Dear Marzuk Ahmed ,

It depends somewhat on the composition of the vesicles but would not advice bath sonication.

For DOPC, DOPG, combinations of both, cardiolipin etc. a succesful procedure described it as follows:

“Large unilamellar vesicles (LUV) were prepared by hydrating dry lipid films, which then were frozen and thawed 10 times and passed subsequently through 400- and 200-nm pore size polycarbonate fdters as described (Hope et al., 1985).”

See for example:

Article Anionic phospholipids are essential for α-helix formation of...

Best regards.

Dear Marzuk Ahmed ,

Somewhat less defined but unilamellar vesicles can be obtained using bath sonication according to the following procedure (see enclosed file for details):

“Liposomes … were prepared by vortexing thoroughly dried lipid mixtures in buffer above the transition temperature and then bath sonicating under N2 for 5 min. Vesicles for use in measurements of probe exchange rates were prepared similarly, but the lipids were initially vortexed in distilled water and bath sonicated for 2 min (to facilitate dispersal as unilamellar vesicles), then mixed with an equal volume of a twofold-concentrated buffer solution, and sonicated for a further 3 min to equilibrate the compositions of the aqueous phases inside and outside the vesicles.”

Best regards.

Dear Rob Keller ,

I want to prepare LUVs in various lipid compositions of PG/PC, for example, 40%DOPG/60%DOPC (mol/mol) LUVs. I have also some confusion relating the time and surrounding temperature while evaporating chloroform by a rotary vacuum pump. On the other hand, some reported works suggest vortexing and sonicating cycles after the addition of buffer (I want to use PIPES buffer) solution to the dry lipid film. The exact number of the vortex period and sonication period is necessary. Besides, the temperature of sonication as it is known to me that the transition temperature for different lipids is different. I can understand from several articles that the extrusion method is good for LUVs synthesis somewhat bath sonication is not clear (I don't want to use probe sonicators). Some articles are attached here. Thank you.

Regards.

Dear Marzuk Ahmed ,

The papers I indicated give a suggestion for a protocol. Your included papers give other suggestions.

But as indicated before and repeated by Lapinski et al.: “The primary advantage of sonication over extrusion is that it is less time-consuming. However, the resulting liposome batch-to-batch mean diameter and size distribution are not as reproducible as those made by extrusion.”

Mendez et al. Indicate ‘mild’ (so bath) sonication after numerous cycles of freeze thaw. In other words, an additional 25 papers will give you at least another 10 different other protocols. So, my advice would be:

1. Choose one and describe this in your paper (which you hopefully end up with after finishing your research): this ensures that others can do the same and know how you did it

2. Vacuum drying at 40 oC like indicated by Mendez will do, but again as long as you strictly follow one protocol just stick to it

3. Temperature for sonication etc. can be ‘simply’ room temperate (this will work with DOPC and DOPG no worry about transition temperature).

So overall message there is no perfect protocol: just pick one and stick to it.

Best regards.

Dear Rob Keller ,

Thank you very much for your precious suggestions.

Dear Marzuk Ahmed ,

I am not sure I fully understand your question, but the preparation of LUVs or SUVs is rather simple. Suppose you depart from multilamellar vesicles (MLV, obtained for example from lipid film hydration), you can simply place your MLVs contained in a glass vial in a bath sonicator. Anything from 2-10 minutes will break your vesicles down forming smaller vesicles.

Note that it is usually the case that sonication generates smaller vesicles (SUVs) than extrusion (LUVs).

If you have access to dynamic light scattering (DLS), it would be important to characterize the size and dispersity of your formed vesicles and check whether you have obtained small vesicles.

Ps.: The temperature is not very important as long as all lipids used have a melting temperature (Tm) significantly lower than room temperature, which is the case for most lipids. If this is not the case, you will need to perform all of the steps (hydration, sonication/extrusion) at at temp higher than the lipid with the highest Tm.

Hope that helps =)

Dear Rafael Lira ,

It is needed to achieve a considerable amount of LUVs than SUVs by bath sonication. My query is that after the hydration of dry lipid film, which steps should I follow to form LUVs from MLVs. As far as I know, there are some vortexing and sonication periods are necessary. Thanks.

In the website of Avanti Polar Lipids (the main company when it comes to lipids) there is a description on how to prepare MLVs

https://avantilipids.com/tech-support/liposome-preparation/lmv

Since it seems you already have the MLVs, there are many ways of achieving smaller vesicles from them.

One is by sonication as in my post before. If you have a bath or a tip sonicator, just sonicate your sample and you get small vesicles. Bath sonication is more common, then if you have access to one, place your MLVs (in a glass vial) in the sonicator for 10 minutes then you have your small vesicles. That's all.

Another way is the so called "freeze and thaw". You just freeze your sample (either in liquid nitrogen or even in the freezer) and thaw it in warm water. You repeat this cycle 10 times and you also get small vesicles.

All of these are very well established methods and a quick google can help you find papers wherein these protocols have been used.

Hope that helps.

Rafael