Flow Cytometric Assessment of Reactive Oxygen Species Generations That are Directly Related to Cellular ZnO Nanoparticle Uptake

Abstract

In this study, a simple flow cytometry protocol to evaluate nanoparticle associated biological response was proposed. Particularly, we have evaluated the effect of surface charge on the cellular nanoparticle associations and nanoparticle-induced apoptosis. Significant enhancement in side scattering intensity was observed for the HeLa cells treated with positively charged (ZnO)-Zn-PLL nanoparticles, suggesting that the (ZnO)-Zn-PLL nanoparticles may induce cell death via adsorption and endocytosis of the nanoparticles. On the other hand, the negatively charged (ZnO)-Zn-PAA nanoparticle seems to cause cell death process indirectly via the released Zn ions, with less contribution from cellular association of nanoparticles. Time- and dose-dependent studies on cellular association of ZnO nanoparticles, and ZnO associated reactive oxygen species generation were also performed for the HeLa cells exposed to the (ZnO)-Zn-PLL nanoparticle, For those cells associated with (ZnO)-Zn-PLL nanoparticle, a significant enhancement in reactive oxygen species generation was observed even at a lower concentration (10 ppm), which was not observable for the results with the whole cell population. By using this approach, we are able to distinguish biological responses (e.g., reactive oxygen species (ROS) generation) directly related to the cellular associations of NPs from those indirectly related to the cellular associations of NPs, such as the cytotoxicity caused by the NP released metal ions.