Investigation of Nanostructures generated in cells

Nanoparticles have established themselves firmly within the biomedical field due to their fine tuneability. Recent work explores NP behaviour in complex biological media lending itself to a growing body of research investigating the biological identity of NPs. Biomolecules have been found to readily adsorb to NPs upon interaction in complex biological media forming a protein shell. Previously viewed as an impediment to NP application in vivo, the protein shell, now referred to as the ‘biological corona’ displays potential for beneficial and independent applications. This has highlighted the need for a sounder understanding of the way in which cells receive, process, and clear nanoparticles and the pathways implicated. This research investigates the ability of magnetic magnetite multicore silica fluorescent nanoparticles to adsorb biomolecules to form a biological corona and further enrich with target EGFP fusion proteins upon administration to and interaction with stable EGFP-transfected, adherent cells in culture. The cell – engineered, biosynthetic particle recovered following internalisation, trafficking and subsequent recycling will be referred to here as a bio nanostructure (BNS). This research builds upon a large body of data encompassing all proteins comprising the BNS generated in HEK-293T cells. Here I will explore several highly abundant RNA binding proteins that are incorporated onto the BNS during cellular processing of the nanoparticle-protein corona. The application of high-resolution confocal laser scanning microscopy, along with a ‘masking’ technique will facilitate observation of EGFP fusion proteins on the BNS coat. The results presented herein support the establishment of a reliable and reproducible method for the fluorescent mapping proteins of interest on cell – generated bio nanostructures. It provides a platform for further investigation of potential subpopulations of cell generated bio nanostructures and the intricate processing networks underlying the export of these bio nanostructures.