ca. 45 mm, whereas that of silver sulfadiazine is ca. 16 mm (where the size of both pellets is ca. 13 mm). This result indicates that the silver/polymer nanofiber has a more effective contact biocidal property than silver sulfadiazine. Silver sulfadiazine slowly releases silver ions as an antimicrobial agent,38 and the diffusion of silver ions might be blocked by the formation of secondary compounds such as AgCl in the Kirby-Bauer test media.39 In the case of silver/polymer nanofiber, the silver nanoparticles can freely diffuse into the test media and act as biocidal agents. In addition, the nanometer-sized polymer nanofiber provides high surface area to contact with bacteria. Furthermore, the PMMA also has a slight antimicrobial property by disrupting cell membranes.40 Therefore, the synthesized silver nanoparticles/PMMA nanofiber has a superior contact antibacterial property to that of silver sulfadiazine. (38) Guo, Z.; Sadler, P. Z. Angew. Chem., Int. Ed. 1999, 38, 1512-1531. (39) Melaiye, A.; Simons, R. S.; Milsted, A.; Pingitore, F.; Wesdemiotis, C.; Tessier, C. A.; Youngs, W. J. J. Med. Chem. 2004, 47, 973-977. (40) Kuroda, K.; DeGrado, W. F. J. Am. Chem. Soc. 2005, 127, 4128-4129.
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Conclusion The synthesized silver nanoparticles/PMMA nanofiber was applied as an antibacterial agent from the viewpoint of releasing silver nanoparticles and contact biocidal activity. From UV-vis analysis, it was concluded that the silver nanoparticles were released from the polymer nanofiber as a function of immersion time. The antibacterial kinetics and MIC test showed that the silver/polymer nanofiber had an enhanced killing rate and effective antimicrobial activity than that of AgNO3 by three times and that of silver sulfadiazine by nine times. In addition, the silver/polymer nanofiber had a superior contact antibacterial property to that of silver sulfadiazine in the modified Kirby-Bauer method. Importantly, the synthesized polymer nanofiber had an excellent biocidal potential against Gram-positive bacteria (S. aureus) as well as Gram-negative bacteria (E. coli). According to these results, it is anticipated that this silver nanoparticles/polymer nanofiber can be used in various applications such as clinical wound dressing, bioadhesive, biofilm, and the coating of biomedical materials. Acknowledgment. This work was supported by the Brain Korea 21 program of the Korea Ministry of Education and Korea Science and Engineering Foundation through the Hyperstructured Organic Materials Research Center. LA703085E
