SnO2-Doped ZnO/Reduced Graphene Oxide Nanocomposites: Synthesis, Characterization, and Improved Anticancer Activity via Oxidative Stress Pathway

Abstract

Background: Therapeutic selectivity and drug resistance are critical issues in cancer therapy. Currently, zinc oxide nanoparticles (ZnO NPs) hold considerable promise to tackle this problem due to their tunable physicochemical properties. This work was designed to prepare SnO₂-doped ZnO NPs/reduced graphene oxide nanocomposites (SnO₂-ZnO/rGO NCs) with enhanced anticancer activity and better biocompatibility than those of pure ZnO NPs.

Materials and methods: Pure ZnO NPs, SnO₂-doped ZnO (SnO₂-ZnO) NPs, and SnO₂-ZnO/rGO NCs were prepared via a facile hydrothermal method. Prepared samples were characterized by field emission transmission electron microscopy (FETEM), energy dispersive spectroscopy (EDS), field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), ultraviolet-visible (UV-VIS) spectrometer, and dynamic light scattering (DLS) techniques. Selectivity and anticancer activity of prepared samples were assessed in human breast cancer (MCF-7) and human normal breast epithelial (MCF10A) cells. Possible mechanisms of anticancer activity of prepared samples were explored through oxidative stress pathway.

Results: XRD spectra of SnO₂-ZnO/rGO NCs confirmed the formation of single-phase of hexagonal wurtzite ZnO. High resolution TEM and SEM mapping showed homogenous distribution of SnO₂ and rGO in ZnO NPs with high quality lattice fringes without any distortion. Band gap energy of SnO₂-ZnO/rGO NCs was lower compared to SnO₂-ZnO NPs and pure ZnO NPs. The SnO₂-ZnO/rGO NCs exhibited significantly higher anticancer activity against MCF-7 cancer cells than those of SnO₂-ZnO NPs and ZnO NPs. The SnO₂-ZnO/rGO NCs induced apoptotic response through the upregulation of caspase-3 gene and depletion of mitochondrial membrane potential. Mechanistic study indicated that SnO₂-ZnO/rGO NCs kill cancer cells through oxidative stress pathway. Moreover, biocompatibility of SnO₂-ZnO/rGO NCs was also higher against normal breast epithelial (MCF10A cells) in comparison to SnO₂-ZnO NPs and ZnO NPs.

Conclusion: SnO₂-ZnO/rGO NCs showed enhanced anticancer activity and better biocompatibility than SnO₂-ZnO NPs and pure ZnO NPs. This work suggested a new approach to improve the selectivity and anticancer activity of ZnO NPs. Studies on antitumor activity of SnO₂-ZnO/rGO NCs in animal models are further warranted.

Keywords: ZnO nanocomposites; better selectivity; breast cancer; caspase-3; improved anticancer activity; reactive oxygen species.

Figure 1

A schematic diagram of SnO₂-ZnO/rGO NCs synthesis.

Figure 2

(A–C) Low resolution TEM images and (D–F) high resolution TEM images of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs. Arrows represent rGO.

Figure 3

Elemental composition of SnO₂-ZnO/rGO NCs assessed through EDS.

Figure 4

SEM elemental mapping of SnO₂-ZnO/rGO NCs. (A) SEM image. (B) Zinc. (C) Tin. (D) Oxygen. (E) Carbon mapping.

Figure 5

(A) XRD spectra of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs. (B) XRD spectra of same samples in the diffraction region of 28–38. (C) XRD spectra of rGO.

Figure 6

(A) optical absorption spectra of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs. (B) Tauc’s plot (αhν)² vs (hν) of the same samples.

Figure 7

Cytotoxic potential of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs in MCF-7 cells. Cells were treated for 24 h to different concentration of these samples (0–200 µg/mL). (A) MTT cell viability. (B) NRU cell viability. (C) Cell morphology after exposure to 50 µg/mL of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs for 24 h. (Scale bar presents 50 µm.) Data represented as mean ±SD of five independent experiments (n=5). *p<0.05 control vs treated groups and ^#p<0.05 pure ZnO NPs vs SnO₂-ZnO/rGO NCs.

Figure 8

Apoptotic potential of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs in MCF-7 cells. Cells were treated for 24 h to different concentration of these samples (10–50 µg/mL). (A) mRNA expression level of CASP3 gene. (B) Activity of caspase-3 enzyme. (C) Quantitative data MMP. (D) Fluorescent cellular images of Rh-123 (MMP indicator) probe after exposure to 50 µg/mL of same samples for 24 h. (Scale bar presents 50 µm.) Data represented as mean ±SD of five independent experiments (n=5). *p<0.05 control vs treated groups and ^#p<0.05 pure ZnO NPs vs SnO₂-ZnO/rGO NCs.

Figure 9

Pro-oxidants generating potential of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs in MCF-7 cells. Cells were treated for 24 h to different concentration of these samples (10–50 µg/mL). (A) Quantitative data ROS level. (B) Fluorescent cellular images of DCF (ROS indicator) probe after exposure to 50 µg/mL of same samples for 24 h. (Scale bar presents 50 µm.) (C) Quantitative analysis of intracellular H₂O₂ level (D) MDA level. Data represented as mean ±SD of five independent experiments (n=5). *p<0.05 control vs treated groups and ^#p<0.05 pure ZnO NPs vs SnO₂-ZnO/rGO NCs.

Figure 10

Antioxidants depleting potential of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs. In MCF-7 cells. Cells were treated for 24 h to different concentration of these samples (10–50 µg/mL). (A) GSH level. (B) GPx activity. (C) SOD activity. (D) CAT activity. Data represented as mean ±SD of five independent experiments (n=5). *p<0.05 control vs treated groups and ^#p<0.05 pure ZnO NPs vs SnO₂-ZnO/rGO NCs.

Figure 11

ROS mediated cytotoxicity. Cells were exposed for 24 h to 50 µg/mL of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs in the presence or absence of NAC. (A) ROS level with or without NAC. (B) Cell viability with or without NAC. Data represented as mean ±SD of five independent experiments (n=5). *Significantly different from the control (p<0.05). ^#Significantly different from ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs groups (p<0.05).

Figure 12

Effect of ZnO NPs, SnO₂-ZnO NPs, and SnO₂-ZnO/rGO NCs on normal human mammary epithelial (MCF10A) cells. Cells were treated for 24 h to different concentration of these samples (0–200 µg/mL), and cell viability and ROS level were determined. (A) MTT cell viability assay and (B) intracellular ROS level. Data represented as mean ±SD of five independent experiments (n=5). *Significantly different from the control (p<0.05).

Figure 13

Possible mechanism of anticancer activity of SnO2-ZnO/rGO NCs.