How to Prepare Multilamellar Liposomes (LMV)

Posted on December 03, 2019

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At Avanti, we’re all about research. That’s why we’re always trying to help streamline yours—keep reading for a brief outline of the protocol for lyophilizing, or freeze-drying, lipid mixtures for the preparation of liposomes.

Preparing Multilamellar Liposomes

First, dissolve your lipids in chloroform.

Then combine the lipids in the appropriate ration.

Using a dry nitrogen stream, very carefully evaporate the organic solvent.

Resuspend your lipid mixture in cyclohexane—if the mixture isn’t completely soluble in the cyclohexane, you can add a small amount of ethanol (1-2% of the cyclohexane volume). Be careful not to use too much ethanol, otherwise your solution won’t freeze.

Freeze the cyclohexane solution using dry ice.

Next, quickly place the frozen mixture on a high vacuum system, or lyophilization system. Generally, “house vacuum” systems are not strong enough for this process. The sample should stay frozen until it’s completely dry—if it begins to thaw out, you’ll know that either your vacuum isn’t strong enough, or there is too much ethanol as mentioned above. A thawed sample won’t produce a white powder, and may bump or foam out of the vial.

Leave your sample on the vacuum system for 3-5 hours or until the sample is completely dry. This time frame will depend on how much cyclohexane was used. The vial shouldn’t feed cold to the touch or smell of cyclohexane when it’s done. This method 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.

Lastly, suspend your lipid mixture in the aqueous buffer and allow the mixture to hydrate above the transition temperature of the lipid for half an hour to an hour. Vortex the mixture occasionally. The buffer temperature should be above the phase transition of the lipid. 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 removed by gel filtration. For more research tips and resources, contact Avanti Polar Lipids today.