I am preparing DC-chol/DOPE=3:2 liposomes, and it is driving me crazy. At first, DC-chol and DOPE was dissolved in chloroform at 3 mg/ml and evaporated under vacuum or 1hr. pH 7.0 or 7.4 20 mM HEPES solution was added and the lipid concentration was 10 mg/mL. After overnight hydration, the lipid film was suspend in the solution. I sonicated this suspension at 300W for 5-10 mins. However, the suspension was still very cloudy and some small 'lipid cake' can be observed. Strange. I try to pass this suspension through a membrane with pores 440 nm in size, but it's all stuck on the membrane. What's wrong?
PE by itself forms the an inverted hexagonal phase at room temperature regardless of the presence of Chol (not sure about DC-Cholesterol) For this reason, it will be very difficult to form liposomes from PE.
You can either:
1. Work below the phase transition of PE (~10C without cholesterol, not sure with)
2. Use a 1:1 PC:PE mixture instead of 100% PE
First, Jeffrey is right that the vesicles will be better if you add a second lipid. You can keep the PE up to about 70%. I would need to know more about what you are trying to do with the vesicles to advise an exact lipid mixture. After drying, add your solution right away, vortex and sonicate.
Thank you Jeffery and Marie. Dc-chol/DOPE liposome is a classical liposome system, created by Leaf Huang. In many papers, it seems that this liposome is very easy to be hydrated. But I've tried many times and i've found this liposome MLV is really hard to reach the so-called 'sufficient hydration' level. I use 20mM HEPES solution and the hydration process takes one to two days. However, I try to pass this suspension through a membrane with pores 440 nm in size, but it seems that most of the 'lipid cake ' stuck on the membrane.
the protocal 'http://geguchadze.com/PDF/protocols/CPonline/Doc/9644-9644.html ' said 'sonicate lipid suspension in bath sonicator until all aggregates disappear' .But this is not the case for DC-chol/DOPE. Even after 10mins of sonication, there is still lots of aggregates.
dilute the liposomes to 1mg/ml. Hydration is concentration dependent
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