Eberhard Neumann

Gene transfer into mouse lyoma cells by electroporation in high electric fields.

Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/‐ 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ‘electroporation model’ the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.

Electroporation and electrofusion in cell biology

1 Dielectrophoresis and Rotation of Cells.- 2 Cellular Spin Resonance.- 3 Dielectrophoresis: Behavior of Microorganisms and Effect of Electric Fields on Orientation Phenomena.- 4 The Relaxation Hysteresis of Membrane Electroporation.- 5 Electrical Breakdown of Lipid Bilayer Membranes: Phenomenology and Mechanism.- 6 Stochastic Model of Electric Field-Induced Membrane Pores.- 7 Theory of Electroporation.- 8 Leaks Induced by Electrical Breakdown in the Erythrocyte Membrane.- 9 Electroporation of Cell Membranes: Mechanisms and Applications.- 10 Electrofusion Kinetics: Studies Using Electron Microscopy and Fluorescence Contents Mixing.- 11 Electrofusion of Lipid Bilayers.- 12 Role of Proteases in Electrofusion of Mammalian Cells.- 13 Electrofusion of Mammalian Cells and Giant Unilamellar Vesicles.- 14 Cell Fusion and Cell Poration by Pulsed Radio-Frequency Electric Fields.- 15 The Mechanism of Electroporation and Electrofusion in Erythrocyte Membranes.- 16 Producing Monoclonal Antibodies by Electrofusion.- 17 Generation of Human Hybridomas by Electrofusion.- 18 Gaining Access to the Cytosol: Clues to the Control and Mechanisms of Exocytosis and Signal Transduction Coupling.- 19 Gene Transfer by Electroporation: A Practical Guide.- 20 Electropermeabilization and Electrosensitivity of Different Types of Mammalian Cells.- 21 Molecular Genetic Applications of Electroporation.- 22 Plant Gene Transfer Using Electrofusion and Electroporation.- 23 Electric Field-Induced Fusion and Cell Reconstitution with Preselected Single Protoplasts and Subprotoplasts of Higher Plants.- 24 Critical Evaluation of Electromediated Gene Transfer and Transient Expression in Plant Cells.- 25 Transformation Studies in Maize and Other Cereals.- 26 Cells in Electric Fields: Physical and Practical Electronic Aspects of Electro Cell Fusion and Electroporation.- 27 External Electric Field-Induced Transmembrane Potentials in Biological Systems: Features, Effects, and Optical Monitoring.

Permeability Changes Induced by Electric Impulses in Vesicular Membranes

SummaryElectric impulses were found to cause transient permeability changes in the membranes of vesicles storing biogenic amines. Release of catecholamines induced by electric fields (of the order of 20 kV/cm and decaying exponentially with a decay time of about 150 μsec) was studied, using the chromaffin granules of bovine adrenomedullary cells as a vesicular model system. Far-UV-absorption spectroscopy was applied to determine the amount of catecholamines released from suspended vesicles. A polarization mechanism is suggested for the induction of short-lived permeability changes caused by electric fields. Such transient changes in permeability may possibly represent a part of the sequence of events leading to stimulated neurohumoral secretion.

Fundamentals of electroporative delivery of drugs and genes

Electrooptical and conductometrical relaxation methods have given a new insight in the molecular mechanisms of the electroporative delivery of drug-like dyes and genes (DNA) to cells and tissues. Key findings are: (1) Membrane electroporation (ME) and hence the electroporative transmembrane transport of macromolecules are facilitated by a higher curvature of the membrane as well as by a gradient of the ionic strength across charged membranes, affecting the spontaneous curvature. (2) The degree of pore formation as the primary field response increases continuously without a threshold field strength, whereas secondary phenomena, such as a dramatic increase in the membrane permeability to drug-like dyes and DNA (also called electropermeabilization), indicate threshold field strength ranges. (3) The transfer of DNA by ME requires surface adsorption and surface insertion of the permeant molecule or part of it. The diffusion coefficient for the translocation of DNA (M(r) approximately 3.5 x 10(6)) through the electroporated membrane is Dm = 6.7 x 10(-13) cm2 s-1 and Dm for the drug-like dye Serva Blue G (M(r) approximately 854) is Dm = 2.0 x 10(-12) cm2 s-1. The slow electroporative transport of both DNA and drugs across the electroporated membrane reflects highly interactive (electro-) diffusion, involving many small pores coalesced into large, but transiently occluded pores (DNA). The data on mouse B-cells and yeast cells provide directly the flow and permeability coefficients of Serva blue G and plasmid DNA at different electroporation protocols. The physico-chemical theory of ME and electroporative transport in terms of time-dependent flow coefficients has been developed to such a degree that analytical expressions are available to handle curvature and ionic strength effects on ME and transport. The theory presents further useful tools for the optimization of the ME techniques in biotechnology and medicine, in particular in the new field of electroporative delivery of drugs (electrochemotherapy) and of DNA transfer and gene therapy.

Electric field mediated gene transfer

Abstract A simple physical chemical method of transferring genes into eucaryotic cells is described. Electric impulses in the intensity range of 5–10 kV/cm with a duration of 5–10 μs were found to appreciably increase the uptake of DNA into cells. After electric field treatment, stable transformants were obtained in a system containing mouse cells deficient in thymidine kinase and a plasmid DNA harbouring the Herpes simplex thymidine kinase gene. The efficiency of transformation well compares with the results of biochemical methods of gene transfer. The electric field technique appears unique in its ease and simplicity.

Stochastic model for electric field-induced membrane pores electroporation

Electric impulses (1-20 kV cm-1, 1-5 microseconds) cause transient structural changes in biological membranes and lipid bilayers, leading to apparently reversible pore formation ( electroporation ) with cross-membrane material flow and, if two membranes are in contact, to irreversible membrane fusion ( electrofusion ). The fundamental process operative in electroporation and electrofusion is treated in terms of a periodic lipid block model, a block being a nearest-neighbour pair of lipid molecules in either of two states: (i) the polar head group in the bilayer plane or (ii) facing the centre of a pore (or defect site). The number of blocks in the pore wall is the stochastic variable of the model describing pore size and stability. The Helmholtz free energy function characterizing the transition probabilities of the various pore states contains the surface energies of the pore wall and the planar bilayer and, if an electric field is present, also a dielectric polarization term (dominated by the polarization of the water layer adjacent to the pore wall). Assuming a Poisson process the average number of blocks in a pore wall is given by the solution of a non-linear differential equation. At subcritical electric fields the average pore size is stationary and very small. At supercritical field strengths the pore radius increases and, reaching a critical pore size, the membrane ruptures (dielectric breakdown). If, however, the electric field is switched off, before the critical pore radius is reached, the pore apparently completely reseals to the closed bilayer configuration (reversible electroporation ).

Control by pulse parameters of electric field-mediated gene transfer in mammalian cells

Electric field-mediated gene transfer in mammalian cells (electrotransformation) depends on the pulsing conditions (field intensity, pulse duration, number of pulses). The effect of these parameters was systematically investigated using the transient expression of the chloramphenicol acetyltransferase and the beta-galactosidase activities in Chinese hamster ovary cells. Pulsing conditions inducing reversible permeabilization of the cell plasma membrane are not sufficient to induce gene transfer. The plasmid must be present during the electric pulse if it is to be transferred across the membrane into the cytoplasm. Only the localized part of the cell membrane brought to the permeabilized state by the external field is competent. Pulse duration plays a key role in the magnitude of the transfer. The field induces a complex reaction between the membrane and the plasmid that is accumulated at the cell interface by electrophoretic forces. This leads to an insertion of the plasmid, which can then cross the membrane.

Kinetics, Statistics, and Energetics of Lipid Membrane Electroporation Studied by Molecular Dynamics Simulations

Membrane electroporation is the method to directly transfer bioactive substances such as drugs and genes into living cells, as well as preceding electrofusion. Although much information on the microscopic mechanism has been obtained both from experiment and simulation, the existence and nature of possible intermediates is still unclear. To elucidate intermediates of electropore formation by direct comparison with measured prepore formation kinetics, we have carried out 49 atomistic electroporation simulations on a palmitoyl-oleoyl-phosphatidylcholine bilayer for electric field strengths between 0.04 and 0.7 V/nm. A statistical theory is developed to facilitate direct comparison of experimental (macroscopic) prepore formation kinetics with the (single event) preporation times derived from the simulations, which also allows us to extract an effective number of lipids involved in each pore formation event. A linear dependency of the activation energy for prepore formation on the applied field is seen, with quantitative agreement between experiment and simulation. The distribution of preporation times suggests a four-state pore formation model. The model involves a first intermediate characterized by a differential tilt of the polar lipid headgroups on both leaflets, and a second intermediate (prepore), where a polar chain across the bilayer is formed by 3-4 lipid headgroups and several water molecules, thereby providing a microscopic explanation for the polarizable volume derived previously from the measured kinetics. An average pore radius of 0.47 +/- 0.15 nm is seen, in favorable agreement with conductance measurements and electrooptical experiments of lipid vesicles.

Mechanism of Electroporative Dye Uptake by Mouse B Cells

The color change of electroporated intact immunoglobulin G receptor (Fc gammaR-) mouse B cells (line IIA1.6) after direct electroporative transfer of the dye SERVA blue G (Mr 854) into the cell interior is shown to be dominantly due to diffusion of the dye after the electric field pulse. Hence the dye transport is described by Ficks first law, where, as a novelty, time-integrated flow coefficients are introduced. The chemical-kinetic analysis uses three different pore states (P) in the reaction cascade (C <==> P1 <==> P2 <==> P3), to model the sigmoid kinetics of pore formation as well as the biphasic pore resealing. The rate coefficient for pore formation k(p) is dependent on the external electric field strength E and pulse duration tE. At E = 2.1 kV cm(-1) and tE = 200 micros, k(p) = (2.4 +/- 0.2) x 10(3) s(-1) at T = 293 K; the respective (field-dependent) flow coefficient and permeability coefficient are k(f)0 = (1.0 +/- 0.1) x 10(-2) s(-1) and P0 = 2 cm s(-1), respectively. The maximum value of the fractional surface area of the dye-conductive pores is 0.035 +/- 0.003%, and the maximum pore number is Np = (1.5 +/- 0.1) x 10(5) per average cell. The diffusion coefficient for SERVA blue G, D = 10(-6) cm2 s(-1), is slightly smaller than that of free dye diffusion, indicating transient interaction of the dye with the pore lipids during translocation. The mean radii of the three pore states are r(P1) = 0.7 +/- 0.1 nm, r(P2) = 1.0 +/- 0.1 nm, and r(P3) = 1.2 +/- 0.1 nm, respectively. The resealing rate coefficients are k(-2) = (4.0 +/- 0.5) x 10(-2) s(-1) and k(-3) = (4.5 +/- 0.5) x 10)(-3) s(-1), independent of E. At zero field, the equilibrium constant of the pore states (P) relative to closed membrane states (C) is K(p)0 = [(P)]/[C] = 0.02 +/- 0.002, indicating 2.0 +/- 0.2% water associated with the lipid membrane. Finally, the results of SERVA blue G cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative delivery of drugs that are similar in structure to SERVA blue G, for instance, bleomycin, which has been used successfully in the new discipline of electrochemotherapy.

Membrane electroporation and direct gene transfer

The direct transfer of genetic material into cells by electroporation can be described in physicochemical terms as an electroporation-resealing hysteresis. The hysteresis concept includes unidirectional state transitions of the membrane, coupled to electrodiffusive migration of DNA through cell wall structures and electroporated plasma membranes. Deeper insight into electroporation phenomena such as electrotransfection, electrofusion and electro-insertion is gained by the inspection of the electrosensitivity and the recovery curves of cell populations as well as by the analysis of the pulse strength-duration relationship. A theoretical framework is developed for an adequate comparison of data obtained with different pulse shapes. The results of the physicochemical analysis of electroporation data not only indicate possible molecular mechanisms but are also instrumental in developing a goal-directed optimization strategy for the various practical applications of electroporation techniques such as electric gene delivery, production of hybridoma cells for antibody secretion or the insertion of immune proteins into the membranes of blood organelles.