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SilVer Nanoparticle Based Formulation for Topical Use articles Table 4. Susceptibility of P. aeruginosa to Individual Agents and Results of Checkerboard Microdilution Studies drug ceftazidime (CF) streptomycin (ST) kanamycin (K) ampiclox (AC) polymyxin B (P) chloramphenicol (CHL) SNP MIC (μg/mL) 0.78 3.12 12.5 1.95 1.56 12.5 3.12 drug combination MIC (μg/mL) CF (0.39) + SNP (0.78) ST (3.12) + SNP (0.39) K (12.5) + SNP (0.39) AC (1.95) + SNP (0.39) P (1.56) + SNP (0.195) CHL (25) + SNP (1.56) FICi interaction 0.75 synergistic 1.125 additive 1.125 additive 1.125 additive 1.0625 additive 2.5 antagonistic protein in control cells). SOD levels were found to be ∼1.4- fold higher for SNP treated cells (4.97 μmol/mg) as compared to unexposed cells (3.47 μmol/mg protein). However, changes observed in the levels of lipid peroxidation and GPx were not statistically significant (p > 0.05). The apoptotic thresholds of SNP were monitored by caspase-3 assay after exposure to SNP for a range of doses (0.39-350 μg/mL), and the results of a representative dose- range study are shown in Figure 10. SNP could induce apoptosis at concentrations in the range of 6.25-250 μg/ mL. The study was complemented by microscopic observa- tions on cells. Exposure to 125 μg/mL (∼(1/2)IC50) SNP resulted in apoptosis (as evidenced by bright green nucleus with condensed or fragmented chromatin under CLSM, upon AO/EB double staining) of cells (Figure 11B). Further, at SNP concentration 500 μg/mL (∼2 × IC50) cells with structurally normal but orange stained nuclei were observed Figure 5. Time kill study performed using SNP against C. albicans at MIC and MFC (error bars not shown as deviations from the mean values are <0.1 × log 10). Figure 6. Inhibition of MMP-2 (gelatinase A) enzyme in the presence of SNP (error bars not shown as deviations from the mean values are <1%). (Figure 11C), suggesting necrosis. Table 5 shows quantitative data on the percentage of live, apoptotic and necrotic cells after exposure to ∼(1/2)IC50 and ∼2 × IC50 SNP as compared to control cells which was obtained using fluo- rescence microscopy after AO/EB double staining. Data showed 67% live cells, 28% apoptotic cells and 5% necrotic cells at SNP concentration of 125 μg/mL (∼(1/2)IC50) whereas 53% live cells, 11% apoptotic cells and 36% necrotic cells were noticed at 500 μg/mL SNP concentration (∼2 × IC50). The above experiments demonstrating the antibacterial, antifungal nature of SNP as well as those proving its anti- inflammatory activity, cytotoxicity, intracellular localization and biochemical changes relating to metal stress response were carried out using aqueous a colloidal suspension of SNP. The following paragraphs describe results of the study on gel formulation containing SNP, i.e., S-gel (Figure 12). Table 6 and Figure 12 show the results of in vitro antibacterial activity of S- gel. The zone of inhibition for S-gel was in the range of 3 to 10 mm at 0.020 mg/g of gel. Maximum antibacterial activity (10 mm) of S-gel was noted against Pseudomonas sp. At higher concentration of silver (0.1 mg/g) S-gel showed a significantly wider zone of Figure 7. Percent viability measured by XTT assay on Hep G2 cells after treatment with SNP (12.5 μg/mL to 400 μg/mL) for 24 h. The data are expressed as mean ( standard deviation (SD) of three independent experiments. An OD value of control cells (unexposed cells) was taken as 100% viability (0% cytotoxicity). Asterisk (*) denotes a statistically significant difference compared to control (p < 0.05). VOL. 6, NO. 5 MOLECULAR PHARMACEUTICS 1395PDF Image | Silver Nanoparticles in Therapeutics: Antimicrobial Gel
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