I am preparing liposomes using the film hydration method (Bangham). However, I need liposomes over 400 nm (size) with a good polydispersity. In my protocol, after hydration of the lipids with PBS, a step of sonication (bath) for 20 seconds is done but this gives as a result very small liposomes (SUV)(between 80 and 160 nm). I'm looking for a new way/method to prepare multilamellar liposomes. There are several methods I've found: shaking (standard shaking procedure?), vortex agitation, sonication with less power, rotary evaporation with beds, and rotary mixer. Do you know a technique to fabricate MLV with a detailed protocol? I will use extrusion through membranes to reduce polydispersity and to obtain liposomes 400 nm. Thank you.
Dear David, either avoid sonication with your method or try with ethanol injection method...
Thanks Giulia but we want to entrap a hydrophobic compound so we prefer MLV over GUV.
Dear Michele, thank you. That's exactly what I'm doing. What do you think about vortexing? Do you have some kind of rpm or time? "You need to apply some kind of mechanical force as vortexing", do you know another type of mechanical force to hydrate the lipids? Thanks. I am going to test vortexing at 2500 rpm for 1 minute. I am trying to make liposomes of 400 nm. I have an extrusor so it could be useful if I obtain liposomes of a size above 1 um.
I have read some bad reviews about using glass beads, for example that they may expulse some small crystals and that is difficult to separete them. I'm going to try vortexing with low rpm and if it's not working I'm going to consider glass beads. Thanks a lot.
Hi David, as I understand from your discussion, your intent is to obtain liposomes with two or more lamellae and with a mean diameter of 400 nm or more. I think that the first part of your protocol is right, but the sonication is what you should abolish from your protocol. If you want to use a conventional method like “thin hydration”, you just have to dissolve phospholipids into an organic solvent, then you make it evaporate and finally you complete the process by gently hydrating the lipid layer with water or PBS, in which you may have dissolved an active principle (hydrophilic). In you hydrate the solution for at least 1 hour at 200 rpm, you will obtain multilamellar liposomes with good polydispersity (dimensions from 300 nm to several microns). If you want to be more precise, you can also use a filter to separate the 400 – 500 nm vesicles from the 20 micron vesicles. The sonication step will disrupt the liposomes, causing their successive re-aggregation by forming very small nanometric dimensions vesicles (50 – 100 nm). So that this final step should be avoided.
Paolo, I did exactly what you said. I obtain liposomes of 1,5 um with a very good polidispersity using rotary evaporation and gentle vortexing. Thanks a lot for your answer.
Really not bad ! Generally the mean diameter also depends on the interaction with the active principle that you encapsulate inside the aqueous core of the liposomes.There are some compound that may enlarge or reduce liposome their volume.
For now they are only blanks (PC, chol and stearylamine) but we plan to encapsulate hydrophobic compounds.
David E. Bautista-Erazo
I am trying to make MLV too. I did the same thing as you did, dissolving in chloroform and evaporating. Then I added my buffer to dissolve the thin film, and vortexed (above phase transition temperature). But it seems that my thin film never really fully re-suspended, I still saw bits of thin film in the solution instead of milky solution. Is this normal? Thank you
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