Hematite iron oxide nanoparticles: apoptosis of myoblast cancer cells and their arithmetical assessment

Abstract

Hematite (α-Fe₂O₃) forms iron oxide nanoparticles (NPs) which are thermally stable and have various electrochemical and optochemical applications. Due to their wide applicability, the present work was designed to form the hematite phase of iron oxide (αFe₂O₃NPs) NPs prepared via a solution process. Their cytological performance was checked with C2C12 cells. The crystalline property of the NPs was examined with X-ray diffraction patterns (XRD) and it was found that the size of the particles formed ranged from 12 to 15 nm. Structural information was also identified via field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM), which again confirmed that the size of each NP is about 12-15 nm. Surface topographical analysis was done via atomic force microscopy (AFM), which reveals that the size of the distance between two particles is in the range of 12 ± 3 nm. The C2C12 cells were cultured in a humidified environment with 5% CO₂ and were checked via a microscope. The αFe₂O₃NPs were used for cytotoxic evaluation against C2C12 cells. A MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was utilized to check the viability of cells in a dose-dependent (100 ng mL^-1, 500 ng mL^-1 or 1000 ng mL^-1) manner. The morphology of the cells under the influence of αFe₂O₃NPs for live and dead cells in a wet environment was confirmed via confocal laser scanning microscopy (CLSM). The apoptosis caused due to the αFe₂O₃NPs was evaluated in presence of caspases 3/7 with GAPDH genes, which confirmed the upregulation that is responsible in caspase 3/7 genes, with treatment of C2C12 at low (500 ng mL^-1) and high (1000 ng mL^-1) doses of αFe₂O₃NPs. Analytical studies were also performed to authenticate the obtained data for αFe₂O₃NPs using parameters such as precision, accuracy, linearity, limits of detection (LOD) and limit of quantitation (LOQ), quantitative recoveries and relative standard deviation (RSD). The analyses play a significant role in investigating the large effect of αFe₂O₃NPs on C2C12 cells.

Fig. 1. The X-ray diffraction pattern of iron oxide nanoparticles (αFe₂O₃ hematite phase).

Fig. 2. Low (a) and high (b) magnification FE-SEM images of αFe₂O₃ hematite iron oxide nanoparticles, respectively. TEM (c) and HR-TEM (d) show the general morphology of the prepared NPs and their fringe distances between two lattices (∼0.233 nm).

Fig. 3. AFM images of prepared αFe₂O₃ hematite iron oxide nanoparticles. General topographical image (a) whereas (b) shows the distance between particles, which is ∼12 nm, (horizontal, vertical and surface distance).

Fig. 4. The FTIR spectra of the prepared iron oxide nanoparticles (αFe₂O₃ hematite phase).

Fig. 5. Microscopic images of the effect on C2C12 of iron oxide αFe₂O₃ (hematite phase) nanoparticles at different concentrations and incubation times.

Fig. 6. The MTT assay of iron oxide nanoparticles (αFe₂O₃ hematite phase) at different concentrations (100 ng mL⁻¹, 500 ng mL⁻¹ and 1000 ng mL⁻¹) and incubation (24, 48 and 72 h) times and with a control solution. Experiments were performed in a triplicate manner.

Fig. 7. mRNA expression of C2C12 cells for caspase 3 in the presence of GAPDH genes, with exposure to NPs at low (500 ng mL⁻¹) and high (1000 ng mL⁻¹) concentrations. Experiments were performed in a triplicate manner.

Fig. 8. mRNA expression of C2C12 cells for caspase 7 in the presence of GAPDH genes, with exposure to NPs at low (500 ng mL⁻¹) and high (1000 ng mL⁻¹) concentrations.

Fig. 9. CLSM images of C2C12 cells at different concentrations of αFe₂O₃ hematite NPs (100 ng mL⁻¹, 500 ng mL⁻¹ and 1000 ng mL⁻¹) with a 24 h incubation period.

Fig. 10. The absorption spectra of αFe₂O₃ NPs (a), C2C12 cells (b), spectra of αFe₂O₃ NPs and C2C12 cells (c).

Fig. 11. The linear calibration graph of αFe₂O₃ NPs.

Fig. 12. Possible proposed schematic mechanism for the interaction of αFe₂O₃NP against C2C12 and their cytological death.