[eng] Biological membranes (BMs) are self-sealing boundaries, which confine the permeability barriers of cells and organelles and provide the means to compartmentalize functions. Apart from being crucial for the cell structure, they provide a support matrix for all the proteins inserted in the cell, acting as channels to exchange mass, energy and information with the environment. BMs mediate several biological functions, such as trafficking, cell division, endocytosis and exocytosis, demanding strong conformational changes of the lipid membrane like fusion, fission or tubes growth. These mechanical requirements are only possible due to the organization of the chemical composition of the lipids into the membrane of each organelle, which is directly linked to the organelle function. Thanks to the dynamic behavior of the membrane, lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched, and as new constituents are added, removed or chemically modified. Differences in structure between the two leaflets and between different areas of the bilayer can be associate to membrane deformation to alter the activities of membrane binding proteins. It is then the correlation between the composition and the packing of the lipids what essentially governs the membrane physicochemical and mechanical properties.
Considering the complex chemical diversity of BMs, model bilayers systems are frequently used to study membrane properties and biological processes. Because of the micro and nanoscale range of domains in BMs, and the consequent need of local techniques to explore BMs at the nanometric level, supported bilayer systems are very manageable platforms, since they retain two-dimensional order and lateral mobility and offer excellent environments for the insertion of membrane proteins. In particular, supported lipid bilayers (SLBs) facilitate the use of surface analytical techniques, being ideal models to study the lipid lateral interactions, the growth of lipid domains, as well as interactions between the lipid membrane and proteins, peptides and drugs, cell signaling, etc.
Several reports demonstrate the wide variety of useful techniques to study supported and non-supported lipid membranes. Thanks to the possibility of working under controlled environment and with distance and force resolution at the nanoscale, atomic force microscopy (AFM) is nowadays a well-established technique for both imaging the morphology and probing the local physical and mechanical properties of SLBs by means of force spectroscopy. However, the resolution given by AFM might be inferior to the one achievable with X-ray (XR) and neutron techniques. In particular, XR techniques such as XR reflectivity (XRR) and grazing incidence XR diffraction (GIXD) are powerful tools to characterize surfaces below the nanoscale, providing structural information in the reciprocal space through the interaction between XR and the sample electronic structure. Still, since these techniques do not involve any mechanical interaction with the specimen, mechanical properties cannot be evaluated with XR.
The general objective of this thesis is to investigate the physicochemical and structural properties of model lipid membranes combining atomic force microscopy (AFM) and spectroscopy (AFM-FS) and X-Ray techniques. The AFM provides the morphological and mechanical information of the SLBs, whereas the XR gives more understandings on the electronic structure of the bilayers. We also propose advanced methodologies based on AFM and XR as well as the coupling of both techniques for local in situ experiments. These technical progresses allow us to study not only the diversity on the chemical composition of the bilayers, but also the effect of small molecules or peptides to the membrane physical and structural properties. In addition, by means of AFM and AFM-FS we also characterize vesicular systems that are not composed by phospholipid molecules, which have a technological application: to act as nanocarriers for drug delivery.[cat] Les membranes biològiques (BMs) són fronteres autosegellants, que limiten les barreres permeables de les cèl·lules i els orgànuls i proporcionen els mitjans necessaris per compartir funcions. A part de ser crucials per l’estructura cel·lular, proporcionen una matriu de suport per a totes les proteïnes que es troben inserides a la cèl·lula, actuant com canals per l’intercanvi de massa, energia i informació amb l’exterior. Les BMs intervenen en moltes funcions biològiques, com el tràfic, la divisió cel·lular, l’endocitosi i l’exocitosi, que exigeixen canvis conformacionals durs en la membrana lipídica com la fusió, la fissió o el creixement de tubs. La correlació entre la composició i l’empaquetament dels lípid regeix les propietats fisicoquímiques de la membrana i la seva estructura mecànica
Considerant la complexa diversitat química de les BMs, sistemes de membranes model són utilitzats sovint per estudiar propietats de membrana. Degut a la heterogeneïtat de les BMs i la conseqüent necessitat de tècniques locals per a explorar BMs a escala nanomètrica, sistemes de bicapes suportades, com les bicapes de lípids suportades (SLBs), s’han proposat com models, ja que conserven l’ordre bidimensional i la mobilitat lateral, oferint ambients excel·lents per a la inserció de proteïnes de membrana.
Diversos informes demostren la gran varietat de tècniques útils per estudiar membranes lipídiques suportades i sense suport. Gràcies a la possibilitat de treballar sota un ambient controlat i amb una resolució nanomètrica en distància i força, la microscòpia de forces atòmiques (AFM) és, avui en dia, una tècnica ben establerta tant per a obtenir una imatge de la morfologia com per mesurar les propietats locals físiques i mecàniques de les SLBs mitjançant modes d’espectroscòpia de forces (AFM-FS). De totes formes, la resolució que s’obté amb l’AFM és inferior a la que es pot obtenir amb tècniques de raigs X (XR) i neutrons.
L’objectiu general d’aquesta tesis és investigar les propietats fisicoquímiques i estructurals de membranes lipídiques model combinant tècniques d’AFM, d’AFM-FS i de XR. També proposem metodologies avançades basades en AFM-FS i XR, així com l’acoblament de les dues tècniques per dur a terme experiments locals in situ. A més, amb AFM i AFM-FS també hem caracteritzat sistemes vesiculars que no contenen fosfolípids, els quals tenen una aplicació tecnològica: actuar com a nanotransportadors per al lliurament de fàrmacs
