Materials Science Engineering C br Preparation of AgNPS
Materials Science & Engineering C 102 (2019) 254–263
2.3. Preparation of AgNPS
About 1 mM silver nitrate (AgNO3 Sigma- Aldrich) solution was added drop wise to the bacterial supernatant with constant stirring by using glass rod. After this, the solution was kept on a shaker for 24 h at 125 rmp for the complete reduction of silver ions that can be visualized by color alteration of the bacterial extract.
2.4. Preparation of AgNPs coated with BSA
The BSA (purchased from Sigma- Aldrich) solution was prepared by using deionized milli –Q water (1% W/V). The 1000 μL of BSA (1% W/ V) solution was added dropwise to the AgNPs solution under constant stirring to 100 mL of silver nanoparticle solution with gentle stirring. The whole solution was transferred to the orbital shaker and kept for 12 h under room temperature at 120 rpm. For this, the methodology was adopted from Cristobal et al. 2015 with slight modification .
2.5. Characterization of AgNPs and capped AgNPs(cAgNPs)
The color change upon addition of AgNO3 solution to the cell-free filtrate was observed by the physical appearance of the solution. These AgNPs and cAgNPs were further characterized to determine the lambda max by UV visible spectroscopy. The FTIR -ATR was used to determine the various proteins involved in the stabilization and reduction of AgNPs and cAgNPs. The FESEM and EDX were used to determine the surface morphology and purity of the AgNPs. The TEM analysis was used to determine the shape, size, and dispersity of AgNPs and cAgNPs. The TGA was used to determine the actual protein content that lost at different temperatures for AgNPs and cAgNPs.
2.6. Stability study of cAgNPs
The prepared cAgNPs were subjected to stability tests. The cAgNPs were incubated for two months at 4 °C. After two months stability was analyzed by UV–vis spectrophotometry and FTIR.
2.7. Anticancer activities of AgNPs and cAgNPs
The breast cancer MCF-7, colon cancer HCT-116, osteosarcoma MG-
63 cell line and normal Prostaglandin E2 3T3 (skin cells) cell lines were pro-cured from national cell line studies, Pune, India. All the four cell lines were grown on minimal essential medium (MEM) as monolayer con-taining 1% (V/V) glutamine, fetal bovine serum (FBS)10% (V/V), pe-nicillin 100 units/mL−1 at 37 °C, streptomycin 100 units/mL−1 (Sigma Aldrich USA) under 5% carbon dioxide environment.
2.7.2. Analysis of cell viability
The cAgNPs were analyzed for its cytotoxic activities against breast cancer MCF-7, colon cancer HCT-116, osteosarcoma MG-63 cell line and normal fibroblast 3T3 cell lines by MTT 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide, tetrazolium) assay. The cAgNPs were mixed and dissolved in double distilled water (ddH2O) with dif-ferent AgNPs and cAgNPs concentration were used from 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, 100 μg/mL, 120 μg/mL, 140 μg/mL and 160 μg/mL to cell culture which were grown on microplates con-taining 96 well and incubate at 37 o C for 24 h. After the growth of cells on the plates were later washed with phosphate buffer solution (PBS) followed by the addition of 100 mL of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide, tetrazolium MTT solution to all 96 wells and further kept it for incubation at 37 °C for 2 to 3 h. Later 100 μL dimethyl sulphoxide (DMSO) was gently added to all wells so that the crystals formed by using MTT dye should solubilize. The whole plate was later transferred on to the orbital shaker for 10 to 15 min in order to properly solubilize the crystals of cancer and normal cells which can
Fig. 1. UV spectrophotometric analysis (A) Bacterial Extract, (B) AgNPs showed peak at 415 nm, (C) AgNPs with BSA showed absorption peak at 420 nm.
be observed by the production of color. The viability of cells were measured by spectrophotometric analysis at 570 nm using the following formula:
Percentage of cell viability (%) = Optical density (OD) of cAgNPs Optical density (OD) of control
The cytotoxic effect of cAgNPs an AgNPs on cancer and normal cells can be calculated by the amount of drug that inhibits/kills 50% of cells and compared with non-treated cells as an IC50 value. Above experi-mentation was performed three times with DMSO as negative control.
Acridine orange (AO)/ethidium bromide (EB) dual staining for apoptosis:
The AO/EB (Sigma Aldrich) dual staining was used to determine the morphological changes that occur to the cells induced by cAgNPs in HCT-116, MCF, and MG-63 cells. To analyze apoptosis in the cells of HCT-116, MCF, and MG-63; the cells were separately plated at 5 × 104 cells/well in the chamber of a six-well plates. At > 90% confluence interval rate, the cells were treated with the required concentration of cAgNPs and incubate for 24 h. The incubated cells were washed with PBS which were fixed in methanol: acetic acid (3:1, v/v) and kept for 10 min. To analyze the mode of cell death, the cancer cells were treated with IC50 concentration of cAgNPs seeded in six-well plates. For the determination of nuclear analysis, the monolayer of cells were washed with phosphate buffer saline and stained with 5 μL of ethidium bromide (100 μg/mL) and 5 μL acridine orange (AO) (100 μg/mL) The change in the morphological change in the stained cells (apoptotic nuclei) were observed/analyzed by using fluorescent microscopy.