DEVELOPMENT AND CHARACTERIZATION OF ASYMMETRIC MODEL MEMBRANES CONTAINING GLYCOLIPIDS

Biological membranes exhibit a complex lipid composition and asymmetric lipid distribution between bilayer leaflets, a feature essential for membrane organization and function. Glycolipids are highly abundant in neuronal plasma membranes, where they are confined to the outer leaflet and play key roles in lipid raft organization, cell recognition and membrane modulation. We investigated three structurally distinct glycolipids: cerebroside, a neutral glycolipid with a single monosaccharide headgroup and high gel-to-fluid phase transition temperature (Tm); sulfatide, containing a sulfate group; and ganglioside, an anionic glycolipid with a bulky oligosaccharide headgroup. Their effects on membrane fluidity, lipid packing, gel-to-fluid phase transition, lateral phase separation and membrane stability were evaluated in symmetric model membranes. Since glycolipids are naturally restricted to the outer leaflet, an asymmetric biomimetic membrane model was developed. Asymmetric Large Unilamellar Vesicles (aLUVs) were prepared by methyl-β-cyclodextrin (mβCD)-mediated lipid exchange. Exchange feasibility was assessed by Isothermal Titration Calorimetry (ITC), characterizing mβCD interactions with the studied lipids. The protocol was optimized using phosphatidylglycerol (PG) as an outer leaflet asymmetry marker, whose negative charge allowed selective lipid incorporation to be confirmed by Zeta Potential measurements. After validation, Differential Scanning Calorimetry (DSC) and Fluorescence Anisotropy were performed using the same assays applied to symmetric LUVs (sLUVs). Compared with sLUVs, aLUVs containing DPPG in the outer leaflet exhibited shifts in thermal profiles and fluorescence anisotropy, demonstrating that lipid asymmetry influences Tm, lipid packing and membrane fluidity. The protocol was applied to prepare aLUVs containing cerebroside, sulfatide or ganglioside exclusively in the outer leaflet (DPPC_in/DPPC:Glycolipid_4:1_out) and their properties were evaluated by DSC and Fluorescence Anisotropy. Compared with sLUVs, asymmetric glycolipid distribution altered the cooperativity and distribution of thermal events without significantly affecting Tm or bilayer packing. Finally, the protocol was adapted to produce Asymmetric Giant Unilamellar Vesicles (aGUVs). Optical Microscopy revealed transient membrane deformations during the interaction between mβCD–lipid complexes and recipient vesicles, consistent with lipid asymmetry establishment. Overall, these results show that glycolipid effects on membrane properties depend on both their chemical structure and transbilayer distribution while establishing a robust protocol for generating asymmetric glycolipid-containing vesicles, expanding the potential of biomimetic membrane studies.