Electric Field Pulses Can Induce Apoptosis

Injection of electric field pulses of high intensity (kV/cm) and short duration (microsecond range) into a cell suspension results in a temporary increase of the membrane permeability due to a reversible electric breakdown of the cell membrane. Here we demonstrate that application of supercritical field pulses between 4.5 and 8.1 kV/cm strength and 40 μsec duration induce typical features of apoptosis in Jurkat T-lymphoblasts and in HL-60 cells including DNA fragmentation and cleavage of the poly(ADP ribose) polymerase. Apoptosis induction did not depend on the presence of any particular electrolyte in the extracellular medium. However, no apoptosis was observed in solutions without a minimum amount of salt. Apoptotic DNA fragmentation was prevented by the caspase inhibitor zVAD. Received: 16 December 1998/Revised: 24 February 1999     

Permeabilization of tumor cells induced by pulsed electric fields in vitro

The permeabilization of tumor cells in vitro under the action of pulsed electric fields with a duration of 6 mks in the range of amplitudes 1-7 kV/cm was studied. In the mode of excitation in the ambience of localized plasma discharge in a chamber of special design, an enhanced damage to cells in suspension was observed. It is assumed that the enhancement is due to the synchronous action of the electric field and acoustic shock wave pulses. In the mode without the plasma breakdown of ambience, when the pulse duration of electric field of intensity of 1-2 kV/cm was increased to 60 mks, the efficiency of permeabilization increases nearly by one order. The experimental results are compared with the known theoretical models of cell membrane electroporation.     

Phase transition of dimyristoylphosphatidylglycerol bilayers induced by electric field pulses

The phase transition of dimyristoylphosphatidylglycerol (DMPG) bilayers has been studied by measurements of light scattering under high electric field pulses. Midpoints of phase transitions have been identified by a clear discontinuity of field induced relaxation amplitudes. We show that the phase transition of DMPG suspensions in monovalent salt is virtually independent of the electric field strength up to approx. 35 kV/cm. A shift of the lipid phase by electric field pulses has been observed, however, for DMPG suspensions in the presence of Ca2+ ions. DMPG suspensions exhibit a jump of the phase transition temperature from 17 degrees C at Ca/DMPG molar ratios r less than 1/7 to 32 degrees C at r greater than 1/7. Field pulses of 60 to 100 microseconds applied to DMPG suspensions with Ca2+ at r greater than 1/7 induce discontinuities of relaxation amplitudes in the temperature range 15 to 22 degrees C in addition to the 'standard' one at 32 degrees C, when the electric field strength is above 15 kV/cm. These results indicate that electric field pulses induce a transition from the phase formed at 'high' Ca(2+)- to the one formed at 'low' Ca(2+)-ion concentrations. Our results are consistent with a dissociation field effect on Ca(2+)-lipid complexes which drives the phase transition.     

Kinetics of permeability changes induced by electric impulses in chromaffin granules

Summary Electric field pulses, ranging in intensity from 20 to 50 kV/cm and in duration from 10 to 40 sec, caused a transient increase in the membrane permeability of chromaffin granules from the bovine adrenal medulla, that led to partial release of granule soluble constituents. This transient permeability change was long-lived, as compared to the pulse duration, and the main part of material efflux occurred after the termination of the pulse. During the latter phase the temporarily increased permeability decayed to its original value, in the absence of the electric field. This indicated that the structural perturbation induced in the membrane was transient and apparently reversible. The release event was characterized by a field-dependent permeability coefficient ranging from 2×10^–4 cm/sec at 30 kV/cm to 3×10^–3 cm/sec at 50 kV/cm. The resealing process of the membrane could be described by two relaxation times, both of which decreased with increasing field strength. ₁ varied from about 3.0 msec at 30 kV/cm to less than 2.0 msec at 50 kV/cm, while ₂ varied from about 100 to about 40 msec in the same interval of field strength. The distribution in the degree of filling of granules that had been partially depleted by an electric field pulse indicated that the population could be considered homogeneous with respect to release.     

The effect of pulse field strength on electric field stimulated biosorption of uranium by Kluyveromyces marxianus IMB3

Summary Improved biosorption of uranium by Kluyveromyces marxianus IMB3 biomass was achieved by increasing the electric field strength of delivered pulses from 1.25kV/cm to 2.5kV/cm. Although this had little or no effect on the maximum biosorption capacity (q_max), at low concentrations of uranium the amount bound to the biomass increased from 70 to 140mg uranium/g biomass. Significant increases in the maximum biosorption capacities (119–180 mg uranium/g biomass) were observed when the pulse field strength was increased from 2.5kV/cm to 3.25kV/cm.     

Electro-fusion of haploid Saccharomyces yeast cells of identical mating type

Yeast protoplasts from the haploid strains 21 a and 111a were exposed to an inhomogeneous alternating field (about 1 kV/cm, 2 MHz). Due to dielectrophoretic aggregation two or more cells with close membrane contact are formed between the electrodes. Cell fusion was observed by application of two field pulses (11 kV/cm, 7 s duration) applied at an interval of 1 s. The intensity of the field pulses is sufficiently high to induce reversible electrical breakdown at membrane sites oriented in the field direction. After a 8 to 14 days incubation period on selection medium two types of fusion products could be isolated: 1) Hybrids with a haploid constitution, respiratory-competent and auxotrophic for histidine. 2) Cells with a diploid cell size and prototrophic for histidine. The genetic analysis for mating types and auxotrophic markers show that in the second case plasmogamy followed by karyogamy had occurred.     

Combination of Microsecond and Nanosecond Pulsed Electric Field Treatments for Inactivation of Escherichia coli in Water Samples

Inactivation of microorganisms with pulsed electric fields is one of the nonthermal methods most commonly used in biotechnological applications such as liquid food pasteurization and water treatment. In this study, the effects of microsecond and nanosecond pulses on inactivation of Escherichia coli in distilled water were investigated. Bacterial colonies were counted on agar plates, and the count was expressed as colony-forming units per milliliter of bacterial suspension. Inactivation of bacterial cells was shown as the reduction of colony-forming units per milliliter of treated samples compared to untreated control. According to our results, when using microsecond pulses the level of inactivation increases with application of more intense electric field strengths and with number of pulses delivered. Almost 2-log reductions in bacterial counts were achieved at a field strength of 30 kV/cm with eight pulses and a 4.5-log reduction was observed at the same field strength using 48 pulses. Extending the duration of microsecond pulses from 100 to 250 μs showed no improvement in inactivation. Nanosecond pulses alone did not have any detectable effect on inactivation of E. coli regardless of the treatment time, but a significant 3-log reduction was achieved in combination with microsecond pulses.     

Effect of high-voltage pulses on the viability of human leucocytes in vitro

Human leucocytes were exposed to high-voltage pulses (transient currents) produced by discharging a capacitor through a test chamber containing the cell suspension then tested for viability using trypan blue. With the pulse discharge times of 1 and 3 μs increases in the number of dyeloaded cells were seen for field strengths above 2.6 kV/cm in the sample. For 0.2-μs pulses the critical field strength was about 5 kV/cm.     

Comparison of Flow Cytometry,Fluorescence Microscopy and Spectrofluorometry for Analysis of Gene Electrotransfer Efficiency

In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.     

Somatic hybridization between human and mouse lymphoblast cells produced by an electric pulse-induced fusion technique

The technique of electric pulse-induced cell fusion (electro-fusion) was used to obtain heterokaryons between normal human lymphoblasts (HSC93) and mouse leukemic lymphoblasts (MCN151). The two types of cells were brought into contact in the cell suspension by dielectrophoresis with an alternating electric field (0.8 kV/cm, 100 kHz) in the presence of calcium ions and pronase E. Cell fusion was induced by giving two successive electric pulses (3.3 and 5 kV/cm, 10 microsec). Prior treatment of human (but not mouse) lymphoblasts with neuraminidase improved fusion efficiency. Differential staining of the two types of cells with Janus Green and Neutral Red showed that about 40% of the viable fused cells underwent heterokaryonic fusion. We concluded that electrofusion is an efficient method for obtaining heterokaryons from human and mouse lymphoblasts.     

Dose-Dependent ATP Depletion and Cancer Cell Death following Calcium Electroporation,Relative Effect of Calcium Concentration and Electric Field Strength

Background

Electroporation, a method for increasing the permeability of membranes to ions and small molecules, is used in the clinic with chemotherapeutic drugs for cancer treatment (electrochemotherapy). Electroporation with calcium causes ATP (adenosine triphosphate) depletion and cancer cell death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy.

Methods

In three human cell lines — H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability was determined after treatment with 1, 3, or 5 mM calcium and eight 99 μs pulses with 0.8, 1.0, 1.2, 1.4 or 1.6 kV/cm. Fitting analysis was applied to quantify the cell-killing efficacy in presence of calcium. Post-treatment intracellular ATP was measured in H69 and SW780 cells. Post-treatment intracellular ATP was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells.

Results

Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (p<0.05) and reduced viability. The 50% effective cell kill was found at 3.71 kV/cm (H69) and 3.28 kV/cm (SW780), reduced to 1.40 and 1.15 kV/cm (respectively) with 1 mM calcium (lower EC50 for higher calcium concentrations). Quinacrine fluorescence intensity of calcium-electroporated U937 cells was one third lower than in controls (p<0.0001).

Conclusions

Calcium electroporation dose-dependently reduced cell survival and intracellular ATP. Increasing extracellular calcium allows the use of a lower electric field.

General Significance

This study supports the use of calcium electroporation for treatment of cancer and possibly lowering the applied electric field in future trials.     

Effect of actin cytoskeleton disruption on electric pulse‐induced apoptosis and electroporation in tumour cells

Electric pulses are known to affect the outer membrane and intracellular structures of tumour cells. By applying electrical pulses of 450 ns duration with electric field intensity of 8 kV/cm to HepG2 cells for 30 s, electric pulse‐induced changes in the integrity of the plasma membrane, apoptosis, viability and mitochondrial transmembrane potential were investigated. Results demonstrated that electric pulses induced cell apoptosis and necrosis accompanied with the decrease of mitochondrial transmembrane potential and the formation of pores in the membrane. The role of cytoskeleton in cellular response to electric pulses was investigated. We found that the apoptotic and necrosis percentages of cells in response to electric pulses decreased after cytoskeletal disruption. The electroporation of cell was not affected by cytoskeletal disruption. The results suggest that the disruption of actin skeleton is positive in protecting cells from killing by electric pulses, and the skeleton is not involved in the electroporation directly.     

A novel method for transformation of intact yeast cells by electroinjection of plasmid DNA

Summary It was found that plasmid DNA (YEp13) can be introduced into intact yeast cells by electric field pulses. The frequency of transformation by this electroinjection method depend upon the initial electric field intensity, the capacitance of the electric discharge capacitor, and the number of pulses applied. A maximum number of transformants (90±20/gDNA) was obtained by three successive pulses with an initial intensity of 5 KV/cm and with a capacitance of 1 F. The present electroinjection method is simple, and transformants can be obtained within 2 to 3 days after transformation treatment.     

Short electric-field pulses convert DNA from "condensed" to "free" conformation

Electric-field pulses of e.g. 20 kV/cm and 100 μs induce a strong decrease in the scattered light intensity of DNA condensed by spermine. Analysis of this effect demonstrates that the decrease of the scattered light intensity results from decondensation of DNA. The decondensation reaction requires an electric-field strength exceeding a threshold value. Complete decondensation can be achieved at field strength that are only slightly higher than the threshold value. The decondensation process is strongly accelerated at high electric-field strengths. At 30 kV/cm, the decondensation time constant is ～8 μs, corresponding to an acceleation factor of 10⁵ relative to the field-free decondensation reaction. The dependence of the time constants on the electric-field strength suggests that the field-induced decondensation is due to a dissociation field effect. The condensation process observed after electric-field pulses at low concentrations of DNA and spermine shows a characteristic induction period, which strongly depends on the spermine concentration. This induction period reflects the time required for the binding of spermine to DNA, until the degree of binding is sufficiently high for the condensation reaction. The fast dissociation of condensed DNA by electric-field pulses together with a relatively long lifetime of the free DNA results in a reaction cycle resembling a hysteresis loop.     

Effect of pulsed electric fields on the activity of neutral trehalase from beer yeast and RSM analysis

The trehalase activity plays an important role in extraction of trehalose from beer yeast. In this study, the effect of pulsed electric field processing on neutral trehalase activity in beer yeast was investigated. In order to develop and optimize a pulsed electric field (PEF) mathematical model for activating the neutral trehalase, we have investigated three variables, including electric field intensity (10-50 kV/cm), pulse duration (2-10 μs) and liquid-solid ratio (20-50 ml/g) and subsequently optimized them by response surface methodology (RSM). The experimental data were fitted to a second-order polynomial equation and profiled into the corresponding contour plots. Optimal condition obtained by RSM is as follows: electric field intensity 42.13 kV/cm, liquid-solid ratio 30.12 ml/g and pulse duration 5.46 μs. Under these conditions, with the trehalose decreased 8.879 mg/L, the PEF treatment had great effect on activating neutral trehalase in beer yeast cells.     

Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric fields

Two pathogenic microorganisms Escherichia coli O157:H7 and Staphylococcus aureus, suspended in peptone solution (0.1% w/v) were treated with 12, 14, 16 and 20 kV/cm electric field strengths with different pulse numbers up to 60 pulses. Pulsed electric field (PEF) treatment at 20 kV/cm with 60 pulses provided nearly 2 log reduction in viable cell counts of E. coli O157:H7 and S. aureus. S. aureus cells were slightly more resistant than E.coli O157:H7 cells. The results related to the effect of initial cell concentration of E. coli O157:H7 on the PEF inactivation showed that more inactivation was obtained by decreasing initial cell concentration. Any possible injury by PEF was also investigated after applying 20 kV/cm electric field to the microorganisms. As a result, it was determined that there was 35.92 to 43.36% injury in E. coli O157:H7 cells, and 17.26 to 30.86% injury in S. aureus cells depending on pulse number. The inactivation results were also described by a kinetic model.     

A High-Power Dielectric Biconical Antenna for Treatment of Subcutaneous Targets

A dielectric biconical antenna (DiBiCA) for radiating subnanosecond pulses to treat subcutaneous tissue was designed, constructed, and tested. It is composed of a conical wave launcher and truncated conical emitter. In between, there is a short cylinder that provides a space for a ring terminating resistor. The material of the antenna has a dielectric constant of 28, so its size is small (length: 7 cm and aperture diameter: 2.2 cm). It was housed in an oil container to withstand high voltages and avoid surface flashover. The radiated electric field, measured in water, increased as the input voltage increased up to 30 kV but leveled off for higher voltages up to 50 kV, presumably because of losses in the antenna dielectric. The maximum field was 1.5 kV/cm for a depth of 5 mm and 1.0 kV/cm for a depth of 20 mm. Although the dielectric loss mechanism remains to be investigated, the antenna can be useful for noninvasive delivery of subnanosecond pulses to induce biological responses on subcutaneous targets. The DiBiCA radiated pulses were shown to change the viabilities of dendritic cells and macrophages for 10-min exposure. Bioelectromagnetics. 2020;41:413–424. © 2020 Bioelectromagnetics Society.     

Nanosecond Electric Pulse Effects on Gene Expression

Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.     

Effects of pulsed electric fields on mouse spleen lymphocytes in vitro

The effects of pulsed electric fields on cell membranes were investigated. In vitro exposure of mouse splenocytes to a single high-voltage pulse resulted in an increase in membrane permeability that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 μs, 3.0 kV/cm pulse resulted in the induction of a 1.26 V transmembrane potential, and elicited a 50% loss of intracellular K⁺. These results are in agreement with previous studies of the effects of pulsed electric fields on erythrocytes and microorganisms. The effect of pulsed electric fields on the functional integrity of lymphocytes was i vestigated by measuring [³H]thymidine incorporation by cells cultured in the presence and absence of various mitogens following exposure to an electrical pulse. No statistically significant effects on the response of mouse spleen lymphocytes to concanavalin A, phytohemagglutinin or lipopolysaccharide were observed following exposure to 2 μs electric pulses at amplitudes of up to 3.5 kV/cm. Exposure to a single 10 μs pulse of 2.4–3.5 kV/cm produced a statistically significant reduction in the response of lymphocytes to lipopolysaccharide stimulation that was attributed to cell death.     

Bioactivity of electric field-pulsed human recombinant interleukin-2 and its encapsulation into erythrocyte carriers

The molecular integrity of human recombinant interleukin-2 (rIL-2), as measured by size exclusion chromatography, was not altered when exposed to high electrical field intensities. In addition, the biological activity was unaffected, as evidenced by the ability of the rIL-2 to stimulate the proliferation (by cell growth assays and tritiated thymidine uptake) and differentiation (by cytotoxicity assay) of human lymphocytes into killer cells. Electroporation conditions chosen for the loading of rIL-2, based upon those which provided for good recovery of carriers and minimal hemoglobin release, involved a lower field intensity (i.e., 6 kV/cm instead of 7 or 8 kV/cm) and multiple pulses (eight pulses, 5 microseconds) rather than a single pulse (40 microseconds). Human erythrocyte carriers consistently encapsulated 5-7.5% of the rIL-2 by electroporation (6 kV/cm, eight pulses, 5 microseconds duration). A rIL-2 concentration of 600,000 U/ml surrounding the erythrocytes during loading resulted in ca. 245,000 U/ml carriers, which represents a therapeutically significant quantity. Thus, rIL-2 shows potential as an encapsulated agent for slow release in the erythrocyte carrier system.