Mycosynthesis of Silver Nanoparticles: Optimization, Characterization and Their Antimicrobial and Cytotoxic Activity

Abstract

Silver nanoparticles (AgNPs) have been synthesized in the past by varying physical and chemical approaches that were neither ecologically safe or economical besides being time consuming. Therefore silver nanoparticles (extracellular) were devised by employing fungi taking all the issues into account. Henceforward, in the present study Aspergillus fumigatus BTCB10 (KY486782) was opted for the mycosynthesis of AgNPs after extensive screening. Silver nanoparticles were further optimized by using a classical method of one factor at a time methodology along with more recent approach Research Surface Methodology (RSM) through central composite design (CCD). Various variables were investigated for producing efficient and size controlled silver nanoparticles comprising of temperature (ºC), light/dark conditions, substrate concentration (mM), biomass weight (g), fungal culture age (days), pH, ratio of cell free filtrate (CFF)/silver nitrate, metal salts (%), surfactants and enhancer, nitrogen sources, carbon sources and enzymes. The most effective parameters were then selected for RSM study which contained concentration of silver nitrate (mM), NaCl (%) and the wet weight of biomass (g). According to the results of one factor design a very stable (-23.4 mV) small size of 0.681 nm AgNPs were obtained in the presence of these conditions i.e. incubation in dark, temperature 25 ºC, AgNO3 concentration 1mM, biomass 7g, fungal culture age 7 days, pH 6, ratio of cell free filtrate (CFF)/silver nitrate (3:2), NaCl 20%. UV-Vis band showed presence of peak at 400 nm and Fourier Transform Infrared (ATR-FTIR) confirmed presence of main functional groups (O-H, C=O). Moreover, Scanning Electron Microscopy (SEM) revealed cubeshaped AgNPs and X-Ray Diffraction (XRD) verified crystalline nature of AgNPs. The RSM studies also reduced the size of silver nanoparticles to 33.23 nm (spherical) with negative zeta potential of -9.91 mV, excitation peak at 420 nm and comprised of wide range of functional groups (O-H, C=C, C≡C, C-Br, C-Cl). A significant F- value (3.91), nonsignificant lack of fit and determination coefficient (R2=0.7786) were also calculated within the study. A tremendous size reduction was observed by one factor design (99.79 %) and RSM (78.7%) compared to nanoparticles produced prior to optimization having an average size of 356 nm. High nitrate reductase activity with value of 179.15 nmol/h/ml was attained; thus indicating a possible relation between enzyme production and AgNPs synthesis. Additionally, antibacterial activity was also monitored with varying concentrations 0.1-7 μg/mL of AgNPs against Pseudomonas aeruginosa BTCB01, Staphylococcus aureus BTCB 02, Escherichia coli BTCB03, Klebsiella pneumoniae BTCB04 and Acientobacter BTCB05. Maximum zone of inhibition was observed with Klebsiella pneumoniae BTCB04 (33mm) at 7 μg/mL and minimum with Pseudomonas aeruginosa BTCB01 and Staphylococcus aureus BTCB 02 (11mm) at 0.1 μg/mL. Combinatorial effect with streptomycin (1 μgmL-1) was also studied with all the strains, Klebsiella pneumoniae BTCB04 showed the maximum zone of 25 mm whereas the minimum zone was observed with Pseudomonas aeruginosa BTCB01 at 11 mm. Lastly MTT assay showed no toxicity against cancer cells (HepG2) with AgNPs (0.25-3.5 μM) attaining highest cell viability of 108.5 % with 3.5 μM, whilst Cisplatin was used as a control to complete the test. Cisplatin alone showed viability of 76.3% at 150 μM and 117 % at 100 μM. Synergistic effect between AgNPs and Cisplatin was also investigated, henceforth lowest cell viability of 44.89% was observed with 1:19 combination. In conclusion both of the methods applied for producing silver nanoparticles proved to be successful in reducing the size of AgNPs by various optimization parameters. Although a very small size was obtained by applying conventional one factor at a time method however RSM methodology reduced the size of AgNPs with only 3 parameters which not only lowered the overall cost but labour and time for the experiments. Antimicrobial activity and MTT assay showed significant results and the synthesized AgNPs that can be used in biomedical field in future as they are safe and economic and dependable.