Cell electrofusion using nanosecond electric pulses.

Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.

Electroporation of intracellular liposomes using nanosecond electric pulses--a theoretical study.

Nanosecond (ns) electric pulses of sufficient amplitude can provoke electroporation of intracellular organelles. This paper investigates whether such pulses could provide a method for controlled intracellular release of a content of small internalized artificial lipid vesicles (liposomes). To estimate the pulse parameters needed to selectively electroporate liposomes while keeping the plasma and nuclear membranes intact, we constructed a numerical model of a biological cell containing a nucleus and liposomes of different sizes (with radii from 50 to 500 nm), which were placed in various sites in the cytoplasm. Our results show that under physiological conditions selective electroporation is only possible for the largest liposomes and when using very short pulses (few ns). By increasing the liposome interior conductivity and/or decreasing the cytoplasmic conductivity, selective electroporation of even smaller liposomes could be achieved. The location of the liposomes inside the cell does not play a significant role, meaning that liposomes of similar size could all be electroporated simultaneously. Our results indicate the possibility of using ns pulse treatment for liposomal drug release.

The role of electrically stimulated endocytosis in gene electrotransfer.

Gene electrotransfer is an established method for transfer of genes into cells, however, the mechanism of transfer of DNA across the cell membrane is still not known. Some studies suggest that DNA is translocated through membrane pores while others propose that DNA enters the cell via electro-endocytosis, but no direct observation was performed. In this paper we investigated the second hypothesis. Cells were stained with membrane dye FM 1-43FX, which is used for observation of endocytosis, and then exposed to electric pulses. We analyzed if endocytosis was stimulated by applying electric pulses with intensities below and above the threshold value for gene electrotransfer. No increase in endocytosis from 20 min or even up to 2h after the pulse delivery was observed, regardless of the electric field strength. These observations do not correlate with electrotransfer efficiency, which increases with field strength and is observed only above the threshold value. Our results suggest that electro-endocytosis is not a crucial mechanism for gene electrotransfer and that the hypothesis of DNA entry by translocation through permeabilized membrane is more plausible. The presented results are important for better understanding of the mechanisms of gene electrotransfer and for its optimization for clinical applications.

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 Ca(2+). 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, DeltaPsi (m)) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of DeltaPsi (m) and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between DeltaPsi (m) 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 DeltaPsi (m) 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.

Measuring the induced membrane voltage with Di-8-ANEPPS.

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by DeltaPhi, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of DeltaPhi on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of DeltaPhi. This video shows the protocol for measuring DeltaPhi using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of DeltaPhi.

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 (Delta Psi) and simulation of cell membrane electroporation. The model was first tested by comparing its results to the time-dependent analytical solution for Delta Psi 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 Delta Psi, the membrane conductivity rapidly increased with time, leading to a modified spatial distribution of Delta Psi. We show that steady-state models are insufficient for accurate description of Delta Psi, 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 Delta Psi 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.

Variability of the minimal transmembrane voltage resulting in detectable membrane electroporation.

We present a study of the variability of the minimal transmembrane voltage resulting in detectable electroporation of the plasma membrane of spherical and irregularly shaped CHO cells (we denote this voltage by ITVc). Electroporation was detected by monitoring the influx of Ca(2+), and the transmembrane voltage was computed on a 3D finite-elements model of each cell constructed from its cross-section images. We found that ITVc was highly variable, particularly in irregularly shaped cells, where it ranged from 512-1028 mV. We show that this range is much too large to be an artifact due to numerical errors and experimental inaccuracies, implying that for cells of the same type and exposed to the same number of pulses with the same duration, the value of ITVc can differ considerably from one cell to another. We also observed that larger cells are in many cases characterized by a higher ITVc than a smaller one. This is in qualitative agreement with the reports that higher membrane curvature facilitates electroporation, but quantitative considerations suggest that the observed variability of ITVc cannot be attributed entirely to the differences in membrane curvature.

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.

Electropermeabilization of dense cell suspensions.

This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 x 10(6) cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 x 10(6) to 400 x 10(6) cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 x 10(6) and 400 x 10(6) cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.

Effect of cell electroporation on the conductivity of a cell suspension.

An increased permeability of a cell membrane during the application of high-voltage pulses results in increased transmembrane transport of molecules that otherwise cannot enter the cell. Increased permeability of a cell membrane is accompanied by increased membrane conductivity; thus, by measuring electric conductivity the extent of permeabilized tissue could be monitored in real time. In this article the effect of cell electroporation caused by high-voltage pulses on the conductivity of a cell suspension was studied by current-voltage measurements during and impedance measurement before and after the pulse application. At the same time the percentage of permeabilized and survived cells was determined and the extent of osmotic swelling measured. For a train of eight pulses a transient increase in conductivity of a cell suspension was obtained above permeabilization threshold in low- and high-conductive medium with complete relaxation in <1 s. Total conductivity changes and impedance measurements showed substantial changes in conductivity due to the ion efflux in low-conductive medium and colloid-osmotic swelling in both media. Our results show that by measuring electric conductivity during the pulses we can detect limit permeabilization threshold but not directly permeabilization level, whereas impedance measurements in seconds after the pulse application are not suitable.

The effect of high frequency electric pulses on muscle contractions and antitumor efficiency in vivo for a potential use in clinical electrochemotherapy.

Muscle contractions present the main source of unpleasant sensations for patients undergoing electrochemotherapy. The contractions are a consequence of high voltage pulse delivery. Relatively low repetition frequency of these pulses (1 Hz) results in separate muscle contractions associated with each single pulse that is delivered. It would be possible to reduce the number of unpleasant sensations by increasing the frequency of electric pulses above the frequency of tetanic contraction, provided that the antitumor efficiency of electrochemotherapy remains the same. These assumptions were investigated in the present paper by measuring the muscle torque at different pulse repetition frequencies and at two different pulse amplitudes in rats and studying the antitumor efficiency of electrochemotherapy at different pulse repetition frequencies on tumors in mice. Measurements of muscle torque confirmed that pulse frequencies above the frequency of tetanic contraction (>100 Hz) reduce the number of individual contractions to a single muscle contraction. Regardless of the pulse amplitude, with increasing pulse frequency muscle torque increases up to the frequency of 100 or 200 Hz and then decreases to a value similar to that after application of a 1 Hz pulse train. Electrochemotherapy in vivo with higher repetition frequencies inhibits tumor growth and is efficient at all pulse frequencies examined (1 Hz-5 kHz). These results suggest that there is a considerable potential for clinical use of high frequency pulses in electrochemotherapy.