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Development of Novel Analytical Methods to assess Nanoparticle cytotoxicity

Periodic Reporting for period 1 - NanoCytox (Development of Novel Analytical Methods to assess Nanoparticle cytotoxicity)

Reporting period: 2015-11-01 to 2017-10-31

In the last decades, biomedical and numerous commercial applications using nanoparticles (NPs) with a size range of 1 to 100 nm have been established due to their unique chemical reactivity and physical properties. However, there are serious concern regarding exposure, distribution and accumulation of NPs in organisms and environment. In particular, the impact of NPs on cells is a key question in biomedical applications as well as in nanotoxicology and numerous analytical methods have been applied to understand the extent and mode of cell-NP interactions. However, such methodologies are still in its infancy to resolve cell-associated NPs uptake and the analysis of NPs at single cell level remains a challenge. So far, the mechanisms of cell-NPs interactions are not well understood yet and the development and validation of new reliable methods that enable the analysis and identification of NPs at single cell level providing information of the number of NPs internalized by cells or externally bound to the cell surface are urgently required.
The NanoCytox project has the main aim to develop analytical methodologies based on inductively coupled plasma mass spectrometry, ICP-MS, to evaluate the quantitative uptake and biological interaction of NPs with individual cells. The following methodologies have been evaluated here:
A. Micro-droplet generator (µDG) for sample introduction of single cells into a quadrupole and/or into a sector field ICP-MS working in single particle mode, (sp)-ICP MS;
B. Mass Cytometry, CyTOF (ICP-TOF MS). This method was originally not described in the NanoCytox project, but during our investigation we found out that it is the most promising one. It should be mentioned that these investigations were performed at the “Deutsche Rheuma Forschungs-Institute” at the Charité who is the owner of such an instrument.
C. Pneumatic nebulization coupled to a quadrupole and/or sector field ICP MS working in a time resolved single particle mode, in the so-called single cell mode, SC-ICP MS. This method was also originally not described in the NanoCytox project, but during our investigation we found out it also promising.
D. Laser ablation (LA) coupled to ICP-MS for spatially resolved bioimaging of the distribution of NPs in single cells upon different NPs incubation experiments.
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.
As stated in this report, most of the training activities were achieved during the course of this fellowship. Moreover, new analytical methodologies, originally not described, have been developed demonstrating important advantages over conventional classic methodologies. During the whole project period, the following achievements can be highlighted:
- The development and validation of an innovative methodology, mass cytometry, that provides multi-variate phenotypic information of individual cells and enables the quantitative analysis of cells-associated to NPs in a single measurement. This methodology allows the analysis of a high number of cells and distinguishes between cells-associated to NPs from those NPs floating in the incubation media. Mass cytometry can also discriminate from death cell and cell fragments events, which otherwise distort the results in toxicological studies.
- The reliability and feasibility of SC-ICP-MS have also been demonstrated for the quantitative analysis of NPs at single cell level. This promising methodology provides immediate information on the NPs content of individual cells and on the related variance in a cell population. SC-ICP-MS presents a huge potential over the most conventional lysate-base assays overcoming some of their limitations. Additionally, the potential advantages of this method have been also demonstrated to study the uptake of a classic contaminant, arsenic, by a different type of cell line providing information on the metal uptake distribution among the cell population and giving further insights into uptake, accumulation and release on single-cell level.
- Furthermore, the potential of LA-ICP-MS for the qualitative analysis of NP associated has also been demonstrated here, although further studies to establish a quantitative approach enabling the determination of the number of AgNPs internalized or externally bound to cells should be continued by the host institution.

Therefore, the results presented in this project will be of high interest for important research fields such as biomedicine, toxicology, food protection, etc. since they demonstrate the potential of these three promising methodologies and contribute to overcome the current demand to develop suitable user-friendly quantitative methodologies for a better understanding of the extent and mode of action of cell-NP interactions.
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