Sub-cytotoxic doses of pharmaceutical silica nanoparticles show significant impact on the proteome of HepG2 cells

Abstract

The ever-growing application of nanoparticles (NPs) in medical and pharmaceutical domains has brought issues related to their toxicity into focus. However, a profound analysis of non-acute, sub-lethal effects of engineered pharmaceutical NPs is often disregarded during such toxicological investigations. Here, two selected NPs were investigated in cultured HepG2 cells in terms of their intracellular localization and the associated impact on pharmacokinetically relevant CYP3A4 isoform, as well as the induced changes observed in the proteome of such cells. Using SILAC (Stable Isotope Labeling by Amino acids in Cell culture)-based mass spectrometry facilitated quantitative proteomics, significant proteomic changes in NP-treated hepatocytes were detected, which were subsequently analyzed via bioinformatic tools. Both, silica NPs (SiO₂ NP) and cargo-free PEGylated stealth liposomes resulted in the induction of CYP3A4-activity up to 150% in a dose-dependent manner, with different time-dependent response-patterns as a function of NP-type after a single treatment. Proteomic analysis revealed that the observed metabolic alterations are only one aspect of the cellular response to NP-exposure. SiO₂NPs (free in cytoplasm) caused extensive changes in the proteome, whereas liposomes (compartmentalized) seemed unproblematic as they accounted for minimal changes in the protein profile. Based on the obtained results, proteomic analyses were revealed to be highly important for the toxicological assessment of NPs. Although sub-toxic concentrations of many NPs are considered as uncritical based on standard toxicological assays, proteomic analysis indicated that drug-free NPs could cause fundamental cellular modifications, which were attributed to different causal networks and regulatory pathways.