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The permeabilization of the live cells membrane by the delivery of electric pulses has fundamental interest in medicine, in particular in tumors treatment by electrochemotherapy. Since underlying mechanisms are still not fully understood, we studied the impact of electric pulses on the biochemical composition of live cells thanks to label-free optical methods: confocal Raman microspectroscopy and terahertz microscopy. A dose effect was observed after cells exposure to different field intensities and a major impact on cell peptide/protein content was found. Raman measurements reveal that protein structure and/or environment are modified by the electric pulses while terahertz measurements suggest a leakage of proteins and other intracellular compounds. We show that Raman and terahertz modalities are a particularly attractive complement to fluorescence microscopy which is the reference optical technique in the case of electropermeabilization. Finally, we propose an analytical model for the influx and efflux of non-permeant molecules through transiently (electro)permeabilized cell membranes. [ABSTRACT FROM AUTHOR]

Electrochemotherapy is now in routine clinical use to treat cutaneous metastases of any histology, and is listed in national and international guidelines for cutaneous metastases and primary skin cancer. Electrochemotherapy is used by dermatologists, surgeons, and oncologists, and for different degrees and manifestations of metastases to skin and primary skin tumours not amenable to surgery. This treatment utilises electric pulses to permeabilize cell membranes in tumours, thus allowing a dramatic increase of the cytotoxicity of anti-cancer agents. Response rates, often after only one treatment, are very high across all tumour types. The most frequent indications are cutaneous metastases from malignant melanoma and breast cancer. In 2006, standard operating procedures (SOPs) were written for this novel technology, greatly facilitating introduction and dissemination of the therapy. Since then considerable experience has been obtained treating a wider range of tumour histologies and increasing size of tumours which was not originally thought possible. A pan-European expert panel drawn from a range of disciplines from dermatology, general surgery, head and neck surgery, plastic surgery, and oncology met to form a consensus opinion to update the SOPs based on the experience obtained. This paper contains these updated recommendations for indications for electrochemotherapy, pretreatment information and evaluation, treatment choices, as well as follow-up. [ABSTRACT FROM AUTHOR]

The chemical consequences of electropulsation on giant unilamellar vesicles (GUVs), in particular the possible oxidation of unsaturated phospholipids, have been investigated by mass spectrometry, flow cytometry and absorbance methods. Pulse application induced oxidation of the GUV phospholipids and the oxidation level depended on the duration of the pulse. Light and O 2 increased the level of pulse-induced lipid peroxidation whereas the presence of antioxidants either in the membrane or in the solution completely suppressed peroxidation. Importantly, pulse application did not create additional reactive oxygen species (ROS) in GUV-free solution. Lipid peroxidation seems to result from a facilitation of the lipid peroxidation by the ROS already present in the solution before pulsing, not from a direct pulse-induced peroxidation. The pulse would facilitate the entrance of ROS in the core of the membrane, allowing the contact between ROS and lipid chains and provoking the oxidation. Our findings demonstrate that the application of electric pulses on cells could induce the oxidation of the membrane phospholipids since cell membranes contain unsaturated lipids. The chemical consequences of electropulsation will therefore have to be taken into account in future biomedical applications of electropulsation since oxidized phospholipids play a key role in many signaling pathways and diseases. [ABSTRACT FROM AUTHOR]

Background: Human mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions. Methods: One or several electric pulses of 100 µs were used to induce Ca2+ spikes caused by the penetration of Ca2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked. Results: According to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability. Conclusions: An easy way to control Ca2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca2+ spikes allowed us to mimic and regulate the Ca2 + oscillations in these cells. Since microsecond electric pulse delivery constitutes a simple technology available in many laboratories, this new tool might be useful to further investigate the role of Ca2+ in human mesenchymal stem cells biological processes such as proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Recently, the use of nanometer liposomes as nanocarriers in drug delivery systems mediated by nanoelectroporation has been proposed. This technique takes advantage of the possibility of simultaneously electroporating liposomes and cell membrane with 10-nanosecond pulsed electric fields (nsPEF) facilitating the release of the drug from the liposomes and at the same time its uptake by the cells. In this paper the design and characterization of a 10 nsPEF exposure system is presented, for liposomes electroporation purposes. The design and the characterization of the applicator have been carried out choosing an electroporation cuvette with 1 mm gap between the electrodes. The structure efficiency has been evaluated at different experimental conditions by changing the solution conductivity from 0.25 to 1.6 S/m. With the aim to analyze the influence of device performances on the liposomes electroporation, microdosimetric simulations have been performed considering liposomes of 200 and 400 nm of dimension with different inner and outer conductivity (from 0.05 to 1.6 S/m) in order to identify the voltage needed for their poration. [ABSTRACT FROM AUTHOR]

Giant unilamellar vesicles (GUV) are widely used cell membrane models. GUVs have a cell-like diameter and contain the same phospholipids that constitute cell membranes. The most frequently used protocol to obtain these vesicles is termed electroformation, since key steps of this protocol consist in the application of an electric field to a phospholipid deposit. The potential oxidation of unsaturated phospholipids due to the application of an electric field has not yet been considered even though the presence of oxidized lipids in the membrane of GUVs could impact their permeability and their mechanical properties. Thanks to mass spectrometry analyses, we demonstrated that the electroformation technique can cause the oxidation of polyunsaturated phospholipids constituting the vesicles. Then, using flow cytometry, we showed that the amplitude and the duration of the electric field impact the number and the size of the vesicles. According to our results, the oxidation level of the phospholipids increases with their level of unsaturation as well as with the amplitude and the duration of the electric field. However, when the level of lipid oxidation exceeds 25 %, the diameter of the vesicles is decreased and when the level of lipid oxidation reaches 40 %, the vesicles burst or reorganize and their rate of production is reduced. In conclusion, the classical electroformation method should always be optimized, as a function of the phospholipid used, especially for producing giant liposomes of polyunsaturated phospholipids to be used as a cell membrane model. [ABSTRACT FROM AUTHOR]

This study investigated whether molecules spontaneously transported inside cells, like glucose derivatives, can also be used as electropermeabilization markers. Uptake of a fluorescent deoxyglucose derivative (2-NBDG) by normal and electropermeabilized cells in culture was analyzed. 2-NBDG was added to DC-3F cell suspensions and cells, exposed or not to eight square-wave electric pulses of 100-μs duration and of appropriate field amplitude at a repetition frequency of 1 Hz or 5 kHz, were incubated at 37 °C. 2-NBDG uptake was temperature-, concentration- and time-dependent in cells submitted or not to the electric pulses. In spite of significant uptake of 2-NBDG mediated by GLUT transporters into nonpermeabilized cells, the electric pulses significantly increased about ten to hundred times the 2-NBDG uptake into the cells. The increase in the field amplitude from 900 to 1,500 V/cm resulted in a progressive increase of 2-NDBG. Our results show that under the conditions of in vivo exposure duration to FDG and the physiological concentration of d-glucose, electric pulses increased 2-NBDG uptake into electropermeabilized cells. Under our experimental conditions, the percentage of permeabilized cells within the population of cells exposed to electric pulses remained at the same level regardless of the pulse frequency used, 1 Hz or 5 kHz. The findings showed that glucose derivatives can also be used to detect electropermeabilized cells exposed to electric pulses. [ABSTRACT FROM AUTHOR]

Survival of mammalian cells is achieved by tight control of cell volume, while transmembrane potential has been known to control many cellular functions since the seminal work of Hodgkin and Huxley. Regulation of cell volume and transmembrane potential have a wide range of implications in physiology, from neurological and cardiac disorders to cancer and muscle fatigue. Therefore, understanding the relationship between transmembrane potential, ion fluxes, and cell volume regulation has become of great interest. In this paper we derive a system of differential equations that links transmembrane potential, ionic concentrations, and cell volume. In particular, we describe the dynamics of the cell within a few seconds after an osmotic stress, which cannot be done by the previous models in which either cell volume was constant or osmotic regulation instantaneous. This new model demonstrates that both membrane potential and cell volume stabilization occur within tens of seconds of changes in extracellular osmotic pressure. When the extracellular osmotic pressure is constant, the cell volume varies as a function of transmembrane potential and ion fluxes, thus providing an implicit link between transmembrane potential and cell volume. Experimental data provide results that corroborate the numerical simulations of the model in terms of time-related changes in cell volume and dynamics of the phenomena. This paper can be seen as a generalization of previous electrophysiological results, since under restrictive conditions they can be derived from our model. [ABSTRACT FROM AUTHOR]

Physical methods of gene (and/or drug) transfer need to combine two effects to deliver the therapeutic material into cells. The physical methods must induce reversible alterations in the plasma membrane to allow the direct passage of the molecules of interest into the cell cytosol. They must also bring the nucleic acids in contact with the permeabilized plasma membrane or facilitate access to the inside of the cell. These two effects can be achieved in one or more steps, depending upon the methods employed. In this review, we describe and compare several physical methods: biolistics, jet injection, hydrodynamic injection, ultrasound, magnetic field and electric pulse mediated gene transfer. We describe the physical mechanisms underlying these approaches and discuss the advantages and limitations of each approach as well as its potential application in research or in preclinical and clinical trials. We also provide conclusions, comparisons, and projections for future developments. While some of these methods are already in use in man, some are still under development or are used only within clinical trials for gene transfer. The possibilities offered by these methods are, however, not restricted to the transfer of genes and the complementary uses of these technologies are also discussed. As these methods of gene transfer may bypass some of the side effects linked to viral or biochemical approaches, they may find their place in specific clinical applications in the future. This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

One of the ways to potentiate antitumor effectiveness of chemotherapeutic drugs is by local application of short intense electric pulses. This causes an increase of the cell membrane permeability and is called electropermeabilization. In order to study the course of tissue permeabilization of a subcutaneous tumor in small animals, a mathematical model was built with the commercial program EMAS, which uses the finite element method. The model is based on the tissue specific conductivity values found in literature, experimentally determined electric field threshold values of reversible and irreversible tissue permeabilization, and conductivity changes in the tissues. The results obtained with the model were then compared to experimental results from the treatment of subcutaneous tumors in mice and a good agreement was obtained. Our results and the reversible and irreversible thresholds used coincide well with the effectiveness of the electrochemotherapy in real tumors where experiments show antitumor effectiveness for amplitudes higher than 900 V/cm ratio and pronounced antitumor effects at 1300 V/cm ratio. [ABSTRACT FROM AUTHOR]

The effectiveness of electrochemotherapy (ECT) in local eradication of tumours in human and veterinary medicine has been proven. ECT consists of increasing the uptake of cytotoxic drugs by means of pulsed electric fields (PEFs) that transiently permeabilise the cell membrane. Still, this tumour treatment includes some drawbacks that are linked to the characteristics of the intense electric pulses (EPs) used. Meanwhile, the emerging field of cancer therapies that are based on the application of non-thermal plasmas (NTP) has recently garnered interest because of their potentialities as rich sources of reactive species. In this work, we investigated the potential capabilities of the combined application of indirect NTP treatment and microsecond PEFs (µsPEFs) to outperform in vitro cell electropermeabilisation, the basis of ECT. Thus, phosphate-buffered saline (PBS) was plasma-treated (pPBS) and used afterwards to explore the effects of its combination with µsPEFs. Analysis of two different cell lines (DC-3F Chinese hamster lung fibroblasts and malignant B16-F10 murine melanoma cells), by flow cytometry, revealed that this combination resulted in significant increases of the level of cell membrane electropermeabilisation, even at very low electric field amplitude. The B16-F10 cells were more sensitive to the combined treatment than DC-3F cells. Importantly, the percentage of permeabilised cells reached values similar to those of cells exposed to classical electroporation field amplitude (1100 V/cm) when the cells were treated with pPBS before and after being exposed only to very low PEF amplitude (600 V/cm). Although the level of permeabilisation of the cells that are treated by the pPBS and the PEFs at 600 V/cm is lower than the level reached after the exposure to µsPEFs alone at 1100 V/cm, the combined treatment opens the possibility to reduce the amplitude of the EPs used in ECT, potentially allowing for a novel ECT with reduced side-effects. [ABSTRACT FROM AUTHOR]

In this paper, we propose a new dynamical model of tissue electroporation. The model is based on equivalent circuit approach at the tissue. Considering two current densities from cells and extracellular matrix, we identify the macroscopic homogenised contribution of the cell membranes. Our approach makes it possible to define a macroscopic homogenised electric field and a macroscopic homogenised transmembrane potential. This provides a direct link between the cell scale electroporation models and the tissue models. Finite element method adapted to the new non-linear model of tissue electroporation is used to compare experiments with simulations. Adapting the phenomenological electroporation model of Leguèbe et al. to the tissue scale, we calibrate the tissue model with experimental data. This makes two steps appear in the tissue electroporation process, as for cells. The new insight of the model lies in the well-established equivalent circuit approach to provide a homogenised version of cell scale models. Our approach is tightly linked to numerical homogenisation strategies adapted to bioelectrical tissue modeling. [ABSTRACT FROM AUTHOR]

Abstract: In our study, we used bleomycin to evaluate the permeabilization caused by nanosecond duration electric pulses (nanopulses). Bleomycin is a non permeant molecule which can be used both as a sensitive and quantitative marker to evaluate cell electropermeabilization. Indeed, the penetration of as few as 500 molecules is sufficient to entail a major biological effect: cell death. We show that one single nanopulse with a duration of 10ns and a field strength of 40kV/cm is sufficient to allow the uptake of at least 500 molecules of bleomycin in 20% of the cells when the external bleomycin concentration is 3μM. When the external bleomycin concentration is reduced by a 100 fold, the same levels of cytotoxicity require an increase of about 25 times in the number of pulses. These results are in favor of the fact that each nanopulse creates new pores or defects on the cell membrane even if most of these pores can reseal between two consecutive pulses. Results also suggest that the cell permeability observed with classical markers when a large number of pulses are delivered results from the large number of nanopores or defects of the cell membrane created by the train of nanopulses. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Electropermeabilization of the cell membrane is a phenomenon caused by exposure of the cell to electric pulses. Permeabilization depends on pulse duration, pulse amplitude, the number of pulses delivered, and also on other experimental conditions. With these parameters properly chosen, the process of permeabilization is reversible and cells return to their normal physiological state. This article describes the development of a model of diffusion-driven transmembrane transport of small molecules caused by electropermeabilization. The process of permeabilization is divided into a short permeabilizing phase that takes place during the pulse, and a longer resealing phase that begins after the end of the pulse. Because both phases of permeabilization are important for uptake of molecules into cells, most of the effort is focused on the optimization of parameters that influence the flow between intracellular and extracellular space. The model describes well the transmembrane transport caused by electropermeabilization, allowing to study the uptake of molecules as a function of elapsed time, voltage and pulse duration. In addition, our results show that the shapes of the curves of cell permeabilization and survival as functions of pulse amplitude can to a large extent be explained by cell size distribution. [Copyright &y& Elsevier]

• Gold nanoparticles (GNPs) enhance electroporative efficacy of electric pulses. • Low amplitude electric pulses bring the GNPs close to the cells by electrophoresis. • Electrophoresis is effective on both GNPs aggregates or on isolated pegylated-GNPs. • GNPs allow to reduce the field amplitude when cell electroporation is sought. The use of conductive nanoparticles (NPs) was previously proposed as a way to locally amplify the electric field (EF) intensity at the cell membrane to enhance cell electroporation. To achieve this, a close distance between the NPs and the cell membrane is mandatory. Here, a new method to improve the contact between NPs and cell surface using the effects of electric pulses (electrophoretic forces) is explored. The effects of two types of electric pulses are analyzed alone or combined in a two-pulse-train protocol on Chinese hamster DC-3F cells. Particularly we used 100 µs duration pulses, low intensity-millisecond pulses and combinations of both. Finally, we studied the use of surface coated NPs (PEGylated) for this application. Our results demonstrate that the delivery of an electric field prior to the electroporation pulses increases the accumulation of NPs around the cell membrane suggesting that NPs are pushed towards the cell surface through electrophoretic forces. This allowed reducing the need for long incubations between cells and NPs to observe an enhancement of electroporation mediated by conductive NPs. Thus low intensity-millisecond pulses can be used to increase the accumulation of either aggregated or individual (i.e. PEGylated) NPs supporting the electrophoretic nature of the observed effects. [ABSTRACT FROM AUTHOR]

The effects of pyroelectricity on cell membrane permeability had never been explored. Pyroelectricity consists in the generation of an electric field in the surface of some materials when a change in temperature is produced. In the present study, tourmaline microparticles, which are known to display pyroelectrical properties, were subjected to different changes in temperature upon exposure to cells in order to induce an electric field at their surface. Then, the changes in the permeability of the cell membrane to a cytotoxic agent (bleomycin) were assessed by a cloning efficacy test. An increase in the permeability of the cell membrane was only detected when tourmaline was subjected to a change in temperature. This suggests that the apparition of an induced pyroelectrical electric field on the material could actually be involved in the observed enhancement of the cell membrane permeability as a result of cell electropermeabilization. [ABSTRACT FROM AUTHOR]

Electric pulses of a few nanoseconds in duration can induce reversible permeabilization of cell membrane and cell death. Whether these effects are caused by ionic or purely dielectric phenomena is still discussed. We address this question by studying the impact of conductivity of the pulsing buffer on the effect of pulses of 12 ns and 3.2 MV/m on the DC-3F mammalian cell line. When pulses were applied in a high-conductivity medium (1.5 S/m), cells experienced both reversible electropermeabilization and cell death. On the contrary, no effect was observed in the low-conductivity medium (0.1 S/m). Possible artifacts due to differences in viscosity, temperature increase or electrochemical reactions were excluded. The influence of conductivity reported here suggests that charges still play a role, even for 12-ns pulses. All theoretical models agree with this experimental observation, since all suggest that only high-conductivity medium can induce a transmembrane voltage high enough to induce pore creation, in turn. However, most models fail to describe why pulse accumulation is experimentally required to observe biological effects. They mostly show no increase of permeabilization with accumulation of pulses. Currently, only one model properly describes pulse accumulation by modeling diffusion of the altered membrane regions. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, we present a novel microfluidic system dedicated to the application of ultra short pulses (i.e. nanopulses) on cells and the visualization of their effects. Cell plasma membranes can be rendered permeable by the use of nanosecond pulsed electric field. In conventional macroscopic electroporation chambers, the typical pulse duration is on the order of several hundred microseconds (micropulses), which allows the penetration of genes or other therapeutic molecules inside the cell. Consequently, electroporation pulses can be used in various clinical applications such as skin cancer treatment. A huge interest recently appeared on the use of ultra-short electric pulses (few nanoseconds) for the treatment of cells, as it was shown to enhance the treatment efficacy compared to micropulses. In this paper, the design, fabrication and biological validation of a microfluidic biochip optimized for the nanoporation of cells is detailed. The developed system presents several advantages such as real time monitoring of the effects of nanoporation on cells, single cell analysis, local generation of extremely high strength electrical fields while using reasonably low voltages and the precise control of electrical and fluidic parameters. To achieve the required spatial homogeneity of the electric field within the fluidic channel, the biochip includes thick electroplated gold electrodes (t =6–30μm). As demonstrated in the paper, the electrical connections between the chip and the nsPEF generator are of prime importance, as it affects directly the quality of the impedance matching i.e. the transfert of energy between the generator and the cells. A study of the effects of these electrical connections on the biochip impedance is detailed. Nanoporation was achieved on living cells within the proposed chip, and characterized through the measurement of the fluorescence decay of intracellular calcein. [Copyright &y& Elsevier]

Abstract: This paper deals with the development of a microfluidic biochip for the exposure of living cells to nanosecond pulsed electric fields (nsPEF). When exposed to ultra short electric pulses (typical duration of 3–10ns), disturbances on the plasma membrane and on the intra cellular components occur, modifying the behavioral response of cells exposed to drugs or transgene vectors. This phenomenon permits to envision promising therapies. The presented biochip is composed of thick gold electrodes that are designed to deliver a maximum of energy to the biological medium containing cells. The temporal and spectral distributions of the nsPEF are considered for the design of the chip. In order to validate the fabricated biochip ability to orient the pulse towards the cells flowing within the exposition channels, a frequency analysis is provided. High voltage measurements in the time domain are performed to characterize the amplitude and the shape of the nsPEF within the exposition channels and compared to numerical simulations achieved with a 3D Finite-Difference Time-Domain code. We demonstrate that the biochip is adapted for 3ns and 10ns pulses and that the nsPEF are homogenously applied to the biological cells regardless their position along the microfluidic channel. Furthermore, biological tests performed on the developed microfluidic biochip permit to prove its capability to permeabilize living cells with nanopulses. To the best of our knowledge, we report here the first successful use of a microfluidic device optimized for the achievement and real time observation of the nanoporation of living cells. [Copyright &y& Elsevier]