CHANGE IN INTRAVESICULAR VOLUME OF LIPOSOMES BY FREEZE-THAW TREATMENT AS STUDIED BY THE ESR STOPPED-FLOW TECHNIQUE

The intravesicular volume of phospholipid vesicles was measured by a spin-label method coupled with the ESR stopped-flow technique. The internal volume was defined as the volume from which a spin-broadening agent, tris(oxalato)chromate ion, but not a spin label, TEMPONE, was excluded. The internal volume of sonicated phospholipid vesicles was about 0.4 μl/mg lipid. Freeze-thaw treatment increased this value 10-50-fold. However, repeated freeze-thaw treatment did not increase the value further. On the other hand, biomembrane vesicles (sarcoplasmic reticulum membrane vesicles) showed no change in internal volume with freeze-thawing. The permeability of the phospholipid vesicle membranes measured as the rate of transmembrane diffusion of the spin-broadening agent into the vesicles was very low even after freeze-thawing. These findings clearly demonstrated that freeze-thaw treatment induced fusion of hundreds of small vesicles to form giant liposomes, the membrane of which was impermeable to the complex ions. Optimal conditions for the formation of giant liposomes were investigated with reference to the composition of the suspension medium and the method of freezing and thawing. Within the range of 0.143-2 M KCl, a concentration of 0.143 M produced the largest increase in internal volume for asolectin vesicles, whereas 2 M was optimal for egg-yolk phosphatidylcholine vesicles. Freezing in either iquidnitrogen or solid CO2/ethanol and thawing in cold air was the best procedure for increasing the internal volume by freeze-thaw treatment. © 1990.