DEVELOPMENT OF NEW LIPOSOME BASED SENSORS

Liposome based sensors are inspired to the sensorial functions of biological membranes; in fact, ligand-receptor interactions mediated by lipid bilayers are involved in physiological activities of biological membranes such as signal transduction and information processing that are based on lipid reorganization. The aim of this work is to explore the possibility of developing a novel liposome based sensor for detecting the presence, and possibly dosing, the activity of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase. These enzymes are involved in the metabolism of pyrimidines and are target of a potent chemotherapic agent, 5−fluorouracil (5-FU), widely employed in the treatment of some of the most frequently occurring malignant solid tumors (breast, colon and skin cancer). The need of rapid and accurate detection of the activity of these enzymes before and during 5-FU treatment to individuate patients who can’t be treated or the optimal doses of treatment for treated patients is a problem of major concern. At present simple, fast, reliable and cheap screening methods to dose the activity of the three enzymes before and during the treatment with 5-FU do not exist. The work reported in this thesis is aimed at developing an optical sensor based on the specific interaction of liposomes with the target enzymes. To this end, liposomes were formulated with both commercial and ad hoc designed lipids to give a fluorescent response upon the interaction with the target enzymes. The interaction of target enzymes with specific liposomes, promoted by 5-FU exposed on the lipid membrane, could induce lipid reorganization and segregation in domains according to lipid component charge features. The lipid reorganization should trigger the response from a pyrene moiety linked to a cationic amphihile embedded in the lipid bilayer. Liposome membranes and Langmuir monolayers composed of different amounts of 1,2- dimyristoyl-sn-glycero-3-phosphatidylcoline (DMPC) or 1,2-dioleoyl-sn-glycero- phosphocholine (DOPC) and a pyrene-containing lipid were investigated to study the influence of the molecular structure and of the amount of components on lipid organization and miscibility. Moreover, the formation of regular lipid organization was investigated in a 3 small range of concentration by fluorescence on liposomes and by Langmuir isotherms on lipid monolayers. Lipid organization is significantly affected by the rigidity of the pyrrolidinium ring of the fluorescent cationic amphiphile and by the balance between repulsive and attractive electrostatic interactions. The different behavior of mixed monolayers containing DMPC or DOPC and the pyrene tagged amphiphile shows that, besides the interactions between lipid headgroups, other parameters such as surface hydration and the nature of the alkyl chains have to be considered to achieve a complete understanding of the physicalchemistry and dynamics of mixed lipid systems. In particular, at certain molar fractions, lipid bilayers present regions organized in regular arrangements, probably hexagonal lattices where the acyl chains form regular patterns around the pyrene labelled chain of the synthetic component. This work allowed individuating the range of component ratio suitable to obtain a detectable fluorescent signal in response to lipid reorganization, further it has demonstrated that the novel 5-FU derivatives are able to interact with the target enzymes when embedded in the liposome bilayer. Both findings are good premises for the development of a sensor for the target enzymes.