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SilVer Nanoparticle Based Formulation for Topical Use articles in clinical settings also has a long history. In 1884, Crede, a German obstetrician, used 1% silver nitrate solution to eliminate blindness caused by postpartum infections in newborns. In 1920s the US Food and Drug Administration approved colloidal silver for wound treatment. With the advent of antibiotics, in the 1940s, research on medical application of silver declined dramatically. However in 1964, Moyer et al.4 first used 0.5% silver nitrate solution in the burn arena, which rekindled the research interest in silver. Four years later in 1968, Fox5 introduced 1% silver sulfa- diazine (SSD) cream, which has become one of the leading topical antimicrobial agents used to treat burn wound infections over the last four decades. At the beginning of 20th century burn wound infections were the major source of morbidity and mortality (over 50%) in burn patients.6 Burn injury disrupts both the normal skin barrier and many of the systemic host defense mechanisms, which makes skin susceptible to microbial colonization resulting in development of burn wound sepsis. The burned skin remains vulnerable to invasive microbial infections of all kinds until complete epithelial repair has occurred. Overuse of antibiotics and failure to apply basic infection control policies and procedures have contributed to high mortality and morbidity among burn wound patients due to infections caused by multidrug-resistant nosocomial patho- gens (e.g., Pseudomonas aeruginosa, methicillin resistant staphylococci, vancomycin-resistant enterococci). Thus topi- cal antimicrobial therapy to control colonization and prolif- eration of microbial pathogens including multidrug-resistant organisms is the most important method of burn wound care. In this respect, antimicrobial agents containing silver (e.g., silver nitrate and silver sulfadiazine) have revolutionized burn wound care and significantly reduced morbidity and mortal- ity.7 Silver therapy, in principle, has many beneficial effects such as (a) multilevel antibacterial effect on cells which considerably reduce the chances of developing resistance, (b) effectiveness against multidrug-resistant organisms and (c) low systemic toxicity. However, silver compounds being used for topical applications, viz., silver nitrate and silver sulfadiazine, may get neutralized by anions (chloride, bicarbonate and protein) in body fluids or cause cosmetic (2) Conrand, A. H.; Tramp, C. R.; Long, C. J.; Wells, D. C.; Paulsen, A. Q.; Conrand, G. W. Ag+ alters cell growth, neurite extension, cardiomyocyte beating, and fertilized egg constriction. AViat. Space EnViron. Med. 1999, 70, 1096–1105. (3) Melaiye, A.; Youngs, W. J. Silver and its application as an antimicrobial agent. Expert Opin. Ther. Pat. 2005, 15, 125–130. (4) Moyer, C. A.; Brentano, L.; Gravens, D. L.; Margraf, H. W.; Monafo, W. W. Treatment of large human burns with 0.5% silver nitrate solution. Arch. Surg. 1965, 90, 812–867. (5) Fox, C. L. Silver sulfadiazine: a new topical therapy for Pseudomonas in burns. Arch. Surg. 1968, 96, 184–188. (6) Reiss, E. R.; Artz, C. P. Current status of research in treatment of burns. Mil. Surg. 1954, 114, 187–190. (7) Fox, C. L., Jr.; Modak, S. M. Mechanism of action of silver sulfadiazine on burn wound infections. Antimicrob. Agents Chemother. 1974, 5, 582–588. abnormality, viz., argyria (blue gray coloration) upon prolonged use, or could arrest the healing process owing to fibroblast and epithelial cell toxicity. Despite these shortcom- ings, silver sulfadiazine is the most popular topical antimi- crobial silver delivery system in use due to nonavailability of safer alternatives. The present study describes our work on providing an alternative silver delivery system, viz., a silver nanoparticle based topical antimicrobial formulation for indications such as burns and wounds. Materials and Methods Synthesis of Silver Nanoparticles (SNP) and Their Characterization. Silver nanoparticles were synthesized by a proprietary process that involves photoassisted reduction of Ag+ to metallic nanoparticles and their biostabilization (United States Patent No. 7514600). The synthesized nano- particles were characterized by UV-vis spectroscopy (Nano- drop ND-1000, USA) and high resolution transmission electron microscopy (Philips, Holland CM 200). Particle size distribution was measured by dynamic light scattering (Zetasizer, Malvern Instruments, U.K.). Studies on stability of SNP were carried out by monitoring the changes in surface plasmon resonance characteristics for 8 weeks. Studies on Bioactivity of SNP. Antibacterial ActiVity: Minimum Inhibitory Concentration (MIC), Minimum Bac- tericidal Concentration (MBC), Time Kill Study, Post Agent Effect (PAE) and Fractional Inhibitory Concentration Index (FICi). Standard bacterial strains (Escherichia coli ATCC 117, Pseudomonas aeruginosa A TCC 9027, Salmonella abony NCTC 6017, Salmonella typhimurium ATCC 23564, Klebsiella aerogenes ATCC 1950, Proteus Vulgaris NCIB 4157, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633 and Staphylococcus epidermidis ATCC 1228) and multidrug-resistant (MDR) clinical isolates (eight strains of Pseudomonas sp, five strains of Staphylococcus sp, six strains of Escherichia coli and three strains of Klebsiella sp.) were used in the MIC and MBC study. MIC and MBC were determined by double dilution technique as described in the NCCLS M7-A5 guidelines. Accordingly, 96-well microtiter plates containing 200 μL Muller Hinton (MH) broth (Hi-Media Mumbai, India) with SNP (in the concentration range of 0.78-50 μg/mL) were inoculated with test strains (final cell density of 1 × 105 CFU/ mL) and incubated at 37 °C for 24 h. The lowest concentra- tion of SNP showing growth inhibition (as seen visually) was considered as the minimum inhibitory concentration. The minimum bactericidal concentration was recorded as the lowest concentration of SNP that showed no growth on MH agar plates after spot inoculation and incubation for 24 h. Assay was performed in triplicate with appropriate controls (uninoculated medium and medium without SNP). For time kill study standard bacterial strains and two representative MDR isolates were used. The cultures were inoculated in 2 mL of MH broth (final cell density of 1 × 105 CFU/mL) supplemented with SNP (at concentrations VOL. 6, NO. 5 MOLECULAR PHARMACEUTICS 1389PDF Image | Silver Nanoparticles in Therapeutics: Antimicrobial Gel
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