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SilVer Nanoparticle Based Formulation for Topical Use articles observed with specific antimicrobial agent-organism com- binations ranges from 0.5 to 6 h. Antibiotics which inhibit protein and nucleic acid synthesis tend to produce longer PAEs, as compared to those which act on cell wall synthe- sis.26 The prolonged PAE ranging from 4 to 11 h for Gram- negative organisms observed with SNP may be attributed to the inactivation of proteins. Direct evidence of protein inactivation by silver nanoparticles has been recently pro- vided by Lok et al.27 Our results suggest that longer dosing interval may be possible because the majority of tested strains have PAE >3 h. We believe that PAE of silver nanoparticles is being demonstrated for the first time. The anti-inflammatory effects of silver ion on a wound have been recognized for centuries. Most of the reports are purely descriptive in nature, identifying the decrease in erythema and increased healing. A number of the biochemi- cal effects of silver on the wound have been documented over a decade ago. There is an increased expression of matrix metalloproteinases (MMPs) in a burn wound and in chronic wounds.28 Excess proteinase activity is now considered to be a major cause of impaired healing by destroying new tissue and growth factors. Matrix metalloproteinases such as collagenases (MMP-1) (MMP-8), gelatinases (MMP-2 and -9), stromelysin (MMP-3) and elastase (MMP-13) are especially found in burn wounds. In the present study a quantitative assay was undertaken using MMP-2 (gelatinase A) and MMP-9 (gelatinase B). As collagenase MMP-1, MMP-8, and MMP-13 have related activities, they are anticipated to behave similarly and produce results similar to those obtained for the gelatinase tested. The dose- dependent inhibition of both MMP-2 and MMP-9 with SNP demonstrates anti-inflammatory response which may enhance wound healing in vivo. In vitro studies on exposure of cell lines (C18-4 germ cell line, BRL 3A liver cell line and PC-12 neuroendocrine cell line) to silver nanoparticles have shown decreased mito- chondrial function and induction of apoptosis or apoptosis- like change of cell morphology.29-31 In the event of entry of nanoparticles into the systemic circulation, they are likely to get distributed throughout the body; specifically in liver, (26) MacKenzie, F. M.; Gould, I. M. The post-antibiotic effect. J. Antimicrob. Chemother. 1993, 32, 519–537. (27) Lok, C. N.; Ho, C. M.; Chen, R.; Qing-Yu, H.; Yiu, Y. W.; Sun, H.; Tam, P. K. H.; Chiu, J. F.; Che, C. M. Proteomic analysis of the mode of antibacterial action of silver nanoparticles. J. Proteome Res. 2006, 5, 915–924. (28) Demling, R. H.; DeSanti, L. Effects of silver on wound manage- ment. Wounds 2001, 13, 4–9. (29) Braydich-Stolle, L.; Hussain, S.; Schlager, J. J.; Hofmann, M. C. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. Toxicol. Sci. 2005, 88, 412–419. (30) Hussain, S. M.; Hess, K. L.; Gearhart, J. M.; Geiss, K. T.; Schlager, J. J. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol. in Vitro 2005, 19, 975–983. (31) Hussain, S. M.; Javorina, A. K.; Schrand, A. M.; Duhart, H. M.; Ali, S. F.; Schlager, J. J. The interaction of manganese nanopar- ticles with PC-12 cells induces dopamine depletion. Toxicol. Sci. 2006, 92, 456–463. which is a major accumulation site for toxic chemicals. Therefore, liver cells (rodent or human origin) represent a useful tool for studying toxicity, drug metabolism, and enzyme induction.32,33 Liver cell line, viz., Hep G2, has been used for assessment of in vitro genotoxicity34 and cytotox- icity35 of many toxic agents as well as nanoparticles.36 Considering these facts, Hep G2 cells were selected for evaluation of in vitro toxicity of SNP. The toxicity of SNP was evaluated by studying specific mitochondrial marker (XTT assay) under control and exposed conditions. Results obtained by us clearly showed decreased mitochondrial function in cells exposed to SNP (12.5-400 μg/mL) in a dose-dependent manner as evidenced in the XTT assay. The IC50 value of SNP was found to be 251 μg/mL. This higher IC50 value is probably an inherent characteristic of Hep G2, because our earlier studies with established cell lines derived from human skin carcinoma and human fibrosarcoma37 show lower IC50 value. Apart from this, studies on localization of silver nanoparticles were carried out by transmission electron microscopy after exposing the cells to SNP, since at this stage, it was not known whether these SNP are interacting with cells physically by getting lodged on the cell surface or are translocated inside the cells. Ultrathin sections of Hep G2 cells exposed to ∼(1/2)IC50 SNP for 24 h, visualized under transmission electron microscope, showed the presence of dark, electron dense, spherical aggregates inside the mitochondria. These structures could probably be silver nanoparticles. Our results are in agreement with earlier reports where nanosized particles of various chemistries were shown to mobilize into mitochon- dria preferentially.38,39 Mitochondria represent the most active cellular redox organelle, and localization of these particles into such redox active centers is expected to cause (32) Davila, J. C.; Acosta, D. Preparation of primary monolayer of postnatal rat liver cells for hepatotoxicity assessment of xenobi- otics. In In Vitro Biological Systems, Tyson, C. A., Frazier, J. M., Eds.; Academic: San Diego, 1993; pp 244-261. (33) Zurlo, J.; Arterburn, L. M. Characterization of a primary hepa- tocyte culture system for toxicological studies. In Vitro Cell DeV. Biol. 1996, 32, 211–220. (34) Bhatt, T. S.; Coombs, M.; DiGiovanni, J.; Diamond, L. Mutagen- esis in Chinese hamster cells by cyclopenta(R)phenanthrenes activated by a human hepatoma cell line. Cancer Res. 1983, 43, 984–986. (35) Babich, H.; Sardana, M. K.; Borenfreund, E. Acute cytotoxicities of polynuclear aromatic hydrocarbons determined in vitro with the human liver tumor cell line Hep G2. Cell Biol. Toxicol. 1988, 4, 295–309. (36) Ponti, J.; Colognato, R.; Franchini, F.; Gioria, S.; Simonelli, F.; Abbas, K.; Rossi, F. Uptake and cytotoxicity of gold nanoparticles in MDCK and HepG2 cell lines. http://www.aist-riss.jp/projects/ nedo-nanorisk/nanorisk_symposium2008/pdf/03_122_en_ROS- SI.pdf. 2008. Accessed Jan 9, 2009. (37) Arora, S.; Jain, J.; Rajwade, J. M.; Paknikar, K. M. Cellular responses induced by silver nanoparticles: In Vitro studies. Toxicol. Lett. 2008, 179, 93–100. (38) Foley, S.; Crowley, C.; Smaihi, M.; Bonfils, C.; Erlanger, B. F.; Seta, P. Cellular localization of a water-soluble fullerene deriva- tive. Biochem. Biophys. Res. Commun. 2002, 294, 116–119. VOL. 6, NO. 5 MOLECULAR PHARMACEUTICS 1399PDF Image | Silver Nanoparticles in Therapeutics: Antimicrobial Gel
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