The solubilization of lipid membranes by Tween-20 is crucial for a number of biotechnological applications, but the mechanistic details remain elusive. Evidence from ensemble assays supports a solubilization model that encompasses surfactant association with the membrane and the release of mixed micelles to solution, but whether this process also involves intermediate transitions between regimes is unanswered. In search of mechanistic origins, increasing focus is placed on identifying Tween-20 interactions with controllable membrane mimetics. Here, we employed ultrasensitive biosensing approaches, including single-vesicle spectroscopy based on fluorescence and energy transfer from membrane-encapsulated molecules, to interrogate interactions between Tween-20 and submicrometer-sized vesicles below the optical diffraction limit. We discovered that Tween-20, even at concentrations below the critical micellar concentration, triggers stepwise and phase-dependent structural remodeling events, including permeabilization and swelling, in both freely diffusing and surface-tethered vesicles, highlighting the substantial impact the surfactant has on vesicle conformation and stability prior to lysis.

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 the 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.

The interaction of Tween-20 with lipid membranes is crucial for a number of biotechnological applications including viral inactivation and membrane protein extraction, but the underlying mechanistic details have remained elusive. Evidence from ensemble assays supports a global model of Tween-20 induced membrane disruption that broadly encompasses association of the surfactant with the membrane surface, membrane fragmentation and the release of mixed micelles to solution, but whether this process involves intermediate and dynamic transitions between regimes is an open question. In search of the mechanistic origins of membrane disruption, increasing focus is put on identifying Tween-20 interactions with highly controllable model membranes. In light of this, and to unveil quantitative mechanistic details, we employed highly interdisciplinary biophysical approaches, including quartz-crystal microbalance with dissipation monitoring, steady-state and time-resolved fluorescence and FRET spectroscopy, dynamic light scattering, fluorescence correlation spectroscopy, wide-field single-vesicle imaging and scanning electron microscopy, to interrogate the interactions between Tween-20 and both freely-diffusing and surface-immobilized model-membrane vesicles. Using ultrasensitive sensing approaches, we discovered that Tween-20 leads to a stepwise and phase-dependent structural remodelling of sub-micron sized vesicles that includes permeabilization and swelling, even at detergent concentrations below the critical micellar concentration. These insights into the structural perturbation of lipid vesicles upon Tween-20 interaction highlight the impact on vesicle conformation prior to complete solubilization, and the tools presented may have general relevance for probing the interaction between lipid vesicles and a wide variety of disruptive agents.

Synthetic lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found vast utility among bio-inspired technologies including drug delivery vehicles. Having a diameter of a few tens to hundreds of nanometers enables such complex processes to be studied at the level of a handful of molecules, conferring benefits in exploring molecular heterogeneity under near-physiological conditions. Vesicle morphology can be modified by changing the mixture of lipids used in creating them, fusing and lysing vesicles using proteins and detergents, or modifying the pH, temperature and ionic strength of the surrounding buffer, enabling the effects of membrane curvature on these processes to be explored. The requirements for experimental control have meant that most vesicle studies are performed under dilute solution conditions in vitro. The use of vesicles in crowded intracellular environments, known to influence the activities of many cellular processes, is limited by our knowledge of how molecular crowders affect vesicle morphology. To tackle this limitation, we used fluorescence spectroscopy, picosecond time correlated single photon counting and single-vesicle imaging to explore the influence of molecular crowding on the structure of freely-diffusing and surface-tethered vesicles fluorescently labelled with DiI and DiD. By quantifying single-molecule Förster resonance energy transfer (smFRET) between the probes, we determined the dependence on vesicle morphology from crowding using the molecular weight crowders sorbitol PEG400, Ficoll400 and PEG8000, identifying a common theme that both low and high molecular weight crowders trigger structural rearrangements of the vesicle that we assign to compaction. A particularly striking observation is that the low molecular weight crowder sorbitol results in irreversible changes to the smFRET efficiency attributed to permanent compaction, whereas the influence of the higher molecular weight crowders was found to be reversible. The effect of crowding perturbation on the architecture of such a reduced system not only emphasizes the power of single-vesicle approaches to probe complex biology, but also illustrates the potential to controllably alter the vesicle volume and radius of curvature for several biotechnological applications.

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