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articles Silver Nanoparticles in Therapeutics: Development of an Antimicrobial Gel Formulation for Topical Use Jaya Jain,† Sumit Arora,† Jyutika M. Rajwade,† Pratibha Omray,‡ Sanjeev Khandelwal,‡ and Kishore M. Paknikar*,† Centre for Nanobioscience, Agharkar Research Institute, G. G. Agarkar Road, Pune 411 004, India, and Nano Cutting Edge Technology PVt. Ltd. 79/87, D. Lad Path, Kalachowki, Mumbai 400 033, India Received February 12, 2009; Revised Manuscript Received May 25, 2009; Accepted May 27, 2009 Abstract: Silver is an effective antimicrobial agent with low toxicity, which is important especially in the treatment of burn wounds where transient bacteremia is prevalent and its fast control is essential. Drugs releasing silver in ionic forms are known to get neutralized in biological fluids and upon long-term use may cause cosmetic abnormality, e.g., argyria and delayed wound healing. Given its broad spectrum activity, efficacy and lower costs, the search for newer and superior silver based antimicrobial agents is necessary. Among the various options available, silver nanoparticles have been the focus of increasing interest and are being heralded as an excellent candidate for therapeutic purposes. This report gives an account of our work on development of an antimicrobial gel formulation containing silver nanoparticles (SNP) in the size range of 7-20 nm synthesized by a proprietary biostabilization process. The typical minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against standard reference cultures as well as multidrug-resistant organisms were 0.78-6.25 μg/mL and 12.5 μg/mL, respectively. Gram-negative bacteria were killed more effectively (3 log10 decrease in 5-9 h) than Gram- positive bacteria (3 log10 decrease in 12 h). SNP also exhibited good antifungal activity (50% inhibition at 75 μg/mL with antifungal index 55.5% against Aspergillus niger and MIC of 25 μg/mL against Candida albicans). When the interaction of SNP with commonly used antibiotics was investigated, the observed effects were synergistic (ceftazidime), additive (streptomycin, kanamycin, ampiclox, polymyxin B) and antagonistic (chloramphenicol). Interestingly, SNP exhibited good anti-inflammatory properties as indicated by concentration-dependent inhibition of marker enzymes (matrix metalloproteinase 2 and 9). The post agent effect (a parameter measuring the length of time for which bacterial growth remains suppressed following brief exposure to the antimicrobial agent) varied with the type of organism (e.g., 10.5 h for P. aeruginosa, 1.3 h for Staphylococcus sp. and 1.6 h for Candida albicans) indicating that dose regimen of the SNP formulation should ensure sustained release of the drug. To meet this requirement, a gel formulation containing SNP (S-gel) was prepared. The antibacterial spectrum of S-gel was found to be comparable to that of a commercial formulation of silver sulfadiazine, albeit at a 30-fold less silver concentration. As part of toxicity studies, localization of SNP in Hep G2 cell line, cell viability, biochemical effects and apoptotic/necrotic potential were assessed. It was found that SNP get localized in the mitochondria and have an IC50 value of 251 μg/mL. Even though they elicit an oxidative stress, cellular antioxidant systems (reduced glutathione content, superoxide dismutase, catalase) get triggered and prevent oxidative damage. Further, SNP induce apoptosis at concentrations up to 250 μg/mL, which could favor scarless wound healing. Acute dermal toxicity studies on SNP gel formulation (S-gel) in Sprague-Dawley rats showed complete safety for topical application. These results clearly indicate that silver nanoparticles could provide a safer alternative to conventional antimicrobial agents in the form of a topical antimicrobial formulation. Keywords: Silver nanoparticles; antimicrobial; formulation; dermal toxicity Introduction For thousands of years, silver and silver ions have been used for their bactericidal properties. Ancient Romans treated * Corresponding author. Mailing address: Agharkar Research Insti- tute, Centre for Nanobioscience, G. G. Agarkar Road, Pune 411 004, India. Fax: +91 20 25651542. E-mail: paknikar@vsnl.com. † Agharkar Research Institute. ‡ Nano Cutting Edge Technology Pvt. Ltd. 1388 MOLECULAR PHARMACEUTICS VOL. 6, NO. 5, 1388–1401 their water with silver coins, a tradition still being continued in many societies and even in space programs for purifying water aboard the Apollo spacecraft,1 MIR space station2 and the Space shuttle.3 The use of silver as an antimicrobial agent (1) Albright, C. F.; Nachum, R.; Lechtman, M. D. Electrolytic silver ion generator for water sterilization in Apollo spacecraft water systems. Apollo applications program. NASA Contract. Rep. 1967; Report Number: NASA-CR-65738. 10.1021/mp900056g CCC: $40.75 2009 American Chemical Society Published on Web 05/27/2009PDF Image | Silver Nanoparticles in Therapeutics: Antimicrobial Gel
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