Exploring the use of Quantum dots as quantitative model delivery agents for small molecules, peptides and proteins

Progress in the biological sciences is leading to the characterization of a wide range of disease processes in molecular detail. However overriding or intervening in these processes in a controlled fashion remains a great challenge and many researchers are now looking beyond the use of individual small molecule inhibitors to the realm of macromolecules and nanoparticle complexes. For small molecules, the lack of cell-specific targeting often limits their reach and they are inherently limited in the scope of their interaction with cells. In contrast, biological macromolecules which comprise of peptide, protein or nucleic acid derived constructs can interface with cellular processes in a broader range of contexts with the potential to confer new paradigms in disease treatment. However, several challenges exist for utilizing macromolecules as therapies including compliance with the immune system and targeting to a site of interest which may be the cell surface or a site within the cell interior. To address these problems recent years have seen an explosion of interest in utilizing nanoparticles as drug delivery systems which exploit the principle that multiple functionalities can be grouped into a single entity to tackle distinct biological barriers. Whilst nanoparticles offer great hope for disease management, there is considerable difficulty in fabricating at the nanoscale and our understanding of the factors which influence nanoparticle cell uptake and processing remains limited. As such there is considerable scope for exploring novel methods for both assembling cargoes into nanoparticle complexes and quantifying their uptake and trafficking in cells. In this work, luminescent nanoparticles known as Quantum dots (QDs) were utilized as a model system for the delivery of peptide, protein and small molecule cargoes. Characterization of QDs in biological media (Chapter 3) revealed that these particles are sensitive to the adsorption of serum proteins to the particle surface in a process known as protein corona formation. This observation inspired an approach to exploit non-covalent association to assemble a hydrophobic small molecule (Chapter 4), and cationic peptide and protein (Chapter 5) cargoes into nanoparticle complexes. In line with recent reports, a recurrent finding from this work identifies the serum protein concentration as a critical factor in determining the performance of these non-covalent nanoparticle complexes in terms of their ability to bind to cells and withstand cargo dissociation. Currently, there are few methods which aim to quantifying intracellular particle trafficking at a single particle level. Here a novel computational method was developed (Chapter 6) based on single particle tracking microscopy to allow the study of individual particles as they undergo transit between cellular compartments. The method demonstrated high specificity and event detection scores using simulated data and the impact of this work relates to the potential to perform high throughput quantification of nanoparticle trafficking at the single particle level.