Although zinc oxide (ZnO) nanoparticles (NPs) have been widely formulated in sunscreens, the relationship between reactive oxygen species (ROS) generation induced by these particles, zinc ions, and cytotoxicity is not clearly understood. This study explores whether these factors can be accurately quantified and related. The study demonstrates a strong correlation between ZnO NP-induced cytotoxicity and free intracellular zinc concentration (R (2) = .945) in human immune cells, indicating a requirement for NP dissolution to precede cytotoxicity. In addition, although direct exposure to ZnO NPs was found to induce cytotoxicity at relatively high concentrations, indirect exposure (via dialysis) was not cytotoxic, even at extremely high concentrations, highlighting a requirement for NP-to-cell contact. Elevated levels of ROS present in NP-exposed cells also correlated to both cytotoxicity and intracellular free zinc. Although the addition of antioxidant was able to reduce ROS, cytotoxicity to ZnO NPs was unaffected, suggesting ROS may be, in part, a result of cytotoxicity rather than a causal factor. This study highlights both the requirement and role of intracellular dissolution of zinc nanomaterials to elicit a cytotoxic response. This response is only partially ROS dependent, and therefore, modification of NP uptake and their intracellular solubility are key components in modulating the bioactivity of ZnO NPs.

The usefulness of zinc oxide (ZnO) nanoparticles has led to their wide distribution in consumer products, despite only a limited understanding of how this nanomaterial behaves within biological systems. From a nanotoxicological viewpoint the interaction(s) of ZnO nanoparticles with cells of the immune system is of specific interest, as these nanostructures are readily phagocytosed. In this study, rapid scanning X-ray fluorescence microscopy was used to assay the number ZnO nanoparticles associated with ∼1000 individual THP-1 monocyte-derived human macrophages. These data showed that nanoparticle-treated cells endured a 400% elevation in total Zn levels, 13-fold greater than the increase observed when incubated in the presence of an equitoxic concentration of ZnCl2. Even after excluding the contribution of internalized nanoparticles, Zn levels in nanoparticle treated cells were raised ∼200% above basal levels. As dissolution of ZnO nanoparticles is critical to their cytotoxic response, we utilized a strategy combining ion beam milling, X-ray fluorescence and scanning electron microscopy to directly probe the distribution and composition of ZnO nanoparticles throughout the cellular interior. This study demonstrated that correlative photon and ion beam imaging techniques can provide both high-resolution and statistically powerful information on the biology of metal oxide nanoparticles at the single-cell level. Our approach promises ready application to broader studies of phenomena at the interface of nanotechnology and biology.

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Nanoparticulate zinc oxide (ZnO) is one of the most widely used engineered nanomaterials and its toxicology has gained considerable recent attention. A key aspect for controlling biological interactions at the nanoscale is understanding the relevant nanoparticle surface chemistry. In this study, we have determined the disposition of ZnO nanoparticles within human immune cells by measurement of total Zn, as well as the proportions of extra- and intracellular dissolved Zn as a function of dose and surface coating. From this mass balance, the intracellular soluble Zn levels showed little difference in regard to dose above a certain minimal level or to different surface coatings. PEGylation of ZnO NPs reduced their cytotoxicity as a result of decreased cellular uptake arising from a minimal protein corona. We conclude that the key role of the surface properties of ZnO NPs in controlling cytotoxicity is to regulate cellular nanoparticle uptake rather than altering either intracellular or extracellular Zn dissolution.