Gorazd Pucihar

Cell membrane electroporation- Part 1: The phenomenon

Each biological cell, trillions of which build our bodies, is enveloped by its plasma membrane. Composed largely of a bilayer (double layer) of lipids just two molecules thick (about 5 nm), and behaving partly as a liquid and partly as a gel, the cell plasma membrane nonetheless separates and protects the cell from its surrounding environment very reliably and stably. Embedded within the lipid bilayer, also quite stably, are a number of different proteins, some of which act as channels and pumps, providing a pathway for transporting specific molecules across the membrane. Without these proteins, the membrane would be a largely impenetrable barrier. Electrically, the cell plasma membrane can be viewed as a thin insulating sheet surrounded on both sides by aqueous electrolyte solutions. When exposed to a sufficiently strong electric field, the membrane will undergo electrical breakdown, which renders it permeable to molecules that are otherwise unable to cross it. The process of rendering the membrane permeable is called membrane electroporation. Unlike solid insulators, in which an electrical breakdown generally causes permanent structural change, the membrane, with its lipids behaving as a two-dimensional liquid, can spontaneously return to its prebreakdown state. If the exposure is sufficiently short and the membrane recovery sufficiently rapid for the cell to remain viable, electroporation is termed reversible; otherwise, it is termed irreversible. Since its discovery [1]���[3], electroporation has steadily gained ground as a useful tool in various areas of medicine and biotechnology. Today, reversible electroporation is an established method for introducing chemotherapeutic drugs into tumor cells (electrochemotherapy) [4]. It also offers great promise as a technique for gene therapy without the risks caused by viral vectors (DNA electrotransfer) [5]. In clinical medicine, irreversible electroporation is being investigated as a method for tissue ablation (nonthermal electroablation) [6], whereas in biotechnology, it is useful for extraction of biomolecules [7] and for microbial deactivation, particularly in food preservation [8]. This article, the first in a series of three focusing on electroporation, describes the phenomenon at the molecular level of the lipid bilayer, and then proceeds to the cellular level, explaining how exposure of a cell as a whole to an external electric field results in an inducement of voltage on its plasma membrane, its electroporation, and transport thorough the electroporated membrane. The second article will review the most important and promising applications of electroporation, and the third article will focus on the hardware for electroporation (pulse generators and electrodes) and on the need for standards, safety, and certification.

Numerical Determination of Transmembrane Voltage Induced on Irregularly Shaped Cells

The paper presents an approach that reduces several difficulties related to the determination of induced transmembrane voltage (ITV) on irregularly shaped cells. We first describe a method for constructing realistic models of irregularly shaped cells based on microscopic imaging. This provides a possibility to determine the ITV on the same cells on which an experiment is carried out, and can be of considerable importance in understanding and interpretation of the data. We also show how the finite-thickness, nonzero-conductivity membrane can be replaced by a boundary condition in which a specific surface conductivity is assigned to the interface between the cell interior (the cytoplasm) and the exterior. We verify the results obtained using this method by a comparison with the analytical solution for an isolated spherical cell and a tilted oblate spheroidal cell, obtaining a very good agreement in both cases. In addition, we compare the ITV computed for a model of two irregularly shaped CHO cells with the ITV measured on the same two cells by means of a potentiometric fluorescent dye, and also with the ITV computed for a simplified model of these two cells.

The influence of medium conductivity on electropermeabilization and survival of cells in vitro

Electropermeabilization and cell death caused by the exposure to high voltage electric pulses depends on the parameters of pulses, as well as the composition of the extracellular medium. We studied the influence of extracellular conductivity on electropermeabilization and survival of cells in vitro. For this purpose, we used a physiological medium with a conductivity of 1.6 S/m and three artificial media with conductivities of 0.14, 0.005, and 0.001 S/m. Measurements of pH, osmolarity, and cell diameter were made to estimate possible side effects of the media on the cells. Our study shows that the percentage of surviving cells increases with the decreasing medium conductivity, while the percentage of electropermeabilized cells remains unaffected. Our results show that cell survival in experiments involving electropermeabilization can be improved by decreasing the medium conductivity. To provide an interpretation of experimental results, we have theoretically estimated the resting transmembrane voltage, the induced transmembrane voltage, the time constant of the voltage inducement, and heating of the cell suspension for each of the media used. These calculations imply that for accurate interpretation of experimental results, both the induced and the resting transmembrane voltage must be considered, taking into account the conductivity and the ionic composition of the extracellular medium.

Kinetics of transmembrane transport of small molecules into electropermeabilized cells.

The transport of propidium iodide into electropermeabilized Chinese hamster ovary cells was monitored with a photomultiplier tube during and after the electric pulse. The influence of pulse amplitude and duration on the transport kinetics was investigated with time resolutions from 200 ns to 4 ms in intervals from 400 micros to 8 s. The transport became detectable as early as 60 micros after the start of the pulse, continued for tens of seconds after the pulse, and was faster and larger for higher pulse amplitudes and/or longer pulse durations. With fixed pulse parameters, transport into confluent monolayers of cells was slower than transport into suspended cells. Different time courses of fluorescence increase were observed during and at various times after the pulse, reflecting different transport mechanisms and ongoing membrane resealing. The data were compared to theoretical predictions of the Nernst-Planck equation. After a delay of 60 micros, the time course of fluorescence during the pulse was approximately linear, supporting a mainly electrophoretic solution of the Nernst-Planck equation. The time course after the pulse agreed with diffusional solution of the Nernst-Planck equation if the membrane resealing was assumed to consist of three distinct components, with time constants in the range of tens of microseconds, hundreds of microseconds, and tens of seconds, respectively.

Role of pulse shape in cell membrane electropermeabilization

The role of the amplitude, number, and duration of unipolar rectangular electric pulses in cell membrane electropermeabilization in vitro has been the subject of several studies. With respect to unipolar rectangular pulses, an improved efficiency has been reported for several modifications of the pulse shape: separate bipolar pulses, continuous bipolar waveforms, and sine-modulated pulses. In this paper, we present the results of a systematic study of the role of pulse shape in permeabilization, cell death, and molecular uptake. We have first compared the efficiency of 1-ms unipolar pulses with rise- and falltimes ranging from 2 to 100 micros, observing no statistically significant difference. We then compared the efficiency of triangular, sine, and rectangular bipolar pulses, and finally the efficiency of sine-modulated unipolar pulses with different percentages of modulation. We show that the results of these experiments can be explained on the basis of the time during which the pulse amplitude exceeds a certain critical value.

The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy

Electrochemotherapy is a technique where electric pulses in combination with chemotherapeutic agents are applied to tumor cells. In general, patients find electrochemotherapy tolerable, in spite of unpleasant sensations associated with contraction of muscles located beneath or in the vicinity of the electrodes. These contractions are due to the intensity of the electric pulses required for effective electropermeabilization of tumor cell membranes. Since a train of eight electric pulses with repetition frequency of 1 Hz is usually applied to the tumors, each pulse in the train excites underlying nerves and provokes muscle contractions. Therefore, for patients involved in electrochemotherapy, the use of pulses with repetition frequency higher than the frequency of tetanic contraction would represent reduced number of muscle contractions and associated unpleasant sensations. Our results of the uptake of Lucifer Yellow into electropermeabilized cells in vitro show that with increased repetition frequency the uptake stays at similar levels even at frequencies up to 8.3 kHz. On the basis of these results the possibilities for the clinical use of pulses with high repetition frequency in electrochemotherapy are considered.

Equivalent Pulse Parameters for Electroporation

Electroporation-based applications require the use of specific pulse parameters for a successful outcome. When recommended values of pulse parameters cannot be set, similar outcomes can be obtained by using equivalent pulse parameters. We determined the relations between the amplitude and duration/number of pulses resulting in the same fraction of electroporated cells. Pulse duration was varied from 150 ns to 100 ms, and the number of pulses from 1 to 128. Fura 2-AM was used to determine electroporation of cells to Ca2+. With longer pulses or higher number of pulses, lower amplitudes are needed for the same fraction of electroporated cells. The expression derived from the model of electroporation could describe the measured data on the whole interval of pulse durations. In a narrower range (0.1-100 ms), less complex, logarithmic or power functions could be used instead. The relation between amplitude and number of pulses could best be described with a power function or an exponential function. We show that relatively simple two-parameter power or logarithmic functions are useful when equivalent pulse parameters for electroporation are sought. Such mathematical relations between pulse parameters can be important in planning of electroporation-based treatments, such as electrochemotherapy and nonthermal irreversible electroporation.

Induced Transmembrane Voltage and Its Correlation with Electroporation-Mediated Molecular Transport

Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, ΔΨm) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of ΔΨm and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between ΔΨm and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of ΔΨm are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.

A Time-Dependent Numerical Model of Transmembrane Voltage Inducement and Electroporation of Irregularly Shaped Cells

We describe a finite-element model of a realistic irregularly shaped biological cell in an external electric field that allows the calculation of time-dependent changes of the induced transmembrane voltage ( DeltaPsi) and simulation of cell membrane electroporation. The model was first tested by comparing its results to the time-dependent analytical solution for DeltaPsi on a nonporated spherical cell, and a good agreement was obtained. To simulate electroporation, the model was extended by introducing a variable membrane conductivity. In the regions exposed to a sufficiently high DeltaPsi, the membrane conductivity rapidly increased with time, leading to a modified spatial distribution of DeltaPsi. We show that steady-state models are insufficient for accurate description of DeltaPsi, as well as determination of electroporated regions of the membrane, and time-dependent models should be used instead. Our modeling approach also allows direct comparison of calculations and experiments. As an example, we show that calculated regions of electroporation correspond to the regions of molecular transport observed experimentally on the same cell from which the model was constructed. Both the time-dependent model of DeltaPsi and the model of electroporation can be exploited further to study the behavior of more complicated cell systems, including those with cell-to-cell interactions.

Non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery

For an effective tissue controlled electropermeabilization as requested for electrochemotherapy and electrogenotherapy, it is very important to have informations about the electric field distribution provided by a defined set of electrodes. Computer simulations using the finite element models approach predicted the associated field distributions and currents. Phantoms made of gels with well-defined electrical conductance were used to measure the current responses of a new electrode geometry (wires), A good agreement between the measured and predicted currents was observed supporting the validity of the prediction for the field distribution. Field distribution was observed to be very localized and highly homogeneous with the new concept of contact wire electrodes. They allowed to focus the field effect along the surface of the tissue to induce a controlled release of drugs or plasmids. Non invasive (contact) electrodes can be moved rapidly on the body and avoid puncturing the skin and the tissue. They can be used for large surface effects, to treat the skin and subcutaneous tumors. The use of contact electrodes after drug or DNA intradermal injection were validated by clinical treatment of large surface skin tumors and by in vivo imaging of permeabilization or of gene expression.