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articles Jain et al. Tris buffer (0.4 M, pH 8.9) was centrifuged at 300g for 15 min. Clear supernatant (500 μL) was mixed with 1 mL of 0.4 M Tris buffer (containing 0.02 M EDTA, pH 8.9), 100 μL of 0.01 M DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] and 100 μL of cell extract. The mixture was incubated at 37 °C for 25 min, and the yellow color developing was read at 412 nm against blank. The enzyme activity was calculated taking the extinction coefficient, 14150 L M-1 cm-1. Lipid peroxidation was estimated as per the method described by Beughe and Aust:15 a mixture of 100 μL of tris buffer (150 mM, pH ) 7.1), 10 μL of ferrous sulfate (100 mM), 10 μL of ascorbic acid (150 mM), 780 μL of distilled water and 100 μL of cell extract was incubated at 37 °C for 15 min. Thiobarbituric acid (0.375%, 2 mL) was then added to the mixture and allowed to react at 100 °C (in water bath) for 15 min. The reaction mixture was then centrifuged (800g for 10 min), and supernatant was read at 532 nm against blank. The enzyme activity was calculated (extinction coefficient value used was 156,000 L M-1 cm-1). For the assessment of apoptosis, caspase-3 colorimetric assays were performed using a standard kit (Sigma, USA) to determine the concentrations of SNP inducing apoptosis and necrosis in cells. Further, to visualize apoptotic/necrotic nuclei, cells were seeded on glass coverslips (18 × 18 mm) placed into 35 mm tissue culture plates 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 SNP at desired concentrations (∼(1/2)IC50 and ∼2 × IC50) were added. Upon incubation for 24 h, cells were washed with PBS and fixed in 4% chilled paraformaldehyde for 20 min and then washed twice with PBS. Cells were stained by adding 1 mL of AO/EB mix (100 μg/mL AO and 100 μg/ mL EB in PBS). After 2 min incubation, cells were washed twice with PBS (5 min each) and visualized under a fluorescence microscope (Zeiss Axio star plus, USA) at 400× magnification with excitation filter 480/30 nm. Three inde- pendent cell counts (counting a minimum of 100 total cells each) were obtained on the basis of differential staining of the nuclei (live cells have a normal green nucleus; early apoptotic cells have bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; necrotic cells display a structurally normal orange nucleus). These results were supported by confocal laser scanning microscopy (CLSM). For this cells were mounted with antifade mounting medium (Chemicon, USA) and observed (at 400× magnification) under confocal laser scanning microscope (CLSM 510, version 2.01; Zeiss, USA) after excitation at 488 nm. Green fluorescence was detected with a band-pass filter ranging between 505 and 530 nm. Simultaneously, red fluorescence was detected using the long pass filter at 585 nm, and superimposition of both green and red fluorescence generated the final images. All the above-mentioned assays were carried out three times, independently. The data obtained were expressed in terms of “mean ( standard deviation” values. Wherever appropriate, the data were also subjected to unpaired two tailed Student’s t test. A value of p < 0.05 was considered as significant. Preparation and Formulation of SNP Containing Gel (S-Gel). In order to use SNP in the form of a topical hydrophilic formulation, two Carbopol based gel formula- tions, viz., containing final silver concentrations of 0.02 mg/g and 0.1 mg/g, respectively, were prepared. These gel formulations, designated as S-gel, were packaged under sterile conditions, labeled with appropriate details and stored at room temperature for further use. In Vitro Antibacterial Activity of S-Gel. A standardized antimicrobial sensitivity test was used to evaluate the antibacterial activity of S-gel against standard bacterial cultures and two representative MDR strains. The test was carried out in Muller-Hinton agar plates according to NCCLS guidelines. For comparison, gel without SNP and 1% silver sulfadiazine (Solvay Pharma, India) was used. Assessment of Toxicity of S-Gel: In Vivo Study. Acute dermal toxicity study was performed following the Organiza- tion for Economic Cooperation and Development (OECD) Guidelines for Testing of Chemicals, Section 4, No. 402 L. Sprague-Dawley rats (5 male and 5 female rats, 12 to 14 weeks old, weighing between 203 and 244 g with healthy intact skin and acclimatized to laboratory conditions for 7 days) were used. Approximately 24 h before application, the hair of each rat was closely clipped with an electric clipper to expose the back from the scapular to the lumbar region. S-gel (0.1 mg/g) was applied uniformly at the dose level of 2000 mg/kg and held in contact with the skin with a porous gauze patch. After the exposure period of 24 h, observations on mortality, intoxication, body weight, and necropsy were recorded for a 14 day period. Results The silver nanoparticles synthesized by the patented process showed a characteristic surface plasmon peak, at 436 nm (Figure 1). Further, the nanoparticles were spherical and in the size range of 7-20 nm (Figure 2) as revealed by high resolution transmission electron microscopy (HRTEM). The data on particle size distribution revealed the presence of particles in the size range of 6.5 to 43.8 nm, with an average size 16.6 nm. The nanoparticles were found to be stable for over two months. Results of susceptibility tests with SNP against reference strains are summarized in Table 1. It could be seen that SNP displayed potent antimicrobial activity against both Gram- positive and Gram-negative organisms. The MICs were in the range 1.56-6.25 μg/mL for all the cultures while the MBCs were 12.5 μg/mL with the exception of S. aureus ATCC 6538P. In the latter case, MBC could not be (14) (15) Saldak, J.; Lindsay, R. H. Estimation of total, protein bound and non-protein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 1968, 25, 192–205. Beuge, J. A.; Aust, A. D. Microsomal lipid peroxidation. Methods Enzymol. 1978, 52, 302–310. 1392 MOLECULAR PHARMACEUTICS VOL. 6, NO. 5PDF Image | Silver Nanoparticles in Therapeutics: Antimicrobial Gel
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