Novel ZnO:Ag nanocomposites induce significant oxidative stress in human fibroblast malignant melanoma (Ht144) cells

Abstract

The use of photoactive nanoparticles (NPs) such as zinc oxide (ZnO) and its nanocomposites has become a promising anticancer strategy. However, ZnO has a low photocatalytic decomposition rate and the incorporation of metal ions such as silver (Ag) improves their activity. Here different formulations of ZnO:Ag (1, 3, 5, 10, 20 and 30% Ag) were synthesized by a simple co-precipitation method and characterized by powder X-ray diffraction, scanning electron microscopy, Rutherford back scattering and diffuse reflectance spectroscopy for their structure, morphology, composition and optical band gap. The NPs were investigated with regard to their different photocatalytic cytotoxic effects in human malignant melanoma (HT144) and normal (HCEC) cells. The ZnO:Ag nanocomposites killed cancer cells more efficiently than normal cells under daylight exposure. Nanocomposites having higher Ag content (10, 20 and 30%) were more toxic compared to low Ag content (1, 3 and 5%). For HT144, under daylight exposure, the IC50 values were ZnO:Ag (10%): 23.37 μg/mL, ZnO:Ag (20%): 19.95 μg/mL, and ZnO:Ag (30%): 15.78 μg/mL. ZnO:Ag (30%) was toxic to HT144 (IC50: 23.34 μg/mL) in dark as well. The three nanocomposites were further analyzed with regard to their ability to generate reactive oxygen species (ROS) and induce lipid peroxidation. The particles led to an increase in levels of ROS at cytotoxic concentrations, but only HT144 showed strongly induced MDA level. Finally, NPs were investigated for the ROS species they generated in vitro. A highly significant increase of (1)O2 in the samples exposed to daylight was observed. Hydroxyl radical species, HO(•), were also generated to a lesser extent. Thus, the incorporation of Ag into ZnO NPs significantly improves their photo-oxidation capabilities. ZnO:Ag nanocomposites could provide a new therapeutic option to selectively target cancer cells.

Keywords: ZnO:Ag nanocomposites; cancer therapy; cytotoxicity; photo-oxidation.

Figure 1

SEM images of the different zinc oxide nanoparticles. (a) ZnO, (b) ZnO:Ag (1%), (c) ZnO:Ag (3%), (d) ZnO:Ag (5%), (e) ZnO:Ag (10%), (f) ZnO:Ag (20%), and (g) ZnO:Ag (30%).

Figure 2

XRD patterns of different types of ZnO:Ag nanoparticles show the ZnO wurzite hexagonal crystalline structure and presence of the cubic crystalline form of Ag. Inset: magnified part of the spectrum showing the decrease in height of the ZnO peaks with an increase in the appearance of the Ag phase.

Figure 3

Characterization of the different ZnO and ZnO:Ag nanocomposites. (a) RBS analysis; atomic composition graph is showing the relative amounts of Zn, O and silver, (b) diffused reflectance spectra, (c) band gap energies of the different nanocomposites.

Figure 4

Effect of ZnO and ZnO:Ag nanocomposites on the viability of HT144 (skin cancer) and HCEC (normal) cells using SRB assay. Exponentially growing cultures were treated with different concentrations of the nanoparticles (25, 50, and 125 µg/mL) for 24 h. Percent viabilities (mean ± SD) were calculated relative to NTC. *p < 0.001, **p < 0.0001 (two tailed t-test) when compared to HCEC.

Figure 5

Comparison of the effect of ZnO and ZnO:Ag nanocomposites on mitochondrial function (MTT reduction) in HT144 (skin cancer) and HCEC (normal) cells. Exponentially growing cultures were treated with different concentrations (5, 12.5, 25, 50, 75, 100 µg/mL) of the nanoparticles and in order to analyze the photo-oxidative effect of nanoparticles, the cultures were exposed to daylight or kept in dark at 37 °C for 15 min and further incubated for 24 h. MTT reduction (mean ± SD) was measured. NTC were included as control. (a) ZnO, (b) ZnO:Ag (10%), (c) ZnO:Ag (20%), and (d) ZnO:Ag (30%). The experiment was performed twice with triplicates of each sample.

Figure 6

Consumptions of the singlet oxygen indicator DPBF mixed with ZnO and ZnO:Ag nanocomposites under the exposure to light and recorded every 30 s. (a) Time course of the natural log of absorption spectrum of DPBF at 410 nm. (b) Quantum yield (mean ± SD) of singlet oxygen. *p < 0.01, **p < 0.0001 (two tailed t-test) in comparison to MB.

Figure 7

TBA assay results for nanoparticle exposure. Data is expressed as percent (%) TBARS (mean ± SD) relative to the NTC sample. *p < 0.0001 (two tailed t-test) when compared to HCEC dark, HCEC light and HT144 dark.

Figure 8

Effect of ROS scavengers mannitol, NaN₃ and DMSO on the photo-oxidative activity of ZnO and ZnO:Ag nanocomposites as measured by MTT assay in HT144 (skin cancer) cells. Viability (mean ± SD) was calculated relative to the NTC samples. (a) ZnO, (b) ZnO:Ag (10%), (c) ZnO:Ag (20%), and (d) ZnO:Ag (30%). *p < 0.01, **p < 0.001, ***p < 0.0001 (two tailed t-test).