Asymmetric giant lipid vesicle fabrication.

Synthetic lipid bilayers have long been used as models of cell membranes. The compositional asymmetry in the eukaryotic plasma membrane is a key chemical characteristic of this membrane that has traditionally been difficult to reproduce in synthetic systems. In this chapter, we describe recent technologies for fabricating compositionally asymmetric giant unilamellar lipid vesicles (GUVs) and provide detailed protocols for a microfluidic-based fabrication technique.

Automated formation of multicomponent-encapuslating vesosomes using continuous flow microcentrifugation.

Vesosomes - hierarchical assemblies consisting of membrane-bound vesicles of various scales - are potentially powerful models of cellular compartmentalization. Current methods of vesosome fabrication are labor intensive, and offer little control over the size and uniformity of the final product. In this article, we report the development of an automated vesosome formation platform using a microfluidic device and a continuous flow microcentrifuge. In the microfluidic device, water-in-oil droplets containing nanoscale vesicles in the water phase were formed using T-junction geometry, in which a lipid monolayer is formed at the oil/water interface. These water-in-oil droplets were then immediately transferred to the continuous flow microcentrifuge. When a water-in-oil droplet passed through a second lipid monolayer formed in the continuous flow microcentrifuge, a bilayer-encapsulated vesosome was created, which contained all of the contents of the aqueous phase encapsulated within the vesosome. Encapsulation of nanoscale liposomes within the outer vesosome membrane was confirmed by fluorescence microscopy. Laser diffraction analysis showed that the vesosomes we fabricated were uniform (coefficient of variation of 0.029). The yield of the continuous flow microcentrifuge is high, with over 60% of impinging water droplets being converted to vesosomes. Our system provides a fully automatable route for the generation of vesosomes encapsulating arbitrary contents. The method employed in this work is simple and can be readily applied to a variety of systems, providing a facile platform for fabricating multicomponent carriers and model cells.

Imaging molecular transport across lipid bilayers.

Low-molecular-weight carboxylic acids have many properties common to small molecule drugs. The transport of these acids across cell membranes has been widely studied, but these studies have produced wildly varying permeability values. Recent reports have even claimed that the transport behavior of these drugs is contrary to the rule of thumb called Overton's rule, which holds that more lipophilic molecules transport across lipid membranes more quickly. We used confocal microscopy to image the transport of carboxylic acids with different lipophilicities into a giant unilamellar lipid vesicle (GUV). Fluorescein-dextran, which acts as a pH-sensitive dye, was encapsulated in the GUV to trace the transport of acid. The GUV was immobilized on the surface of a microfluidic channel by biotin-avidin binding. This microchannel allows the rapid and uniform exchange of the solution surrounding the GUV. Using a spinning-disk confocal microscope, the entire concentration field is captured in a short (<100 ms) exposure. Results show that more lipophilic acids cross the bilayer more quickly. A finite difference model was developed to simulate the experimental process and derive permeabilities. The permeabilities change with the same trend as literature oil-water partition coefficients, demonstrating that Overton's rule applies to this class of molecules.

Microfluidic fabrication of asymmetric giant lipid vesicles.

We have developed a microfluidic technology for the fabrication of compositionally asymmetric giant unilamellar vesicles (GUVs). The vesicles are assembled in two independent steps. In each step, a lipid monolayer is formed at a water-oil interface. The first monolayer is formed inside of a microfluidic device with a multiphase droplet flow configuration consisting of a continuous oil stream in which water droplets are formed. These droplets are dispensed into a vessel containing a layer of oil over a layer of water. The second lipid monolayer is formed by transferring the droplets through this second oil-water interface by centrifugation. By dissolving different lipid compositions in the different oil phases, the composition of each leaflet of the resulting lipid bilayer can be controlled. We have demonstrated membrane asymmetry by showing differential fluorescence quenching of labeled lipids in each leaflet and by demonstrating that asymmetric GUVs will bind an avidin-coated surface only when biotinylated lipids are targeted to the outer leaflet. In addition, we have demonstrated the successful asymmetric targeting of phosphatidylserine lipids to each leaflet, producing membranes with a biomimetic and physiologically relevant compositional asymmetry.