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Biocidal Activity of Plasma Modified Electrospun Polysulfone Mats Functionalized with Polyethyleneimine-Capped Silver Nanoparticles Jessica D. Schiffman,*,†,§ Yue Wang,‡ Emmanuel P. Giannelis,‡ and Menachem Elimelech† † ‡
Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520-8286, United States Department of Materials Science and Engineering, Cornell University, Bard Hall, Ithaca, New York 14853, United States ABSTRACT: The incorporation of silver nanoparticles (AgNPs) into polymeric nanofibers has attracted a great deal of attention due to the strong antimicrobial activity that the resulting fibers exhibit. However, bactericidal efficacy of AgNP-coated electrospun fibrous mats has not yet been demonstrated. In this study, polysulfone (PSf) fibers were electrospun and surface-modified using an oxygen plasma treatment, which allowed for facile irreversible deposition of cationically charged polyethyleneimine (PEI)-AgNPs via electrostatic interactions. The PSfAgNP mats were characterized for relative silver concentration as a function of plasma treatment time using ICP-MS and changes in contact angle. Plasma treatment of 60 s was the shortest time required for maximum loss of bacteria (Escherichia coli) viability. Time-dependent bacterial cytotoxicity studies indicate that the optimized PSfAgNP mats exhibit a high level of inactivation against both Gram negative bacteria, Escherichia coli, and Gram positive bacteria, Bacillus anthracis and Staphylococcus aureus.
’ INTRODUCTION Intensive research into antibacterial agents continues to be a pressing necessity as both microbial resistance and infectious diseases remain. Microorganisms continue to contaminate the surfaces of medical devices, hospital and dental equipment, water purification systems, food packaging, and textiles.13 Hence, there exists a strong demand for the development of a broad spectrum of antibacterial materials that can deliver those agents. Since bacterial colonization and subsequent biofilm formation occurs on virtually any surface, the antibacterial materials should be robust and readily applicable to a variety of surface topographies. The bactericidal effect of silver nanoparticles (AgNPs) against a wide range of microorganisms is well-documented. Surprisingly, despite the widespread use of AgNPs, the mechanism responsible for their cytotoxicity is not fully understood. Numerous studies conclude that dissolved Ag+ ions dictate the toxicity of nanosilver.4,5 One possibility is that the silver ions react with and, consequently, inactivate the thiol groups of vital enzymes.6 In contrast, literature has also reported that relying on the release of Ag+ ions alone is not significant enough to result in toxicity. Direct contact between the bacteria and nanoscale silver is required.7,8 One physiochemical property of AgNPs, which has proven especially powerful toward increasing their toxicity, is nanoparticle diameter. Smaller AgNPs (99%.49 13162
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contact with the PSfAgNP mats. Baweja et al.53 and Eby et al.54 observed a complete inactivation and >3 log removal of B. anthracis after utilizing paint containing silver encapsulated within zeolites and colloidal silver coatings, respectively. It should be noted that, in these cases, the viability of the spores was not significantly affected as B. anthracis is known to produce highly stable spores that remain viable for decades.53,55
Figure 7. Fluorescence-based toxicity assay results for various bacteria in contact with 60 s plasma-treated electrospun PSfAgNP mats and asspun PSf mats (control, indicated by hatch marks). Cell suspensions of E. coli, Staphylococcus aureus (S. aureus), and Bacillus anthracis (B. anthracis) were incubated for 15 min in an isotonic solution (0.9% NaCl, pH 5.7) at 37 °C. Error bars indicate one standard deviation.
Inactivated E. coli cells displayed in Figure 6B show a distinct difference in cell morphology compared to viable cells. Inactivated cells exhibit membranes that appear wrinkled and deflated. As previously discussed, the mechanism of Ag ion toxicity toward microorganisms remains under debate. In addition to concentration dependence, Sondi and Salopek-Sondi48 demonstrated that monodispersed (12 nm) AgNPs caused pits to form on the E. coli when tested in liquid LB medium after 4 h. The pits allowed for the accumulation of AgNPs within the cell membrane, which resulted in cell death. It has also been suggested50 that irregularly shaped pits in the outer cell membrane result from metal depletion. This changes the permeability of the membrane and allows for lipopolysaccharide molecules and membrane proteins to be released. Kim et al.35 suggest that the antimicrobial activity of AgNPs results from the formation of free radicals and the subsequent free radical-induced membrane damage. Due to the high surface area-tovolume ratios of our small-diameter (23 nm) AgNPs, there is an abundance of silver atoms available on the surface of the nanoparticles, which we suspect contributes to cell membrane disruption. PSfAgNP Mats are Effective Biocidal Materials for Gram Positive and Gram Negative Bacteria. The antimicrobial effects of the PSfAgNP mats against two Gram-positive bacteria, S. aureus and B. anthracis, are displayed in Figure 7. After 15 min of contact with the PSfAgNP mats, loss of viability was observed to be 66 ( 1% for S. aureus and 68 ( 5% for E. coli; these results were not statistically equivalent. The findings are consistent with previous studies on nanofiber mats containing AgNPs, which report higher inactivation rates for Gram-negative E. coli than for Gram-positive S. aureus.22,51 The classification of bacteria as Gram-positive versus Gram-negative stems from their different membrane structures and, in particular, the thickness of the peptidoglycan layer. This layer between the cytoplasmic membrane and the outer membrane of Gram-negative bacteria is ∼2 to 3 nm, whereas Gram-positive bacteria have a much thicker, 30 nm peptidoglycan layer, but lack the outer membrane.35,52 It is for this reason that oftentimes the efficacy of antimicrobial agents is lower for Gram-positive than for Gram-negative bacteria. The fluorescence-based viability assay determined that more than 99% of B. anthracis cells became unviable after 15 min of
’ CONCLUSION Fibrous PSf mats have been successfully functionalized with in-house-synthesized, PEI-capped AgNPs using a simple postproduction plasma treatment process. After AgNP attachment, the morphology of the fibers within the PSfAgNP mats remains consistent with as-spun mats, continuous and cylindrical. By using E. coli as a model Gram-negative bacteria and correlating the findings with observed contact angle plateau and observed metal count trends, plasma treatment time for the PSfAgNP mats was optimized. Plasma-treated (60 s) PSfAgNP mats displayed high bioactivity against E. coli, S. aureus, and B. anthracis. Research into composite delivery systems that maximize the efficiency of active bactericidal agents continues to be a pressing necessity as infectious disease and bacterial outbreaks remain. The present work demonstrates that electrospun PSf mats that have been surface-modified with PEI-capped AgNPs offer potential applicability as broad-spectrum biocidal coatings. ’ AUTHOR INFORMATION Corresponding Author
*schiff
[email protected]. Present Addresses §
Department of Chemical Engineering, University of Massachusetts, 686 North Pleasant Street, Amherst, Massachusetts 01003, United States
’ ACKNOWLEDGMENT This publication was based on work supported by Award No. KUS-C1-018-02, made by King Abdullah University of Science and Technology (KAUST). We acknowledge use of facilities at the Cornell NanoScale Facility, a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (Grant ECS-0335765). ’ REFERENCES (1) Spellberg, B.; Guidos, R.; Gilbert, D.; Bradley, J.; Boucher, H. W.; Scheld, W. M.; Bartlett, J. G.; Edwards, J. Clin. Infect. Dis. 2008, 46, 155–164. (2) Cho, K.-H.; Park, J.-E.; Osaka, T.; Park, S.-G. Electrochim. Acta 2005, 51, 956–960. (3) Dunne, W. M., Jr. Clin. Microbiol. Rev. 2002, 15, 155–166. (4) Navarro, E.; Piccapietra, F.; Wagner, B.; Marconi, F.; Kaegi, R.; Odzak, N.; Sigg, L.; Behra, R. Environ. Sci. Technol. 2008, 42, 8959–8964. (5) Miao, A.-J.; Schwehr, K. A.; Xu, C.; Zhang, S.-J.; Luo, Z.; Quigg, A.; Santschi, P. H. Environ. Pollut. 2009, 157, 3034–3041. (6) Russell, A. D.; Hugo, W. B. 7 Antimicrobial Activity and Action of Silver. In Progress in Medicinal Chemistry, Ellis, G. P., Luscombe, D. K., Eds.; Elsevier: London, 1994; Vol. 31, pp 351370. (7) Fabrega, J.; Fawcett, S. R.; Renshaw, J. C.; Lead, J. R. Environ. Sci. Technol. 2009, 43, 7285–7290. (8) Kawata, K.; Osawa, M.; Okabe, S. Environ. Sci. Technol. 2009, 43, 6046–6051. 13163
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