Single cell ICP-MS for the assessment of potential nephroprotectors against cisplatin

Abstract

The use of cisplatin chemotherapy is often limited by the occurrence of various side effects, with renal toxicity being one of the most serious. In the present work, a single cell ICP-MS (scICP-MS) methodology was optimised to evaluate the cellular uptake of cisplatin in the presence of three potential nephroprotectors such as chitosan-stabilised selenium nanoparticles (Ch-SeNPs), selenomethionine (SeMet) and methionine (Met). Human telomerase reverse transcriptase-immortalised renal proximal tubular epithelial cells (RPTEC/TERT1) and human cervical cancer cells (HeLa) were employed with this aim. In both cell lines, a decrease in the intracellular Pt levels when using SeMet and Met as coadjuvants was revealed, involving less toxicity in renal cells but no reduction in the anticancer effect after measurement of cell viability by MTT assays. In contrast, Ch-SeNPs had no effect on the internalisation of the Pt-drug but enhanced its antitumour efficacy with no additional damage to kidney cells. This would allow decreasing cisplatin doses which would in turn reduce nephrotoxicity risk. Se determination by scICP-MS was also done to study the cell uptake of the selenocompounds, in addition to transmission electron microscopy (TEM) analysis of Ch-SeNPs internalisation. The effects of both SeMet and Ch-SeNPs were confirmed despite the Pt-drug was shown to induce a decrease in cell uptake. Results were compared by two different scICP-MS settings (a conventional introduction system and a special configuration for intact cells), as well as with the classical digestion-based bulk analysis. Our results demonstrate the potential of scICP-MS for metallomic cellular studies to improve cisplatin-based therapies.

Keywords: Cisplatin; Kidney protection; Methionine; Selenium nanoparticles; Selenomethionine; Single cell ICP-MS.

Fig. 1

Microscopy images of RPTEC/TERT1 by TEM analysis: control cells (a), cells after 24 h exposure to Ch-SeNPs (b, d), and cells after 24 h exposure to uncovered SeNPs (c, e). Ch-SeNPs (d) and uncapped SeNPs (e) internalised by cells and located in different cell compartments such as the cytoplasm, vacuoles or mitochondria

Fig. 2

Cell viability results from MTT assays of 24 (a) and 48-h cultures (b) of RPTEC/TERT1 and HeLa after being exposed to cisplatin alone or combined with Ch-SeNPs, SeMet or Met. Values are represented as the mean ± the standard deviation (n = 5). Statistically significant differences were considered for treatments marked with *(p < 0.05)

Fig. 3

Plots corresponding to scICP-MS measurements of ¹⁹⁵Pt in RPTEC/TERT1 exposed to cisplatin and Ch-SeNPs for 24 h after one (a, b, c) or two pellet washes (d, e, f): variation of the number of single cell events (a, d), the average element content per cell (b, e) and the extracellular element concentration (c, f) with the dilution factor of the cell suspensions. All the results are presented as the mean ± the standard deviation (n = 3). The ratios of detected single cell events to expected events (%) for each dilution are shown in plots a and d

Fig. 4

Plots corresponding to scICP-MS measurements of ⁷⁸Se in RPTEC/TERT1 exposed to cisplatin and Ch-SeNPs for 24 h after one (a, b, c) or two pellet washes (d, e, f): variation of the number of single cell events (a, d), the average element content per cell (b, e) and the extracellular element concentration (c, f) with the dilution factor of the cell suspensions. All the results are presented as the mean ± the standard deviation (n = 3). The ratios of detected single cell events to expected events (%) for each dilution are shown in plots a and d

Fig. 5

Histograms of the mass distribution obtained by scICP-MS for the intracellular content of Se corresponding to 24 h cultures of RPTEC/TERT1 with uncapped SeNPs (a), selenite (b), Ch-SeNPs (c), SeMet (d), cisplatin + Ch-SeNPs (e) and cisplatin + SeMet (f). The estimated numbers of SeNPs per cell were indicated for cultures with uncapped SeNPs (a) and Ch-SeNPs (c)

Fig. 6

Histograms of the mass distribution obtained by scICP-MS for the intracellular content of Pt corresponding to 24-h cultures of RPTEC/TERT1 with cisplatin (a), cisplatin + Met (b), cisplatin + Ch-SeNPs (c) and cisplatin + SeMet (d)

Fig. 7

Graphs comparing the intracellular contents (a, b) and the number of registered single cell events (c, d) during scICP-MS measurements of ¹⁹⁵Pt (a, c) and.⁷⁸Se (b, d) in RPTEC/TERT1 exposed for 24 and 48 h to cisplatin, Ch-SeNPs, SeMet, cisplatin + Ch-SeNPs, cisplatin + SeMet and cisplatin + Met. All the results are presented as the mean value ± the standard deviation (n = 3)