Intracellular Dissolution of Silver Nanoparticles: Evidence from Double Stable Isotope Tracing

To track transformations of silver nanoparticles (AgNPs) in vivo, HepG2 and AS49 cells were cocultured with two enriched stable Ag isotopes ((107)AgNPs and (AgNO3)-Ag-109) at nontoxic doses. After enzymatic digestion, (107)AgNPs, ionic Ag-107(+) and Ag-109(+) in exposed cells could be separated and quantified by liquid chromatography combined with ICP-MS. We found that ratios of Ag-107(+) to total Ag-107 and proportions of Ag-107(+)/in cells increased gradually after exposure, proving that the Trojan-horse mechanism occurred, i.e., AgNPs released high contents of Ag+ after internalization. While the presence of Ag-109(+) (5 and 100 mu g/L) has little influence on the uptake of (107)AgNPs (0.1 and 2 mg/L), the presence of (107)AgNPs at a high dose (2 mg/L) dramatically increases the ingestion of Ag-109(+), even though (107)AgNPs at a low dose (100 mu g/L) showed negligible effects on the internalization of Ag-109(+). Cellular homeostasis may be perturbed under sublethal exposure of (107)AgNPs, and thus enhanced uptake of Ag-109(+). Our findings suggest that the widely adopted control experiments in toxicology studies, culturing organisms with AgNO3 at the same concentration of Ag+ in the AgNP exposure medium, may underestimate uptake of Ag+ and thus cannot exclude suspected toxic effects of Ag+ at high AgNP exposure doses.