Emerging investigator series: Long-term exposure of amorphous silica nanoparticles disrupts the lysosomal and cholesterol homeostasis in macrophages

Amorphous silica nanoparticles (ASiNPs) are generally considered to be biocompatible with limited acute toxicity. These nanoparticles were therefore exploited in diverse nanoproducts (e.g. foods and cosmetics) and may be released into environments in daily life. This has led to substantial concerns on their affects by long-term exposure. Herein, Raw264.7 cells were subjected to long-term exposure of ASiNPs for assessment of the potential cytotoxicity. Two ASiNPs with diameters of 50 ~ 100 nm (ASiNP1) and 450 ~ 500 nm (ASiNP2) were synthesized and comprehensively characterized. The acquired cells were denoted as Raw264.7-NP1 (ASiNP1 treatment) and Raw264.7-NP2 (ASiNP2 treatment) after long-term exposure. Both the small and large ASiNPs showed similar cellular distributions in lysosomes. We therefore examined the impacts of ASiNPs in lysosomal density, size and function by microscopy imaging and proteomics analysis. As a result, Raw264.7-NP1 cells displayed 2.3-fold higher lysosomal density and 149% increments of average lysosomal area than parent cells. Proteomics analysis of lysosomes revealed 25 significantly changed proteins closely relating to 5 cellular effects, including autophagy and lipid metabolism. Confocal imaging and immunoblotting of LC-3 proteins indicated an accumulation of autophagosomes in Raw264.7-NP1 cells. Elevation of cholesterol was also demonstrated in Raw264.7-NP1 cells by visualization and quantification of cellular cholesterols. Notably, the cholesterol elevation in Raw264.7-NP1 cells could be ameliorated by acceleration of autophagic flux (rapamycin treatment) but aggregated by blockage of autolysosome formation (chloroquine treatment), indicating the critical role of autophagic flux in lipid metabolism. Overall, the discovery of these hazard effects raised warnings for the long-term uses of ASiNP-based nanoproducts and may facilitate the decision making on environmental exposure thresholds of ASiNPs.