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  • taining different concentrations mg mL of MgONPs or Ag MgONP

    2020-08-07

     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 Clozapine-N-oxide 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.
    2.6. Cell nuclear morphology
    DAPI staining was applied to study the cell nucleus morphology according to the methods described elsewhere [38] with modiÞca-tions. The PC-3 cells were grown in the 30 mm confocal glass bot-tom dish containing the RPMI 1640 medium for 24 h and then it was washed with PBS. Then, the cells were treated with an IC50 concentration of NPs samples for 24 h. At the end of the treat-ments, the cells were again washed with PBS then Þxed in 4% formaldehyde for 4 min at ambient temperature. For the DAPI staining 300 mL of the 300 nM of DAPI solution was laid on the cells for 5 min at room temperature in dark then it was washed again with PBS for several times to remove the residual dye and observed under using the ßuorescence microscope with excitation of 358 nm and emission of 461 nm.
    2.7. Flow cytometer assay
    The NPs induced apoptosis was determined using the FITC Annexin V/Dead cell apoptosis kit according to the manufacturer instructions using the ßow cytometer (BD FACS Calibur, BD USA). In brief, the PC-3 cells (1 104 cells mL 1) were grown in the 6 well plates (coster) for 24 h in humidiÞed 5% of CO2 incubator. After reaching the 80% of conßuence the cells were washed with PBS, the medium was replaced with NPs (IC50) incorporated med-ium and incubated for 24 h then the cells were harvested for the ßow cytometer analysis.
    2.8. Statistical analysis
    Experiments were performed three times and the descriptive statistics, one-way ANOVA (p < 0.05; p < 0.01) and post hoc test was done by use of the statistical software SPSS version 16.0 (SPSS, Chicago, IL). The results were shown as mean ± standard error.
    3. Results and discussion
    Biogenic NPs are signiÞcantly used in biomedical, food and chemical industrial applications due to their remarkable properties [40,41]. Green synthesized unaccompanied AgNPs and MgONPs are reportedly cause the cytotoxicity and antimicrobial effect [17,42] but there is no work describing accompanied biogenic
    Fig. 1. Transmission electron microscopic imaging of MgONPs (a), Electron image for EDS mapping (b), EDS layered image (c), EDS mapping of Mg (d) Energy-dispersive X-ray spectroscopy analysis (e).
    Fig. 2. Transmission electron microscopic imaging of Ag-MgONPs (a), Electron image for EDS mapping (b), EDS layered image (c), EDS mapping of Mg (d) Energy-dispersive X-ray spectroscopy analysis (e).
    Ag-MgONPs on cytotoxicity and antimicrobial effects. Although a study reports the sol-gel preparation of Ag-doped MgONPs and their antibacterial activity, its synergetic cytotoxic effects against cancer cells are unknown [36]. Trichoderma strains are a renowned source for various bioactive enzymes and metabolites with thera-peutic activities against various pathogens and diseases [43Ð45]. Moreover, its cell-free extracts are known to synthesise the silver and chitosan NPs [14,46,47]. Furthermore, this extracts-based fabrication of Ag-MgONPs are not reported. Herein we used the 
    Trichoderma MCFE for the Þrst time to synthesise the Ag-MgONPs in an eco-friendly manner for enhanced cancer cell ablation.
    3.1. Synthesis and characterization of nanoparticles