In vivo electrical impedance measurements during and after electroporation of rat liver

Electroporation is used for in vivo gene therapy, drug therapy and minimally invasive tissue ablation. Applying electrical pulses across cells can have a variety of outcomes; from no effect to reversible electroporation to irreversible electroporation. Recently, it has been proposed that measuring the passive electrical properties of electroporated tissues could provide real time feedback on the outcome of the treatment. Here we describe the results from the impedance characterization (single dispersion Cole model) for up to 30 min of the electroporation process in in vivo rat livers (n=8). The electroporation sequence consisted of 8 pulses of 100 micros with a period of 100 ms. Half of the animals were subjected to field magnitudes considered to have reversible effects (R group, E=450 V/cm) whereas for the other half irreversible field amplitudes were applied (I group, E=1500 V/cm). As expected, there was an immediate increase of conductivity (R group Deltasigma/sigma(t=0)=9+/-3%; I group Deltasigma/sigma(t=0)=43+/-1%). However, the overall long term pattern of change in conductivity after electroporation is complex and different between reversible and irreversible groups. This suggests the superposition of different phenomena which together affect the electrical properties.     

Real time electroporation control for accurate and safe in vivo non-viral gene therapy

In vivo cell electroporation is the basis of DNA electrotransfer, an efficient method for non-viral gene therapy using naked DNA. The electric pulses have two roles, to permeabilize the target cell plasma membrane and to transport the DNA towards or across the permeabilized membrane by electrophoresis. For efficient electrotransfer, reversible undamaging target cell permeabilization is mandatory. We report the possibility to monitor in vivo cell electroporation during pulse delivery, and to adjust the electric field strength on real time, within a few microseconds after the beginning of the pulse, to ensure efficacy and safety of the procedure. A control algorithm was elaborated, implemented in a prototype device and tested in luciferase gene electrotransfer to mice muscles. Controlled pulses resulted in protection of the tissue and high levels of luciferase in gene transfer experiments where uncorrected excessive applied voltages lead to intense muscle damage and consecutive loss of luciferase gene expression.     

Improvement of the quality of self assembled bilayer lipid membranes by using a negative potential

Cell electropermeabilization (also termed cell electroporation) is nowadays a routine technique used in biochemical and pharmacological studies for the in vitro introduction of nonpermeant molecules into living cells. But electric pulses can be used as well in vivo for the delivery of drugs or DNA into cells of tissues. This review then gives an updated overview of the therapeutic perspectives of cell electropermeabilization in vivo, in particular of the antitumour electrochemotherapy (i.e., the combination of a cytotoxic nonpermeant drug with permeabilizing electric pulses delivered to the tumours) and of in vivo DNA electrotransfer for gene therapy. After a short summary of the present knowledge on cell electropermeabilization (particularly in vivo), the basis, the present achievements, and the challenges of electrochemotherapy are described and discussed, which includes an overview of still open questions and an update on recent clinical trials. DNA electrotransfer for gene therapy is an emerging field in which results are rapidly accumulating. Present knowledge on DNA electrotransfer mechanisms, as wel as the potentialities of DNA electrotransfer to become an efficient non-viral approach for gene therapy, are reviewed.     

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.     

Electroporation of subcutaneous mouse tumors by rectangular and trapezium high voltage pulses

The artificial electrotransfer of bioactive agents such as drugs, peptides or therapeutical nucleic acids and oligonucleotides by membrane electroporation (MEP) into single cells and tissue cells requires knowledge of the optimum ranges of the voltage, pulse duration and frequency of the applied pulses. For clinical use, the classical electroporators appear to necessitate some tissue specific presetting of the pulse parameters at the high voltage generator, before the actual therapeutic pulsing is applied. The optimum pulse parameters may be derived from the kinetic normal mode analysis of the current relaxations due to a voltage step (rectangular pulse). Here, the novel method of trapezium test pulses is proposed to rapidly assess the current (I)/voltage (U) characteristics (IUC). The analysis yields practical values for the voltage U(app) between a given electrode distance and pulse duration t(E) of rectangular high voltage (HV) pulses, to be preset for an effective in vivo electroporation of mouse subcutaneous tumors, clamped between two planar plate electrodes of stainless steel. The IUC of the trapezium pulse compares well with the IUC of rectangular pulses of increasing amplitudes. The trapezium pulse phase (s) of constant voltage and 3 ms duration, following the rising ramp phase (r), yields a current relaxation which is similar to the current relaxation during a rectangular pulse of similar duration. The fit of the current relaxation of the trapezium phase (s) to an exponential function and the IUC can be used to estimate the maximum current at a given voltage. The IUC of the falling edge (phase f) of the trapezium pulse serves to estimate the minimum voltage for the exploration of the long-lived electroporation membrane states with consecutive low-voltage (LV) pulses of longer duration, to eventually enhance electrophoretic uptake of ionic substances, initiated by the preceding HV pulses.     

Simulation and experimental demonstration of the electric field assisted electroporation microchip for in vitro gene delivery enhancement

Simulation and experimental demonstration of the in vitro gene delivery enhancement using electrostatic forces and electroporation (EP) microchips were conducted. Electroporation is a technique with which DNA molecules can be delivered into cells using electric field pulses. This study demonstrates that plasmid DNA can be attracted to the cell surfaces at the specific regions using an electrostatic force. Therefore, the DNA concentration on the cell surface is dramatically increased, which highly enhances the gene transfection efficiency compared to that without an attracting-electric field. The electrostatic force can be designed into specific regions, where the DNA plasmids are attracted to, to provide the region-targeting function. In this micro-device, the top electrode and the interdigitated electrodes provided the DNA attracting-electric field, and the interdigitated electrodes provided adequate electric fields for the electroporation process on the chip surface. Using the EP microchip, cells could be manipulated in situ without detachment if adherent cells were used for electroporation. Five different cells of two different types, primary cell and cell line, were successfully transfected under multi-pulse or single pulse electric field stimulation without applying an attracting-electric field. This study simulated and analyzed the electric field distributions at the DNA attracting and electroporation processes, and successfully demonstrated that the electrostatic force attracted DNA plasmids to specific regions and highly enhanced the gene delivery. In summary, this EP microchip should provide many potential applications for gene therapy.     

Theoretical analysis of the thermal effects during in vivo tissue electroporation

Tissue electroporation is a technique that facilitates the introduction of molecules into cells by applying a series of short electric pulses to specific areas of the body. These pulses temporarily increase the permeability of the cell membrane to small drugs and macromolecules. The goal of this paper is to provide information on the thermal effects of these electric pulses for consideration when designing electroporation protocols. The parameters investigated include electrode geometry, blood flow, metabolic heat generation, pulse frequency, and heat dissipation through the electrodes. Basic finite-element models were created in order to gain insight and weigh the importance of each parameter. The results suggest that for plate electrodes, the energy from the pulse may be used to adequately estimate the heating in the tissue. However, for needle electrodes, the geometry, i.e. spacing and diameter, and pulse frequency are critical when determining the thermal distribution in the tissue.     

Micro pulsed radio-frequency electroporation chips

Electroporation (EP) is one of the most important physical methods in biotechnology, which employs electrical pulses to transiently permeabilize cell membranes. In this study, a new micro pulsed radio-frequency electroporation cell (microPREP) chip was fabricated using a lift-off technique and SU-8 photolithography. The biological tests were carried out using three different plant protoplasts (cabbage, spinach and oil rape) on the micro EP chip and a pulsed RF electric field was applied to the microchip. The variations of fluorescent intensity and cell viability as functions of the electric pulse amplitude and duration time during the electroporation process were studied in detail at the single-cell level. Using such chip design and test method, one can easily optimize the efficiency and cell viability. Also, a large amount of statistical data can be quickly obtained. Finally, results of this parametric study were presented in the "phase diagram", from which the critical electric field for inducing single-cell electroporation under different conditions can be clearly determined.     

Feasibility study for cell electroporation detection and separation by means of dielectrophoresis

Electroporation is a phenomenon during which exposure of a cell to high voltage electric pulses results in a significant increase in its membrane permeability. Aside from the fact that after the electroporation the cell membrane becomes more permeable, the cells' geometrical and electrical properties change considerably. These changes enable use of the force on dielectric particles exposed to non-uniform electric field (dielectrophoresis) for separation of non-electroporated and electroporated cells. This paper reports the results of an attempt to separate non-electroporated and electroporated cells by means of dielectrophoresis. In several experiments we managed to separate the non-electroporated and electroporated cells suspended in a medium with conductivity 0.174 S/m by exposing them to a non-uniform electric field at a frequency of 2 MHz. The behaviour of electroporated cells exposed to dielectrophoresis raises the presumption that in addition to conductivity, considerable changes in membrane permittivity occur after the electroporation.     

An efficient and high-throughput electroporation microchip applicable for siRNA delivery

Here we report a novel electroporation microchip with great performance and compatibility with the standard multi-well plate used in biological research. The novel annular interdigitated electrode design makes it possible to achieve efficient cell transfection as high as 90% under low-strength electrical pulses, thereby circumventing the many adverse effects of conventional cuvette-type and previously reported microchip-based electroporation devices. Using this system, we demonstrated substantially improved cell transfection efficacy and viability in cultured and primary cells, for both plasmid and synthetic siRNA. Improvements of this system open new opportunities for high-throughput applications of siRNA technology in basic and biomedical research.     

Pulse Duration Dependent Asymmetry in Molecular Transmembrane Transport Due to Electroporation in H9c2 Rat Cardiac Myoblast Cells In Vitro

Electroporation (EP) is one of the successful physical methods for intracellular drug delivery, which temporarily permeabilizes plasma membrane by exposing cells to electric pulses. Orientation of cells in electric field is important for electroporation and, consequently, for transport of molecules through permeabilized plasma membrane. Uptake of molecules after electroporation are the greatest at poles of cells facing electrodes and is often asymmetrical. However, asymmetry reported was inconsistent and inconclusive—in different reports it was either preferentially anodal or cathodal. We investigated the asymmetry of polar uptake of calcium ions after electroporation with electric pulses of different durations, as the orientation of elongated cells affects electroporation to a different extent when using electric pulses of different durations in the range of 100 ns to 100 µs. The results show that with 1, 10, and 100 µs pulses, the uptake of calcium ions is greater at the pole closer to the cathode than at the pole closer to the anode. With shorter 100 ns pulses, the asymmetry is not observed. A different extent of electroporation at different parts of elongated cells, such as muscle or cardiac cells, may have an impact on electroporation-based treatments such as drug delivery, pulse-field ablation, and gene electrotransfection.     

Electric field-mediated transport of plasmid DNA in tumor interstitium in vivo

Local pulsed electric field application is a method for improving non-viral gene delivery. Mechanisms of the improvement include electroporation and electrophoresis. To understand how electrophoresis affects pDNA delivery in vivo, we quantified the magnitude of electric field-induced interstitial transport of pDNA in 4T1 and B16.F10 tumors implanted in mouse dorsal skin-fold chambers. Four different electric pulse sequences were used in this study, each consisted of 10 identical pulses that were 100 or 400 V/cm in strength and 20 or 50 ms in duration. The interval between consecutive pulses was 1 s. The largest distance of transport was obtained with the 400 V/cm and 50 ms pulse, and was 0.23 and 0.22 microm/pulse in 4T1 and B16.F10 tumors, respectively. There were no significant differences in transport distances between 4T1 and B16.F10 tumors. Results from in vivo mapping and numerical simulations revealed an approximately uniform intratumoral electric field that was predominantly in the direction of the applied field. The data in the study suggested that interstitial transport of pDNA induced by a sequence of ten electric pulses was ineffective for macroscopic delivery of genes in tumors. However, the induced transport was more efficient than passive diffusion.     

Electroporation of transplantable tumour for the enhanced accumulation of photosensitizers

The aim of this study was to verify whether electroporation could increase the accumulation of the hydrophilic photosensitizers: aluminium phthalocyanine tetrasulphonate (AlPcS(4)) and chlorin e(6) (C e(6)) in tumour tissue. The experiment was performed in vivo using hybrid mice (C57Bl/CBA) bearing hepatoma A22 (MH-A22) tumours transplanted in the right haunch. The time dependence of the fluorescence intensity of administered photosensitizers was measured after the ordinary and electrically stimulated delivery. The obtained fluorescence spectroscopy results implied the tumour being affected by an electrical field in a way, which led to a higher accumulation of both photosensitizers (AlPcS(4) and C e(6)) in the periphery of the tumour and it superficial layer. Our pilot study suggests that electroporation could be considered as a useful procedure seeking for the more effective application of photodynamic tumour treatment.     

Theoretical analysis of localized heating in human skin subjected to high voltage pulses

Electroporation, the increase in the permeability of bilayer lipid membranes by the application of high voltage pulses, has the potential to serve as a mechanism for transdermal drug delivery. However, the associated current flow through the skin will increase the skin temperature and might affect nearby epidermal cells, lipid structure or even transported therapeutic molecules. Here, thermal conduction and thermal convection models are used to provide upper and lower bounds on the local temperature rise, as well as the thermal damage, during electroporation from exponential voltage pulses (70 V maximum) with a 1 ms and a 10 ms pulse time constant. The peak temperature rise predicted by the conduction model ranges from 19 degrees C for a 1 ms time constant pulse to 70 degrees C for the 10 ms time constant pulse. The convection (mass transport) model predicts a 18 degrees C peak rise for 1 ms time constant pulses and a 51 degrees C peak rise for a 10 ms time constant pulse. The convection model compares more favorably with previous experimental studies and companion observations of the local temperature rise during electroporation. Therefore, it is expected that skin electroporation can be employed at a level which is able to transport molecules transdermally without causing significant thermal damage to the tissue.     

Cyclic capillary electrophoresis

A strategy is described here for increasing both the resolution and the flexibility of capillary electrophoresis performed in a sieving medium of ungelled polymer. This strategy is based on analysis and, sometimes, re-analysis that is done in several stages of constant-field electrophoresis. Enhancement-stages are between the analysis-stages. An enhancement-stage (i) increases the separation between peaks, while (ii) moving DNA molecules in the reverse direction. An enhancement-stage is based on an electrophoretic ratchet generated by a pulsed electrical field that can be zero-integrated. The ratchet-generating pulses are longer than the field pulses that have previously been used to improve the resolution of DNA molecules. No limit has been found to the resolution enhancement achievable. Apparently, diffusion-induced peak broadening is inhibited and, in some cases, may be reversed by the ratchet. The enhancement-stages are critically dependent on the electrical field-dependence of a plot of electrophoretic mobility as a function of DNA length. To generate the pulsed electrical field, a computer-controlled system with a time resolution of 30 microseconds has been developed. Programming is flexible enough to embed other pulses within ratchet-generating pulses. These other pulses can be either the previously used, shorter field-inversion pulses or high-frequency periodic oscillations previously found to sharpen peaks.     

Perturbation of human skin due to application of high voltage

Electroporation is believed to be the effect that greatly enhances the transport of water-soluble molecules across the stratum corneum (SC) by application of short high voltage pulses. However, electroporation was originally a phenomenon investigated at the level of cell and model membranes, which is only partially comparable to the complicated structure of the stratum corneum. Here, we show, that electroporation is accompanied by other effects, which may be primarily involved in creation of new pathways and altering existing pathways, respectively. Experimental evidence shows that the dramatic increase in skin permeability is due to synergistic effect of electric field and heating by high local current density. Heating starts at small spots, not related to a visible skin structure and results in a propagating heat front. The phase transition of the SC lipids plays a major role in skin permeability during the pulse. The permeability after a high voltage pulse correlates well with the surface area showing a permanent low electrical resistance after pulsing. The main transport of water-soluble molecules is facilitated by the electric field due to the electrophoretic driving force in conjunction with the high permeability due to the breakdown of the multilamellar system of the SC lipids.     

Effects of application voltage and cathode and anode positions at electroporation on the in vitro permeation of benzoic acid through hairless rat skin

The enhancing effect of electroporation on the in vitro skin permeation of benzoate was evaluated. Needle and ring electrodes made of Ag/AgCl were connected to an electrical power source, which produced exponentially decaying pulses. The needle electrode was kept in contact with the skin surface, and the ring electrode was positioned either on or under the skin. The electrical pulse was applied to abdominal hairless rat skin at 150-600 V every minute from 4 to 6 h during the 10-h permeation experiment. Skin permeation of benzoate was promoted by electroporation and the effect was increased by application of a higher voltage. No immediate recovery to the control flux, however, was observed for high voltage groups after turning off the voltage application. When the cathode and anode were separated by the skin membrane by setting in the epidermal and dermal sides, respectively, an iontophoretic effect may also play a role in benzoate flux. These results indicated that the drug permeation by electroporation is the result of passive diffusion and an iontophoretic effect as well as the electroporation effect.     

Oxidative Effects during Irreversible Electroporation of Melanoma Cells—In Vitro Study

Irreversible electroporation (IRE) is today used as an alternative to surgery for the excision of cancer lesions. This study aimed to investigate the oxidative and cytotoxic effects the cells undergo during irreversible electroporation using IRE protocols. To do so, we used IRE-inducing pulsed electric fields (PEFs) (eight pulses of 0.1 ms duration and 2–4 kV/cm intensity) and compared their effects to those of PEFs of intensities below the electroporation threshold (eight pulses, 0.1 ms, 0.2–0.4 kV/cm) and the PEFs involving elongated pulses (eight pulses, 10 ms, 0.2–0.4 kV/cm). Next, to follow the morphology of the melanoma cell membranes after treatment with the PEFs, we analyzed the permeability and integrity of their membranes and analyzed the radical oxygen species (ROS) bursts and the membrane lipids’ oxidation. Our data showed that IRE-induced high cytotoxic effect is associated both with irreversible cell membrane disruption and ROS-associated oxidation, which is occurrent also in the low electric field range. It was shown that the viability of melanoma cells characterized by similar ROS content and lipid membrane oxidation after PEF treatment depends on the integrity of the membrane system. Namely, when the effects of the PEF on the membrane are reversible, aside from the high level of ROS and membrane oxidation, the cell does not undergo cell death.     

Electrotransformation of Saccharomyces cerevisiae protoplast by a plasmid of Escherichia coli

The experiments reported here are aimed at obtaining optimum conditions for gene transformation after electric field pulses. Saccharomyces cerevisiae DBY746 is used as the recipient strain for shuttle plasmid (YRp group). From the relationship between the optimum electric field conditions and the transformation efficiency it is discovered that the maximum transformation efficiency appears at a wide pulse length of 400 μs with an electric field strength of 4 kV/cm, yielding up to 273 transformants/μg DNA. The electroporation unit used in the experiment is a home-made set featuring simplicity, readiness and practicality.