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articles Jain et al. corresponding to MIC and MBC for the respective cultures) and incubated for 8 h. Aliquots (0.1 mL) were removed at hourly intervals, serially diluted, and total viable counts (TVC) on MH agar plates were determined after incubation at 37 °C for 24 h. Kill curves were constructed by plotting the log CFU against time. The post agent effect (PAE) was estimated according to a method described by Craig and Gudmundson.8 One milliliter saline suspensions of standard bacterial strains and two representative MDR isolates (1 × 107 CFU/mL) were exposed to SNP (at concentrations 10 times MIC of respec- tive strains) and incubated at 37 °C for 1 h. At the end of the exposure period, cultures were centrifuged (3000g for 5 min) and diluted 1:1000 in nutrient broth. TVC before SNP exposure and immediately after dilution (zero time), and at hourly intervals until tube turbidity reached a No. 1 McFar- land standard, were then determined. Growth controls with inoculum but without SNP were included with each experi- ment. PAE was defined according to the formula PAE ) T - C, where T is the time required for viability count of an SNP exposed culture to increase by 1 log10 unit above counts taken immediately after dilution and C is the corresponding time for the growth control. The fractional inhibitory concentration index (FICi) for combination of SNP and commonly used antibiotics (i.e., streptomycin, chloramphenicol, kanamycin, polymyxin B sulfate, ampiclox and ceftazidime) was determined using Pseudomonas aeruginosa ATCC 9027. MIC for each of the antibiotics was first estimated, and subsequently, the frac- tional inhibitory concentration of a combination of drug and SNP was determined by the checkerboard micro titration method8 in a 96-well microtiter plate using MH broth. The plates were incubated at 37 °C for 18 h, and results were recorded visually as growth/no growth. The FIC was calculated as follows: MICA in combination FIC*A ) MIC A FIC*B ) MICB in combination MICB where A ) antibiotic and B ) SNP FICi ) FICA + FICB The interaction was defined as synergistic if the FICi was e0.5, as additive if the FICi was >0.5 to 1.0, as indifferent if the FICi was >1.0 to 2.0, and as antagonistic if the FICi was >2.0. Antifungal ActiVity of SNP: MIC, Minimum Fungicidal Concentration (MFC), Time Kill Study and PAE. To assess the antifungal activity of SNP, a laboratory isolate of Aspergillus niger was used. The assay was carried out in 100 mm × 15 mm Petri plates containing 4 mL of potato (8) Craig, W. A.; Gudmundsson, S. The postantibiotic effect. In Antibiotics in laboratory medicine, 4th ed.; Lorian, V. Ed., Williams and Wilkins Co.: Baltimore, MD, 1996. dextrose agar (PDA) supplemented with three different doses of SNP (50, 100, and 200 μg/mL). A. niger was spot inoculated on PDA plates, which were then incubated at 28 °C for 72 h. The diameter of the mycelial colony developing on the SNP containing plates was measured and compared with the diameter of the colony obtained on control plates (without SNP). The inhibition of fungal growth was calcu- lated as follows: antifungal index (%) ) (1 - D /D ) × 100 ab where Da is the diameter of the growth zone in the test plates and Db is the diameter of growth zone in the control plate. In another experiment, the minimum inhibitory concentra- tion (MIC) of SNP for C. albicans ATCC 2091 was determined in 96-well microtiter plates containing 200 μL of YES (yeast extract 0.5 g% and glucose 1 g%, pH 5.6) broth supplemented with SNP (0.78-50 μg/mL) at an initial inoculum density of 2 × 104 CFU/mL. The microtiter plates were incubated at 37 °C and were scored visually for growth/ no growth after 24 h. For time kill study, C. albicans was inoculated (final cell density of 2 × 104 CFU/mL) in 2 mL of YES broth supplemented with appropriate amounts of SNP (at concen- tration corresponding to MIC and MFC) according to the procedure outlined above. The PAE was determined by the viable plate count method as described earlier for the bacterial strains. Matrix Metalloproteinases (MMP) Inhibition: In Vitro Studies. The gelatinase inhibition test was conducted using a kit based assay (Colorimetric Assay Kit for Drug DiscoverysAK-410-A/AK-408-A BIOMOL QuantiZyme Assay System, USA) in a 96-well microtiter plate as per the manufacturer’s instructions. Different concentrations of SNP (6.25-100 μg/mL) were added to an assay mixture contain- ing MMP enzyme (MMP-2 or MMP-9; 9 mU/μL). The plates were incubated at 37 °C for 30 min to allow enzyme inhibitor reaction, and then appropriately diluted (in assay buffer) colorimetric substrate (Ac-PLG-[2-mercapto-4-methyl-pen- tanoyl]-LGOC2H5; 100 μM) was added and plates were read at 412 nm in a microplate reader (μQuant, BioTek Instru- ments, USA). The activity of the enzyme in the presence of SNP was compared to baseline activity (no inhibitor). The assay was also conducted in the presence of an inhibitor (1.3 μM NNGH, a prototype control inhibitor provided with the kit), which served as positive control. In Vitro Cytotoxicity and Biochemical Mechanisms Involved in Interactions of SNP with Liver Cells. Hep G2 (human hepatocellular carcinoma) cells were obtained from National Center for Cell Sciences (NCCS), Pune, India. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with L-glutamine (4 mM), penicillin (100 units/ mL), streptomycin (100 μg/mL) and 10% (v/v) heat inacti- vated fetal bovine serum (growth medium). Cells were maintained in 5% CO2 humidified incubator at 37 °C. During subculture, cells were detached by trypsinization when they reached 80% confluency and split (1:4). Growth medium was changed every 3 days. 1390 MOLECULAR PHARMACEUTICS VOL. 6, NO. 5PDF Image | Silver Nanoparticles in Therapeutics: Antimicrobial Gel
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