During the whole period of the project, the methodologies A-D were investigated and compared for the quantitative analysis of silver NPs, AgNPs. For this, several incubation experiments of different cell lines of toxicological interest (THP-1 macrophages and monocytes, 3T3 fibroblasts, A549) with 50 nm AgNPs were assayed for further study of their cytotoxicity.
Then, the main analytical features of each methodology were optimized and a calibration scheme based on the analysis of standard AgNPs (50nm) was developed. The most appropriated methodologies resulted to be mass cytometry, CyTOF, and pneumatic nebulization coupled to a sector field ICP-MS working in a time resolved single particle mode, SC-ICP-MS. Both methodologies were high-throughput techniques and allow the quantitative analysis of AgNPs in very low, i.e. realistic doses where other conventional methods can be hardly applied. Below the main analytical advantages and results obtained after apply both methodologies for the quantitative analysis of cell-associated to AgNPs are briefly described as follow:
- By one hand, SC-ICP-MS has important advantages as it requires minimal sample preparation, is fast, selective and very sensitive, provides information on the NPs content of individual cells and on the related variance in a population of cells. SC-ICP-MS could be a new promising tool to track, assess and understand the uptake of AgNPs by individual cells. The application of this methodology to monitor the uptake of AgNPs by THP-1 cell in a time manner dose dependent demonstrated that the AgNPs uptake was dependent on the exposure dose and time. Moreover, the results reveal that there was a large cell-to-cell variance the AgNPs uptake by THP-1 demonstrating that not all cell responds alike inside a cell population since some cells can have a significantly higher uptake rate compared to a mean value while some cells did not take any AgNPs at all.
- By the other hand, the power of mass cytometry is based on the combination of NPs quantitation and multi parametric analysis of cell characteristics. Mass cytometry is a very fast technology allowing for up to 500 cells to be analyzed per second and it can discriminate live and dead cells as well as cell fragments, which otherwise would distort the results in toxicological studies. Mass cytometry results demonstrated that AgNPs uptake by THP-1 was dependent on exposure dose and time as well and provided important additional information such as the number of free AgNPs outside a cell. This methodology should be easily transferable to other types of NPs. Therefore, we propose that this approach should become a valuable new quantification approach in nanotoxicology.
