We have been using phospholipid vesicle systems to study the induction, distribution, and lifetime of electric field-induced membrane pores. We have also explored the possible use of loaded vesicle systems as reaction vessels to study fast reactions as the vesicles can be made to rupture in the microsecond time scale by applying short electric pulses to initiate the mixing reaction. We can now prepare large, stable, unilamellar vesicles from Di-oleoylphosphatidylcholine (DOPC), with diameters ranging from 25-40 micrometers. In our electroporation studies using membrane-labeled vesicles with potential sensitive dyes, we found the following sequence of events: (1) Within 30 msecs after the pulse, hyperpolarization (+) and depolarization (-) of the membrane potential facing the electrodes, as well as physical deformation of the spherical vesicle in the direction of the applied field is observed. (2) Between 30-60 msec, two, large membrane pores (several micrometer in diameter) are observed facing the electrodes. These pores are created by the expulsion of some portion of the phospholipid bilayer from the vesicle membrane. (3) Between 60-90 msecs, the pores are completely resealed. Measurement of the vesicle size before and after the electric pulse revealed that up to 9% of the membrane surface is lost when the pores were created. These sequence of events are unlike what is known in electroporation of cell membranes and suggests a role for non lipid membrane components in maintaining partial membrane integrity. In experiments designed to use the vesicles as reaction vessels, we have thus far obtained preliminary data on the rate of Calcien release from loaded vesicles. However, there is lack of sensitivity with our current detection system to be of use with reacting systems with much lower quantum yields. We are working on ways to increase the sensitivity.
