Bio-Orthogonal Manipulation of Cellular Membranes for Biological Applications
O'Brien, Paul J.
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Recent breakthroughs using novel chemistry and nanotechnology methodologies for cellular biology applications have spurred tremendous interest in the development of new technologies to manipulate cell-lines or cellular products for biotechnology applications, without complicated or permanent techniques used in academia and industry. Small molecule drugs, proteins, deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) have been employed to manipulate and probe cellular behaviour with the intention of elucidating healthy and diseased molecular pathways for understanding human health. This research focused on the application of liposomal delivery of bioorthogonal chemoselective chemistry to cellular membranes to control biological functions. These smart membranes were used to investigate cellular adhesion, cell spheroid aggregation kinetics and incorporation of ligands. In Chapters 2 and 3, NMR spectroscopy, oxime reaction kinetics and oxime cell surface engineered cell adhesion was correlated and probed under microfluidic conditions, while the robustness of adhesion and flexibility of cluster and tissue formation was established using live cell technique microfluidics. Using this as a base strategy, we developed a general method for the microfluidic manipulation and CSE of cells for dual labelling and flexible delivery of nucleic acids and small molecule ligands (Paul OBrien et al. 2017, in preparation). In Chapter 4, the bioorthogonal liposomal strategy originally utilized for cell adhesion and cell membrane ligand integration was modified and extended to transfect mammalian cell lines with nucleic acids, limiting the use of cationic charge as a mild and general method to tag and target cells in monocultures and co-cultures. The method was characterized using microscopy, protein production quantification and targeting in complex co-cultures for selective internalization. Finally, in Chapter 5 a new chemical moiety termed dialdehyde was designed and synthesized for the easy and mild conjugation of primary amine containing molecules. This conjugation strategy was used to deliver small molecule ligands and macromolecules to bacterial and mammalian cell membranes using a generalized liposomal formation method. The dialdehyde was characterized and modified to contain two dialdehyde moieties with a Polyethylene Glycol (PEG) linker for crosslinking proteins, inorganic surface functionalization, organic bead assembly and cell aggregation for tissue formation.