br Synthesis and characterization of

2.2. Synthesis and characterization of nanoparticles
Nanoparticles (MgONPs and Ag-MgONPs) were synthesized using the MCFE of strain SKCGW013. For the preparation of MCFE, the strain SKCGW013 was grown in a 250 mL Erlenmeyer ßask containing 100 mL of potato dextrose broth (PDB) in a shaker with agitation of 180 rpm at 28 LC for 4 days. Mycelial biomass was col-lected by Þltration using Whatman No.1 Þlter paper and then washed with sterile distilled water to remove the excess medium residues. A 20 g of wet weight of mycelial biomass was dissolved in 100 mL of Milli-Q deionized water and stirred for 60 min at 180 rpm in 28 LC using a shaker, then Þltered again using the Whatman No.1 Þlter paper. For the synthesis of MgONPs, the 2, 5 and 10 mM of Mg(NO3)2 6H2O was dissolved in 100 mL of MCFE in 250 mL ßask and the solution was kept at 40 LC under darkness for 24 h then the NaOH solution was adding dropwise [37]. For the synthesis of Ag-MgONPs, 2, 5 and 10 mM of AgNO3 and 2, 5 and 10 mM of Mg (NO3)2 6H2O were dissolved in 100 mL of MCFE incu-bated in at 40 LC under darkness for 24 h. The both of synthesized NPs were prepared for the characterization according to the meth-ods described previously [17] and studied by a high throughput techniques including FTIR (PerkinElmer Paragon 500, USA), XPS (Thermo ScientiÞcTM K-AlphaTM), XRD (XÕpert-pro MPD- PANalytical, Netherland), operated at 40 keV with Cu ja radiation in h-2h, FESEM with EDS (S-4300/HITACHI), TEM with EDS (JEOL-JSM 1200EX, Japan). The particle size was analyzed using PSA (Malvern Mastersizer 2000, Britain).
2.3. Cytotoxicity assay
MgONPs or Ag-MgONPs induced cytotoxicity in the PC-3 cells were investigated using a rapid WST assay kit. In brief, the PC-3 cells were cultured in 5 mL of RPMI 1640 incorporated with 10% of FBS and 1% of SB 431542 solutions in a T25 ßask in humidiÞed 5% of CO2 incubator for 24 h. After reaching 80Ð90% conßuence, the cells were harvested by trypsinization, followed by these healthy cells (2 104 cells. mL 1) were dissolved in RPMI 1640 and seeded into 96 well plates (coster) as 1 mL per well. Then the 96 well plates were kept in 5% of CO2 incubator for 24 h to allow the cells to attach in the bottom of the well. After 24 h, the cells were washed with PBS and replaced RPMI1640 medium con-  taining different concentrations (0Ð1000 mg mL 1) of MgONPs or Ag-MgONPs and further incubated for 24 h in 5% of CO2 incubator. Finally, the cytotoxicity was measured using the EZ-CYTOX indica-tor according to the manufacturerÕs instructions. The cells were counted by trypan blue dye exclusion method using a hemocy-tometer under a bright Þeld microscope (Olympus, CKX53 culture microscope, Japan).
The nuclear changes and apoptotic body formation of the PC-3 cells were visualized using the ßuorescence microscope [38]. The NPs treated or untreated PC-3 cells were harvested and washed with the PBS. Then, the 25 mL of cell suspension (0.5 106 - cells mL 1) were incubated with 1 mL of 1:1 of AO and EB for 5 min. The cells were again washed with PBS several times to remove the residual stain then visualized under the ßuorescence microscope (Olympus, CKX53 culture microscope, Japan).
2.5. Determination of ROS generation
The ROS generation was measured in PC-3 cells by DCFH-DA staining method according to the method described earlier [39] with slight modiÞcations. In brief, the PC-3 cells (2 104 - cells mL 1) were seeded in 30 mm confocal glass bottom dish (SPL life sciences, Republic of Korea) containing RPMI 1640 med-ium and allowed to attach the cells. For the NPs treatment, after the 24 h, the cells were washed with PBS and medium replaced with NPs (IC50 concentration) incorporated medium and incubated for 24 h. After the incubation period, the cells were again washed with cold PBS and added the DCFH-DA (50 mm). Then it was incu-bated for 30 min at 37 LC. Finally, the cells washed with the PBS and maintained in 1 mL of PBS. The ROS production assessed using the ßuorescence microscope with excitation of 488 nm and emis-sion of 530 nm.