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SilVer Nanoparticle Based Formulation for Topical Use articles Cell viability was tested using sodium 3′-[1-(phenylami- nocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) assay, based on the cleavage of yellow tetrazolium salt XTT by metabolically active cells to form an orange formazan dye which was quantified using a microplate reader (Biorad, model No. 680). XTT assay was performed according to the manufacturer’s instructions with appropriate controls. Cells were seeded in 96-well microtiter plates (1 × 104 cells/200 μL of growth medium/ well) followed by overnight incubation. Supernatants from the wells were aspirated out, and fresh aliquots of growth medium (containing SNP in desired concentrations in the range of 3.12-400 μg/mL) were added. After 24 h, supernatants were aspirated out and the cell monolayers in the wells were washed with 200 μL of PBS (0.1 M, pH 7.4). Subsequently, XTT reagent (70 μL) was added in each well and incubated for 5 h, and absorbance at two wavelengths (415 nm for soluble dye and 630 nm for cells) was recorded using the microplate reader. Concentration of SNP showing 50% reduction in cell viability (i.e., IC50 value) was then calculated. For performing transmission electron microscopy, cells were plated into a 35 mm tissue culture plate at a density of 2 × 105 cells (in 2 mL of growth medium). After overnight growth, supernatants from the culture plates were aspirated out and fresh aliquots of growth medium containing ∼(1/2)IC50 SNP were added. For control experiments, medium without nanoparticles was used. Upon incubation for 24 h, the cells (about 80% confluent) were trypsinized and pelleted by centrifugation at 500g for 5 min. Cell pellets were washed with phosphate buffer (0.1 M, pH 7.4). Cell pellets were fixed using 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h followed by postfixation in 1% osmium tetroxide (Agar Scientific, Stansted Essex, England, U.K.), for 1.5 h. Cell pellets were dehydrated through a series of ethanol concen- trations (20%, 30%, 40%, 50%, 60%, 70%, 90%) followed by treatment with 2% uranyl acetate in 95% ethanol (Enblock stain) for 1 h and further dehydration with 100% ethanol for 1 h. Cell pellets were finally treated with propylene oxide (twice for 15 min each) followed by 1:1 propylene oxide: Araldite resin for overnight. Cell pellets were infiltrated with fresh Araldite resin (3 changes with a gap of 3 to 4 h). Cell pellets were subsequently embedded in Araldite resin at 60 °C for 48 h and ultrathin sections (70-80 nm) were cut with glass knives in an ultramicrotome (LEICA EM UC6, Netherlands). The sections were mounted on copper grids and stained with 1% aqueous uranyl acetate and 0.2% lead citrate.9 The stained sections were scanned with an electron microscope (Technai G2 Spirit Biotwin, Netherlands) for ultra structural observations at 80 kV. For biochemical assays preparation of cell extract was done as follows: 75 cm2 flasks containing 15 mL of growth (9) Reynolds, E. S. The use of lead citrate at high pH as an electron- opaque stain in electron microscopy. J. Cell Biol. 1963, 17, 208– 212. medium were seeded with ca. 1 × 107 cells. After overnight growth the cells were challenged with ∼(1/2)IC50 SNP (125 μg/mL for Hep G2 cells) and incubated for 24 h. The cells (about 80% confluent) were then trypsinized and pelleted by centrifugation at 500g for 5 min. The cell pellet was washed with PBS (0.1 M, pH 7.4), resuspended in 500 μL of chilled homogenizing buffer (250 mM sucrose, 12 mM Tris-HCl, 0.1 mM DTT, pH 7.4) and lysed using Dounce homogenizer. The lysate was centrifuged (8000g, 10 min, 4 °C), and the supernatant (cell extract) was used in various biochemical assays. Protein concentration in the cell extract was estimated by the Bradford method.10 Catalase activity was measured by the method described by Aebi.11 In this, absorbance (240 nm) of 1 mL of reaction mixture containing 0.8 mL of H2O2 phosphate buffer (H2O2 diluted 500-fold with 0.1 M phosphate buffer of pH 7), 100 μL of cell extract, and 100 μL of distilled water was recorded for 4 min against blank (H2O2 phosphate buffer). Change in absorbance per min (i.e., ∆A240) was then calculated and used in the estimation of enzyme activity (extinction coefficient value used was 39.4 L M-1 cm-1). Superoxide dismutase (SOD) activity was measured ac- cording to the method described by Kono.12 Briefly, the reaction mixture (2.1 mL) contained 1924 μL of sodium carbonate buffer (50 mM), 30 μL of nitrobluetetrazolium (1.6 mM), 6 μL of Triton X-100 (10%) and 20 μL of hydroxy- lamine-HCl (100 mM). Subsequently 100 μL of cell extract was added and absorbance (560 nm) was read for 5 min against blank (reaction mixture without cell extract). Change in absorbance per min (i.e., ∆A560) was calculated and used in the estimation of enzyme activity (extinction coefficient value used was 9920 L M-1 cm-1). Glutathione peroxidase activity was measured as described by Flohe and Gunzler.13 The reaction mixture contained 1.2 mL of potassium phosphate buffer (0.1 M, pH 7), 200 μL of GSH (10 mM), 10 μL of sodium azide (200 mM), 200 μL of cell extract and 200 μL of glutathione reductase (2.4 U/mL). Following incubation at 37 °C for 10 min, 200 μL of NADPH (1.5 mM) and 200 μL of H2O2 (1.5 mM) were added. Absorbance (340 nm) was recorded for 4 min against blank (reaction mixture sans cell extract), and change in absorbance per min (i.e., ∆A340) was calculated. The enzyme activity was calculated (extinction coefficient value used was 6220 L M-1 cm-1). Total reduced glutathione (GSH) content was measured as described by Saldak and Lindsay.14 The reaction mixture containing 1.2 mL of EDTA (0.02 M), 1 mL of distilled water, 250 μL of 50% trichloroacetic acid, and 50 μL of (10) (11) (12) (13) Bradford, M. A. rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. Aebi, H. Catalase. Methods Enzymol. 1984, 2, 673–684. Kono, Y. Generation of Superoxide radical during auto-oxidation of dyhydroxylamine and an assay for Superoxide dismutase. Arch. Biochem. Biophy. 1978, 186, 189–195. Flohe, L.; Gunzler, W. A. Assays of glutathione peroxidase. Methods Enzymol. 1984, 105, 114–121. VOL. 6, NO. 5 MOLECULAR PHARMACEUTICS 1391PDF Image | Silver Nanoparticles in Therapeutics: Antimicrobial Gel
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