Crowding induced morphological changes in synthetic lipid vesicles determined using smFRET

Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found utility among bio-inspired technologies including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro , and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely-diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of vesicle architecture in vitro , with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding. ### Competing Interest Statement The authors have declared no competing interest.