Quantification of the Uptake of Silver Nanoparticles and Ions to HepG2 Cells

The toxic mechanism of silver nanoparticles (AgNPs) is still debating, partially because of the common cooccurence and the lack of methods for separation of AgNPs and Ag+ in biological matrices. For the first time, Triton-X 114-based cloud point extraction (CPE) was proposed to separate AgNPs and Ag+ in the cell lysates of exposed HepG2 cells. Cell lysates were subjected to CPE after adding Na2S2O3, which facilitated the transfer of AgNPs into the nether Triton X-114-rich phase by salt effect and the preserve of Ag+ in the upper aqueous phase through the formation of hydrophilic complex. Then the AgNP and Ag+ contents in the exposed cells were determined by ICP-MS after microwave digestion of the two phases, respectively. Under the optimized conditions, over 67% of AgNPs in cell lysates were extracted into the Triton X-114-rich phase while 94% of Ag+ remained in the aqueous phase, and the limits of detection for AgNPs and Ag+ were 2.94 mu g/L and 2.40 mu g/L, respectively. This developed analytical method was applied to quantify the uptake of AgNPs to the HepG2 cells. After exposure to 10 mg/L AgNPs for 24 h, about 67.8 ng Ag were assimilated per 10(4) cells, in which about 10.3% silver existed as Ag+. Compared to the pristine AgNPs (with 5.2% Ag+) for exposure, the higher ratio of Ag+ to AgNPs in the exposed cells (10.3% Ag+) suggests the transformation of AgNPs into Ag+ in the cells and/or the higher uptake rate of Ag+ than that of AgNPs. Given that the toxicity of Ag+ is much higher than that of AgNPs, the substantial content of Ag+ in the exposed cells suggests that the contribution of Ag+ should be taken into account in evaluating the toxicity of AgNPs to organisms, and previous results obtained by regarding the total Ag content in organisms as AgNPs should be reconsidered.