Assessing the Effects of Silver Nanoparticles on ARPE-19 Cells via High-Throughput Phenotypic Profiling with the Cell Painting Assay.
Increasing commercialization of silver nanoparticles (AgNPs) has resulted in elevated opportunity for human exposure and outpaced traditional risk assessment approaches that rely on in vivo testing. Therefore, efficient methods are needed to evaluate potential hazards of AgNPs. Previous studies identified particle size and surface charge as determinants of AgNP toxicity, which has primarily been attributed to reactive oxygen species generation. Those studies have generally focused on cytotoxicity or targeted cellular effects, potentially missing critical sub-cytotoxic effects. Here, high throughput phenotypic profiling (HTPP) with the Cell Painting assay was used to characterize the effects of 12 distinct AgNPs (= 30 µg/mL) on organelle morphology in human retinal pigmented epithelial cells (ARPE-19). Three coatings (branched polyethyleneimine, polyvinylpyrrolidone, citrate) were selected for their distinct surface charges and tested at 4 different sizes (40, 60, 80, 100 nm) to determine the effects of these properties on toxicity. HTPP was conducted in conjunction with a transcriptomic profiling experiment involving 60 nm particles of all coating types. This facilitated evaluation of mechanisms of action underlying morphological effects observed using HTPP. A cell viability and apoptosis assay was also run in parallel and used to exclude cytotoxic treatments from phenotypic and transcriptomic analyses. HTPP revealed concentration-dependent emergence of sub-cytotoxic phenotypic profiles that clustered by coating type, suggesting surface charge to be more influential than particle size in the determination of AgNP toxicity. The transcriptomic data highlighted oxidative stress and cell cycle alterations as fundamental underlying toxicity mechanisms of AgNPs. Overall design: For the RNA-Seq assay, dosing was performed manually by removing half of the cell culture media from each flask and replacing it with an equivalent volume of 2X concentrated test particle or fresh cell culture media as a negative control. Initial 1 mg/mL AgNP solutions (polyethyleneimine (Ag-BPEI), polyvinylpyrrolidone (Ag-PVP) and citrate (Ag-Citrate) were diluted in media such that the final concentrations applied to cells were 0.3, 1, 3, 10, and 30 µg/mL. There were six technical replicates of each nanoparticle treatment and eleven negative controls. For this assay, cells were exposed for 24 hours.