Fluorescent nanoparticles: a probe to study the molecular trafficking of somatostatin

Thesis submitted for the degree of Doctor of Philosophy, Macquarie University, Dept. of Physics and Astronomy, 2012.Includes bibliographical references.1. General introduction and outline -- 2. Fluorescence - basics, imaging and nanoparticles -- 3. Related biological aspects -- 4. Experimental methodology -- 5. Talking to the cells -- 6. Non-specific nanoparticle internalization -- 7. Biofunctionalizing fluorescent nanodiamonds -- 8. Tagging cell-signaling molecules with fluorescent nanoparticles -- 9. Summary and prospects -- Appendices.Somatostatin is a biological molecule that serves important physiological functions in a human body, including the regulation of blood pressure and growth. These functions are furnished by a set of complex interactions between somatostatin and other participating biomolecules. The main goal of this Ph.D. project was to develop tools based on fluorescent nanoparticles that can report on these interactions at a molecular scale. Fluorescent nanoparticles offer the advantages of protein-comparable size, high detection sensitivity, biocompatibility, and physical, chemical and photophysical stabilities, in comparison with many conventional molecular probes. Three key milestones of this project included: (i) Elucidating and harnessing the interactions of nanoparticles with biological molecules and cells. In particular, two types of nanoparticle-cell interactions were investigated: non-specific interactions, characterized by poorly controllable parameters, such as nanoparticle size, charge and surface chemical groups; and specific interactions, characterized by a precise communication protocol of a particular cell type with a nanoparticle surface, grafted with a specific ligand. As a result of these investigations, the non-specific interaction channel was inhibited, whereas the somatostatin-specific interaction channel was activated. (ii) Development of a universal biofunctionalization methodology to assemble robust, versatile nanoparticular and biomolecular modules. Specifically, an affinity molecular pair based biofunctionalization platform was developed, which allowed a 'pot-mix' procedure to attach biomolecules onto nanoparticle surfaces. (iii) Design, synthesis and demonstration of a biologically active nanoparticle-somatostatin complex. This included two stages: characterization of the receptor-binding activity of somatostatin ligands loaded with a fluorescent tag, such as a red fluorescent protein; and introduction and testing of a two-stage nanoparticle biofunctionalization and receptor-mediated internalization procedure, which allowed to circumvent the loss of the somatostatin-nanoparticle biological activity. Identication and rectification of the loss of biological activity has been one of the core accomplishments. Such an assembly has been demonstrated to be suitable for observing the somatostatin-cell interactions. A comprehensive, multi-stage program for tagging fluorescent nanoparticles to a targeting biomolecule has been incepted and carried out to a successful completion. The prospects of deploying fluorescent nanoparticle labeling technology is far-reaching, ranging from in vivo investigations of neuronal machinery to tumor diagnosis and treatment. This work is believed to provide a tangible contribution to nanotechnology and life sciences.Mode of access: World Wide Web.1 online resources (xiv, 276 pages) illustration