Dear everyone,
I am trying to prepare the liposomes and face technical problem.
I am using Avestin Liposofast extruder and 400 nm membrane. When I prewash membrane with PBS everything seems working fine. I lose 50 um on the first passage through the membrane. However, in the next passages I receive the same volume. When I further fill the syringes with my liposome solution, everything leaks out. I don't see the leakage in the window, so I suspect that it comes from luer lock. The filter supports are not clogged. May be some of you have experienced something similar and can share suggestions how I can fix this problem?
I am preparing the liposomes as follows: prepare thin film in rotovapor from 20 mg/ml of POPC in chloroform, hydrate it in PBS using sonication bath. Then I do 3 freeze-thaw cycles in liquid N2 and try to extrude.
What temperature are you using for extrusion? Di you have an ultrasonic processor?
Hi Veronica, have you tried chromatography of your liposomes using a PD-10 or similar gel filtration columns?
Intact liposomes will be separated from soluble materials. You collect the liposomes and sonicate them with the compound you want to incorporate and repeat the gel filtration until you get the liposomes with the concentrations of the material you want.
Dear Steingrimur, thank you for your answer. I will consider the chromatography. It definitely will be useful for the next step, I need to learn the chromatography, have never done it before. Extrusion seemed to be so easy, who knew that it will take so much effort. Hopefully, I will get it done soon.
Dear Omar, thank you for your answer! I am doing the extrusion at room temperature (should be fine for POPC). I have tip sonicator, is that what you mean?
Do not run freeze and thaw cycles, use the tip sonicator instead to reduce size and then extrude.
Veronica Zelmanovich hi i am trying to prepare liposomes using the Avanti mini extruder. I too am facing leakage after passing the solution twice. Did you find a solution to this??
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