UBC Theses and Dissertations
Studies on the antiatherogenic properties of liposomes Rodrigueza, Wendi V.
There has been a wealth of prior literature demonstrating that the infusion of phospholipid causes the reversal of experimentally induced atherosclerosis in animals. However, the development of liposomal therapies to manage atherosclerosis is only now being considered seriously. The studies presented in this thesis were aimed at confirming the antiatherogenic properties of phospholipid using a pharmaceutically acceptable formulation of liposomes capable of pre-clinical development. The work can be divided into three main areas of investigation. In order to test the capacity of liposomes to act as a "sink" for cholesterol, the relative rates of movement of sterol were measured between leaflets and between vesicles. The kinetics of movement of cholesterol and cholesterol sulphate (CS) between vesicles were studied using donor vesicles of various size ranging from 40-250 nm, composed of egg phosphatidylcholine (EPC)/Sterol/N-palmitoyl dihydrolactosylcerebroside (75:10:15 mol ratio). Trace amounts of [³H]-sterol in donor vesicles was used to monitor sterol transfer into a 10 fold excess of EPC large unilamellar vesicles (LUV) of 100 nm diameter. Following the addition of a lectin, the two populations of vesicles were separated by centrifugation. The rate constants for efflux and transbilayer diffusion for both sterol molecules were determined after fitting kinetic data to a three compartment model. The rate of intermembrane exchange for CS was considerably faster than for cholesterol in all liposomes tested. Using the kinetic model, a rate of transbilayer movement for cholesterol and CS was estimated. In both cases, it was found to be slower than the rate of efflux from the surface of vesicles. The results from this chapter suggest that the greatest rate of cholesterol uptake in vivo should be achieved using unilamellar vesicles with greatest surface area and the smallest diameter. The second group of studies examined the capacity of different liposomal preparations (as studied in chapter two), to mobilize cholesterol in vivo. This involved measuring the ability of liposomes to increase plasma cholesterol concentrations in mice. Surprisingly, LUV, composed of EPC, were found to be approximately twice as effective at mobilizing cholesterol than small unilamellar vesicles (SUV) of the same composition. This is because the increase in plasma cholesterol is proportional to the residence time of vesicles in the circulation. LUV with a diameter of approximately 100 nm, accumulate the most sterol during 24 h in the animal model tested here. Vesicles composed of phospholipid in a gel state at physiological temperatures, gave rise to a smaller increase in plasma cholesterol compared to liquidcrystalline vesicles. A significant decrease in the ratio of cholesterol-to-phospholipid in the lipoprotein pool was observed following liposome infusion, whereas cholesterol levels and turnover in erythrocytes remained constant. These results indicate that net transfer of cholesterol into liposomes in vivo occurs more extensively from the lipoprotein cholesterol pool than from the erythrocyte cell membrane pool. This is consistent with the hypothesis of Williams et al. (1984) that liposomes enhance reverse cholesterol transport by generating cholesterol-poor HDL particles that can extravasate and promote more sterol efflux from peripheral tissues. In the final groups of studies presented here, the antiatherogenic properties of EPC LUV of 100 nm diameter were tested in an experimental model for atherosclerosis in which rabbits were fed a diet rich in cholesterol. Forty eight rabbits were divided into two diet groups fed standard rabbit chow or fed a diet enriched in cholesterol (0.5% by weight) to induce the formation of atherosclerotic plaques. Prior to the initiation of phospholipid therapy, the cholesterol diet was ceased and all animals were returned to standard rabbit chow. The treatment protocol involved a total of 10 bolus injections of vesicles at a phospholipid dose of 300 mg/kg or an equivalent volume of saline, with one injection given to each animal every 10 days. Liposomal injections brought about a large movement of cholesterol into the blood pool and resulted in a significant reduction in the cholesterol content of aortas as well as the degree of surface plaque involvement in atherosclerotic animals. Most notably, the thoracic aorta exhibited a 48% reduction in tissue cholesterol content per gram of protein compared to tissue from saline-treated controls. Histochemical analyses revealed that aortas from animals receiving the repeated infusions of phospholipid, displayed less cholesterol deposits in lesions, and a moderate reduction in the ratio of intimal/medial thickness. This liposome-mediated regression of atheroma was observed despite persistent elevation of plasma cholesterol levels after removal of the animals from the high cholesterol diet. These results support previous observations and suggest that phospholipid infusion may indeed be a useful therapy in the management of atherosclerosis.
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