It's true that including freeze thawing can increase encapsulation during liposome preparation. It's done by alternatingly putting your sample into liquid nitrogen and at room temperature or 37°C. It should be noted that it's not the same as freeze drying (lyophilisation) - this is most often destructive for liposomes unless you add a cryoprotectant. However, encapsulation efficiency will most likely still be relatively low if there's no direct interaction between your peptide and the lipids. Encapsulation efficiency could be increased by using opposite charges on peptide and lipids (e.g. cationic peptide/anionic lipids or vice versa) or by adding a lipid tail to your peptide. Concerning the size, it is very hard to have a defined size by just using freeze-thawing - you'll have to be very lucky with your system for that to work. A combination of freeze-thawing and extrusion might do the trick.

Hi,

Have a look at attached chapter-in-book (Protocol) comparing different methods of liposome and nanoliposome manufacture. Let me know any questions.

Hello Christina,

Thank you so much for your response.

I wanted to confirm my procedure- I would take my mixture of lipids with a solvent in a round bottom flask. Pass it through rotary evaporation to develop a thin lipid moon layer. Next hydrate with solvent and sonicate the solution and obtain a turbid solution. Next I would freeze and lyophilise the solution to obtain liposomes. I will encapsulate my drug into the liposome by hydrating it again with a drug. Continue the freezing and lyophilization step and repeat the previous step again to obtain a turbid mixture.

My questions are-

1. What do you suggest where should I include my extrusion step? Should I include it before or after the freeze thaw method? Also should the extrusion be done in colder conditions to maintain maximum encapsulation efficiency? I have seen in literature that there is liposome loss when its extruded and my drug of choice is temperature sensitive.

2. While freezing and lyophilising- I would put my sample in liquid nitrogen and thaw it and next lyophilise to a powder and hydrate it, later repeat the step again? or just freeze thaw it alternatively?

The first hydration step of your procedure makes no sense to me - with the hydration and then lyophilization you would just get a less smooth lipid film than the one you started with. Why not directly hydrate with the drug solution? Otherwise it looks solid - hydration, freeze-thawing, sonication. I would not dry it down after you have added the drug. For question 1: Extrusion should always come as the last step and it should be done above the phase transition temperature of your lipids. Depending on the temperature sensitivity profile of your drug, it might not be suitable for you to use. For question 2: Just freeze and thaw alternatingly.

Thank you Christina.

I will directly hydrate the moon layer lipid film with the drug solution and I will not dry it down after I have added the drug.

Because the drug is temperature sensitive and extrusion might not work. Do you suggest any other possible ways which could help me achieve a required particle size (200nm).

If you cannot use extrusion, it will be more of a trial and error game. I would suggest to check different sonication times and strengths at first. This has an influence on size and depending on your system you might be able to tune it to reach your desired size. Defined size can also be reached by using a microfluidics system, but it also has to be tuned to your specific composition, so it might be difficult to set up and expensive if you don't have the equipment. Testing different formation techniques (other than thin film hydration) might also be a way to achieve this. The heating methods mentioned above are likely not suitable for your problem, but you could look into ethanol injection or reverse phase evaporation, for example.

Thank you so much for your suggestions Christina.

Thank you Mozafari Sir. I will look into the Nanoliposomes pdf that you have shared.