Matrix effect of vesicle formation as investigated by cryotransmission electron microscopy

It has been recently reported that under certain conditions the size distribution of vesicles being formed upon addition of fresh surfactant to an aqueous solution is strongly affected by the presence of preformed and narrowly distributed vesicles. in particular, the final size distribution is strongly biased toward the size distribution of the initial vesicles (the so-called matrix effect). On the basis of a novel experimental approach, we present hete an investigation of the matrix effect and the corresponding fission processes of oleic acid/oleate vesicles and mixed POPC/oleic acid/oleate vesicles (POPC = 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), This novel approach is based on two complementary techniques: the use of cryotransmission electron microscopy (cryo-TEM) and the entrapment of ferritin which can be visualized by cryo-TEM in the initial preformed POPC liposomes. Addition of micellar oleate to an equimolar amount of 100 nm extruded POPC liposomes results in the formation of mixed vesicles with a new size and ferritin distribution. This can be determined by cryo-TEM, and two main findings have thus been obtained. On one hand, the matrix effect has been substantiated; i.e., the size distribution of the preformed liposomes strongly affects the final size distribution. However, and surprisingly, the final suspension 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 these small vesicles were not present in the initial population of preformed vesicles and the fact that some of them contain ferritin molecules, brings one to the conclusion that they have derived from fission processes of larger ferritin-containing vesicles. More generally, this cryo-TEM-based investigation also sheds light on the basic properties of oleic acid/oleate vesicles formed by spontaneous vesiculation, for example, the most probable size (main peak around 40-60 nm) and the surprisingly small unilamellarity (of the order of 1.02).