Anticancer effect of zinc oxide nanoparticles prepared by varying entry time of ion carriers against A431 skin cancer cells in vitro

Abstract

Although, zinc oxide nanoparticles (ZRTs) as an anti-cancer agent have been the subject of numerous studies, none of the reports has investigated the impact of the reaction entry time of ion-carriers on the preparation of ZRTs. Therefore, we synthesized variants of ZRTs by extending the entry time of NaOH (that acts as a carrier of hydroxyl ions) in the reaction mixture. The anti-proliferative action, morphological changes, reactive oxygen species (ROS) production, and nuclear apoptosis of ZRTs on human A431 skin carcinoma cells were observed. The samples revealed crystallinity and purity by X-ray diffraction (XRD). Scanning electron microscopy (SEM) images of ZRT-1 (5 min ion carrier entry) and ZRT-2 (10 min ion carrier entry) revealed microtubule like morphology. On prolonging the entry time for ion carrier (NaOH) introduction in the reaction mixture, a relative ascent in the aspect ratio was seen. The typical ZnO band with a slight shift in the absorption maxima was evident with UV-visible spectroscopy. Both ZRT-1 and ZRT-2 exhibited non-toxic behavior as evident by RBC lysis assay. Additionally, ZRT-2 showed better anti-cancer potential against A431 cells as seen by MTT assay, ROS generation and chromatin condensation analyses. At 25 μM of ZRT-2, 5.56% cells were viable in MTT test, ROS production was enhanced to 166.71%, while 33.0% of apoptotic cells were observed. The IC₅₀ for ZRT-2 was slightly lower (6 μM) than that for ZRT-1 (8 μM) against A431 cells. In conclusion, this paper presents a modest, economical procedure to generate ZRT nano-structures exhibiting strong cytotoxicity against the A431 cell line, indicating that ZRTs may have application in combating cancer.

Keywords: MTT assay; malignant cell lines; reactive oxygen species; sol-gel synthesis; zinc oxide nanoparticles.

FIGURE 1

XRD characterization of ZRT samples prepared by varying reaction time of ion-carriers on the synthesis of zinc oxide nanoparticles: ZRT-1 and ZRT-2.

FIGURE 2

SEM image of ZRT samples prepared by varying reaction time of ion-carriers on the synthesis of zinc oxide nanoparticles (ZRTs): (A) ZRT-1 and (B) ZRT-2.

FIGURE 3

UV-Visible spectra of ZRT samples prepared by varying reaction time of ion-carriers on the synthesis of zinc oxide nanoparticles: ZRT-1 and ZRT-2.

FIGURE 4

(A) Hemolytic activity of ZRTs nanoparticles: The extent of damage caused to red blood cells by the ZRT was measured as percent lysis of total erythrocytes used in the individual sample. (B): In vitro cytotoxicity assay: Dose–response effects of ZRTs nanoparticles on cytotoxicity against Vero cells.

FIGURE 5

(A,B) Morphological view of live and dead cells of human epidermoid carcinoma A431 cell lines treated with control, 5 μM, 10 μM and 25 μM concentration of zinc oxide nano-particles, ZRT-1 and ZRT-2, respectively.

FIGURE 6

MTT assay: Percent cell viability of human epidermoid carcinoma A431 cells at 24 h. Values are expressed as mean ± SEM of at least three independent experiments, *p < 0.05 as compared with their respective control.

FIGURE 7

(A,B): Photomicrographs showing intra-cellular ROS generation in human epidermoid carcinoma A431 cell lines induced by control, 5 μM, 10 μM, and 25 μM concentration of zinc oxide nano-particles, ZRT-1 and ZRT-2, respectively after 12 h incubation and stained with DCFH-DA. (C) Graph showing extent of ROS generation expressed as the percentage of fluorescence intensity relative to the control. Values are expressed as mean ± SEM of at least three independent experiments, *p < 0.05 as compared with their respective control.

FIGURE 8

(A,B): Human epidermoid carcinoma A431 cell lines were treated with control, 5 μM, 10 μM and 25 μM concentration of zinc oxide nano-particles, ZRT-1 and ZRT-2, respectively and stained with DAPI (C) Representative graphs showing the numerical data of the percent apoptotic cells against the concentration of zinc oxide nano-particles, ZRT-1 and ZRT-2. Values are expressed as mean ± SEM of at least three independent experiments, *p < 0.05 as compared with their respective control.