Did you heat sample? If your phospholipids have phase transition temperature, heating above the phase transition temperature would facilitate the lipid hydration. However, if your liposomes are unstable, liposomes form large aggregation and then precipitate soon. In this case, the problem might be dispersion stability of liposomes.

In our lab, we prepared liposomes with PC and Cholesterol as their major components. As the mol ratio of Cholestrol to PC increase above 1:5, we saw some aggregation as well. As you have mentioned before reducing Cholesterol content may minimize the aggregation. If the aggregation still persist I suggest you to measure zeta potential of your liposomes to show electrostatic stability of your liposomes.Non-Pegylated liposomes with zeta potential close to zero is not stable in suspension. Then diluting your liposomes or changing ionic strength of your buffer may reduce the aggregation.

First of all, how fresh is your DPhPc?

Properties of diphytanoyl phospholipids at the air-water interface by Yasmann and Sukharev (DOI: 10.1021/la503800g) use a similar method to prepare DPhPc liposomes with some slight, but significant differences. Consider them when comparing with your methods.

Hi Mohamed Khedawy,

Thank you for your answer and your suggestions. The lipid I am using is DPhPC which does not exhibit a detectable gel to liquid crystalline phase transition from -120°C to +120°C. During hydration, I am heating my sample at 65 deg.C. Also, I am not using any cholesterol here, and the ionic solution strength is 150 mM NaCl which is already high.

Hi Gamid Abatchev

Thank you for your answer and the reference you provided it is really helpful. My lipids are very fresh. They have been purchased within the past month.

Best,

Rami