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.
- Lipids (usually egg PC and 1-2 mole % dye)
- Wide-mouth screw top glass vials, ~ 10 mL
- ICN 7x detergent
- Millipore water
- Absolute ethanol
- 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
- 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).
- 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.
- Dry the lipids down with N2(g). Ideally, the lipids will form a thin uniform film over much of the interior glass surface.
- Dry the lipids under vacuum for 1 hour. If this is done in a desiccator, do not use desiccant.
- Rehydrate the lipids in buffer or Millipore water to ~10 mg/mL. Vortex the solution to redissolve as much lipid material as possible. Allow lipids to rehydrate for 15 min before extruding.
- Clean the components of extruder. Rinse pieces with absolute ethanol, allow to dry on Kimwipes, and repeat.
- Assemble the extruder with filter supports (4 total) and polycarbonate membranes (2 total). See extruder instructions for more details.
- Rinse the assembled extruder with Millipore water three times to remove any air bubbles.
- 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).
- Store the vesicles at 4ºC.
- Rinse the disassembled extruder first with water, then with ethanol. Allow to dry on Kimwipes.
- 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.
- 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.
- It is important to use lipid compositions which are in the liquid phase at the temperature which the vesicles are formed. Since egg PC is fluid to ~-10C, vesicles of egg PC can be created at room temperature and stored at 4C. Other compositions may require handling at different temperatures.
Acknowledgements: This procedure was developed in Steve Boxer’s laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.
References: Cremer & Boxer J. Phys. Chem. B 1999
Last updated: Caroline Ajo-Franklin 06 August 2007.