Matrix effect of vesicle formation as investigated by cryotransmission electron microscopy

It has been recently reported that under certain conditions the size distri bution of vesicles being formed upon addition of fresh surfactant to an aqu eous solution is strongly affected by the presence of preformed and narrowl y distributed vesicles. in particular, the final size distribution is stron gly biased toward the size distribution of the initial vesicles (the so-cal led "matrix effect"). On the basis of a novel experimental approach, we pre sent hete an investigation of the matrix effect and the corresponding fissi on processes of oleic acid/oleate vesicles and mixed POPC/oleic acid/oleate vesicles (POPC = 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), This n ovel approach is based on two complementary techniques: the use of cryotran smission electron microscopy (cryo-TEM) and the entrapment of ferritin whic h can be visualized by cryo-TEM in the initial preformed POPC liposomes. Ad dition of micellar oleate to an equimolar amount of 100 nm extruded POPC li posomes results in the formation of mixed vesicles with a new size and ferr itin distribution. This can be determined by cryo-TEM, and two main finding s have thus been obtained. On one hand, the matrix effect has been substant iated; i.e., the size distribution of the preformed liposomes strongly affe cts the final size distribution. However, and surprisingly, the final suspe nsion contains a large amount of vesicles with a diameter between 20 and 40 nm, i.e., significantly smaller than the preformed ones. The fact that the se small vesicles were not present in the initial population of preformed v esicles and the fact that some of them contain ferritin molecules, brings o ne to the conclusion that they have derived from fission processes of large r ferritin-containing vesicles. More generally, this cryo-TEM-based investi gation also sheds light on the basic properties of oleic acid/oleate vesicl es formed by spontaneous vesiculation, for example, the most probable size (main peak around 40-60 nm) and the surprisingly small unilamellarity (of t he order of 1.02).