Production of Isolated Giant Unilamellar Vesicles under High Salt Concentrations

Abstract

The cell membrane forms a dynamic and complex barrier between the living cell and its environment. However, its in vivo studies are difficult because it consists of a high variety of lipids and proteins and is continuously reorganized by the cell. Therefore, membrane model systems with precisely controlled composition are used to investigate fundamental interactions of membrane components under well-defined conditions. Giant unilamellar vesicles (GUVs) offer a powerful model system for the cell membrane, but many previous studies have been performed in unphysiologically low ionic strength solutions which might lead to altered membrane properties, protein stability and lipid-protein interaction. In the present work, we give an overview of the existing methods for GUV production and present our efforts on forming single, free floating vesicles up to several tens of μm in diameter and at high yield in various buffer solutions with physiological ionic strength and pH.

Keywords: double emulsion; electroformation; giant unilamellar vesicle; lipid membrane; microfluidic jetting; micropipette aspiration; model membrane system; swelling.

Figure 1

Overview (not complete) over lipid membrane model systems ranging from flat lipid bilayers to differently sized liposomes. SUV, small unilamellar vesicle; LUV, large unilamellar vesicle; MLV, multilamellar vesicle; MVV, multivesicular vesicle; GUV, giant unilamellar vesicle.

Figure 2

Schematic illustration of vesicle formation by natural swelling. (A) Lipids dissolved in an organic solvent; (B) Evaporation of the solvent and self-assembly of the amphiphilic lipid molecules into several stacks of bilayers; (C) Hydration of the dried lipid film with aqueous solution; (D) Swelling of the lipid film into vesicles.

Figure 3

Electroformation using Pt wires as electrodes. (A1) Schematic illustration of a vertical chamber geometry (cross section). The chamber consists of a plastic ring glued to a cover glass. The chamber is filled with aqueous solution (e.g., sucrose) and a lid with two Pt wires coated with lipids is placed on top. Application of an AC electric field leads to GUV swelling and detachment. After formation, the lid can be replaced with a chamber elongation. In case of sucrose as swelling solution, glucose can be added to the observation buffer which leads to sinking of the GUVs to the bottom for observation and easy harvesting. (A2) Photographic images of the vertical chamber. (A3) GUVs in sucrose/glucose solution at the bottom of the growing chamber (lipid composition: DOPC with 0.02 mol-% Atto532-DOPE). (B1) Schematic illustration of a horizontal chamber geometry (top view). A plastic ring is glued onto a cover glass. The platinum wires with the lipids are inserted into a plastic ring very close to the edge of the ring to be in small distance to the glass (see inset labeled with side view). (B2) Photographic image of the horizontal chamber. (B3) Observation of GUV growth during electroformation in sucrose (lipid composition: DOPC with 0.02 mol-% Atto532-DOPE).

Figure 4

Electroformation using ITO coated glasses. (A1) Schematic illustration of an ITO chamber. Two ITO coated glasses with conducting sides facing each other are separated by a spacer. Connecting the ITO layers via copper tape to a function generator leads to swelling of the GUVs. (A2) Photographic image of an ITO chamber. A plastic frame with magnets holds the chamber closely together. (A3) Observation of GUV growth during electroformation in sucrose (lipid composition: DOPC with 0.02 mol-% Atto532-DOPE). (B1) Schematic illustration of a cover glass with interdigitated ITO electrodes with 200 μm spacing. (B2) Photographic image of the glass with interdigitated ITO electrodes connected with wires to a function generator. (B3) Observation of GUVs grown on the interdigitated electrodes (lipid composition: DOPC with 0.02 mol-% Atto532-DOPE).

Figure 5

GUVs prepared by electroformation: (A) in deionized water and (B) in 300 mM sucrose after applying 10 Hz and 2 Vpp for 2 h (lipid composition: DOPC with 0.02 mol-% Atto532-DOPE).

Figure 6

GUVs formed by electroformation for 2 h at 2 V_pp and different frequencies for Tris buffer, Bilayer buffer and PBS. Lipid composition: DOPC and 0.02 mol-% Atto532-DOPE. Scale bars: 10 μm.

Figure 7

Effect of ITO layer degradation on the GUV yield (Tris buffer, 300 Hz, 2Vpp): (A) GUVs formed on new and (B) on used and discolored ITO glasses (lipid composition: DOPC with 0.02 mol-% Atto532-DOPE).

Figure 8

Schematic illustration of polymer-assisted swelling: Upon hydration of a dried lipid film, vesicles swell at an enhanced rate due to an accelerated buffer flow from below.

Figure 9

GUVs formed by PVA-assisted swelling in Tris buffer, Tris buffer + 150 mM NaCl, Bilayer buffer and PBS (left to right): Vesicles attached to the lipid film focused on the lipid film (upper row), a few micrometers above (middle a row) and detached vesicles in solution (lower row). Lipid composition: DOPC and 0.02 mol-% Atto532-DOPE. Scale bars: 10 μm.

Figure 10

GUVs prepared by PVA-assisted swelling in Bilayer buffer and transferred into an observation chamber. (A) Vesicle cluster detached by tipping against the growing chamber. (B) Vesicle with membrane defects detached by sonication. Lipid composition: DOPC and 0.02 mol-% Atto532-DOPE.

Figure 11

GUV production by the double emulsion method. (A) Photographic image with overlaid schematic illustration of GUV formation using double emulsion: Flow of an aqueous solution (inner flow) into an oil phase flow containing lipids (middle flow) leads to bulging at the tip of a tapered capillary. Introducing a flow of another aqueous solution (outer flow) in between the outer and middle capillary walls into another tapered capillary causes a tear-off resulting in double emulsion droplets, which are collected by the right capillary. (B) Photographic image of the whole configuration including a schematic illustration of the PDMS adapters for connecting the capillaries and for flow inlet of the different solutions.

Figure 12

DOPC-GUVs formed from double emulsion droplets.

Figure 13

Micropipette aspiration and injection. (A) Detached GUV prepared in Bilayer buffer on PVA and aspirated by a micropipette; (B) Insertion of a nanopipette (200 nm opening) into an aspirated GUV.