Protein-based nanostructures as carriers for photo-physically active molecules in biosystems

In nature, many proteins function as carriers, being able to bind, transport and possibly release a ligand within a biological system. Protein-based carriers are interesting systems for drug delivery, with the remarkable advantage of being water-soluble and, as inherent components of biosystems, highly bio-compatible. This work focuses on the use of protein-based carriers for the delivery of hydrophobic photo-physically active molecules, whose structure and chemical properties lead to spontaneous association of the two components. The self-assembly of stable nanostructures formed by the combination of the protein and the photo-active molecule was assessed spectroscopically. It was demonstrated that the association with the carrier generally induce an improvement of the photo-physical activity of the compounds under physiological conditions, by preventing aggregation of the compounds. The behavior of the nanostructures was then analyzed in the presence of bacterial cells in suspension or cultured tumor cells. Preventing aggregation is particularly important for photosensitizing molecules (PS). When photo-excited, these molecules react with the molecular oxygen present in solution, leading to the generation of cytotoxic reactive oxygen species in a reaction called photosensitization, which is exploited to inactivate pathogen microbes or tumor cells. The efficacy of the photosensitization-based inactivation of cells induced by the PS delivered with the protein-based carrier was evaluated in relation with the photo-physical parameters and correlated to the images collected with fluorescence microscopy techniques. Additionally, the increased fluorescence emission from the protein-bound PS Hypericin was successfully exploited for imaging the cellular distribution of this molecule with subdiffraction resolution by means of super-resolution stimulated emission depletion (STED) microscopy. Some clear advantages in the use of protein-based carrier for the delivery of PSs have been identified for the photo-treatment of cells, in comparison with the unbound PS. Photo-active metal-organic compounds belonging to the class of so-called triplet emitters were also investigated for their potential as luminescent reporters for bio-imaging techniques. The major problem concerning the use of these compounds is related to their poor spectroscopic stability, due to hydrophobic aggregation. It was successfully demonstrated that the combination with a protein carrier allows to overcome this problem and to obtain a stable, water-soluble, minimally-invasive system, suitable as luminescent probe that is potentially useful for bio-imaging.