Growth and transformation of vesicles studied by ferritin labeling and cryotransmission electron microscopy

An investigation of the processes of growth and division of oleic acid/oleate and POPC/oleic acid/oleate vesicles (POPC stands for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) is presented. Preliminarily, kinetic turbidimetric studies show that the very slow process of spontaneous formation of vesicles from oleote micelles is dramatically accelerated by the presence of a small amount of performed POPC vesicles ([POPC]/[oleic acid + oleate] = 1:25), suggesting in the latter case a quite different process of vesicle formation. The changes in the size distribution of preformed vesicles is then studied by cryotransmission electron microscopy (cryo-TEM) and by using an entrapped protein, ferritin, which, due to its dense iron core, can be detected by cryo-TEM. In this way, the size distributions of filled (ferritin-containing) and empty vesicles were determined. Addition of oleate to preformed POPC vesicles resulted in a shift of the size distribution of the ferritin-containing vesicles toward larger diameters, together with an increase in the number of vesicles. Most of these vesicles were empty, unilamellar, and often smaller than the preformed ones. This suggests the generation of new vesicles through a fission process of larger vesicles, which arise from the preformed vesicles. Oleic acid and oleate molecules produced in situ through the hydrolysis of oleic anhydride (added as a supernatant to an aqueous suspension containing preformed ferritin-containing oleic acid/oleate vesicles) led to a broadening of the vesicle size distribution and an increase of the percentage of empty vesicles. All of the data taken together give evidence of the growth of vesicles upon addition of fresh surfactant, as well as evidence of fission processes of larger vescicles that lead to a large number of small vesicles.