Hi,
I'm attempting to experimentally determine POPC liposome-water partitioning coefficients of a range of chemicals. For the preparation of liposomes (large unilamellar vesicles) i'm using the extrusion technique through 100 nm filters. (https://avantilipids.com/divisions/equipment/)
Briefly my (intended) protocol consists of dissolving POPC in chloroform and blow dry with nitrogen and vacuum. I then re-suspend the dry film in PBS to 4-10mM and leave shaking overnight. At this stage a lot people apply multiple cycles of cycles of freezing and thawing in liquid nitrogen or CO2/ethanol bath, but i don't have the material to perform this. How crucial is this step for the preparation of liposomes ? and will it affect the water-liposome partitioning ?
Thank you,
Alex
Hello Alexandre,
You can extrude directly.
Make sure that when you shake your POPC, they are under a nitrogen (or Ar) blanket, especially if you do it overnight - though an hour should be more than enough.
Extruded liposomes are not really unilamellar, the population tends to be oligolamellar. You can determine the average number of lamellae by NMR.
HI,
I would actually say that freeze thaw in part is used to decrease multilamellarity. For POPC it could be around 10-15% of the vesicles that are multilamellar without this step. The important thing is to extrude at least 5 times... We typically do 11. And even more importantly is to use the solution fresh, as POPC vesicles tend to aggregate over time. To increase vesicle stability you could add some negatively charged lipid.
Good luck with your work!
Marité
Hi all,
Thank you so much for the tips, really helpful. Van Du Nguyen, it is considerably easier to do decreasing size filtration, thank you for that.
As i said i'm unable to perform freeze thaw cycles, but at least now i know some of the implications of that (e.g. 10-15% multilamellar) so it will be easier to compare and explain differences i may encounter with literature values.
Thank you
Hi, Antonius,
Sorry, didn't see the question -
It is true that the lamellarity problem is bigger with larger vesicles, at least for PCs. But, my impression is that a few OLVs or MLVs will sneak through somehow...
We don't use vesicles that are more than two weeks old, stored at 4 C under Ar.
@Alexandre -
Actually, all you need is liquid nitrogen and a dewar - and appropriate protective gear to work with the liquid nitrogen... Drop the eppendorf with liposomes in, wait till it freezes, take it out and thaw in a water bath. Repeat.
@Marité - do you see aggregation (size increase in the DLS or aggregates in the TEM), or precipitation (white powdery substance at the bottom of the tube)? We extrude 30 - 50 times, and I don't recall any problems with POPC liposomes. We did have occasional aggregation problems we attributed to contamination, but that was independent of the lipids we used.
@Ilya - We see both.... precipitation can occur upon keeping in the fridge for zwitterionic lipids in buffers over a few days, even for vesicles that have been tip sonicated down to 20-30 nm. We see aggregation overtime as well (as measured by DLS). We do not use old lipid solutions, all fresh or within a day. Maybe it is a matter of eliminating larger vesicles to start, I guess your size distribution is quite sharp upon so many extrusion steps!
Worse is for saturated lipids with high Tm, off course.
Hi all. Thank you for the tips and useful discussions. I managed to get hold of some liquid nitrogen and compared liposomes with (1) and without (2) the freeze-thaw step.
In terms of particle size (measured on a Zetasizer Nano-Zs) we found no significant difference between the two. (1) - 208nm, 0.24 PdI, (2) - 203nm, 018PdI.
We also performed a quick partitioning experiment and the only difference we observed was higher variability for values obtained with (2). this might be due to increased multilamellarity as the system did not reach equilibrium.
Extrusion 13x trough 100nm filters.
https://doi.org/10.1016/j.bbamem.2016.09.020
Hi....may be useful ....