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

Abstract

It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic 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 31P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters < or =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 exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion 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 applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles.

Influence of preparation path on the formation of discs and threadlike micelles in DSPE-PEG2000/lipid systems

“…Another effect of freeze-thawing was the decrease in liposome size found in the EPC/DSPE-PEG 2000 sample. This is a documented effect of freeze-thawing for liposomes in the liquid crystalline phase, which has been shown to apply also for liposomes containing a relatively high amount of PEG-lipids [21][22][23][24].…”

Optimization, formulation, and characterization of multiflavonoids-loaded flavanosome by bulk or sequential technique

“…Though sonication aids in arranging the lipid molecule into lipid bilayer vesicles, 46 the fusion of two or more phytosomes through sonication or bulk loading of compounds (M1, M2, and M3) leads to aggregation of vesicles that results in heterogeneous formulation with the coexistence of small and large unilamellar vesicles. 47,48 In contrast, M4 flavonosomes yielded an average particle size of 375.93±33.61 nm with a narrow monodisperse index of 0.286±0.02, most likely due to sequential addition of flavonoids during thin-film hydration followed by direct nanoprecipitation without bulk loading or sonication.…”

Stratum corneum lipid liposome-encapsulated panomycocin: preparation, characterization, and the determination of antimycotic efficacy against <em>Candida</em> spp. isolated from patients with vulvovaginitis in an in vitro human vaginal epithelium tissue model

“…In previous studies where liposomes were loaded by a freeze-thaw method in a buffer solution, similar morphological observations were also reported. 36,37 Both types of liposomes had hydrodynamic sizes larger than their static sizes observed in TEM images. This is because in DLS analyses liposomes are dynamic, freely active in solution, and interacting with solvents.…”