Nanoscale structural and mechanical properties of lipid bilayers in air environment / Propietats estructurals i mecàniques a la nanoescala de les bicapes lipídiques en aire

Abstract

Cell membranes are 2-D heterogeneous fluid systems that show nanoscale structures of great interest because of its importance in membrane functions. Due to the inherent complexity of natural membranes, the study of model membranes is essential to obtain important information about membranes. The small lateral size and tiny variation of the height of the lipid structures require the use of nanoscale characterization techniques. However, the requirement of liquid environment to preserve the integrity of the membrane limits the number of techniques that can be applied in the study of the physical and chemical properties of biomembranes. The objective of the present thesis was to develop a procedure to prepare model lipid bilayers stable in air environment and showing physicochemical properties as close as possible to their equivalent in liquid media. For the first experiments DOPC was selected as model to adapt the existing protocols for hydrated spin-coated samples to the new protocol for dried samples. In the structural study it was demonstrated that the dewetting pattern described by previous authors not only depends on the proximity of the layer to the substrate because also depends on the lipid concentration on the coating solution. Apart from that the presence of a continuous monolayer in contact with the mica substrate was demonstrated contrary to previous results with other lipids. The force spectroscopy measurements represented the first study on single bilayers in air and surprisingly demonstrated that the lipid layers in air presented similar mechanical properties than hydrated samples. Secondly the preparation protocol was adapted for phospholipids with different characteristics, the saturated phosphocholines. Contrary to unsaturated lipids, which present in general a high fluidity at ambient temperature, this is not always true for saturated lipids. For phosphocholines with short hidrocarbonated chains the melting temperature is low and then they are fluid, but for long hidrocarbonated chains the melting temperature is high and they can present a non-fluid behavior. For this reason different saturated lipids with different chain lengths were studied (DLPC, DMPC, DPPC and DSPC). The results with these lipids demonstrate that the conventional protocol of spin-coating induces the interdigitation of certain areas of the samples for the cases of DPPC and DSPC. The effect of the presence of alcohols and lateral tension were studied, being the rotation speed determined as causative of this phenomenon. Finally, we studied the more important case of multicomponent samples. In this study, ternary samples made of DOPC, Sphingomyelin and Cholesterol, relevant for lipid raft models, were selected. The corresponding binary samples with Cholesterol were also studied to determine separately the effect of Cholesterol in each of the components (DOPC and SM). Results unambiguously showed that air stable multicomponent lipid bilayers can be prepared by the spin coating technique with structural and mechanical properties remarkably resembling those of the equivalent systems in liquid media, specially on what concerns phase segregation phenomena. In particular, and more importantly, we showed that the ternary mixtures of DOPC/Chol/SM under dry conditions showed also the presence of lipid rafts. In summary, the present thesis has showed that it is possible to prepare lipid bilayer model systems morphologically stable in dry air conditions that present similar topography and mechanical properties than hydrated samples. Therefore it opens the possibility to characterize these systems with nanoscale techniques that until now have not been applied to them, thus offering the possibility to clarify physicochemical properties of lipid bilayer model systems that still today remain unexplored in spite of the vast literature of lipid bilayer model systems.

Les membranes cel•lulars són fluids en 2D heterogenis que presenten estructures a la nanoescala (dominis) de gran interès degut a la seva importància en les funcions de membrana. Com a conseqüència de la inherent complexitat de les membranes naturals, els estudis amb membranes naturals són essencials per tal d’obtenir informació sobre les propietats fisicoquímiques de les membranes. No obstant això, el requeriment de medi líquid, per preservar la integritat de la bicapa lipídica, limita el nombre de tècniques que es poden aplicar en l'estudi de les propietats físiques i químiques de les biomembranes. L'objectiu d'aquesta tesi és desenvolupar un procediment per preparar bicapes lipídiques model estables en aire i que mostrin propietats fisicoquímiques similars a la dels seus equivalents en medi líquid. Per aquest fi es va utilitzar la tècnica d’spin-coating per preparar les mostres, tant monocomponent (fosfolípids insaturats i saturats) com multicomponent (barreges binàries i models de lipid raft). Les mostres varen ser caracteritzades amb microscòpia de forces atòmiques (AFM) i es va demostrar la seva similitud amb les mostres hidratades tant mecànicament com estructuralment.

En resum, enquesta tesi ha demostra que és possible preparar sistemes l model de bicapa lipídica morfològicament estables en aire sec que presenten topografia similar i les propietats mecàniques que les mostres hidratades. Per tant, s'obre la possibilitat de caracteritzar aquests sistemes a nanoescala amb tècniques que fins ara no s'han aplicat a ells, oferint així la possibilitat d'aclarir les propietats fisicoquímiques de sistemes lipídics model bicapa que encara avui romanen inexplorades tot i la vasta literatura del model de bicapa lipídica sistemes.