| Summary: | This thesis is focused on the effects of osmotic pressure and transbilayer area asymmetry on the morphology and stability of model lipid vesicle systems. These forces have been implicated in many diverse biological functions including membrane fusion, maintenance of cellular shape and membrane trafficking. The osmotic stability of these large unilamellar vesicles (LUVs) in the present of plasma has also been investigated. This is of practical interest as these vesicles are currently being used as in vivo drug carriers.
The LUVs used in this thesis are made by the extrusion procedure which involves repeatedly passing an aqueous lipid dispersion through small pore sized filters. The morphology of the resulting egg phosphatidylcholine: cholesterol (55:45,mol:mol) LUVs formed by the extrusion technique (LUVETs) is found to be predominantly non-spherical, a property which has important effects on their osmotic properties. In particular, the initial influx of water that results from exposure of these vesicles to a hypo osmotic solution is first accomodated by the vesicles "rounding up" to maximize their volume to surface area ratio. Further studies show that osmotically induced vesicle lysis is a very rapid event with most of the solute release occurring within the first 30 seconds. However. lysis results in only partial release of solute such that a residual osmotic gradient results. This residual gradient is similar to the gradient required to initiate lysis. The maximum residual osmotic gradients were measured for LUVET systems with different mean diameters (90 to 340 nin). These results indicate that the osmotic properties of LUVETs obey Laplace's law for a spherical vesicle, relating the pressure difference across a close elastic membrane to the membrane tension. Osmotic lysis studies were also conducted in the presence of plasma with palmitoyloleoylphosphatidylcholine:cholesterol (55:45. mol:mol) LUVETs. Plasma was
found to enhance solute release. However, both the residual and threshold osmotic gradients are reduced to the same extent. The plasma component responsible for the reduction in the membrane lysis tension is demonstrated to be the lipoproteins, with the high density lipoproteins exerting the greatest effect. The third area of investigation concerns the morphological consequences of imbalances between the surface areas of the vesicle's inner and outer monolayers as examined by cryo-electron microscopy techniques. Surface area imbalances are generated by inducing net transbilayer transport of DOPG indioleoylphosphatidylcholine: dioleoylphosphatidylglycerol (DOPC:DOPG. 9:1. mol:mol)vesicles in response to transmembrane pH gradients. It is shown that when DOPG is transported from the inner monolayer to the outer monolayer. initially invaginated LUVETs are transformed to long narrow tubular structures. or spherical structures with one or more tubular extensions. Conversely. when DOPG is transported from the outer monolayer to the inner monolayer of non-invaginated LUVETs, a reversion to invaginated structures is observed. These results are consistent with proposals that factors leading to imbalances in monolayer surface areas could play important roles in intracellular membrane transport processes. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate
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