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

To track transformations of silver nanoparticles (AgNPs) in vivo, HepG2 and A549 cells were cocultured with two enriched stable Ag isotopes (¹⁰⁷AgNPs and ¹⁰⁹AgNO₃) at nontoxic doses. After enzymatic digestion, ¹⁰⁷AgNPs, ionic ¹⁰⁷Ag⁺ and ¹⁰⁹Ag⁺ in exposed cells could be separated and quantified by liquid chromatography combined with ICP-MS. We found that ratios of ¹⁰⁷Ag⁺ to total ¹⁰⁷Ag and proportions of ¹⁰⁷Ag⁺/ ¹⁰⁹Ag⁺ 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 ¹⁰⁹Ag⁺ (5 and 100 μg/L) has little influence on the uptake of ¹⁰⁷AgNPs (0.1 and 2 mg/L), the presence of ¹⁰⁷AgNPs at a high dose (2 mg/L) dramatically increases the ingestion of ¹⁰⁹Ag⁺, even though ¹⁰⁷AgNPs at a low dose (100 μg/L) showed negligible effects on the internalization of ¹⁰⁹Ag⁺. Cellular homeostasis may be perturbed under sublethal exposure of ¹⁰⁷AgNPs, and thus enhanced uptake of ¹⁰⁹Ag⁺. 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.