Atomic force microscopy to elucidate lipidic membranes enhanced by engineered liposomes

Resum

This thesis is aimed at the study and generation of an engineered formulation able to fuse and carry drugs or other components into the cells. For the study, the targeted cells selected have been HeLa cells and prior to its use, a model membrane mimicking the real HeLa’s lipidic membrane has been developed. Starting from the basic components, from a bottom-up development point of view, different phospholipids have been studied and compared to find the proper construction blocks for the liposomes, as well as the effects of cholesterol on these phospholipids has been studied. After selecting the desired composition, a HeLa mimicking membrane mixture has been developed in order to test the fusion events from the engineered liposomes and to try to understand the fusion process before starting the in vitro assays with real HeLa cells. As for the in vitro assays, the engineered liposomes have demonstrated to be able to fuse into the membrane as well as to carry and liberate a model drug (methotrexate) into the cells demonstrating that the engineered liposomes are able to work efficiently as nanocarriers. Across all the thesis, one technique has been constantly present, Atomic Force Microscopy (AFM) as it provides the possibility to perform studies from the smallest samples like the study of lipidic monolayers to bigger samples like the HeLa cells. Also allowing for physicochemical observations of any of these samples using the force spectroscopy mode allowing to probe the samples and gaining insight on the nanomechanics of the samples studied. Several techniques have been used for this purpose, from the ones that helped study the physicochemical properties of the lipids like the Langmuir-Blodgett trough, to more complex ones to determine the fusion effects of the engineered liposomes like Fluorescence Resonance Energy Transfer (FRET) or visualization techniques like AFM or confocal microscopy and even viability techniques to test viability on the HeLa cells. Finally, we have developed and demonstrated the capabilities of the engineered liposomes to fuse with cells, being able based on its physicochemical properties to act as nanocarriers. the HeLa mimicking mixture has been validated for mimicking the actual HeLa lipidic membrane.