Liposome Preparation

This protocol is recommended as a general guide for preparation of liposomes from lyophilized lipids. Additional considerations and common troubleshooting points are addressed in our Liposome FAQ.

Protocol

This protocol is recommended as a general guide for preparation of liposomes from lyophilized lipids. Additional considerations and common troubleshooting points are addressed in our Liposome FAQ.

1. Dissolve lipids in chloroform and combine in the appropriate ratios.
2. Evaporate chloroform from lipid mixtures using a dry nitrogen stream or vaccum (eg. SpeedVac). Dried lipid mixture can be stored for up to 6 months at –20 ºC.
3. Re-suspend 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.
4. Freeze cyclohexane solution using dry ice or place at –20 ºC for 30 min to 1 hour depending on volume of cyclohexane used.
5. 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 until the sample is completely dry. The vial should not feel cold to the touch or smell of cyclohexane when removed from the vacuum.
   • This produces a dry, white powder, which readily suspends in water and dried lipid mixture can be stored for up to 6 months at –20 ºC.
6. Suspend the lipid mixture in the aqueous buffer (buffer temperature should be above the phase transition of the lipid. See Liposome FAQ) and allow the mixture to hydrate above the transition temperature of the lipid for 30-60 minutes (vortexing occasionally).
   • For encapsulating water-soluble compounds in the liposome the same protocol is followed except compound is dissolved in the aqueous buffer before reconstituting the dry lipid. The external compound (not encapsulated) can be removed by gel filtration. 

Notes

1. An alternative method is to skip steps 3-5 and resuspend the lipid film produced by evaporating the chloroform into the appropriate aqueous buffer. See step 6.
2. All solvent solutions should be brought to room temperature before applying to lipids. Please read the entire protocol before beginning.
3. When selecting lipids to use for liposome formation, please consider the following properties: phase transition temperature, charge, stability, and source. These can all affect lipid packing, permeability, and how liposomes are handled and stored. 
4. Once formed liposomes may be stored in aqueous solutions buffer or as a lyophilized powder. Stability when stored in solution will be affected by many factors including pH, temperature, and the addition of cryoprotectants or antioxidants.
5. Liposomes should not be confused with micelles. Liposomes are made up of a lipid bilayer with an aqueous core whereas micelles have a lipid monolayer with a fatty acid core. They are physically and functionally distinct from one another.
6. Liposomes may range in size from approximately 30 nm up to 2 microns. Liposome size is best measured by dynamic light scattering which can provide the average size of the liposome population as well as how uniform the distribution is. 
7. Liposome size and integrity may be affected by repeated freeze-thawing, therefore to obtain a uniform size distribution it is best to use sonication or extrusion. 
8. Sonication of the liposome preparation should be done in a water bath at a temperature above the transition temperature of the predominant lipid in the liposome for up to 10 minutes. 

FAQ

Echelon Biosciences carries a variety of lipids that can be used to generate liposomes in vitro. Addtional details are available in our Liposome FAQ. 

References

1. Sundberg, E. et al. (2019) Syndecan-1 Mediates Sorting of Soluble Lipoprotein Lipase with Sphingomyelin-Rich Membrane in the Golgi Apparatus. Developmental Cell VOLUME 51, ISSUE 3, P387-398
2. Aceb́ron, I. et al. (2020) Structural basis of Focal Adhesion Kinase activation on lipid membranes. EMBO J 39:19
3. Frank Szoka, Jr. & Demetrios Papahadjopoulos, (1980), Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes). Ann. Rev. Biophys. Bioeng., 9:467-508