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Abstract

This thesis examines the properties of large unilamellar lipid vesicles (LUVs) having transmembrane pH gradients (ΔpH). These pH gradients Induce proton movement across the membrane and can result in accumulation of amines into the vesicles. A major focus of the thesis is to develop a quantitative approach to describe the response of drugs which are lipophilic amines, as well as protons, to these pH gradients. Large, stable transmembrane pH gradients (ΔpH) of up to 3.5 units are shown to be detectable in LUVs by examining the transmembrane distribution of [¹⁴]C-methy4lamine. This approach is subject to artefacts in situations where the interior buffering capacity is low, where the interior vesicle volume changes due to osmotic effects, or where probe redistribution is too slow to be practical. These problems are generally easily overcome in liposomal systems. It is demonstrated that transmembrane pH gradients provide a practical method of entrapping drugs in lipid vesicles. It is shown that the anticancer drug doxorubicin accumulates into LUVs with an acidic interior via permeation of the neutral form of the drug. The critical dependence of translocation rates on pH, temperature and lipid composition allow manipulation of drug loading and release to achieve desired characteristics. A model incorporating vesicle volume, buffering capacity, drug partitioning and other factors is shown to describe the accumulation of doxorubicin in response to a ΔpH. This results in the conclusion that more than 95% of the encapsulated doxorubicin is partitioned into the inner monolayer for a 100 nm vesicle. Representative basic drugs from different drug classes also accumulate into vesicles in response to an acidic interior, although the extent of uptake varies considerably. Finally, the unique nature of the transbilayer movement of protons (or equivalents) was examined in well buffered lipid systems with large (3 unit) pH gradients. Development of a transmembrane electrical potential had a half-time of about 12 mm in EPC LUVs at 25°C, with an activation energy near 11 kcal/mol, near the activation energy of water transport. Further, model membrane systems were developed which exhibited stable membrane potentials without induced pH gradients. or stable pH gradients without induced membrane potentials.