Study of the fusion mechanism of cationic fusogenic liposomes and their potential as carrier system

Abstract

Liposomes have been used as a drug delivery system in biological media. However, conventional liposomes typically enter the cell via endocytic routes, making it difficult to deliver the encapsulated material to the intracellular medium. To optimize direct intracellular delivery, a fusogenic liposomal system was recently developed. This system has presented high fusion efficiency with both biomimetic lipid vesicles and cells. These fusogenic liposomes are composed of equal fractions of DOTAP, a cationic lipid, and DOPE, a lipid with a curvature that is able to promote fusion, and a smaller fraction of a fluorescent lipid. In this project, we intend to extend studies with model systems to understand the role of lipid composition and membrane phase in the mechanism and fusion efficiency, and also to test the intracellular delivery of these systems in cancer cell culture. The DOTAP and DOPE lipids will be replaced by analogous lipids with other hydrocarbon tails (different length and degree of unsaturation) to test the role of the membrane phase in the fusion efficiency. On the other hand, the composition of the cellular mimetic system (small liposomes-LUVs and giant liposomes-GUVs) will also be varied to include cholesterol-containing systems and in biologically relevant phases, such as liquid-ordered and liquid-disordered phases. In particular, a fusogenic system which is triggered when the system reaches the physiological temperature will be tested. The thermal behavior of the lipid mixtures will be evaluated by Differential Scanning Calorimetry (DSC). The melt efficiency will be quantified by FRET efficiency measurements using confocal microscopy, Isothermal Titration Calorimetry (ITC) and zeta potential measurements and Dynamic Light Scattering (DLS). The fusion mechanism and efficiency will be discussed and compared with membrane properties that are relevant to the membrane fusion process, such as line tension, diffusion coefficient and bending modulus. Finally, the delivery capacity of materials encapsulated in the fusogenic liposomes will be evaluated both in biomimetic systems (GUVs) and in cancer cell culture. (AU)