Internalization of carbon black and maghemite iron oxide nanoparticle mixtures leads to oxidant production

The risk of potential human exposure to mixed nanomaterials in consumer, occupational, and medicinal settings is increasing as nanomaterials enter both the workplace and the marketplace. In this study, we investigated the toxicity of mixed engineered carbon black (ECB) and maghemite iron oxide (Fe2O3) nanoparticles in a cellular system to understand the mechanism of toxicity and potential methods of toxicity mitigation. Lung epithelial cells (A549) were exposed to mixed Fe2O3 and ECB nanoparticles, mixed Fe2O3 and ECB nanoparticles with the addition of l-ascorbic acid, and mixed Fe2O3 and surface-oxidized engineered carbon black (ox-ECB) nanoparticles. The nanoparticles were characterized using transmission electron microscopy, nitrogen adsorption surface area measurement (BET), X-ray diffraction, and surface charge measurement. The carbon black nanoparticles were also characterized with a reductive capacity assay and by X-ray photoelectron spectroscopy (XPS). The cellular uptake of nanoparticles was analyzed via transmission electron microscopy and fluorescence microscopy; the cellular uptake of iron was quantified using inductively coupled plasma mass spectrometry (ICP-MS). Both the MTT assay and the ethidium homodimer and calcein AM live/dead assay were used to measure cellular proliferation and cytotoxicity, respectively. The dichlorofluorescein diacetate (DCFH-DA) assay was used to measure the intracellular generation of reactive oxygen species. Results show that both Fe2O3 and ECB (or Fe2O3 and ox-ECB) were co-internalized in intracellular vesicles. Additionally, after exposure to the mixture of nanoparticles, the amount of acidified lysosomes increased over time. The cellular uptake of Fe2O3 nanoparticles was unaffected by mixing with ECB. Significant oxidant production occurred in cells exposed to mixed Fe2O3 and ECB, but not in cells exposed to mixed Fe2O3 and ox-ECB or in cells exposed to Fe2O3 and ECB with the addition of ascorbic acid. Furthermore, exposure to mixed Fe2O3 and ECB nanoparticles yielded a dose-dependent decrease in the level of cellular proliferation (MTT assay) and a decrease in cellular viability (ethidium homodimer and calcein AM live/dead assay) that were not seen in the Fe2O3 and ox-ECB scenario. The results support the hypothesis that exposure to mixed Fe2O3 and ECB nanoparticles produces oxidants that are mediated by the surface reductive capability of ECB when both particle types are colocalized in acidic cellular compartments. This oxidant production mechanism may lead to oxidative stress, but it can be mitigated by an antioxidant such as ascorbic acid or by surface treatment of the ECB to decrease its surface reductive capacity.