Description of Work Packages

WP1 – Supply of Materials

Within task 1 all particle samples and non-biological materials will be distributed to the project partners along with data of their physical-chemical pre-characterization. For this purpose on the one hand pre-characterized reference-particles from earlier BMBF-sponsored projects (NanoCare, NanoGEM, NanoBioDetect) will be included into the studies. On the other hand, industrially relevant and not yet pre-characterized particle species will be analyzed.

WP2 – Supply and Characterization of Particle Laden Tissue Samples

Within this task, the organ samples will be selected and pre-characterized, that will be analyzed in the project. Particle laden cells will be identified by means of histological and immunocytochemical methods. In addition, newly developed microscopic methods will be applied, e.g. the combined Dark-Field-Raman-Microscopy. For these analyses mostly particle-laden tissues samples from forerunner projects (NanoCare, NanoGEM, NanoGRAVUR, NanoBiotedect) will be used. Examples on respective studies can be found here (https://www.mdpi.com/2079-4991/7/12/441).

With respect to regulatory toxicology the overall goal is the measurement of local particle concentration in tissue samples used for routine histology. A well suited SOP could be appended to the existing OECD conform methodology, as it allows the retrospective and localized analysis of particle burden. With such an approach additional animal tests can be avoided.

WP3 – Supply and Characterization of Particle Laden Cells

WP3 aims on the establishment of new cell models that allow the realistic mimicking of the cellular particle uptake as measured in vivo. An example of this kind of biotest is the alveolar macrophage assay (https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s12951-016-0164-2). The focus of this work is on cells of the lung, the liver, the kidney and the spleen. In these cells a peripheral accumulation of nanoparticles has been detected. Within this work package, emphasis is given to the newly developed digital microscopic holography (http://www.hh.um.es/Abstracts/Vol_33/33_5/33_5_417.htm). This technique allows the observation on the development of cell cultures over long time periods without the need of external marker. Findings from literature may considered as well, if the cell type and particle burden had been identified.

WP4 – LA-ICP-MS

The further development of Laser Ablation – Inductively Coupled Plasma – Mass Spectrometry (LA-ICP-MS) is a core part of the project NanoBioQuant. LA-ICP-MS is a high-sensitive and element-selective method for the direct analysis of solid samples. Its lateral resolution is defined by the obtainable focus of the laser beam, which is currently in the lower µm-range. Quantification is performed with help of calibration standards, that are ablated using the identical parameters also used for the tissue sample. At low measurement time (10 µs) the identification of signals derived from individual nanoparticles is possible.

For the analysis of toxicological relevant effect markers (e.g. alteration of lipids in the lung; https://particleandfibretoxicology.biomedcentral.com/articles/10.1186/s12989-018-0267-z) Matrix Assisted Laser Desorption Ionization — Mass Spectrometry (MALDI-MS) will be applied to the samples and will be combined with the other methods in the project.

WP5 – ToF-SIMS

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a high-performance  method for the chemical analysis of surfaces, thin layers and nanostructures. It offers information on the elemental composition of the sample (detection limit: ppm to ppb range). Furthermore, molecules in the mass range up to 1000 u can be detected (detection limit: fmol range). In ideal cases, ToF-SIMS offers a lateral resolution of 16 nm. The recent development of Argon-Cluster-Sources allows for the removal of complex organic materials with low destruction of the underlying material. With this approach, a depth resolution of 5 nm is achieved. Applying the dual-beam-approach (alternating the removal of material and the analysis of the sample) ToF-SIMS offers to image organic layer structures and three dimensional structures with high lateral resolution as well as high mass resolution. An example on the use of ToF-SIMS in the field of nanotoxicology can be found here (https://doi.org/10.3390/nano8080571, https://doi.org/10.3390/nano8010044).

To establish ToF.SIMS as a routine method in the field of tissue analysis, the parallel detection of organic and inorganic substances has to be improved. The throughput of samples should be increased by automation and acceleration of the analysis and data-evaluation processes. Finally, the feasibility of the quantification of the analytical results is aspired.