A Comprehensive Characterization of Surface-Assembled Populations of Giant Liposomes using Novel Confocal Microscopy-Based Methods

Giant liposomes, or giant unilamellar vesicles (GUVs), are thin, semi-permeable,man-made compartments that often serve as models of the cell plasma membrane due to their sizes (1−100 ?m) and molecular composition (composed of lipids). GUVs have proven useful for understanding a variety of different biophysical phenomena such as lipid membrane organization, membrane protein function, and cytoskeletal mechanics. A variety of different formation methods have been developed to try to optimize the populations of GUVs produced. However, information that allows for the direct comparison of the sizes and yields of the GUVs obtained from the different methods is lacking. In my dissertation, I describe my work on the development of a novel confocal microscopy-based technique that allows for the characterization of the populations of GUVs produced from the most commonly employed surface-assisted assembly methods. Through the development and standardization of careful protocols that allow for the quantification of ?(100,000) vesicles per sample, I characterize the surface-assembled populations of GUVs in comprehensive sets of experiments. From this work, I show novel discoveries including i) the use of nanocellulose paper as a surface to obtain GUVs, ii) the effect of substrate properties on the formation of GUVs, iii) the modulations of ionic strength technique to allow high yields of GUVS to be obtained using physiological salts, and iv) the effect of osmolytes on the formation of GUVs. The results from these quantitative experiments has led to the development of the budding and merging thermodynamic model which describes the mechanism of GUV formation. Overall, the discoveries pave the way for the largescale production of GUVs for biophysical studies as well as towards more practical applications of GUVs such as for compartments for targeted drug delivery or synthetic cells