Preparation, Characterization, And Application of Liposomes in the Study of Lipid Oxidation Targeting Hydroxyl Radicals

In the onset of many chronic illnesses including Parkinson’s, Alzheimer’s, and cardiovascular diseases, there is evidence to support the delicate balance between prooxidant and antioxidant species is shifted in favor of the former. Under these conditions, many reactive oxygen species (ROS) including hydroxyl radicals, are generated. Hydroxyl radicals formed in close proximity to DNA, nucleotides, proteins, and lipids rapidly oxidize these biological molecules in a nonspecific way. However, their toxicity is limited by their short lifetimes. Currently, the mechanism by which hydroxyl radicals are involved in the onset of many illnesses, particularly with regard to lipid peroxidation, has yielded some controversy in the literature. Conventional studies which generate hydroxyl radicals with Fenton chemistry through bolus additions of iron and hydrogen peroxide do not mimic conditions found physiologically because there is a steady-state concentration of hydrogen peroxide concentration found in normal cellular systems. Also, former reports that used fluorescent fatty acids or free probes intercalated within liposomal membranes did not have the probes covalently attached to the phospholipids making up the liposomes. Thus, the actual placement of the probes over the analysis time may vary with experimental conditions. The objective of this research project was to prepare, characterize, and employ liposomes as models for cell membranes during free radical oxidation. Also, compared to the popularly-used technique of electron spin resonance, (ESR), our aim was to use a fluorescence-based approach which yielded the advantages of high sensitivity, fast analysis time, and less expensive equipment requirements. Degradation of fluorescently-tagged liposomes with probes covalently bound to the phospholipids was correlated with the ability of hydroxyl radicals and other possible reactive oxygen species to penetrate into the liposomes to deeper into the lipophilic layer. However, alone this experimental setup may not fully define the mechanistic role of hydroxyl radicals in lipid oxidation. Thus, a complementary approach embracing the use of MALDI-TOF mass spectrometry, lipophilic scavenger studies, and the effects of cholesterol and temperature allow a deeper understanding of the radically-driven oxidation of lipids. It was determined that hydroxyl radicals were generated and reacted with three fluorescent probes