Plasma membrane charging of Jurkat cells by nanosecond pulsed electric fields

The initial effect of nanosecond pulsed electric fields (nsPEFs) on cells is a change of charge distributions along membranes. This first response is observed as a sudden shift in the plasma transmembrane potential that is faster than can be attributed to any physiological event. These immediate, yet transient, effects are only measurable if the diagnostic is faster than the exposure, i.e., on a nanosecond time scale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to nsPEFs of 60 ns and amplitudes from 5 to 90 kV/cm with a temporal resolution of 5 ns by means of the fast voltage-sensitive dye Annine-6. The measurements suggest the contribution of both dipole effects and asymmetric conduction currents across opposite sides of the cell to the charging. With the application of higher field strengths the membrane charges until a threshold voltage value of 1.4–1.6 V is attained at the anodic pole. This indicates when the ion exchange rates exceed charging currents, thus providing strong evidence for pore formation. Prior to reaching this threshold, the time for the charging of the membrane by conductive currents is qualitatively in agreement with accepted models of membrane charging, which predict longer charging times for lower field strengths. The comparison of the data with previous studies suggests that the sub-physiological induced ionic imbalances may trigger other intracellular signaling events leading to dramatic outcomes, such as apoptosis.     

The effects of intense submicrosecond electrical pulses on cells

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.     

Electrofusion between heterogeneous-sized mammalian cells in a pellet: potential applications in drug delivery and hybridoma formation.

High-efficiency electrofusion between cells of different sizes was achieved by application of fusing electric pulses to cells in centrifuged pellets. Larger target cells (Chinese hamster ovary or L1210 cells) were stacked among smaller human erythrocytes or erythrocyte ghosts by sequential centrifugation at 700 g to form five-tier pellets in a specially designed centrifugation-electrofusion chamber. The membranes of erythrocytes and ghost were labeled with fluorescent membrane dye (1,1' dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine (Dil)), and the contents of ghosts were loaded with water-soluble fluorescent dye (42-kDa fluorescein isothiocyanate dextran (FITC-dextran)), to monitor heterogeneous cell fusion. Fusion efficiency was assayed by the extent of either membrane dye mixing or contents (FITC-dextran) mixing with target cells. Four rectangular electric pulses at 300 V and 80 microseconds each were found to give the optimal fusion results of approximately 80% heterogeneous fusion by the content-mixing assay and approximately 95% by the membrane-dye-mixing assay. Cell viability remained greater than 80% after electrofusion. Because of the electric breakdown of cell membranes at the beginning of the pulse, the pellet resistance and hence the partial voltage across the pellet reduced rapidly during the remaining pulse time. This voltage redistribution favored the survival of fused cells. The limited colloidal-osmotic swelling of cells in pellets enhanced cell-cell contact and increased the pellet resistance after each pulse. As a result, the partial voltage across the pellet was restored when the next pulse was applied. This redistribution of pulse voltage in the pellet system permitted the breakdown of cell membranes at a lower applied voltage threshold than that required for electrofusion of cells in suspension or in dielectrophoretic cell chains. The cell viability and soluble dye retention within cells (FITC-dextran) remained at the same high levels for 3 h when the cells were incubated in respective culture media with serum at 37 degrees C. Viability and dye retention decreased significantly within 30 min when cells were incubated in phosphate-buffered saline without serum. The pellet technique was applied to form hybridomas by fusion of larger SP2/0 murine myelomas with smaller naive mouse lymphocytes. An optimum of 173 +/- 70 hypoxanthine aminopterin thymidine (HAT)-selected clones of the hybridomas was obtained from 40,000 SP2/0 cells and 1.5 x 10(6) lymphocytes used in each trial. This high-efficiency fusion technique may be adapted to mediate drug and gene transfer to target cells ex vivo as well as to form hybrid cells with limited cell sources.     

Dielectric Breakdown of Cell Membranes

With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 · 10⁶ V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector voltage (or the electric field strength in the orifice) depends on the membrane composition (or the intrinsic membrane potential) as revealed by measuring the critical voltage in E. coli B harvested from the logarithmic and stationary growth phases. The critical detector voltage increased by about 30% for a given volume on reaching the stationary growth phase.     

Aspects of plant plasmalemma charging induced by external electric field pulses

The charging of the plasmalemma is a necessary condition for permeabilization of the plasma membrane (electroporation) in response to external electric field exposure. Common theories explain this permeabilization by formation of pores in the lipid bilayer. Using pulsed laser fluorescence microscopy, we measured the charging process of the membrane during the application of an external electric field with a temporal resolution of 5 ns. Visualization of the charging process of protoplasts plasma membrane (Nicotiana tabacum Bright Yellow 2) was achieved by staining of the plasma membrane with the voltage-sensitive fluorescent dye ANNINE-6. Measurements on membranes exhibiting negligible membrane permeabilization confirm the sine-shaped azimuthal distribution of the membrane voltage predicted by the relation of Cole. At higher membrane voltages, enhanced pore formation allows for the exchange of charge carriers, leading to deviations from the sine-shaped curve progression, i.e., a saturation of the membrane voltage at membrane segments facing the electrodes. Additionally, measurements on protoplasts exposed to multiple successive pulses indicate that the recovery of the membrane seems to be a fast process, occurring within seconds after termination of the external electric field pulse.     

Calcium bursts induced by nanosecond electric pulses

We report here real-time imaging of calcium bursts in human lymphocytes exposed to nanosecond, megavolt-per-meter pulsed electric fields. Ultra-short (less than 30 ns), high-field (greater than 1 MV/m), electric pulses induce increases in cytosolic calcium concentration and translocation of phosphatidylserine (PS) to the outer layer of the plasma membrane in Jurkat T lymphoblasts. Pulse-induced calcium bursts occur within milliseconds and PS externalization within minutes. Caspase activation and other indicators of apoptosis follow these initial symptoms of nanosecond pulse exposure. Pulse-induced PS translocation is observed even in the presence of caspase inhibitors. Ultra-short, high-field, electroperturbative pulse effects differ substantially from those associated with electroporation, where pulses of a few tens of kilovolts-per-meter lasting a few tens of microseconds open pores in the cytoplasmic membrane. Nanosecond pulsed electric fields, because their duration is less than the plasma membrane charging time, develop voltages across intracellular structures without porating the cell.     

Electrical behavior and pore accumulation in a multicellular model for conventional and supra-electroporation

Extremely large but very short (20 kV/cm, 300 ns) electric field pulses were reported recently to non-thermally destroy melanoma tumors. The stated mechanism for field penetration into cells is pulse characteristic times faster than charge redistribution (displacement currents). Here we use a multicellular model with irregularly shaped, closely spaced cells to show that instead overwhelming pore creation (supra-electroporation) is dominant, with field penetration due to pores (ionic conduction currents) during most of the pulse. Moreover, the model's maximum membrane potential (about 1.2 V) is consistent with recent experimental observations on isolated cells. We also use the model to show that conventional electroporation resulting from 100 microsecond, 1 kV/cm pulses yields a spatially heterogeneous electroporation distribution. In contrast, the melanoma-destroying pulses cause nearly homogeneous electroporation of cells and their nuclear membranes. Electropores can persist for times much longer than the pulses, and are likely to be an important mechanism contributing to cell death.     

Electric field driven jetting: an emerging approach for processing living cells

This paper reports for the first time the ability to process living cellular materials by means of electrified jets at electric field strengths of up to 2 kV/mm. Bio-suspensions containing living human Jurkat cells at different concentrations were processed via this jetting approach. The jetting process was carried out at an electric field strength between 0.67 kV/mm and 2 kV/mm, corresponding to an applied voltage of 10-30 kV between two electrodes approximately 15 mm apart. The Jurkat cells were jetted under sterile conditions, collected in petri dishes and incubated for 24 and 48 hours. During and after incubation, cells were assessed for survival and structural damage; cells were found to be unharmed and to retain their integrity under all electric field strengths examined. At all field strengths jetting took place in the unstable mode. Good correlation was observed between droplet distribution plots generated by way of laser spectroscopy and estimated values from measurements of droplet relics.     

Nanosecond pulsed electric field generators for the study of subcellular effects

Modeling and experimental studies have shown that pulsed electric fields of nanosecond duration and megavolt per meter amplitude affect subcellular structures but do not lead to the formation of large pores in the outer membrane. This "intracellular electromanipulation" requires the use of pulse generators which provide extremely high power but low energy pulses. In this study, we describe the concept of the required pulsed power sources, their design, operation, and the necessary diagnostics. Two types of pulse generators based on the Blumlein line principle have been developed and are described here. One system is designed to treat a large number of cells in cuvettes holding volumes from 0.1 to 0.8 ml. Pulses of up to 40 kV amplitude, with a duration of 10 ns and a rise time close to 1 ns can be applied to the cuvette. For an electrode gap of 1 mm this voltage corresponds to an average electric field of 40 MV/m. The second system allows for real time observation of individual cells under a microscope. It generates pulses of 10-300 ns duration with a rise time of 3.5 ns and voltage amplitudes up to 1 kV. Connected to a microreactor with an electrode gap of 100 microm, electric fields up to 10 MV/m are applied.     

Dependence of the electrical breakdown voltage on the charging time inValonia utricularis

Summary Charge-pulse experiments were performed on giant algal cells ofValonia utricularis. For a charging time of 420 sec the breakdown voltage is about 750 mV (18°C), a value that is in close agreement with earlier results obtained with current pulses (Coster & Zimmermann, 1975;J. Membrane Biol. 22:73). If the membrane is charged to the breakdown voltage in a shorter time, the breakdown voltage is found to be a function of the duration of the charge pulses. Whereas towards smaller pulse lengths down to 10 sec only a small, but significant, increase in the breakdown voltage is observed (1.1 V at 10 sec pulse length and 18°C), a strong increase in the breakdown voltage is found for even shorter charging times. For a pulse length of 800 nsec the breakdown voltage has a value of about 2.4 V (18°C) and a plateau seems to be reached for a pulse duration of 500 nsec. The influence of temperature on the breakdown voltage as observed for short charging times is very similar to that reported earlier for current pulses of 500 sec duration. For charge pulses of 1 to 2 sec duration the breakdown voltage decreases from 3.6 V at 3°C to 1.6 V at 25°C by more than a factor of two.Voltage relaxation studies in the low-field range suggest that the time constants of the two membranes arranged in series, tonoplast and plasmalemma, are similar. From this, it is suggested that both membranes show electrical breakdown, whereby the breakdown voltage of a single membrane is probably half the value of the total breakdown voltage. Its dependence on pulse length is therefore considered to be an intrinsic property of one single membrane. The strong dependence of the breakdown voltage on the charging time of the membrane further supports the interpretation of the breakdown phenomenon on the basis of the electro-mechanical model proposed earlier. In this model it is assumed that the electrical and mechanical compressive forces are counter balanced by elastic restoring forces within the membrane. However, towards very short pulses (less than 800 nsec), where a plateau seems to be reached, other processes may be generated by the application of the electric field. We discuss whether one of these processes is the ion movement through the membranes induced by a high electric field (Born energy).     

Leukemic cell intracellular responses to nanosecond electric fields

Intense, nanosecond (ns) pulsed electric fields (PEFs) are known to affect the intracellular structures of cells. The probability of preferentially inducing subcellular effects increases with decreasing pulse length while effects on the plasma membrane are diminished. This has been demonstrated by applying electrical pulses of 60 and 10 ns duration with electric field intensities of up to 6.5 MV/m to HL-60 cells. Using confocal microscopy, PEF-induced changes in the integrity of the plasma membrane and nucleus were measured by recording fluorescence changes with propidium iodide (PI) and acridine orange (AO), respectively. Results suggest that high voltage, nsPEFs target the nucleus and modify cellular functions while plasma membrane effects are delayed and become smaller as pulse duration is shortened. Cell viability was not affected by these pulses. In spite of the high pulsed electric fields, thermal effects can be neglected because of the ultrashort pulse duration. The results suggest application of this ultrashort pulse technology to modulate nuclear structure and function for potential therapeutic benefit.     

Transmembrane potential measurements on plant cells using the voltage-sensitive dye ANNINE-6

The charging of the plasma membrane is a necessary condition for the generation of an electric-field-induced permeability increase of the plasmalemma, which is usually explained by the creation and the growth of aqueous pores. For cells suspended in physiological buffers, the time domain of membrane charging is in the submicrosecond range. Systematic measurements using Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) protoplasts stained with the fast voltage-sensitive fluorescence dye ANNINE-6 have been performed using a pulsed laser fluorescence microscopy setup with a time resolution of 5 ns. A clear saturation of the membrane voltage could be measured, caused by a strong membrane permeability increase, commonly explained by enhanced pore formation, which prevents further membrane charging by external electric field exposure. The field strength dependence of the protoplast’s transmembrane potential V _M shows strong asymmetric saturation characteristics due to the high resting potential of the plants plasmalemma. At the pole of the hyperpolarized hemisphere of the cell, saturation starts at an external field strength of 0.3 kV/cm, resulting in a measured transmembrane voltage shift of ?V _M?=??150 mV, while on the cathodic (depolarized) cell pole, the threshold for enhanced pore formation is reached at a field strength of approximately 1.0 kV/cm and ?V _M?=?450 mV, respectively. From this asymmetry of the measured maximum membrane voltage shifts, the resting potential of BY-2 protoplasts at the given experimental conditions can be determined to V _R?=??150 mV. Consequently, a strong membrane permeability increase occurs when the membrane voltage diverges |V _M|?=?300 mV from the resting potential of the protoplast. The largest membrane voltage change at a given external electric field occurs at the cell poles. The azimuthal dependence of the transmembrane potential, measured in angular intervals of 10° along the circumference of the cell, shows a flattening and a slight decrease at higher fields at the pole region due to enhanced pore formation. Additionally, at the hyperpolarized cell pole, a polarization reversal could be observed at an external field range around 1.0 kV/cm. This behavior might be attributed to a fast charge transfer through the membrane at the hyperpolarized pole, e.g., by voltage-gated channels.     

Lateral electrophoresis and diffusion of Concanavalin A receptors in the membrane of embryonic muscle cell

A uniform electric field of 10 V/cm applied across the surface of embryonic toad Xenopus muscle cells results in the asymmetric accumulation of concanavalin A (Con A) receptors toward one side of the cells within 10 min, as visualized by postfield fluorescent Con A labeling. This field produces an extracellular voltage difference of 20 mV across these 20-microns wide cells. The effect is reversible in two respects: (a) Additional exposure of the cell to the same field of opposite polarity for 10 min completely reverses the asymmetric accumulation to the other side of the cell. (b) Relaxation occurs after the removal of the field and results in complete recovery of the uniform distribution in 30 min. Both the accumulation and the recovery movements are independent of cell metabolism, and appear to be electrophoretic and diffusional in nature. The threshold field required to induce a detectable accumulation by the present method is between 1.0 and 1.5 V/cm (corresponding to a voltage difference of 2-3 mV across a 20-microns wide cell). The electrophoretic mobility of the most mobile population of nonliganded Con A receptors is estimated to be about 2 x 10(-3) microns/s per V/cm, while their diffusion coefficient is in the range of 4-7 x 10(-10) cm2/s. Extensive accumulation of the Con A receptors by an electric field results in the formation of immobile aggregates. The Con A receptors appear to consist of a heterogeneous population of membrane components different in their charge properties, mobility, and capability in forming aggregates.     

Calcium waves induced by large voltage pulses in fish keratocytes.

Intracellular calcium waves in fish keratocytes are induced by the application of electric field pulses with amplitudes between 55 and 120 V/cm and full width at half-maximum of 65-100 ms. Calcium concentrations were imaged using two-photon excited fluorescence microscopy (Denk et al., 1990 Science. 248:73-76; Williams et al. 1994 FASEB J. 8:804-813) and the ratiometric calcium indicator indo-1. The applied electric field pulses induced waves with fast calcium rise times and slow decays, which nucleated in the lamellipodium at the hyperpolarized side of the cells and, less frequently, at the depolarized side. The effectiveness of wave generation was determined by the change induced in the membrane potential, which is about half the field strength times the cell width in the direction of the field. Stimulation of waves began at voltage drops across the cell above 150 mV and saturated at voltage drops above 300 mV, where almost all cells exhibited a wave. Waves were not induced in low-calcium media and were blocked by the nonselective calcium channel blockers cobalt chloride and verapamil, but not by specific organic antagonists of voltage-sensitive calcium channel conductance. Thapsigargin stopped wave propagation in the cell body, indicating that calcium release from intracellular stores is necessary. Thus a voltage pulse stimulates Ca2+ influx through calcium channels in the plasma membrane, and if the intracellular calcium concentration reaches a threshold, release from intracellular stores is induced, creating a propagating wave. These observations and the measured parameters (average velocity approximately 66 micron/s and average rise time approximately 68 ms) are consistent with a wave amplification model in which[equation, see text] determines the effective diffusivity of the propagating molecules, D approximately 300 micron2/s (Meyer, 1991. Cell. 64:675-678).     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.     

High electric field effects on the cell membranes of Halicystis parvula

The electrical breakdown behavior of the giant algal cell Halicystis parvula was studied in order to predict the optimum conditions for electrically induced cell-to-cell fusion. Using the charge pulse technique, the membranes were charged at different pulse lengths to the maximum voltage V_c. Because of a reversible, high-conductance state of the membrane (electrical breakdown), it was not possible to exceed the critical membrane breakdown potential. The breakdown voltage exhibited a strong dependence on the charging time (pulse length) between 10 s and 100 s. Below 10 s the breakdown voltage of the two membranes, tonoplast and plasmalemma, assumed a constant value of about 1.9 V, whereas above a pulse length of about 100 s the breakdown voltage was nearly constant with a value of about 0.6 V. The extreme values for the breakdown voltage at very short and at very long charging times agree fairly well with results which have been obtained on cells of Valonia utricularis and planar lipid bilayer membranes. However, the pulse length dependence of the breakdown voltage was found to be quite different in H. parvula. In addition, the membrane conductance increase during breakdown in H. parvula cells is much more pronounced than in membranes of V. utricularis, but similar to lipid bilayer membranes. From this result it is suggested that the membrane structure of H. parvula is quite different from V. utricularis (larger lipid domains).     

Increased binding of liposomes to cells by electric treatment.

The influence of electric field treatments on the interaction of large unilamellar vesicles (liposomes) with animal cells was monitored by the fluorescence assay based on the use of the liposomes loaded by a dye 1-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). It was shown that application of a short electric pulse (100 microseconds of 3-4 kV/cm) to the suspension of cells in presence of vesicles resulted in significant (more than 2 times) increase of the fluorescence associated with cells. The pH-sensitivity of the excitation spectrum of the dye and its interaction with the quencher were used to determine the nature of the phenomenon as the increase of the liposome binding onto the cell surface but not the consequence of a promotion of liposome uptake into the cells by endocytosis. The higher affinity for the liposome caused by the electric field has a lifetime of several minutes. The possible relation of the effect described to the electroporation of cell membranes and to macroscopic changes in membrane structure is discussed.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10³–10⁴ V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strenght it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yields a mean value of about 1.6 V for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 °C as shown by incorporation of ¹³¹I-labeled albumin and repeated dielectric breakdown.     

Selective electrofusion of conjugated cells in flow.

Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells and cell pairs are first optically analyzed in a normal flow channel and then forced to flow through a Coulter orifice. If the optical analysis indicates that a cell pair is present, an electric pulse is applied across the orifice to induce fusion. The pulsed cell pairs were subsequently analyzed using normal and confocal microscopy to evaluate fusion induction. It appears that fusion can be induced in about 10% of pulsed cell pairs when one electric pulse with a duration of 10-15 microseconds and an effective electric field strength of 4-8 10(5) V/m is used.     

Assessment of electroporation by flow cytometry

BACKGROUND: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation. METHODS: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin. RESULTS: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells. CONCLUSIONS: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.