NanoBio: Making Unilamellar Vesicles

=PREPARATION OF SMALL UNILAMELLAR VESICLES=

Abstract
This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion.

Reagents

 * Lipids (usually egg PC and 1-2 mole % dye)
 * Wide-mouth screw top glass vials, ~ 10 mL
 * ICN 7x detergent, i.e MP Biomedicals catalog # 097667093
 * Millipore water
 * Acetone
 * Chloroform
 * Absolute ethanol
 * N2(g)
 * Kimwipes

Equipment

 * 50 uL Hamilton syringe, to be used for lipids only
 * 250 uL Hamilton syringe, to be used for lipids only
 * Hot plate
 * Extruder, e.g. mini-extruder from Avanti Polar Lipids
 * Filter supports
 * Polycarbonate membranes
 * Clean dessicator free from desiccant
 * Vortex

Procedure

 * 1) Clean glass vials. Immerse vials in 20-30% 7x detergent (70-80% distilled water) that has been heated until clear (~90ºC) for 5 minutes.  Rinse thoroughly with double distilled water.  Rinse three times with acetone, then three times with chloroform.  Dry with N2(g).
 * 2) Add appropriate quantities of lipids in chloroform to clean glass vials. It is recommended that you keep a set of syringes to be used only for lipids.
 * 3) Dry the lipids down with N2(g). Ideally, the lipids will form a thin, transparent, uniform film over much of the interior glass surface.
 * 4) Dry the lipids under vacuum for 1 hour. If this is done in a desiccator, do not use desiccant.
 * 5) Rehydrate the lipids in buffer or Millipore water to ~10 mg/mL. Vortex the solution to redissolve as much lipid material as possible. The lipid/aqueous solution should result appear cloudy. Allow lipids to rehydrate for 15 min before extruding.
 * 6) Clean the components of extruder. Rinse pieces with absolute ethanol, allow to dry on Kimwipes, and repeat.
 * 7) Assemble the extruder with filter supports (4 total) and polycarbonate membranes (2 total). See extruder instructions for more details.
 * 8) Rinse the assembled extruder with Millipore water three times to remove any air bubbles.
 * 9) Pass the lipid solution ~20 times through the polycarbonate membranes. You should feel slight resistance when pushing the syringes, and the lipid solution should become transparent (vs. opaque).
 * 10) Store the vesicles at 4ºC.
 * 11) Rinse the disassembled extruder first with water, then with ethanol. Allow to dry on Kimwipes.

Critical Steps

 * Cleanliness of the glass vials is very important. It is crucial that there is no visible aqueous liquid in the glass vials before addition of the lipids in chloroform.
 * Having a uniform film of lipids on the interior of the glass vial makes rehydration of the lipids easier.

Troubleshooting

 * The inability to form a uniform lipid film can be an indication that the glass vial is not sufficiently clean.
 * Sometimes excessive pressure can build up in the extruder, and passing the lipid solution through the membrane requires a great deal of force. When this occurs, remove the lipid solution from the syringe, re-assemble of the extruder with fresh filter supports and polycarbonate membranes, and re-extrude the lipid solution.
 * If the lipid solution does not become clear and/or it requires very, very little force to pass the lipid solution through the extruder, then it is possible that the polycarbonate membranes have developed a tear. After completing 20 extrusions, disassemble the polycarbonate and check for a tear. If one is present, re-extrude the lipid solution.

Acknowledgements
This procedure was developed in Steve Boxer's laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.