I'm trying to separate the encapsulated active compound from the non-entrapped active compound in my liposomes. Do you have a protocol or some recommendations about dialysis? Centrifugation failed since my liposomes have a size of 300 nm.
If the unincorporated active compound is water-soluble, then you can dialyze it away from liposomes. Since the liposomes are very large, they will remain inside. I like to use Slide-a-lyzer dialysis cassettes (Thermo-Fisher) with 10,000 or 20,000 molecular weight cutoff, but you could also use tubing. Dialyze against a large volume with fast stirring. It may take a few days. Change the dialysis bath at least once a day.
My unincorporated active compound is highly hydrophobic (Vitamin D, dexamethasone). I am going to put the membrane over my a little tube of 2mL. But the cut off is 3500 (do you think is bad?). Do you think 2 mL of sample in a 50 mL falcon tube is a large volume? I wanted to change the receptor buffer at 10 min, 30 min, 2 hours, 6 hours and then daily for 2 or 3 days. What do you think?
(1) For a 2-ml sample I would recommend using a dialysis cassette. (2) I would use a higher molecular weight cutoff tubing (10,000 or higher), since the liposomes won't go through no matter what cutoff you use, but higher cutoff gives faster dialysis. (3) I think it won't work because of the hydrophobicity of the compound, but you can check by seeing whether you can dialyze the compound without liposomes. (4) Dialysis is a slow process, so there is no point in changing the bath after only a few minutes or hours. Twice in the 1st day is sufficient, then once a day for each subsequent day.
I have cholesterol in un-loaded liposomes (control liposomes). But the liposomes encapsulating active are: DSPC:stearylamine:active 7:2:1. Active is vitamin D, or curcumine or dexamethasone. Thanks a lot I was also thinking that dialysis may be complicated :/.
We want to make them cationic so we are using stearylamine. I'm going to do calorimetry as you say. And I am not using cholesterol in my active liposomes since my active ingridients are also sterols.
Hi David. Alternatively, you could use the Amicon 10 kDa filter. This should retain your liposomes.
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