Electric pulses of intensity in kilovolts per centimeter and of duration in microseconds to milliseconds cause a temporary loss of the semipermeability of cell membranes, thus leading to ion leakage, escape of metabolites, and increased uptake by cells of drugs, molecular probes, and DNA. A generally accepted term describing this phenomenon is "electroporation." Other effects of a high-intensity electric field on cell membranes include membrane fusions, bleb formation, cell lysis... etc. Electroporation and its related phenomena reflect the basic bioelectrochemistry of cell membranes and are thus important for the study of membrane structure and function. These phenomena also occur in such events as electric injury, electrocution, and cardiac procedures involving electric shocks. Electroporation has found applications in: (a) introduction of plasmids or foreign DNA into living cells for gene transfections, (b) fusion of cells to prepare heterokaryons, hybridoma, hybrid embryos... etc., (c) insertion of proteins into cell membranes, (d) improving drug delivery and hence effectiveness in chemotherapy of cancerous cells, (e) constructing animal model by fusing human cells with animal tissues, (f) activation of membrane transporters and enzymes, and (g) alteration of genetic expression in living cells. A brief review of mechanistic studies of electroporation is given.
A study of the voltage induction of transient pores in phospholipid bilayer vesicles is reported. Unilamellar vesicles (dipalmitoylphosphatidylcholine), with a size distribution of 100 +/- 30 nm, were prepared by the method of Enoch & Strittmatter [Enoch, H., & Strittmatter, P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 145]. The vesicles loaded with [14C]sucrose and suspended in a mixture of 150 mM NaCl and 272 mM sucrose (both are the isotonic solvent for erythrocytes) were exposed to an intense electric field in the range of 20--40 kV/cm, with a field decay time of 5--15 micro second. A transient leakage of sucrose label was detected when the field strength exceeded 30 kV/cm. After the field was removed, no slow leakage of the tracer molecules occurred during a 65-h incubation period at the room temperature (23 +/- 2 degrees C). The leakage is attributed to the field-induced transmembrane potential, but not other effects such as the Joule heating or the shock wave associated with the voltage discharge. When the potential exceeded a threshold value of 200 mV, corresponding to an applied field strength of 30 kV/cm, there was a dielectric breakdown of the bilayer structure. Pores which allowed passage of sucrose were formed, transiently. Experiments show that these pores were fully reversible, and no global and permanent damages to the vesicle bilayer were detected. The implication of this membrane potential triggered conducting state of lipid bilayers to biological functions of cells is discussed.
Exposure of human erythrocytes, under isotonic conditions, to a high voltage pulse of a few kV/cm leads to total hemolysis of the red cells. Experiments described herein demonstrate that the hemolysis is due to the effect of the electric field. Neither the effect of current nor the extent of the rapid Joule-heating to the suspending medium shows a direct correlation with the observed hemolysis. Voltage pulsation of the erythrocyte suspension can induce a transmembrane potential across the cell membrane and, at a critical point, it either opens up or creates pores in the red cells. In isotonic saline the pores are small. They allow passage of potassium and sodium ions but not sucrose and hemoglobin molecules. The pores are larger in low ionic conditions and permit permeation of sucrose molecules, but under no circumstances can hemoglobin leak out as the direct result of the voltage pulse. Kinetic measurements indicate that the hemolysis of the red cells follows a stepwise mechanism: leakage of ions leads to an osmotic imbalance which in turn causes a colloidal hemolysis of the red cells.
Dielectrophoresis and electrorotation are commonly used to measure dielectric properties and membrane electrical parameters of biological cells. We have derived quantitative relationships for several critical points, defined in Fig. A 1, which characterize the dielectrophoretic spectrum and the electrorotational spectrum of a cell, based on the single-shell model (Pauly, H., and H.P. Schwan, 1959. Z. Naturforsch. 14b:125-131; Sauer, F.A. 1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). To test these equations and to obtain membrane electrical parameters, a technique which allowed simultaneous measurements of the dielectrophoresis and the electrorotation of single cells in the same chamber, was developed and applied to the study of Neurospora slime and the Myeloma Tib9 cell line. Membrane electrical parameters were determined by the dependence of the first critical frequency of dielectrophoresis (fct1) and the first characteristic frequency of electrorotation (fc1) on the conductivity of the suspending medium. Membrane conductances of Neurospora slime and Myeloma also were found to be 500 and 380 S m-2, respectively. Several observations indicate that these cells are more complex than that described by the single-shell model. First, the membrane capacities from fct1 (0.81 x 10(-2) and 1.55 x 10(-2) F m-2 for neurospora slime and Myeloma, respectively) were at least twice those derived from fc1. Second, the electrorotation spectrum of Myeloma cells deviated from the single-shell like behavior. These discrepancies could be eliminated by adapting a three-shell model (Furhr, G., J. Gimsa, and R. Glaser. 1985. Stud. Biophys. 108:149-164). Apparently, there was more than one membrane relaxation process which could influence the lower frequency region of the beta-dispersion. fct1 of Myeloma in a medium of given external conductivity were found to be similar for most cells, but for some a dramatically increased fct1 was recorded. Model analysis suggested that a decrease in the cytoplasmatic conductivity due to a drastic ion loss in a cell could cause this increase in fct1. Model analysis also suggested that the electrorotation spectrum in the counter-field rotation range and fc1 would be more sensitive to conductivity changes of the cytoplasmic fluid and to the influence of internal membranes than would fct1, although the latter would be sensitive to changes in capacitance of the cytoplasmic membranes.
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