br Several previous studies reported that the microbial or
Several previous studies reported that the microbial or plants extracts derived compounds such as carbohydrates, proteins, triterpenoids, steroids, and ßavonoids are act as capping or reduc-ing agent for the synthesis of the metallic NPs and Trichoderma
Fig. 3. FTIR analysis of functional group modiÞcations in cell free extract (MCFE), magnesium oxide nanoparticles (MgONPs), and silver coated magnesium oxide nanoparticles (Ag- MgONPs).
strains are able to produce the variety of novel metabolites may involve in synthesis of NPs [14,43,48,49]. The formation of the Ag-MgONPs was conÞrmed by color changes from pale yellow to brown and the MgONPs was conÞrmed through the observation of precipitations (Supplementary Fig. 1). The surface morphology of synthesized MgONPs was visualized using the FESEM and the
results showed the agglomeration colloids and porous organiza-tion, spherical in shape and crystal nature of MgO NPs (Fig. 1a), and it is agreement with earlier reports [42,50,51]. Followed by SEM-EDS studies indicated the presence of the Mg and O forms, it further conÞrms the effective synthesis of the MgONPs (Supple-mentary Fig. 2a and b). On the other hand, FESEM images showed the colloidal agglomeration of nano-sized and subsequent forma-tion of the spherical and oval shaped of Ag-MgONPs (Supplemen-tary Fig. 3a). These FESEM results are in accordance with morphological characteristics of sol-gel based generation of Ag-doped MgONPs  with modiÞcations due to the capping of MCFE. The SEM-EDS for Ag-MgONPs indicated the presence of the Ag, Mg and O forms in the synthesized materials (Supplemen-tary Fig. 3b and c). Fig. 1(aÐe) shows the TEM images, EDS map-ping, and the chromatogram of MgONPs synthesized by MCFE. The results presented the needle-shaped MgONPs crystals, and it is in accordance with an earlier report . The EDS results showed the presence of the Mg and O in the specimen of MgONPs, which is agreement with SEM-EDS study. Followed by the TEM-EDS results of Ag-MgONPs showed the hexagonal and spherical structured nanocrystals (Fig. 2a and b). The EDS results conÞrm the existence of Ag, Mg, and O in mapping as well as in chro-matogram were accordance with SEM-EDS (Fig. 2cÐe). The PSA results revealed the average size of 15.09 nm for Ag-MgONPs and 13.68 nm for MgONPs (Supplementary Fig. 4a,b). The higher size of Ag-MgONPs was attributed due to the capping of Ag on the sur-face of the MgONPs. r> In the present study, FTIR analysis was attributed to Þnding out the functional Phosal 50 PG from MCFE which responsible for the
the increased interplanar spacing of Ag-MgONPs at diffraction indexed (1 1 1) (Supplementary Fig. 5), which was a strong evi-dence for Ag embedded in MgO [36,53].
Fig. 5. Effect of MgONPs and Ag-MgONPs treatments on PC3 cells. bright Þeld microscopic observation of morphological changes (aÐc), live and dead cells staining by AO/EB (dÐf), ROS generation (gÐi), observation of nucleolus damage by DAPI staining (jÐl).
Fig. 6. Flow cytometer-based determination of apoptosis stages in PC3 cells untreated (a) treated with MgONPs (b) and Ag-MgONPs (c); upper left-necrosis cells; upper right-dead cells; Lower left-live cells; Lower right- apoptosis cells.
3.4. Cytotoxicity assay
Cytotoxicity of these two metallic NPs was tested against PC-3 cells using WST assay. The cell viability was signiÞcantly decreased with the decrease of the NPs concentration (Supplementary Fig. 7). The calculated inhibitory concentration (IC50) was 125 mg mL 1 for Ag-MgNPs and 218.75 mg mL 1 for MgONPs. This results demon-strated the Ag-MgNPs induced the more cell death than the MgONPs due to the presence of the silver ions which reported as a cytotoxic agent against various cancer cells lines such as HT 29 cells , A549, Hep2, HeLa, COLO 205 and SH-SY5Y . More-over few earlier studies are indicated that the silver-doped NPs easily penetrate through the cell membrane and induce the free radicals, ROS production and trigger the nucleus damage . Hence in the present study, we further studied the effect of IC50 concentrations of these two NPs on cell death by AO/EB staining, ROS generation followed by nucleus damage by DAPI staining. The results exhibited that treatment of Ag-MgONPs induces the more apoptosis necrosis (Fig. 5aÐf), the ROS production (Fig. 5gÐ
i) cellular and nuclear membrane damage than the MgONPs (Fig. 5jÐl). Flow cytometer results demonstrated that the necrosis cells (4.51%), dead cells (3.15%), live cells (90.56%), apoptosis cells (1.78%) in untreated cells (Fig. 6a). The Ag-MgONPs treated showed the necrosis cells (48.67%), dead cells (7.3%), live cells (33.16%), apoptosis cells (11.14%) (Fig. 6b). For MgONPs treated showed the necrosis cells (8.22%), dead cells (1.86%), live cells (88.94.16%); apoptosis cells (0.98%) (Fig. 6c). These results revealed a higher range of necrosis and dead cells in Ag-MgONPs treated one than the MgONPs treated, which agrees with all the cytotoxicity related assay of this study. It is known that the MgONPs induce active oxygen, reactive oxygen species [16Ð18] and AgNPs ions penetrate through the cancer cell membrane and stop the meta-bolic activity . Therefore the Ag-embedded MgONPs stronger in inducing the cell death through the interactions with cancer molecules such as proteins, DNA and block the replication of can-cer cells through ROS production and activating nucleus damage related apoptosis signaling pathway [59Ð62].