If you sonicate more time, you could obain formation of more liposomes I think. You have to centrifugate because the pellet from your centrifugation is not formed of liposomes, but of multilamellar vesicles still. If the solution is not clear, it is because these vesicles are cloudy in your sample. A liposome (only) solution looks clear.

But I have a question for you now!? When you hydrate the lipid film, you gel multilamelar vesicles enclosing your peptide. When you sonicate, they reorganise to form liposomes; how do you ensure they still enclose a lot of your peptide?

Also I am doing a similar protocole, but not to enclose peptides, instead I add a membrane anchored protein. I found that sonication was much more difficult in presence of the protein, somehow it interferes with the liposome formation. i dont know if your peptide could have a similar effect?

What do you mean by "difficult" during sonication in presence of the protein? In my case it becomes milky solution either with or without the peptide. After centrifugation it is characterized by DLS and TEM to be liposomes, but I do not know how many liposomes there are. Do you have other methods to charaterize them?

As for your question, I thought the peptide is uniformly dispersed in solution, so any single vesicle, either MLV or single layer liposome, may somehow enclose some peptide. But the fact is the poor encapsulation ratio... Do you have any suggestions?

Thanks!

download this pdf.

http://www.pharmaxchange.info/notes/ceutics/liposomes.pdf

may be useful for you.

As per my experience, you optimize centrifugation speed and time(5000 to 20,000 rpm).

I also characterize by DLS, and yes, if there is just one average size around say 50-60nm, then everything is liposomes of that size; the vesicles that dont form liposomes show a second population of bigger size in the DLS measurement, if some remain present after sonication.

In my cae I have to sonicate longer with protein to obtain liposomes, but it does not happen to you. The liposomes solution are normally clear, except if you have a high concentration (more than 2mM I would say).

Not many suggestions for encapsultion, I am not so familiar with that unfortunately. Is it suitable to use liposome formation via extrusion for enclosing molecules as well?

Now I somewhat suspect that maybe the neutral surface of PC makes the liposome aggregate easily, thus low yield. I am now trying the reverse-phase evaporation method, which theoretically makes a higher encapsulation ratio.

Thanks for all the advice from you two!

As Anabel has suggested your procedure produces multilamellar vesicles which have a very low entrapped volume per vesicle. To increase your yield, you should try to make unilamellar vesicles. the easiest way to do this is through extrusion (heat the vesicles above the phase transition temperature of your lipids before extruding). The switch from multilamellar to unilamellar vesicles should increase the yield around 10 fold if you have sufficiently concentrated vesicles before extruding (around 10 mg/ml).

The concentration of lipid during vesicle formation is one of the dominant factors affecting your encapsulation efficiency. If extrusion is not possible for some reason you can try freeze thaw/cycles which will reduce the number of bilayer leaflets per mulitlamellar vesicle.

I will try the extrusion and see how it works with encapsulation ratio. By the way, I find that after incubation at room temperature, the un-centrifuged milky solution turns into semi-transparent one with white lipid precipitates. What are those precipitates? Should they be discarded or collected altogether for extrusion?

Many thanks again for all the suggestions :)

Hi Jenny,

You should watch this video to understand what multilamellar vesicles are and why you get low encapsulation.

https://www.youtube.com/watch?v=vGz-qDE3Go4

After leaving your solution at room temp overnight you will see that the white percipitate is basically liposomes that are huge, 1um-10um. your solution clears, because they sank down. The reason you get 60nm liposome is because after the huge ones sank down all you are left with in solution are the little tiny ones.

Had you used a probe sonication on your solution all these enormous liposomes would turn to around 30nm liposomes. I never did this but know someone who uses it routinely, down side is some of the metal from the probe is deposited in the solution, and its not a clean technique.

Using extrusion you can get a uniform distribution of liposomes, but at high lipid concentrations this will get very difficult to extrude by hand (it also depends on lipid types, your PC:Ch combo should be easy). Also flash freezing in liquid nitrogen and defrosting as suggested previously will also break down the onion like multilamellar vesicles (also as seen in video) but it will not give you a set liposome size like in extrusion.

the more lipids you have the more of your peptide you'll get encapsulated.

I haven't seen that proteins interfere with liposome formation, but then again this depends on protein size, liposome size, lipid composition, buffers ect ect.

good luck !

A few points based on the above question/ comments:

(1) To answer your first question, I don't know why would would centrifuge out your lipid. If you want the small liposomes, simply extrude your sample through a 100 or 50 nm membrane. It seems like you would be removing a large amount of lipid since multilamellar liposomes are formed during your first step and no downsizing is done. If you do extrude, make sure the temperature is above the Tm of the lipid, otherwise, you will have major lipid loss and difficulty in extrusion.

(2) You should add a charged lipid to stabilize the formulation, otherwise, as you have noted, you get a large degree of large vesicle formation (vesicle fusion) -- and this will be lost if you centrifuge.

(3) What is the ultimate goal? (unilamellar, multilamellar, size preference?) If you have 26 mM lipid, the total amount of encapsulation is very low (depends highly on average particle size). see www.http://liposomemodel.com/.

(4) Run some tests to see if the peptide is stable under sonication, heating, or any other test you would perform. This should be done before any work on encapsulation is performed, otherwise, you may be wasting lipid and your time.

(5) It is difficult to "see" the particles.. but if the solution is turbid/white or has a sediment... you have very large particles (>200nm).. a slight "bluish" tint is a good indication of a monodispersed,