Single-Cell ICP-MS in Combination with Fluorescence-Activated Cell Sorting for Investigating the Effects of Nanotransported Cisplatin(IV) Prodrugs.

The combined use of fluorescence-activated cell sorting (FACS) and single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) is reported, for the first time, in this work. It is applied to evaluate the differences between the cellular uptake of ultrasmall iron oxide nanoparticles (FeNPs) loaded with cisplatin(IV) prodrug (FeNPs-Pt(IV)) and cisplatin regarding cell viability. For this aim, FACS is applied to separate viable, apoptotic, and necrotic A2780 ovarian cancer cells after exposing them to the nanotransported prodrug and cisplatin, respectively. The different sorted cell populations are individually analyzed using quantitative SC-ICP-MS to address the intracellular amount of Pt. The highest Pt intracellular content occurs in the apoptotic cell population (about 2.1 fg Pt/cell) with a narrow intercellular distribution when using FeNPs-Pt(IV) nanoprodrug and containing the largest number of cells (75% of the total). In the case of the cisplatin-treated cells, the highest Pt content (about 1.6 fg Pt/cell) could be determined in the viable sorted cell population. The combined methodology, never explored before, permits a more accurate picture of the effect of the intracellular drug content together with the cell death mechanisms associated with the free drug and the nanotransported prodrug, respectively, and opens the door to many possible single-cell experiments in sorted cell populations.

Intracellular Biotransformation of Ultrasmall Iron Oxide Nanoparticles and Their Effect in Cultured Human Cells and in Drosophila Larvae In Vivo.

A systematic investigation on the cellular uptake, intracellular dissolution, and in vitro biological effects of ultra-small (<10 nm) iron hydroxide adipate/tartrate coated nanoparticles (FeAT-NPs) was carried out in intestinal Caco-2, hepatic HepG2 and ovarian A2780 cells, and the nucleotide excision repair (NER) deficient GM04312 fibroblasts. Quantitative evaluation of the nanoparticles uptake, as well as their transformation within the cell cytosol, was performed by inductively coupled plasma mass spectrometry (ICP-MS), alone or in combination with high performance liquid chromatography (HPLC). The obtained results revealed that FeAT-NPs are effectively taken up in a cell type-dependent manner with a minimum dissolution after 3 h. These results correlated with no effects on cell proliferation and minor effects on cell viability and reactive oxygen species (ROS) production for all the cell lines under study. Moreover, the comet assay results revealed significant DNA damage only in GM04312 cells. In vivo genotoxicity was further studied in larvae from Drosophila melanogaster, using the eye-SMART test. The obtained results showed that FeAT-NPs were genotoxic only with the two highest tested concentrations (2 and 5 mmol·L−1 of Fe) in surface treatments. These data altogether show that these nanoparticles represent a safe alternative for anemia management, with high uptake level and controlled iron release.

Capabilities of Single Cell ICP-MS for the Analysis of Cell Suspensions from Solid Tissues.

Single cell elemental (SC) analysis of isogenic cell cultures can be done using inductively coupled plasma (ICP-MS) detection. However, 2D cell cultures are just models to simplify the complexity of real tissue samples. Here, we show for the first time the capabilities of the technique (SC-ICP-MS) to analyze single cell suspensions of isolated cells from tissues. An optimized cocktail of proteolytic and collagenolytic enzymes was applied in a single preparation step with cellular yields up to 28% using 0.5 g of fresh rat spleen and liver, respectively. The retrieved cells revealed adequate morphology and stability to be examined by SC-ICP-MS. Quantitative elemental analysis of P, S, Cu, and Fe from disaggregated cells from rat spleen and liver tissues revealed levels of Fe of 7-16 fg/cell in the spleen and 8-12 fg/cell in the liver, while Cu was about 3-5 fg/cell in the spleen and 1.5-2.5 fg/cell in the liver. Evaluation of the transmembrane protein transferrin receptor 1 (TfR1) expression levels in disaggregated cells was also conducted by using a Nd-labelled antibody against this cell surface biomarker. Quantitative results showed significantly lower expression in the disaggregated cells than in the cell model HepG2, in agreement with the overexpression of this biomarker in tumor cells. In this proof of concept study, the tissue disaggregation protocol has shown to maintain the elemental intracellular content of cells as well as the presence of relevant antigens. This opens a completely new area of research for SC-ICP-MS in tissue samples as a complementary strategy with validation capabilities.