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Global Translational Medicine ZnO NPs induce apoptosis in MG63 cells

the resulting residue was obtained and completely dried 2.7. Cytotoxicity of ZnO NPs on MG63 cells
for further analysis. The cytotoxic effect of produced ZnO NPs on MG63 and

2.3. Ultraviolet (UV)-vis spectra and size distribution Vero cells was measured by MTT assay. The MG63 and Vero
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analyses cells were inoculated (1 × 10 cells/well) in a 96-well plate
and cultivated for 24 h in a humified environment. Except
After being harvested by centrifugation, the ZnO NPs for control, the cells were introduced to increasing dosages
were examined using UV-vis spectral analysis. The UV-vis of ZnO NPs for 24 h. Further, the cells were subsequently
spectrophotometer was used to record a spectral reading exposed to MTT (100 µL, 5.0 mg/mL in PBS) for 4 h in
of amalgamated ZnO NPs at 300 – 700 nm wavelength dark conditions to form formazan crystals. The resulting
(Hitachi, model U-2800). A light scattering crystallite size formazans in the wells were diluted by 100 µL of DMSO.
analyzer was used to explore the size and distribution of The optical density value of the well was scrutinized by a
the prepared nanoparticles. After ultrasonication, ZnO microplate reader (Tecan Multimode Reader, Austria).
NPs were placed into a sample stage, and then the size and Regression analysis was used to determine the ZnO NP
its distribution were estimated using a computer-based concentrations.
dynamic light scattering analyzer.
2.8. Biochemical assays
2.4. Transmission electron microscopy (TEM) and The biochemical assays for lipid peroxidation (LPO),
elemental analyses superoxide dismutase (SOD), catalase (CAT), and
The produced ZnO NPs were mixed in water, which was glutathione peroxidase (GPx) were conducted to analyze
deionized previously, and one drop of the mixture was the LPO and endogenous antioxidant activity in ZnO
placed on the sample stage and dried under a vacuum NPs-treated MG63 cells. The cell suspension was obtained
before being coated with carbon. Tecnai G-10, an 80kV by trypsinization after treatment and processed for
transmission electron microscope, was used to obtain biochemical studies. The LPO biomarker, thiobarbituric
micrographs of carbon-coated nanoparticles. The produced acid reactive substances (TBARS) activity was assessed
[13]
ZnO NPs were deposited on a section of micro-glass slip by adapting the procedure suggested previously . The
closed by a carbon layer for energy dispersive X-ray (EDX) antioxidant activity in MG63 cells was determined by
studies, and then permitted to dehydrate at 37°C. The quantifying the antioxidant markers SOD, CAT, and GPx
mimicked elements were studied using the spectrum of by adapting the aforementioned protocols [14-16] . The cells
EDX (BrukerAXS Incorporation., USA). that remained untreated were designated as control.
2.5. X-ray crystallography (XRD) and Fourier- 2.9. Determination of ROS
transform infrared spectroscopy (FTIR) The ZnO NPs-induced ROS generation in MG63 cells was
examinations determined by staining the cells with DCFH-DA, which was
then oxidized into fluorescent dichlorofluorescein (DCF).
The ZnO NPs were thoroughly dehydrated for XRD The cells were inoculated and grown (1 × 10 cells) for 24 h
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analysis before being bonded in an XRD condition by in six-well plate. After treatment with 15, 30, and 45 µg/mL
the prominent 30 kV at 20 mA between and radiation of ZnO NPs for 24 h, the cells were administered 100 µL of
through an XRD analyzer (Philips Model PW1252/36). DCFH-DA for 10 min under dark conditions, then the cells
The scanning temperature range was set from 20°C to were observed under a fluorescence microscope equipped
80°C. FTIR measurements were used to investigate the with appropriate filters. The cells were trypsinized and the
surface functional group of chemicals coupled with phyto fluorescence intensity of the cell suspension was estimated
produced ZnO NPs (Shimadzu 8201PC, Japan). The sample using a spectrofluorometer (Shimadzu RF-5301 PC). The
was mixed with 10 mL of deionized water and examined in untreated wells served as a control group.
KBr pellets using FT-IR with a resolution of 4 cm .
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2.10. Examination of apoptotic morphological
2.6. Cell culture changes
The osteosarcoma MG63 and normal Vero cell lines were The ZnO NPs-mediated apoptotic sign represents the
obtained from ATCC. The cells were cultivated in DMEM hallmark of morphological alterations in MG63 cells and
37°C in a 5% CO and 95% air environment (humidified this sign was confirmed by staining with AO and EtBr .
[17]
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incubation). The S. xanthocarpum-stabilized ZnO NPs The cells were inoculated in six-well plate and incubated in
were dissolved in dimethyl sulfoxide (DMSO) before being humified incubator, and treated with various doses (15, 30,
treated with the cells. 45 µg/mL) of ZnO NPs for 24 h under the same humified

Volume 1 Issue 1 (2022) 3 https://doi.org/10.36922/gtm.v1i1.34