Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and P-31-nuclear magnetic resonance

It has been reported that repetitive freeze-thaw cycles of aqueous suspensi ons of dioleoylphosphatidylcholine form vesicles with a diameter smaller th an 200 nm. We have applied the same treatment to a series of phospholipid s uspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoyl phosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band P-31 NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters less than or equal to 30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From 31P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an expon ential size distribution was of the order of 100 nm after 10 freeze-thaw cy cles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawi ng does not have the same efficiency for all lipid mixtures. As found alrea dy by others, fragmentation into small vesicles requires the presence of sa lt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. I f applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusio n of sonicated unilamellar vesicles into larger vesicles only in pure water . These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The appli cability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles.