Deformation and instability of membrane structure of phospholipid vesicles caused by osmophobic association: mechanical stress model for the mechanism of poly(ethylene glycol)-induced membrane fusion

Abstract

The mechanism of poly(ethylene glycol)-induced fusion of phospholipid vesicles was studied based on the "osmophobic association" theory which was recently proposed both theoretically [Ito, T., Yamazaki, M., and Ohnishi, S. (1989) Biochemistry 28, 5626-5630] and also experimentally [Yamazaki, M., Ohnishi, S., and Ito, T., (1989) Biochemistry 28, 3710-3715]. Osmophobic association and fusion were detected by measuring the light scattering of the vesicle suspension; the former was detected from the increase in light scattering induced by the addition of PEG, and the latter was from the irreversibility of the increase in light scattering. Threshold concentrations of PEG were required not only for osmophobic association but also for fusion. The threshold concentration for fusion depended on the molecular weight of PEG and also on the electrostatic repulsive interaction between phospholipid vesicles, which was manipulated by the use of vesicle with negative surface charge; increasing the molecular weight of PEG lowered the threshold concentration, and increasing the electrostatic repulsive interaction raised it. In addition, a transient leakage of internal contents from the vesicles was observed at the concentration that caused fusion. When the surface charge of the vesicle was varied, the threshold for fusion coincided with that for osmophobic association, provided that the latter exceeded 22 wt % of PEG 6000. However, when the threshold for osmophobic association was less than 22 wt %, the threshold for fusion remained .apprx. 22 wt %, irrespective of the difference in the threshold for osmophobic association. Electron microphotographs of quick-frozen replicas of egg yolk phosphatidylcholine vesicles showed that the vesicles in the aggregate caused by PEG-induced osmophobic association were deformed to increase their area of contact with the adjacent vesicles. According to the analysis based on the osmophobic association theory, the mechanical force (f) that causes the deformation of the vesicle (deformation force) is counterbalanced by the thermodynamic force due to osmophobic association, increasing with increased concentrations of PEG, but it is little affected by the electrostatic repulsive interaction between the vesicles. On the basis of the results described above, a "mechanical stress model" is proposed for the mechansim of PEG-induced fusion. Membranes that are tightly associated by osmophobic association are mechanically strained by the deformation force f. Consequently, the membrane structure becomes unstable at increased concentrations of PEG, and above a critical concentration, .apprx. 22 wt % of PEG 6000, destruction of the bilayer structure into a leaky membrane structure may cause fusion.