Asymmetric giant unilamellar vesicles as model systems for membrane biophysics

Lipid bilayer asymmetry is a fundamental feature of biological membranes. The asymmetric distribution of diverse lipid species between leaflets is known to
influence phase behavior and interactions with proteins. However, its role in determining membrane organization and dynamics remains poorly understood, partly due to the experimental difficulty of generating, controlling and characterizing asymmetric model membranes. Here, we develop an experimental approach to generate controlled asymmetry in giant unilamellar vesicles (GUVs) via cyclodextrin-mediated lipid exchange. This method enables selective modification of the outer leaflet while preserving vesicle integrity. Key experimental parameters, including cyclodextrin loading and post-exchange purification, were optimized, with density-driven separation providing efficient isolation of GUVs without mechanical disruption. Membrane asymmetry was assessed using Förster Resonance Energy Transfer (FRET) between NBD-PE and Rhod-PE probes. These asymmetric membranes can be used to study interactions with proteins and nanoparticles, allowing evaluation of how membrane asymmetry influences binding, insertion and membrane response under controlled experimental conditions. In addition, they provide a model system to investigate how compositional imbalance between leaflets affects membrane organization, including domain formation and vesicle morphology.