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

Abstract

The toxic mechanism of silver nanoparticles (AgNPs) is still debating, partially because of the common co-occurrence 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 μg/L and 2.40 μg/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.

Silver Nanoparticles Induced RNA Polymerase-Silver Binding and RNA Transcription Inhibition in Erythroid Progenitor Cells

“…However, we circumvented this issue by looking at the portions of Ag ions and nAg particles inside the cells using the technique of cloud point extraction. 29 We followed this optimized method of cloud point extraction for separation of nAg particles and Ag ions in mammalian cells (a schematic diagram described in Figure 6A), as previously described. 29 We demonstrated that over 82.1% of Ag existed in the form of nanoparticles, with less than 17.9% as Ag ions inside MEL cells ( Figure 6B).…”

“…29 We followed this optimized method of cloud point extraction for separation of nAg particles and Ag ions in mammalian cells (a schematic diagram described in Figure 6A), as previously described. 29 We demonstrated that over 82.1% of Ag existed in the form of nanoparticles, with less than 17.9% as Ag ions inside MEL cells ( Figure 6B). Similar to our finding, Yu and colleagues documented that about 10.3% of Ag presented as ions in exposed Hep G2 cells.…”

“…We recently established a customized protocol of cloud point extraction for separation of nAg particles and Ag ions in mammalian cells, and this method was successfully used to quantify nAg particles and Ag ions in Hep G2 cells. 29 We thus followed this method to quantify nAg particles and Ag ions in MEL cells with modifications. 29 To avoid possible dissolution of nAg by HNO 3 during pH adjustment, we prepared tubes with 9 mL of ultrapure H 2 O at pH 3.4 supplemented with 0.2 mL of Na 2 S 2 O 3 and 0.3 mL of Triton X-114 (TX-114) prior to collection of cells.…”

Separation and Determination of Silver Nanoparticles

“…The interference of Ag + was negligible when the concentration of Ag + was lower than two times that of AgNPs [5]. It was successfully applied to determine AgNPs and Ag + in environmental samples such as commercial available products [7], HepG2 cells [9] and municipal wastewater [10,11]. Previous study reported the speciation analysis of AgNPs and Ag + in six antibacterial products based on CPE [7].…”

“…As a result, nanoscale silver particles in the effluent was relatively low (< 12 ng/L), indicating that wastewater treatment plants were not the pollution source of nanoscale silver particles to the aquatic system. The CPE method allows us to quantify the uptake of AgNPs and Ag + to cells as well [9]. Under the optimized conditions, AgNPs and Ag + can be readily separated and quantified in HepG2 cells.…”