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Global Translational Medicine ZnO NPs induce apoptosis in MG63 cells

conditions. Further, the cells were exposed to a 1:1 ratio for 1 h at 37 °C. The caspase expressions were examined by
of AO/EB for 30 min and then gently splashed with PBS. detecting the optical density value at 405 nm wavelength
Then, the cells were observed under the fluorescence using a spectrophotometric plate reader (BioRad, Tokyo,
microscope (Olympus, Japan). Japan), in adherence with steps described by Huang
et al. The assays were conducted in three independent
[18]
2.11. DAPI staining experiments.
The ZnO NPs-induced nuclear condensation in MG63 cells
were assessed by staining the cell with a DAPI stain. The 2.14. Western blot analysis
cells were inoculated (1 × 10 cells) and seeded in six-well The cell suspension was obtained by trypsinization after a
5
plates for 24 h. Then, the cells were exposed to 15, 30, 24-h treatment with ZnO NPs. The collected suspension
and 45 µg/mL of ZnO NPs for 24 h. The cells were gently was processed for centrifugation and the pellet was
washed and fixed with paraformaldehyde before being collected. After rinsing with cold PBS, the pellet was
cleaned and fixed again with 70% ethanol. After that, the lysed using RIPA lysis buffer (Pierce Biotechnology, IL,
cells were exposed to DAPI (1 mg/mL) and kept for 20 min USA). The protein separation was achieved on a 10%
in the dark. The treated ZnO NPs and untreated control SDS-PAGE and then transferred onto a polyvinylidene
cells were observed under the fluorescence microscope for difluoride membrane. After that, the membrane was
nuclear condensation. blocked with 5% bovine serum albumin for 2 h at room
temperature. Monoclonal primary antibodies (p53,
2.12. Determination of DNA damage (comet assay)
Bcl-2, Bax, P13K, AKT, mTOR, LC3, beclin-1, P62,
The gel electrophoresis method was used to determine and β-actin 1:1000) were used to probe the membrane
the ZnO NPs-induced DNA damage in MG63 cells. The overnight at 4°C. The probed membrane was treated for
cells were obtained after administration with various 1 h with secondary antibodies that were horseradish
doses (15, 30, and 45 µg/mL) of ZnO NPs. Then, the peroxidase-conjugated. By following the manufacturer’s
harvested cell suspension was transferred into a 1× instructions, the protein bands were visualized using
PBS solution. After that, 10 mL of the suspensions a chemiluminescence detection ECL kit (Amersham
were transferred to low melting 0.5 % agarose (60 mL), Biosciences, Buckinghamshire, UK).
which was then laden into slides and allowed to solidify
correctly. The slides were submerged in ice-cold lysis 2.15. Statistical analysis
buffer for 1 h at 4°C after full solidification and then The mean ± standard deviation of three replicates was
left for DNA to loosen up in electrophoresis solution used to display all results. One-way ANOVA analysis was
for 30 min. The electrophoresis setup was operated at a used to analyze the significant differences between various
continuous voltage of 22 V and 200 mA. Using 0.4 M Tris groups using GraphPad Prism 5. P < 0.05, P < 0.01, and
(pH 7.5), the slides were neutralized for 10 min, and then P < 0.001 were considered as statistically significance
fixed using ethanol (70%). Further, slides were exposed between different groups.
to 0.5 mg/mL of ethidium bromide for 20 min in a
dark room. The epifluorescence microscope captured 3. Results
the images using a 40× objective lens aided by a digital 3.1. UV-vis and particle size distribution analysis
camera.
The optical characteristics of metal oxide nanoparticles
2.13. Determination of caspase 3, 8 and 9 activities are highly dependent on the size, shape, and interaction
The ZnO NPs-induced modulations in caspase 3, 8, and of the constituents on the nanoparticles. After 48 h of
9 expressions in MG63 cells were assessed by adapting incubation, the reaction solution changed from pale yellow
caspase assay as per manufacturer’s instructions. The to brown, indicating that ZnO NPs had been synthesized.
MG63 cells were inoculated and grown in six-well plates The increased strong absorption arises at 380 nm in the
for 24 h before being supplemented with different doses of UV-vis spectra readings affirms the amalgamation of ZnO
ZnO NPs (15, 30, and 45 g/mL) for another 24 h. After the NPs (Figure 1A). The formed ZnO NPs ranged from 5 to
cells were collected, the cells were lysed by utilizing a lysis 60 nm and had a mean size of 21.62±7.45 nm in diameter
buffer containing 1 mM EDTA, 10 mM EGTA, 50 mM (Figure 1B).
Tris-HCl, 10 mM digitonin, and 2 mM DTT. Further,
the lysates were collected by centrifugation at 15,000 ×g 3.2. TEM and EDX analysis
for 1 h at 4°C, and then treated with caspase-3, 8, and 9 The TEM observation shows that the ZnO NPs were
specific substrates in a 96-well plate with reaction buffer spherical and cylindrical. The ZnO NPs size had ranged

Volume 1 Issue 1 (2022) 4 https://doi.org/10.36922/gtm.v1i1.34