Self-powered electroporation using a singularity-induced nano-electroporation configuration

This study demonstrates the feasibility of creating a self-powered (galvanic) electroporation device using the singularity-induced nano-electroporation configuration. Using this configuration, the electric field in a galvanic electrochemical cell can be amplified and used for electroporation. A secondary current distribution model of a self-powered electroporation device shows that the device can create both reversible and irreversible electroporation-inducing electric field magnitudes, and generate a small amount of power.     

The importance of electric field distribution for effective in vivo electroporation of tissues.

Cells exposed to short and intense electric pulses become permeable to a number of various ionic molecules. This phenomenon was termed electroporation or electropermeabilization and is widely used for in vitro drug delivery into the cells and gene transfection. Tissues can also be permeabilized. These new approaches based on electroporation are used for cancer treatment, i.e., electrochemotherapy, and in vivo gene transfection. In vivo electroporation is thus gaining even wider interest. However, electrode geometry and distribution were not yet adequately addressed. Most of the electrodes used so far were determined empirically. In our study we 1) designed two electrode sets that produce notably different distribution of electric field in tumor, 2) qualitatively evaluated current density distribution for both electrode sets by means of magnetic resonance current density imaging, 3) used three-dimensional finite element model to calculate values of electric field for both electrode sets, and 4) demonstrated the difference in electrochemotherapy effectiveness in mouse tumor model between the two electrode sets. The results of our study clearly demonstrate that numerical model is reliable and can be very useful in the additional search for electrodes that would make electrochemotherapy and in vivo electroporation in general more efficient. Our study also shows that better coverage of tumors with sufficiently high electric field is necessary for improved effectiveness of electrochemotherapy.     

Three dimensional electrode array for cell lysis via electroporation

Microfabricated devices for cell lysis have demonstrated many advantages over conventional approaches. Among various design of microdevices that employ electroporation for cytolysis, most utilize Ag/AgCl wires or 2D planar electrodes. Although, simple in fabrication the electric field generated by 2D electrodes decays exponentially, resulting in rather non-uniform forcing on the cell membrane. This paper investigates the effect of electric field generated by 3D cylindrical electrodes to perform cell lysis via electroporation in a microfluidic platform, and compared with that by 2D design. Computational results of the electric field for both 2D and 3D electrode geometries showed that the 3D configuration demonstrated a significantly higher effective volume ratio-volume which electric field is sufficient for cell lysis to that of net throughflow volume. Hence, the efficacy of performing cell lysis is substantially greater for cells passing through 3D than 2D electrodes. Experimentally, simultaneous multi-pores were observed on leukocytes lysed with 3D electrodes, which is indicative of enhanced uniformity of the electric field generated by 3D design. Additionally, a single row of 3D electrode demonstrated a substantially higher lysing percentage (30%) than that of 2D (8%) under that same flow condition. This work should aid in the design of electrodes in performing cell lysis via electroporation.     

In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution.

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.     

Effect of Twisted Fiber Anisotropy in Cardiac Tissue on Ablation with Pulsed Electric Fields

BackgroundAblation of cardiac tissue with pulsed electric fields is a promising alternative to current thermal ablation methods, and it critically depends on the electric field distribution in the heart.MethodsWe developed a model that incorporates the twisted anisotropy of cardiac tissue and computed the electric field distribution in the tissue. We also performed experiments in rabbit ventricles to validate our model. We find that the model agrees well with the experimentally determined ablation volume if we assume that all tissue that is exposed to a field greater than 3 kV/cm is ablated. In our numerical analysis, we considered how tissue thickness, degree of anisotropy, and electrode configuration affect the geometry of the ablated volume. We considered two electrode configurations: two parallel needles inserted into the myocardium (“penetrating needles” configuration) and one circular electrode each on epi- and endocardium, opposing each other (“epi-endo” configuration).ResultsFor thick tissues (10 mm) and moderate anisotropy ratio (a = 2), we find that the geometry of the ablated volume is almost unaffected by twisted anisotropy, i.e. it is approximately translationally symmetric from epi- to endocardium, for both electrode configurations. Higher anisotropy ratio (a = 10) leads to substantial variation in ablation width across the wall; these variations were more pronounced for the penetrating needle configuration than for the epi-endo configuration.For thinner tissues (4 mm, typical for human atria) and higher anisotropy ratio (a = 10), the epi-endo configuration yielded approximately translationally symmetric ablation volumes, while the penetrating electrodes configuration was much more sensitive to fiber twist.ConclusionsThese results suggest that the epi-endo configuration will be reliable for ablation of atrial fibrillation, independently of fiber orientation, while the penetrating electrode configuration may experience problems when the fiber orientation is not consistent across the atrial wall.     

A Numerical Investigation of the Electric and Thermal Cell Kill Distributions in Electroporation-Based Therapies in Tissue

Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs). Electrochemotherapy (ECT) and gene therapy based on gene electrotransfer (EGT) both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE) results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent unnecessary damage to surrounding healthy tissue, critical vascular structures, and/or adjacent organs.     

Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential.

Transmembrane potential was induced in a sea urchin egg by applying a microsecond electric pulse across the cell. The potential was imaged at a submicrosecond time resolution by staining the cell membrane with the voltage-sensitive fluorescent dye RH292. Under moderate electric fields, the spatial distribution of the induced potential as well as its time dependence were in accord with the theoretical prediction in which the cell membrane was regarded as an insulator. At higher field intensities, however, the potential apparently did not fully develop and tended to saturate above a certain level. The saturation is ascribed to the introduction of a large electrical conductance, in the form of aqueous openings, in the membrane by the action of the induced potential (electroporation). Comparison of the experimental and theoretical potential profiles indicates that the two regions of the membrane that opposed the electrodes acquired a high membrane conductance of the order of 1 S/cm2 within 2 microseconds from the onset of the external field. The conductance was similar in the two regions, although permeability in the two regions of the membrane long after the pulse treatment appeared quite different.     

Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells

We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 μs pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.     

Stretchable Zn-Ion Hybrid Capacitor with Hydrogel Encapsulated 3D Interdigital Structure

To enhance the areal energy density of current flexible energy storage devices, hybrid capacitors combining the advantages of supercapacitors and batteries are proposed and further enhanced by incorporating the 3D interdigital structure design. However, uneven electric field distribution and hindered ion diffusion kinetics due to the non-electroactive components in these devices limit the enhancement of areal electrochemical performances when expanding the electrodes longitudinally. Herein, hydrogels with high ionic conductivity and high mechanical stability are designed to accommodate Zn²⁺-containing electrolytes and integrated with Ti₃C₂T_x-MXene electrodes to assemble flexible Zn-ion hybrid capacitors (ZIHCs). Fully encapsulated by ionic conductive hydrogels, 3D interdigital electrodes enable omnidirectional ion transport and unimpeded ionic accessibility, facilitating adequate electrode reactions, rapid energy storage, and uniform energy distribution. Hence, the all-hydrogel-encapsulated ZIHC achieved a 50-fold increase in capacitance with a quadrupled electrode thickness, exhibiting a large areal capacitance of 1432 mF cm⁻² and an energy density of 389.7 µWh cm⁻² without sacrificing power density and rate performance. Finite element simulations further illustrate the uniform distribution of potential, electric field intensity, and energy density in this structure. In addition, the device shows great stability under deformation, excellent adhesion, and underwater workability, demonstrating great promise for next-generation wearable energy storage devices.     

A new approach to the determination of cardiac potential distributions: application to the analysis of electrode configurations

This paper presents a mathematical model and new solution technique for studying the electric potential in a slab of cardiac tissue. The model is based on the bidomain representation of cardiac tissue and also allows for the effects of fibre rotation between the epicardium and the endocardium. A detailed solution method, based on Fourier Series and a simple one-dimensional finite difference scheme, for the governing equations for electric potential in the tissue and the blood, is also presented. This method has the advantage that the potential can be calculated only at points where it is required, such as the measuring electrodes. The model is then used to study various electrode configurations which have been proposed to determine cardiac tissue conductivity parameters. Three electrode configurations are analysed in terms of electrode spacing, placement position and the effect of including fibre rotation: the usual surface four-electrode configuration; a single vertical analogue of this and a two probe configuration, which has the current electrodes on one probe and the measuring electrodes on the other, a fixed distance away. It is found that including fibre rotation has no effect on the potentials measured in the first two cases; however, in the two probe case, non-zero fibre rotation causes a significant drop in the voltage measured. This leads to the conclusion that it is necessary to include the effects of fibre rotation in any model which involves the use of multiple plunge electrodes.     

Reduction of electrode polarization capacitance in low-frequency impedance spectroscopy by using mesh electrodes

Dielectric measurements of biological samples are obscured by electrode polarization, which at low frequencies dominates over the actual sample response. Reduction of this artifact is especially necessary in studying interactions of electric field with biological systems in the α-dispersion range. We developed a method to reduce the influence of electrode polarization by employing mesh instead of solid electrodes as sensing probes, thereby reducing the area of the double layer. The design decreases the electrode-electrolyte contact area by almost 40% while keeping the bulk sample capacitance the same. Interrogation electric fields away from the electrode surface and sensitivity are unaffected. Electrodes were microfabricated (600μm×50μm, spacing of 100μm) with and without mesh holes 7.5μm×7.5μm in size. Simulations of electric field performed using Comsol Multiphysics showed non-uniformity of the electric field within less than 1.5μm from the electrode surface, which encompasses the double layer region, but at greater distance the solid and mesh electrodes gave the same results. Mesh electrodes reduced capacitance measurements for water and KCl solutions of different concentrations at low frequencies (<10kHz), while higher frequency capacitance remained the same for both electrode types, confirming our hypothesis that this design leaves the electric field mainly unaffected. Impedance measurements at low frequencies for water and mice heart mitochondrial suspension were lower for mesh than for solid electrodes. Comsol simulations confirmed these results by showing that mesh electrodes have a greater charge density than solid electrodes, which affects conductance. These electrodes are being used for mitochondrial membrane potential studies.     

Thick Electrode Batteries: Principles,Opportunities, and Challenges

The ever‐growing portable electronics and electric vehicle markets heavily influence the technological revolution of lithium batteries (LBs) toward higher energy densities for longer standby times or driving range. Thick electrode designs can substantially improve the electrode active material loading by minimizing the inactive component ratio at the device level, providing a great platform for enhancing the overall energy density of LBs. However, extensive efforts are still needed to address the challenges that accompany the increase in electrode thickness, not limited to sluggish charge kinetics and electrode mechanical instability. In this review, the principles and the recent developments in the fabrication of thick electrodes that focus on low‐tortuosity structural designs for rapid charge transport and integrated cell configuration for improved energy density, cell stability, and durability are summarized. Advanced thick electrode designs for application in emerging battery chemistries such as lithium metal electrodes, solid state electrolytes, and lithium–air batteries are also discussed with a perspective on their future opportunities and challenges. Finally, suggestions on the future directions of thick electrode battery development and research are suggested.     

Numerical modeling of in vivo plate electroporation thermal dose assessment

Electroporation is an approach used to enhance the transport of large molecules to the cell cytosol in which a targeted tissue region is exposed to a series of electric pulses. The cell membrane, which normally acts as a barrier to large molecule transport into the cell interior, is temporarily destabilized due to the development of pores in the cell membrane. Consequently, agents that are ordinarily unable enter the cell are able to pass through the cell membrane. Of possible concern when exposing biological tissue to an electric field is thermal tissue damage associated with joule heating. This paper explores the thermal effects of various geometric, biological, and electroporation pulse parameters including the blood vessel presence and size, plate electrode configuration, and pulse duration and frequency. A three-dimensional transient finite volume model of in vivo parallel plate electroporation of liver tissue is used to develop a better understanding of the underlying relationships between the physical parameters involved with tissue electroporation and resulting thermal damage potential.     

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).     

Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge

The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversible and reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnostic substances into the cells and tissues.     

Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations

In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field‐induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Bioelectromagnetics 34:253–263, 2013. © 2012 Wiley Periodicals, Inc.     

In Vivo Muscle Electroporation Threshold Determination: Realistic Numerical Models and In Vivo Experiments

In vivo electroporation is used as an effective technique for delivery of therapeutic agents such as chemotherapeutic drugs or DNA into target tissue cells for different biomedical purposes. In order to successfully electroporate a target tissue, it is essential to know the local electric field distribution produced by an application of electroporation voltage pulses. In this study three-dimensional finite element models were built in order to analyze local electric field distribution and corresponding tissue conductivity changes in rat muscle electroporated either transcutaneously or directly (i.e., two-plate electrodes were placed either on the skin or directly on the skeletal muscle after removing the skin). Numerical calculations of electroporation thresholds and conductivity changes in skin and muscle were validated with in vivo measurements. Our model of muscle with skin also confirms the in vivo findings of previous studies that electroporation “breaks” the skin barrier when the applied voltage is above 50?V.     

Contactless method for studying the parameters of the electrode region of an RF discharge

A nonintrusive contactless method for studying the parameters of the electrode region of a capacitive low-pressure RF discharge is proposed. The method involves the measurements of dc and ac electric voltages at the elements of the discharge circuit with subsequent calculations of both the electrostatic potential drop across the electrode sheath and the sheath thickness by using relations derived in the paper. For a collisionless electrode sheath, the density of the positive-ion current onto the electrode and the charge density at the plasma boundary are determined. It is shown experimentally that the method can be successfully applied to studying capacitive RF discharges with inner or outer electrodes.     

3D Stationary electric current density in a spherical tumor treated with low direct current: an analytical solution

Electrotherapy with direct current delivered through implanted electrodes is used for local control of solid tumors in both preclinical and clinical studies. The aim of this research is to develop a solution method for obtaining a three-dimensional analytical expression for potential and electric current density as functions of direct electric current intensity, differences in conductivities between the tumor and the surrounding healthy tissue, and length, number and polarity of electrodes. The influence of these parameters on electric current density in both media is analyzed. The results show that the electric current density in the tumor is higher than that in the surrounding healthy tissue for any value of these parameters. The conclusion is that the solution method presented in this study is of practical interest because it provides, in a few minutes, a convenient way to visualize in 3D the electric current densities generated by a radial electrode array by means of the adequate selection of direct current intensity, length, number, and polarity of electrodes, and the difference in conductivity between the solid tumor and its surrounding healthy tissue.     

Effect of Blood Vessel Segmentation on the Outcome of Electroporation-Based Treatments of Liver Tumors

Electroporation-based treatments rely on increasing the permeability of the cell membrane by high voltage electric pulses applied to tissue via electrodes. To ensure that the whole tumor is covered with sufficiently high electric field, accurate numerical models are built based on individual patient anatomy. Extraction of patient''s anatomy through segmentation of medical images inevitably produces some errors. In order to ensure the robustness of treatment planning, it is necessary to evaluate the potential effect of such errors on the electric field distribution. In this work we focus on determining the effect of errors in automatic segmentation of hepatic vessels on the electric field distribution in electroporation-based treatments in the liver. First, a numerical analysis was performed on a simple ''sphere and cylinder'' model for tumors and vessels of different sizes and relative positions. Second, an analysis of two models extracted from medical images of real patients in which we introduced variations of an error of the automatic vessel segmentation method was performed. The results obtained from a simple model indicate that ignoring the vessels when calculating the electric field distribution can cause insufficient coverage of the tumor with electric fields. Results of this study indicate that this effect happens for small (10 mm) and medium-sized (30 mm) tumors, especially in the absence of a central electrode inserted in the tumor. The results obtained from the real-case models also show higher negative impact of automatic vessel segmentation errors on the electric field distribution when the central electrode is absent. However, the average error of the automatic vessel segmentation did not have an impact on the electric field distribution if the central electrode was present. This suggests the algorithm is robust enough to be used in creating a model for treatment parameter optimization, but with a central electrode.