Understanding the uptake mechanisms, biological fate and biodistribution of ENMs is fundamental to comprehending their possible toxicological endpoints. The objective of this work package is to study the translocation of ENMs across different barriers at cellular, tissue, organ and organism level as a function of their physicochemical properties for a set of ENMs.

“The objective of this work package is to study the translocation of ENMs across different barriers at cellular, tissue, organ and organism level”

Outcomes of the workpackage

The study of the uptake mechanism, biological fate and biodistribution of ENMs is crucial to understand toxicological endpoints of ENMs. The objective of WP9 was to study the translocation of ENMs across biological barriers at cellular, organs and organism levels in relation to the physical-chemical properties of the ENMs, in particular with their chemical functionalization.

The fate of ENMs was studied at cellular level. Intracellular uptake studies were conducted employing different techniques: Ion beam microscopy, flow cytometry, transmission electron microscopy, the CytoViva hyperspectral imaging system, confocal Raman microspectroscopy. The surface functionalization and more specifically PEGylation of ENM was confirmed to be a reasonable strategy to reduce the dissolution of ENM in acidic environments (e.g. CuO NPs), also reducing cytotoxicity, and the generation of reactive oxygen species (ROS). Cell homeostasis was affected by ENMs, as in the case of intrinsic concentration of iron and zinc considerably changed in cells exposed to CdTe and CuO. Surface functionalization affected the interaction of ENMs with cellular compartments. For example, while carboxylated CdTe NPs were mainly found also co-localized with the endoplasmic reticulum, PEGylated NPs were found associated to mitochondria and amine NPs with lipid bodies (Raman studies). For some ENMs was observed increased aggregation during cellular incubation (e.g. TiO2-PEG, TiO2-COOH and Ag-PEG) and the colloidal destabilization of the ENMs was correlated to a lower cellular uptake.

Translocation at higher complexity level was investigated across endothelial and placental barriers. In the blood-tissue barrier the integrity of the endothelial glycocalyx modulated translocation/uptake of ENMs in vitro. Furthermore, Au and quantum dots translocation from the blood stream into atherosclerotic plaques was characterized and the interaction between ENMs and the plaques showed to be dependent on the ENMs-protein corona formation.

Ex vivo placenta perfusion studies were performed for several NPs, which during the experiments generally underwent physico-chemical changes as agglomeration, dissolution and precipitation. The role of the surface functionalization showed to be again of primary importance as for Au NPs, which crossed the trophoblast barrier into the placental tissue in a size-dependent manner but can be effectively blocked by grafting PEG-moieties to the Au NPs. From a toxicological point of view, the exposure of placenta to CuO and CdTe NPs resulted in an interference with the release of placental hormones (e.g., beta-hCG).

Finally, translocation was studied at organism level. Single Photon Emission Computed Tomography and Positron Emission Tomography were used to directly follow the uptake, distribution and release of radiolabeled ENMs in animal models. Also in this case the surface functionalization was fundamental in determining the biological fate of the ENMs. Some ENMs (e. g. Ag NPs) could always be efficiently radiolabeled while other could not (MWCN), other could be labeled only when functionalized by a certain surface group (Au-NH2 and Au-PEG). The different surface chemistry for the ENMs resulted in distinct biodistribution and release patterns.

The outcomes from WP9 confirmed the role of core nature, size, surface coating as determinant for ENMs translocation across biological barriers from the cellular level to tissue, organ and organism level. Moreover, some of the developed models (ex vivo placental perfusion models and cultured endothelial cells expressing glycocalyx) showed to give relevant toxicological indication as the importance of avoiding exposure to CuO and CdTe NPs during pregnancy and the importance of endothelial glycocalyx integrity in in vivo in microvessels.