Project 4 (UCD)
Project 4: Connection of the nature of NP in biofluids to the cellular uptake, translocation & subsequent biological impacts
Fellow: Bogumila Reidy (ESR4), 36 months
Tutor: Dr. Iseult Lynch (UCD)
Immediately upon contact with biological systems, including upon dispersion in cell culture media, NP are surrounded by a layer of biomolecules which confer a biological identity onto the NP, and it is this corona of biomolecules that interacts with the cellular machinery and likely determine the fate and behaviour of nanomaterials. Thus, identification of key biomarkers of uptake, trafficking and sub-cellular localisation and their connection to biological impacts will be the aim of this project. Another end-point will be the impact of Ag NP on protein fibrillation in situ in cells, depending on the proteins identified as having the strongest interactions with the NP.
To establish the connection between the NP biomolecule corona and uptake and subsequent functional impacts, a PhD project performed by an ESR will utilize the same panel of cell lines as used in project 1 (stably transfected with cytokine-promoter sequences linked to the luciferase gene) will be used here. Coronas will be identified following uptake and localisation, by fractionating the cells and mass spectrometric fingerprinting of the cells pre- and post-exposure to the NP, as well as semi-quantitative assessment of the proteins bound to the NP pre- and post-exposure using, for example, iTRAQ. The hypothesis of this work is that the proteins that interact with the different Ag NP will determine their uptake and subsequent transport and localisation in cells, which will be followed using EM. By identifying the key transport proteins by mass spectrometry and semi-quantitative proteomics, and correlating these with sub-cellular localisation and cellular impacts, we can identify biomarkers of uptake, localisation and immune response. To further investigate the correlation between bound protein specificity and amounts with particle uptake mechanism and trafficking, electron microscopy approaches and stereology will be used to quantitate the sub-cellular distribution of the NP. GFP and other specific organelle co-staining will be used to assess spatial localisation of such effects around particles in situ and will provide essential confirmation of the role of particular corona proteins in the accumulation and distribution of the various NP and point towards biomarkers of specific trafficking pathways.
The main skills acquired and training given will be in the areas of in situ characterisation of nanoparticles, proteomics and mass spectrometry approaches, including semi-quantitative approaches, electron microscopy, stereology, and the use of advanced microscopy methods for the characterisation of engineered NP both in vitro and in vivo. Secondments to partners 1 (reporter gene methods), 5 (role of metal binding proteins in nanotoxicology), 8 (localisation in neurons), 9 (methods for NP visualisation in biological entities).
In WP1 (tasks 1.1, 1.2 and 1.3), WP3 (task 3.1) and WP4 (tasks 4.2 and 4.5).