Following is a brief outline of the protocol for lyophilizing (freeze-drying) lipid mixtures for preparation of liposomes:

• Dissolve the lipids in chloroform.

• Combine the lipids in the appropriate ratio.

• Carefully evaporate the organic solvent using a dry nitrogen stream.

• Resuspend the lipid mixture in cyclohexane. If the mixture is not completely soluble in the cyclohexane, add a small amount of ethanol (1-2% of the cyclohexane volume). Do not use too much ethanol as the solution will not freeze with excessive ethanol present.

• Freeze the cyclohexane solution using dry ice.

• Quickly place the frozen mixture on a high vacuum system (lyophilization system). Generally, "house vacuum" systems are not strong enough for this process. The sample should remain frozen until it is completely dry; if the sample begins to thaw, either the vacuum is not strong enough or there is too much ethanol present. A thawed sample will not produce a white powder and it may bump or foam out of the vial.

• Leave the sample on the vacuum system for 3-5 hours (depending on how much

cyclohexane was used) or until the sample is completely dry (the vial should not feel cold to the touch or smell of cyclohexane when removed from the vacuum).

Note: This produces a dry, white powder which readily suspends in water. An alternative method is to resuspend the lipid film produced by evaporating the chloroform using the appropriate aqueous buffer.

• Suspend the lipid mixture in the aqueous buffer (buffer temperature should be above the phase transition of the lipid) and allow the mixture to hydrate above the transition temperature of the lipid for 30-60 minutes (vortexing occasionally).

This procedure yields large, multilamellar vesicles (LMV or MLV). For water soluble compounds to be entrapped, the same protocol is followed except the compound is dissolved in the aqueous buffer used to reconstitute the dry lipid and the external compound (not encapsulated) is remove by gel filtration.

Reference:

Frank Szoka, Jr. & Demetrios Papahadjopoulos, (1980), "Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes)", Ann. Rev. Biophys. Bioeng., 9:467-508.