Have u tried to change the ratio of POPE: POPG or change the lipidic components as a whole? What about your buffer system? Have you checked the solubility of API in the buffer system?

Check my research article published on Current Drug Delivery entitled "Enhancing the Intestinal Permeation of the Chondroprotective Nutraceuticals Glucosamine Sulphate and Chondroitin Sulphate Using Conventional and Modified Liposomes"

DOI: 10.2174/1567201815666180123100148

Hello Brandt ,

Could you say something about your buffer composition? Specifically, do you have divalent cations? That might be one problem.

Check this out: http://www.pnas.org/content/pnas/suppl/2013/03/21/1301442110.DCSupplemental/pnas.201301442SI.pdf

Thanks you Ahmed and Ilya for your comments. Im using a potassium buffer KCL 150 mM and HEPES 10 mM (pH 7.2), This buffer has no divalent ions.

I dont have much options on changing the lipid ratio, since I want to compare it to other systems. Ahmed, could you share a full text of your article?

Tnaks for the link Ilya. Maybe i should try low pH to see if it helps.

Francesco, thanks for your suggestions. Yes i paint the lipids film, and then i use a rotavapor to dry the excess chloroform. I can´t use water as I am studying dye release with a certain toxin. I will try the hydration at 60 degrees as you suggest.

I suggest increasing temperature and rotation speed.

Ok, Thanks David. I have tried higher temperatures, but I will try the faster rotation speed.

It also depends on your hydophilic molecule or the ionic strenght of the hyration medium. For example, I had cationic lipid film to form cationic liposomes and I tried to hydrate with a solution of CpG which is a polyanion but when it was highly concentrated (400 ug/mL), it didn't really hydrate... even a higher speeds or temperature. We tried less concentration (200 ug/mL) and it was succesful. Also, pH is important.

Ok. Thanks alot

Any updates Brandt? Can you clarify what you mean when you say you "have not gotten liposomes"? How do you determine if you have "gotten liposomes" or not?

Hi Graham.

Using POPG:POPE 75:25, I have not been able to hydrate the lipids, and thus, have not gotten any luck with obtaining LUVs. I found an article that explains the hydration problem due to high H-bonding between PE and PG (Murzyn et al. 2005). I aslo found that PE:PG liposomes can be made at pH 9 since at this pH, the amine of the head groups are largely deprotonated, the lipid is charged, the vesicles cannot aggregate, and the lipid cannot form the HII, phase (Siegel and Epand, 1997). I finally gave up on this system, since my toxin for dye leakage experiments function at pH7.2.

Hi Marité.

I have not tried H20, since buffer is necessary for my assays.

Thanks for your suggestion.

Ok. Thank you very much. I will, since this system is the most adequate for my assasy

Hi, Dhanaraju. To remove the excess fluorescent probe I use gel filtration. Prepare a sephadex 75 column by hydrating and washing the powder in water. Add the slurry to the column and leave to settle until it is packed. Equilibrate the colum with the buffer of choice (note that the calcium may affetct the quality of your liposomes you collect). The liposomes should come out first and the dye after.

In my case, the liposomes have a dark red colour, so I can trace their movement through the column. If you cannot see them, you will need to collect all the fractions and test them. Make sure there are no air bubbles in the column or the column' s tap. I dont think milky is good. Did you extrude before passing through the column?

Ok. After extrusion if your liposome mix does not turn clear, it might be due to lipid aggregation. thats the case I have with POPE:POPG, and I have not solved it yet. I check my liposomes filled with calcein (fluorescent dye) by calcein leakage. You could use dynamic light scattering also.