The equivalence of different types of electric pulses for electrochemotherapy with cisplatin - an in vitro study.

BACKGROUND: Electrochemotherapy (ECT) is a treatment involving the administration of chemotherapeutics drugs followed by the application of 8 square monopolar pulses of 100 µs duration at a repetition frequency of 1 Hz or 5000 Hz. However, there is increasing interest in using alternative types of pulses for ECT. The use of high-frequency short bipolar pulses has been shown to mitigate pain and muscle contractions. Conversely, the use of millisecond pulses is interesting when combining ECT with gene electrotransfer for the uptake of DNA-encoding proteins that stimulate the immune response with the aim of converting ECT from a local to systemic treatment. Therefore, the aim of this study was to investigate how alternative types of pulses affect the efficiency of the ECT. MATERIALS AND METHODS: We performed in vitro experiments, exposing Chinese hamster ovary (CHO) cells to conventional ECT pulses, high-frequency bipolar pulses, and millisecond pulses in the presence of different concentrations of cisplatin. We determined cisplatin uptake by inductively coupled plasma mass spectrometry and cisplatin cytotoxicity by the clonogenic assay. RESULTS: We observed that the three tested types of pulses potentiate the uptake and cytotoxicity of cisplatin in an equivalent manner, provided that the electric field is properly adjusted for each pulse type. Furthermore, we quantified that the number of cisplatin molecules, resulting in the eradication of most cells, was 2-7 × 107 per cell. CONCLUSIONS: High-frequency bipolar pulses and millisecond pulses can potentially be used in ECT to reduce pain and muscle contraction and increase the effect of the immune response in combination with gene electrotransfer, respectively.

Genetically engineered HEK cells as a valuable tool for studying electroporation in excitable cells.

Electric pulses used in electroporation-based treatments have been shown to affect the excitability of muscle and neuronal cells. However, understanding the interplay between electroporation and electrophysiological response of excitable cells is complex, since both ion channel gating and electroporation depend on dynamic changes in the transmembrane voltage (TMV). In this study, a genetically engineered human embryonic kidney cells expressing NaV1.5 and Kir2.1, a minimal complementary channels required for excitability (named S-HEK), was characterized as a simple cell model used for studying the effects of electroporation in excitable cells. S-HEK cells and their non-excitable counterparts (NS-HEK) were exposed to 100 µs pulses of increasing electric field strength. Changes in TMV, plasma membrane permeability, and intracellular Ca2+ were monitored with fluorescence microscopy. We found that a very mild electroporation, undetectable with the classical propidium assay but associated with a transient increase in intracellular Ca2+, can already have a profound effect on excitability close to the electrostimulation threshold, as corroborated by multiscale computational modelling. These results are of great relevance for understanding the effects of pulse delivery on cell excitability observed in context of the rapidly developing cardiac pulsed field ablation as well as other electroporation-based treatments in excitable tissues.

Characterization of Experimentally Observed Complex Interplay between Pulse Duration, Electrical Field Strength, and Cell Orientation on Electroporation Outcome Using a Time-Dependent Nonlinear Numerical Model.

Electroporation is a biophysical phenomenon involving an increase in cell membrane permeability to molecules after a high-pulsed electric field is applied to the tissue. Currently, electroporation is being developed for non-thermal ablation of cardiac tissue to treat arrhythmias. Cardiomyocytes have been shown to be more affected by electroporation when oriented with their long axis parallel to the applied electric field. However, recent studies demonstrate that the preferentially affected orientation depends on the pulse parameters. To gain better insight into the influence of cell orientation on electroporation with different pulse parameters, we developed a time-dependent nonlinear numerical model where we calculated the induced transmembrane voltage and pores creation in the membrane due to electroporation. The numerical results show that the onset of electroporation is observed at lower electric field strengths for cells oriented parallel to the electric field for pulse durations ≥10 µs, and cells oriented perpendicular for pulse durations ~100 ns. For pulses of ~1 µs duration, electroporation is not very sensitive to cell orientation. Interestingly, as the electric field strength increases beyond the onset of electroporation, perpendicular cells become more affected irrespective of pulse duration. The results obtained using the developed time-dependent nonlinear model are corroborated by in vitro experimental measurements. Our study will contribute to the process of further development and optimization of pulsed-field ablation and gene therapy in cardiac treatments.

Gene Electrotransfer into Mammalian Cells Using Commercial Cell Culture Inserts with Porous Substrate.

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we expand the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. We first use multiscale numerical modeling to determine the pulse parameters, substrate pore size, and other factors that are expected to result in successful gene electrotransfer. Based on the numerical results, we design a simple device combining an insert with substrate containing pores with 0.4 µm or 1.0 µm diameter, a multiwell plate, and a pair of wire electrodes. We test the device in three mammalian cell lines and obtain transfection efficiencies similar to those achieved with conventional bulk electroporation, but at better cell viability and with low-voltage pulses that do not require the use of expensive electroporators. Our combined theoretical and experimental analysis calls for further systematic studies that will investigate the influence of substrate pore size and porosity on gene electrotransfer efficiency and cell viability.

Models of electroporation and the associated transmembrane molecular transport should be revisited.

Electroporation has become a powerful tool for nonviral delivery of various biomolecules such as nucleic acids, proteins, and chemotherapeutic drugs to virtually any living cell by exposing the cell membrane to an intense pulsed electric field. Different multiphysics and multiscale models have been developed to describe the phenomenon of electroporation and predict molecular transport through the electroporated membrane. In this paper, we critically examine the existing mechanistic, single-cell models which allow spatially and temporally resolved numerical simulations of electroporation-induced transmembrane transport of small molecules by confronting them with different experimental measurements. Furthermore, we assess whether any of the proposed models is universal enough to describe the associated transmembrane transport in general for all the different pulse parameters and small molecules used in electroporation applications. We show that none of the tested models can be universally applied to the full range of experimental measurements. Even more importantly, we show that none of the models has been compared to sufficient amount of experimental data to confirm the model validity. Finally, we provide guidelines and recommendations on how to design and report experiments that can be used to validate an electroporation model and how to improve the development of mechanistic models.

Microtrap array on a chip for localized electroporation and electro-gene transfection.

We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidic device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecules using propidium iodide and large molecules using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).

Identification of electroporation sites in the complex lipid organization of the plasma membrane.

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon termed electroporation is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrhythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study, we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. Additionally, by analysing poration kinetics with Bayesian survival analysis we show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

Revisiting the role of pulsed electric fields in overcoming the barriers to in vivo gene electrotransfer.

Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.

Actin networks regulate the cell membrane permeability during electroporation.

Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials.

Pulsed Electric Fields Can Create Pores in the Voltage Sensors of Voltage-Gated Ion Channels.

Pulsed electric fields are increasingly used in medicine to transiently increase the cell membrane permeability via electroporation to deliver therapeutic molecules into the cell. One type of event that contributes to this increase in membrane permeability is the formation of pores in the membrane lipid bilayer. However, electrophysiological measurements suggest that membrane proteins are affected as well, particularly voltage-gated ion channels (VGICs). The molecular mechanisms by which the electric field could affects these molecules remain unidentified. In this study, we used molecular dynamics simulations to unravel the molecular events that take place in different VGICs when exposing them to electric fields mimicking electroporation conditions. We show that electric fields can induce pores in the voltage-sensor domains (VSDs) of different VGICs and that these pores form more easily in some channels than in others. We demonstrate that poration is more likely in VSDs that are more hydrated and are electrostatically more favorable for the entry of ions. We further show that pores in VSDs can expand into so-called complex pores, which become stabilized by lipid headgroups. Our results suggest that such complex pores are considerably more stable than conventional lipid pores, and their formation can lead to severe unfolding of VSDs from the channel. We anticipate that such VSDs become dysfunctional and unable to respond to changes in transmembrane voltage, which is in agreement with previous electrophysiological measurements showing a decrease in the voltage-dependent transmembrane ionic currents after pulse treatment. Finally, we discuss the possibility of activation of VGICs by submicrosecond-duration pulses. Overall, our study reveals a new, to our knowledge, mechanism of electroporation through membranes containing VGICs.

DNA-membrane complex formation during electroporation is DNA size-dependent.

Size of DNA molecules governs their interaction with the cell membrane during electroporation and their subsequent transport inside the cell. In order to investigate the effect of DNA size on DNA-membrane interaction during electroporation, cells are electro-pulsed with DNA molecules; 15 bp, 25 bp, 50 bp, 100 bp and 1000 bp (bp = base pairs). Within the experimental parameter space, DNA-membrane complexes or DNA aggregates are observed at the cell membrane for DNA molecules containing 25 or more base pairs. No aggregates are observed for DNA molecules containing 15 bp. For all DNA sizes, direct access to the cytoplasm is observed, however the amount translocated decays with the size. The observed dependency of DNA aggregate formation on the size of the DNA molecules is consistent with the Onsager's theory of condensation of anisotropic rod-like molecules.

DNA translocation to giant unilamellar vesicles during electroporation is independent of DNA size.

Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25-20 000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery.

Response of an actin network in vesicles under electric pulses.

We study the role of a biomimetic actin network during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic network provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

Membrane Electroporation and Electropermeabilization: Mechanisms and Models.

Exposure of biological cells to high-voltage, short-duration electric pulses causes a transient increase in their plasma membrane permeability, allowing transmembrane transport of otherwise impermeant molecules. In recent years, large steps were made in the understanding of underlying events. Formation of aqueous pores in the lipid bilayer is now a widely recognized mechanism, but evidence is growing that changes to individual membrane lipids and proteins also contribute, substantiating the need for terminological distinction between electroporation and electropermeabilization. We first revisit experimental evidence for electrically induced membrane permeability, its correlation with transmembrane voltage, and continuum models of electropermeabilization that disregard the molecular-level structure and events. We then present insights from molecular-level modeling, particularly atomistic simulations that enhance understanding of pore formation, and evidence of chemical modifications of membrane lipids and functional modulation of membrane proteins affecting membrane permeability. Finally, we discuss the remaining challenges to our full understanding of electroporation and electropermeabilization.

The contribution of lipid peroxidation to membrane permeability in electropermeabilization: A molecular dynamics study.

Electroporation or electropermeabilization is a technique that enables transient increase in the cell membrane permeability by exposing cells to pulsed electric field. However, the molecular mechanisms of the long-lived cell membrane permeability, which persists on the minutes time scale after the pulse treatment, remain elusive. Experimental studies have suggested that lipid peroxidation could present a mechanism of this prolonged membrane permeabilization. In this study we make the first important step in quantifying the possible contribution of lipid peroxidation to electropermeabilization. We use free energy calculations to quantify the permeability and conductance of bilayers, containing an increasing percentage of hydroperoxide lipid derivatives, to sodium and chloride ions. We then compare our calculations to experimental measurements on electropermeabilized cells. Our results show that the permeability and conductance increase dramatically by several orders of magnitude in peroxidized bilayers. Yet this increase is not sufficient to reasonably account for the entire range of experimental measurements. Nevertheless, lipid peroxidation might be considered an important mechanism of prolonged membrane permeabilization, if exposure of cells to high voltage electric pulses leads to secondary lipid peroxidation products. Our analysis calls for experimental studies, which will determine the type and amount of lipid peroxidation products in electropermeabilized cell membranes.

The role of gel-phase domains in electroporation of vesicles.

Transient permeabilisation of the cell membrane is a critical step to introduce drugs or DNA into living cells, yet challenging for both biological research and therapeutic applications. To achieve this, electroporation (or electropermeabilisation) has become a widely used method due to its simplicity to deliver almost any biomolecule to any cell type. Although this method demonstrates promise in the field of drug/gene delivery, the underlying physical mechanisms of the response of the heterogeneous cell membrane to strong electric pulses is still unknown. In this study, we have investigated the role of gel-phase lipids in the electroporation of binary giant unilamellar vesicles (GUVs), composed from DPPC (gel-phase) and DPhPC (fluid-phase) lipids (molar ratio 8:2 and 2:8). We have observed that the exposure to electric pulses leads to expel of fluid-phase lipids and concomitant decrease in GUV size, whereas the gel-phase domains become buckled. Based on experiments on pure fluid-phase and gel-phase GUVs, we have found that fluid-phase lipids can be expelled by electrical forces and the highly viscous gel-phase lipids cannot. Moreover, our analyses suggest that pore formation occurs primarily in fluid-phase domains and that the pore size is similar in all GUVs containing fluid-phase lipids, irrespective of the gel-phase percentage.

Lipid vesicles in pulsed electric fields: Fundamental principles of the membrane response and its biomedical applications.

The present review focuses on the effects of pulsed electric fields on lipid vesicles ranging from giant unilamellar vesicles (GUVs) to small unilamellar vesicles (SUVs), from both fundamental and applicative perspectives. Lipid vesicles are the most popular model membrane systems for studying biophysical and biological processes in living cells. Furthermore, as vesicles are made from biocompatible and biodegradable materials, they provide a strategy to create safe and functionalized drug delivery systems in health-care applications. Exposure of lipid vesicles to pulsed electric fields is a common physical method to transiently increase the permeability of the lipid membrane. This method, termed electroporation, has shown many advantages for delivering exogenous molecules including drugs and genetic material into vesicles and living cells. In addition, electroporation can be applied to induce fusion between vesicles and/or cells. First, we discuss in detail how research on cell-size GUVs as model cell systems has provided novel insight into the basic mechanisms of cell electroporation and associated phenomena. Afterwards, we continue with a thorough overview how electroporation and electrofusion have been used as versatile methods to manipulate vesicles of all sizes in different biomedical applications. We conclude by summarizing the open questions in the field of electroporation and possible future directions for vesicles in the biomedical field.

Flow of DNA in micro/nanofluidics: From fundamentals to applications.

Thanks to direct observation and manipulation of DNA in micro/nanofluidic devices, we are now able to elucidate the relationship between the polymer microstructure and its rheological properties, as well as to design new single-molecule platforms for biophysics and biomedicine. This allows exploration of many new mechanisms and phenomena, which were previously unachievable with conventional methods such as bulk rheometry tests. For instance, the field of polymer rheology is at a turning point to relate the complex molecular conformations to the nonlinear viscoelasticity of polymeric fluids (such as coil-stretch transition, shear thinning, and stress overshoot in startup shear). In addition, nanofluidic devices provided a starting point for manipulating single DNA molecules by applying basic principles of polymer physics, which is highly relevant to numerous processes in biosciences. In this article, we review recent progress regarding the flow and deformation of DNA in micro/nanofluidic systems from both fundamental and application perspectives. We particularly focus on advances in the understanding of polymer rheology and identify the emerging research trends and challenges, especially with respect to future applications of nanofluidics in the biomedical field.

Quantification of cell membrane permeability induced by monopolar and high-frequency bipolar bursts of electrical pulses.

High-frequency bipolar electric pulses have been shown to mitigate undesirable muscle contraction during irreversible electroporation (IRE) therapy. Here, we evaluate the potential applicability of such pulses for introducing exogenous molecules into cells, such as in electrochemotherapy (ECT). For this purpose we develop a method for calculating the time course of the effective permeability of an electroporated cell membrane based on real-time imaging of propidium transport into single cells that allows a quantitative comparison between different pulsing schemes. We calculate the effective permeability for several pulsed electric field treatments including trains of 100µs monopolar pulses, conventionally used in IRE and ECT, and pulse trains containing bursts or evenly-spaced 1µs bipolar pulses. We show that shorter bipolar pulses induce lower effective membrane permeability than longer monopolar pulses with equivalent treatment times. This lower efficiency can be attributed to incomplete membrane charging. Nevertheless, bipolar pulses could be used for increasing the uptake of small molecules into cells more symmetrically, but at the expense of higher applied voltages. These data indicate that high-frequency bipolar bursts of electrical pulses may be designed to electroporate cells as effectively as and more homogeneously than conventional monopolar pulses.

Properties of lipid electropores II: Comparison of continuum-level modeling of pore conductance to molecular dynamics simulations.

Electrical conductance of an aqueous pore in the lipid bilayer has an important role in the process of membrane electroporation, i.e., formation of pores induced by electric pulses. In our present study we compare the pore conductance as predicted by a theoretical model based on the continuum Poisson-Nernst-Planck theory to the pore conductance obtained with molecular dynamics simulations (Casciola et al., Bioelectrochemistry 109:108-116, 2016). Our analysis demonstrates that the Poisson-Nernst-Planck model is able to quantitatively predict the dependence of the pore conductance on the pore radius when considering the toroidal shape of the pore. In order to correctly describe the difference in the pore conductance for Cl and Na ions (the pore selectivity), however, it is necessary to take into account the electric double layer next to the lipid-water interface and the electroosmotic flow through the pore. We further show that simplified analytical descriptions of pore conductance can lead to incorrect predictions of the pore size extracted from experimental measurements, and can affect the predictions of electroporation models. Overall, this study demonstrates that continuum modeling can be efficiently used as complementary method to molecular scale models for investigating lipid pores.