Electropermeabilization (Electroporation)

Electropermeabilization or electroporation is the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane. These pores are commonly called "electropores." Their presence allows molecules, ions, and water to pass from one side of the membrane to the other. As the left side bar shows, the electropores are located primarily on the surfaces of cells which are closest to the electrodes. If the electric field pulse has the proper parameters, then the "electroporated" cells can recover (the electropores reseal spontaneously), and the cells can continue to grow. The time for the pathways to form is about one microsecond. The time to reseal is minutes.

The use of electroporation became very popular through the 1980s because it was found to be an exceptionally practical way to place drugs, DNA or other molecules into cells. In the late 1980s, scientists began to use electroporation for applications in multi-cellular tissue.

Research has shown that the induction of electropores is affected by three major factors (see figure below). First, cell-to-cell biological variability causes some cells to be more sensitive (dashed line) to electroporation than other cells (solid line). Second, for electropores to be induced, the product of the pulse amplitude and the pulse duration has to be above a lower limit threshold (at "A" and "B" for sensitive and resistant cells, respectively). Third, pore number and effective pore diameter increase with the product of "amplitude" and "duration." Although other factors are involved, this threshold is now understood to be largely dependent on a fourth factor, the reciprocal of cell size. If the upper limit threshold is reached (at "C" and "D" for sensitive and resistant cells, respectively), pore diameter and total pore area are too large for the cell to repair by any spontaneous or biological process. The result is irreversible damage to the cell.

                                              To prevent this damage, pulse protocols are thus empirically developed to be at some 

                                               point between "B" and "C."

 

Because the mechanism of electroporation is not well understood, the developmentof protocols for a particular application has usually been achieved empirically, by adjusting pulse parameters (amplitude, duration, number, and inter-pulse interval).

Early research on electro-pore-mediated transport across membranes assumed that simple thermal motion (i.e. diffusion) propelled molecules through electropores. Research in the late 1980s and early 1990s showed that certain experimental conditions and parameters of electrical pulses may be capable of causing many more molecules to move per unit time than simple diffusion. For example, there is good evidence (Dimitrov and Sowers, 1990) [1] that molecular flow is in the direction of the arrow in the sidebar but there is also good evidence (Sukharev, et al., 1992) [2] that DNA movement is in the opposite direction of the arrow in the sidebar. This implies that electroporation has polarity dependence. Although this apparent contradiction will have to be resolved by future basic research, it clearly suggests that pulse generators with polarity-adjustable electrical parameters are necessary for protocol development.

An additional important consideration is that during the electroporation pulse, the electric field causes electrical current to flow through the cell suspension or tissue. Biologically relevant buffers for cells, bathing media, and fluid in extra-cellular space in tissues contain ionic species at concentrations high enough to cause high electric currents to flow. These currents can lead to dramatic heating which is biologically unacceptable. Principles of physics suggest that the early part of exponentially decaying pulses does most of the membrane porating but the late part continues to heat the medium. One way to minimize the heating is to use a relatively high amplitude short duration rectangular wave pulse instead of an exponentially-decaying pulse. A second strategy is to use two short-duration pulses instead of one pulse with a duration equal to the sum of the two short pulses.

There have been two main waveform categories of porating pulses: exponentially decaying, and rectangular wave. These waveform qualities were a matter of customary electrical engineering principles and the fact that pulse generators designed for one waveform usually could not deliver the other waveform. Moreover, only a few side-by-side studies were conducted which showed a fundamental and universal superiority of one waveform over another. In cases where there is evidence that an exponentially decaying pulse may have an advantage for a particular application, a protocol which delivers two pulses, one which is high in amplitude and short in duration followed by a second which is low in amplitude but long in duration, may simulate the effects of the exponentially-decaying pulse or even provide an improved result.

 

 

Acterization of Cell Membrane

Electroporation

 

T. R. Gowrishankar, R. C. Lee
The University of Chicago
and
J. C. Weaver
Massachusetts Institute of Technology

Scientific Problem

The plasma membrane of a cell serves the vital function of partitioning the molecular contents of the cytoplasm from its external environment. These membranes are largely composed of amphiphilic lipids which self-assemble into highly insulating structures and thus present a large energy barrier to transmembrane ionic transport.

However, the lipid matrix can be disrupted by a strong external electric field leading to an increase in transmembrane conductivity and diffusive permeability. These effects are the result of formation of aqueous pores in the membrane, which also alter the electrical potential across the membrane. These events are encountered in practice, both by design and by accident.

Electroporation of cell membranes is used as a tool in injecting drugs and DNA into the cell (Tsong, 1991). Electroporation is also the basic mechanism of tissue injury in high-voltage electric shock (Lee, 1994).

The objective of this project is to develop a model to characterize the molecular events involved in electroporation. Electroporation occurs as a result of the reorientation of lipid molecules of the bilayer membrane to form hydrophilic pores in the membrane.

The distribution of such pores, both in terms of size and number, determine the electrical properties of the cell membrane. Changes in pore radius are effected by surface tension forces on the pore wall, diffusion of water molecules into and out of the pore and an electric field induced force of expansion.

Pore distribution in the presence of an external electric field can be described by Smoluchowski's equation (Barnett and Weaver, 1991). In order to estimate the changes in pore distribution and membrane electrical properties, the partial differential equation was solved subject to appropriate boundary conditions.

The Crank-Nicolson method of implicit finite difference scheme was used to solve the partial differential equation (Mü, 1990). The tridiagonal matrix was partitioned into p subsystems, each processor thus requiring to work on n/p equations where n is the order of the original matrix.

Results

The molecular events underlying electroporation determine the kinetics of opening and closing of membrane pores. The opening and closing of pores occur as a result of the rotation of lipid molecules that form the pore walls.

The movement of lipid molecules in electroporation is characterized in the theoretical model by the diffusion coefficient of lipid molecules in pore radius space. The relaxation of transmembrane current following the removal of the external pulse occurs as a result of the reorientation of the lipid molecules to close the membrane pores or shrink them.

The post-field relaxation time constant of transmembrane current for different transmembrane potentials was determined from voltage clamp measurements .

 

 

Figure 1: Post-field relaxation time constant of transmembrane current for different transmembrane potentials.

 

The relaxation time constants determined from voltage clamp measurements of transmembrane current at different transmembrane potentials were used to estimate the diffusion coefficient of lipid molecules in pore radius space. Theoretical estimations of relaxation time constants obtained from numerical simulation of electroporation on SP1 were used to fit the experimental data.

The decrease in relaxation time constant with transmembrane potential is related to the size and distribution of membrane pores prior to the termination of the pulse. Theoretical estimations of relaxation time constants were used to fit the experimental data. The diffusion coefficient was estimated from this fit to be around 5x10e-16 m2/sec.

 

Membrane Component D(m2/sec)
Gramicidin C on artificial BLM 3-6xe-12
Lipid analog on mouse 3T3 cells 2-4xe-13
Class I MHC on HDF 1-2xe-13
Con-A on mouse 3T3 5-10xe-15
aqueous electropores 5xe-16

Table 1: Range of diffusion coefficient of membrane components reported in the literature.

 

The diffusion coefficient of pores in radius space is orders of magnitude smaller than most membrane components.

The diffusion coefficient of pores in radius space (D) was estimated from relaxation time constants determined from experimental measurements and model simulations to be 5x10e-16 m2/sec.

The estimated value of D is lower than the estimates for other membrane components .

This suggests that certain pores are stable over a few msec after the pulse is turned off owing to a slower rotation of lipid molecules following electroporation resulting in a rectification of transmembrane current. The two orders of magnitude reduction in diffusion coefficient following electroporation may be attributed to the decreased diffusivity of lipids in the presence of stable pore-protein complexes.

The diffusion coefficient estimated from the experimental measurements was used in simulating electroporation at different transmembrane potentials. The evolution of pore distribution is shown for different transmembrane potentials.

 

Figure 2: Total number of pores at different transmembrane potentitals.

 

The abrupt decrease in the number of pores immediately following the pulse is indicative of the spontaneous shrinking of small pores.

The abrupt decrease in the number of pores immediately following the pulse is indicative of the spontaneous shrinkage of small pores.

Further decrease in the number of pores indicates the decrease in size of larger pores after the pulse is removed.

The simulation results agree with experimental measurements which show that the transmembrane potential threshold for electroporation is around -240 mV. Below this threshold, pore creation and destruction occur at a comparable rate resulting in a negligible increase in transmembrane current during the pulse.

The corresponding membrane conductance profiles for different transmembrane potentials are also shown .

 

 

 

Figure 3: Membrane Conductance at different transmembrane potentials.

 

The orders of magnitude increase in membrane conductance reflects the large increase in the total number of pores.

An order of magnitude increase in the total number of pores is reflected by a corresponding increase in membrane conductance. This large increase in membrane conductance for large magnitude pulses removes the ionic gradient across the membrane.

Maintenance of this ionic gradient requires metabolic energy.

A continuous loss of energy will lead to cell death. Thus, it is essential that the membrane pores be sealed in order to restore the ionic gradient and thus the metabolic energy of the cell in case of membrane injury by electric field.

References

1.
Tsong, T. Y. 1991. Electroporation of cell membranes. Biophys. J. 60:297-306.
 
2.
Lee, R. C. 1994. In Advances in Electromagnetic Fields in Living Systems. Vol. 1. Ed.: J. C. Lin. 81-127. Plenum Press, New York:NY.
 
3.
Barnett, A. and J. C. Weaver. 1991. Electroporation: a unified, quantitative theory of reversible electrical breakdown and mechanical rupture in artificial planar bilayer membranes. Bioelectrochem. Bioenerg. 25:163-182.
 
4.
Mü, S. M. 1990. A Method to Parallelize Tridiagonal Solvers. Proc. 5th Dist. Mem. Conf. South Carolina. Eds: T. L. Huntsberger and B. A. Huntsberger. IEEE Press.
 
[1] Dimitrov, D.S., and Sowers, A.E., (1990) Membrane electroporation - fast molecular exchange by electroosmosis. Biochimica et Biophysica Acta 1022: 381-392.
[2] Sukharev SI, Klenchin VA, Serov SM, Chernomordik LV and Chizmadzhev YA, (1992) Electroporation, and electrophoretic DNA transfer into cells: The effect of DNA interaction with electropores, 1992, Biophys J. 63: 1320-1327.
 

References, General Electroporation

Books

Nickoloff, Jac A., ed. (1995) Electroporation Protocols for Microorganisms, Methods in Molecular Biology, Volume 47, (Humana Press, Totowa, New Jersey), 372 pp.

Nickoloff, Jac A., ed. (1995) Animal Cell Electroporation and Electrofusion Protocols, Methods in Molecular Biology, Volume 48. (Humana Press, Totowa, New Jersey). 369 pp.

Nickoloff, Jac A., ed. (1995) Plant Cell Electroporation and Electrofusion Protocols, Methods in Molecular Biology, Volume 55. (Humana Press, Totowa, New Jersey). 205 pp.

E. A. Disalvo and S.A. Simon, eds. (1995) Permeability and Stability of Lipid Bilayers (CRC Press, Boca Raton), p 105-121.

Chang, D.C., Chassy, B.M., Saunders, J.A. and Sowers, A.E., eds. (1992) Guide to Electroporation and Electrofusion, (Academic press, San Diego), 581 pp.

Neuman, E., Sowers, A.E., and Jordan, C.A.., eds. (1989) Electroporation and Electrofusion in Cell Biology, (Plenum Press, New York) 581 pp.

Journal Articles

Bartoletti, D. C., Harrison, G. I., & Weaver, J. C. (1989). The number of molecules taken up by electroporated cells: quantitative determination. FEBS Lett., 256, 4-10.

Canatella, P. J., Karr, J. F., Petros, J. A., & Prausnitz, M. R. (2001). Quantitative study of electroporation-mediated molecular uptake and cell viability. Biophys.J, 80, 755-764.

Djuzenova, C. S., Zimmermann, U., Frank, H., Sukhorukov, V. L., Richter, E., & Fuhr, G. (1996). Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells. Biochim.Biophys.Acta, 1284, 143-152.

Klenchin VA, Sukharev SM, Chernomordik LV, Chizmadzhev YA, Electricaly induced DNA uptake by cells is a fast process involving DNA electrophoresis, 1991, Biophys J. 60:804-811

Neumann, E., Kakorin, S., & Toensing, K. (1999). Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem.Bioenerg., 48, 3-16.

Neumann, E., Toensing, K., Kakorin, S., Budde, P., & Frey, J. (1998). Mechanism of electroporative dye uptake by mouse B cells. Biophys.J., 74, 98-108.

Sukharev, S. I., Klenchin, V. A., Serov, S. M., Chernomordik, L. V., & Chizmadzhev, Y. (1992). Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys.J., 63, 1320-1327.

Wolf, H., Rols, M. P., Boldt, E., Neumann, E., & Teissie, J. (1994). Control by pulse parameters of electric field-mediated gene transfer in mammalian cells. Biophys.J., 66, 524-531.

Zerbib, D., Amalric, F., & Teissie, J. (1985). Electric field mediated transformation: isolation and characterization of a TK+ subclone. Biochem.Biophys.Res.Commun., 129, 611-618.