ELECTROFUSION OF CELL-SIZE LIPOSOMES

Cell size liposomes of egg phosphatidylcholine (PC), trans-acylated egg phosphatidylethanolamine (PE), bovine brain phosphatidylserine (PS) and egg phosphatidylglycerol, suspended in 2.5% of polyethylene glycol (M(I) 8000), Ficoll (M(I) 400 000) or Dextran (M(I), 71 200) were aligned by dielectrophoresis and fused by applying a 1.7 kV/cm pulse of varying duration. Because the internal and external media of liposome suspension can be controlled, and the surface charge is known, the results can be described mathematically. The fusion yields (FY) at different pulse length were measured by microscopy. The FY curves were sigmoidal, with a common minimally required pulse length of 19 mu s, and shifted to longer pulse length with increasing external media conductivity or vesicle surface charge. The types of polymer in solutions had little effect. The minimal required pulse length was interpreted to be the minimum rise time for the smallest vesicle capable of reaching the bilayer breakdown potential induced by the pulse, and the sigmoidal shape FY curves represented the cumulative vesicle size distribution curve. The shifting of FY curves upon changing media conductivity or vesicle surface charge were quantitatively accounted for by the balance of pulse-induced dipole-dipole attraction and electrostatic repulsion. Deviation from sigmoidal shape FY curves in the cases of charged liposomes was explained by increasing electrostatic repulsion due to vesicle deformation under pulse-induced dipole pressure. The data confirm the hypothesis that membrane potential breakdown is a pre-requisite or minimum requirement for electrofusion, and support our earlier proposition that pulse-induced dipole force plays an important role in the electrofusion process, and that electrostatic repulsion posts additional barrier to electrofusion.