The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Damijan Miklavčič - One of the best experts on this subject based on the ideXlab platform.
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membrane electroporation and electropermeabilization mechanisms and models
Annual Review of Biophysics, 2019Co-Authors: Tadej Kotnik, Lea Rems, Mounir Tarek, Damijan MiklavčičAbstract: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.
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Sequential finite element model of tissue electropermeabilization.
IEEE Transactions on Biomedical Engineering, 2017Co-Authors: Davorka Sel, Danute Batiuskaite, David Cukjati, Lluis M. Mir, Tomaž Slivnik, Damijan MiklavčičAbstract:Permeabilization, when observed on a tissue level, is a dynamic process resulting from changes in membrane permeability when exposing biological cells to external electric field (E). In this paper we present a sequential finite element model of E distribution in tissue which considers local changes in tissue conductivity due to permeabilization. These changes affect the pattern of the field distribution during the high voltage pulse application. The presented model consists of a sequence of static models (steps), which describe E distribution at discrete time intervals during tissue permeabilization and in this way present the dynamics of electropermeabilization. The tissue conductivity for each static model in a sequence is determined based on E distribution from the previous step by considering a sigmoid dependency between specific conductivity and E intensity. Such a dependency was determined by parameter estimation on a set of current measurements, obtained by in vivo experiments. Another set of measurements was used for model validation. All experiments were performed on rabbit liver tissue with inserted needle electrodes. Model validation was carried out in four different ways: 1) by comparing reversibly permeabilized tissue computed by the model and the reversibly permeabilized area of tissue as obtained in the experiments; 2) by comparing the area of irreversibly permeabilized tissue computed by the model and the area where tissue necrosis was observed in experiments; 3) through the comparison of total current at the end of pulse and computed current in the last step of sequential electropermeabilization model; 4) by comparing total current during the first pulse and current computed in consecutive steps of a modeling sequence. The presented permeabilization model presents the first approach of describing the course of permeabilization on tissue level. Despite some approximations (ohmic tissue behavior) the model can predict the permeabilized volume of tissue, when exposed to electrical treatment. Therefore, the most important contribution and novelty of the model is its potentiality to be used as a tool for determining parameters for effective tissue permeabilization.
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resistive heating and electropermeabilization of skin tissue during in vivo electroporation a coupled nonlinear finite element model
International Journal of Heat and Mass Transfer, 2011Co-Authors: Natasa Pavselj, Damijan MiklavčičAbstract:Abstract The use of electric pulses to increase cell membrane permeability – electroporation – has, among other applications also been used on skin for (a) enhanced transdermal molecular delivery or (b) the delivery of drugs or DNA into viable skin cells. Based on finite element numerical method, we theoretically described skin electropermeabilization and the amount of heating in and around an electrically created pore in the stratum corneum (SC). With the model, we address both, electrical as well as thermal effects on skin tissue, specifically for electrode design and pulse protocols we used for gene electrotransfer in vivo (already published results), where plasmid DNA was injected intradermally with a syringe and external plate electrodes were used for pulse delivery. Theoretical results obtained with the model show no significant further thermal expansion of the aqueous pore for our specific pulse protocol (one short high voltage pulse: 400 V, 100 μs + one longer low voltage pulse: 80 V, 400 ms), as well as no thermal damage to the tissue. With some modifications to the protocol, electroporation could be used to (a) create pores in the SC through which to transport the DNA, and then (b) introduce the DNA into viable skin cells.
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non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery
Journal of Controlled Release, 2009Co-Authors: Serge Mazeres, Muriel Golzio, Y Tamzali, Davorka Sel, Damijan Miklavčič, Gorazd Pucihar, Justin TeissiéAbstract:For an effective tissue controlled electropermeabilization as requested for electrochemotherapy and electrogenotherapy, it is very important to have informations about the electric field distribution provided by a defined set of electrodes. Computer simulations using the finite element models approach predicted the associated field distributions and currents. Phantoms made of gels with well-defined electrical conductance were used to measure the current responses of a new electrode geometry (wires), A good agreement between the measured and predicted currents was observed supporting the validity of the prediction for the field distribution. Field distribution was observed to be very localized and highly homogeneous with the new concept of contact wire electrodes. They allowed to focus the field effect along the surface of the tissue to induce a controlled release of drugs or plasmids. Non invasive (contact) electrodes can be moved rapidly on the body and avoid puncturing the skin and the tissue. They can be used for large surface effects, to treat the skin and subcutaneous tumors. The use of contact electrodes after drug or DNA intradermal injection were validated by clinical treatment of large surface skin tumors and by in vivo imaging of permeabilization or of gene expression.
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Blumlein Configuration for High-Repetition-Rate Pulse Generation of Variable Duration and Polarity Using Synchronized Switch Control
IEEE Transactions on Biomedical Engineering, 2009Co-Authors: Matej Rebersek, Matej Kranjc, Denis Pavliha, Tina Batista-napotnik, Danilo Vrtacnik, Slavko Amon, Damijan MiklavčičAbstract:Blumlein generators are used in different applications such as radars, lasers, and also recently in various biomedical studies, where the effects of high-voltage nanosecond pulses on biological cells are evaluated. In these studies, it was demonstrated that by applying high-voltage nanosecond pulses to cells, plasma membrane and cell organelles are permeabilized. As suggested in a recent publication, the repetition rate and polarity of nanosecond high-voltage pulses could have an important effect on the electropermeabilization process, and consequently, on the observed phenomena. Therefore, we designed a new Blumlein configuration that enables a higher repetition rate of variable duration of either bipolar or unipolar high-voltage pulses. We achieved a maximal pulse repetition rate of 1.1 MHz. However, theoretically, this rate could be even higher. We labeled endocytotic vesicles with lucifer yellow and added propidium iodide to a cell suspension for testing the cell plasma membrane integrity, so we were able to observe the permeabilization of endocytotic vesicles and the cell plasma membrane at the same time. The new design of pulse generator was built, verified, and also tested in experiments. The resulting flexibility and variability allow further in vitro experiments to determine the importance of the pulse repetition rate and pulse polarity on membrane permeabilization - both of the cell plasma membrane as well as of cell organelle membranes.
Justin Teissié - One of the best experts on this subject based on the ideXlab platform.
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intravital microscopy at the single vessel level brings new insights of vascular modification mechanisms induced by electropermeabilization
Journal of Controlled Release, 2012Co-Authors: Elisabeth Bellard, Gregor Sersa, Justin Teissié, Bostjan Markelc, Sandrine Pelofy, Francois Le Guerroue, Maja CemazarAbstract:Electroporation/electropermeabilization, i.e. the result of the application of electric pulses to tissues, is a physical method for delivery of exogenous molecules into cells. It is effective particularly for compounds with limited transmembrane transport. In vivo, electropermeabilization facilitates the delivery of chemotherapeutic drugs into tumor cells that is the basic mechanism of the antitumor effectiveness of electrochemotherapy. This therapy has also blood flow modifying effects in tissues. The aim of our present study was to understand and explain the effects of electropermeabilization on the dynamics (vasomotricity, permeability and recovery) of subcutaneous blood vessels towards different size of molecules. These features were measured in C57Bl/6 mice via a dorsal skin fold window chamber, using fluorescently labeled dextrans of different sizes, intravital fluorescence microscopy imaging and specific image analysis. Application of electric pulses on the skin in vivo resulted in a rapid increase in vascular permeability that gradually recovered to basal levels at different times post-treatment, depending on dextran size. Simultaneously, the immediate constriction of the blood vessels occurred that was more pronounced for arterioles compared to venules. This vasoconstriction of arterioles results in a transient "vascular lock". The increased permeability of small vessels walls whatever the dextran size associated with delayed perfusion explains the improved delivery of the intravenous injected molecules (i.e. drugs, gene delivery) into the tissues induced by electropermeabilization in vivo.
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new insights in the gene electrotransfer process evidence for the involvement of the plasmid dna topology
Current Gene Therapy, 2012Co-Authors: Jeanmichel Escoffre, Muriel Golzio, Justin Teissié, Cyril Favard, Biliana Nikolova, Laetitia Mallet, Julien Henri, Iana Tsoneva, M RolsAbstract:Electropermeabilization is a non-viral method that can be used to transfer plasmid DNA (pDNA) into cells and tissues. According the applications and considered tissues, this safe method can be less efficient than the viral approaches. Biophysical mechanisms of gene electrotransfer are not entirely known. Contrary to small molecules that have direct and fast access to the cytoplasm, pDNA is electrophoretically pushed towards the permeabilized membrane where it forms a complex before being transferred into the cytoplasm. In order to understand the biophysical mechanisms of gene electrotransfer and in this way to improve it, we investigated the dependence of the topology of pDNA i.e. linear versus supercoiled on both pDNA/membrane interaction and gene expression. Our results revealed that: i) even if pDNA/membrane interactions are only slightly affected by the topology of pDNA, ii) gene transfer and expression are strongly influenced by it. Indeed, the linearization of pDNA leads to a decrease in the transfection level.
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non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery
Journal of Controlled Release, 2009Co-Authors: Serge Mazeres, Muriel Golzio, Y Tamzali, Davorka Sel, Damijan Miklavčič, Gorazd Pucihar, Justin TeissiéAbstract:For an effective tissue controlled electropermeabilization as requested for electrochemotherapy and electrogenotherapy, it is very important to have informations about the electric field distribution provided by a defined set of electrodes. Computer simulations using the finite element models approach predicted the associated field distributions and currents. Phantoms made of gels with well-defined electrical conductance were used to measure the current responses of a new electrode geometry (wires), A good agreement between the measured and predicted currents was observed supporting the validity of the prediction for the field distribution. Field distribution was observed to be very localized and highly homogeneous with the new concept of contact wire electrodes. They allowed to focus the field effect along the surface of the tissue to induce a controlled release of drugs or plasmids. Non invasive (contact) electrodes can be moved rapidly on the body and avoid puncturing the skin and the tissue. They can be used for large surface effects, to treat the skin and subcutaneous tumors. The use of contact electrodes after drug or DNA intradermal injection were validated by clinical treatment of large surface skin tumors and by in vivo imaging of permeabilization or of gene expression.
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time dependence of electric field effects on cell membranes a review for a critical selection of pulse duration for therapeutical applications
Radiology and Oncology, 2008Co-Authors: Justin Teissié, M Rols, Jeanmichel Escoffre, Muriel GolzioAbstract:Background. Electropulsation is one of the non-viral methods successfully used to transfer drugs and genes into living cells in vitro as in vivo. This approach shows promise in field of gene and cellular therapies. This presentation first describes the temporal factors controlling electropermeabilization to small molecules (< 4kDa) and then the processes supporting DNA transfer in vitro. The description of in vitro events brings our attention on the processes occurring before (s), during (ms) and after electropulsation (ms to hours) of DNA and cells. They all appear to be multistep events with well defined kinetics. They cannot be described as just punching holes in a lipid matrix in a two states process. Conclusions. The faster events (may be starting on the ns time scale) appear to be under the control of the external field while the slower ones are linked to the cell metabolism. Investigating the associated collective molecular reorganization by fast kinetics methods and molecular dynamics simulation will help in their safe developments for the in vivo processes and their present and potential clinical applications.
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electrochemotherapy in veterinary oncology
Journal of Veterinary Internal Medicine, 2008Co-Authors: Maja Cemazar, Natasa Tozon, Gregor Sersa, Y Tamzali, R Lowe, Damijan Miklavčič, Justin TeissiéAbstract:Electropermeabilization is a method that uses electric field pulses to induce an electrically mediated reorganization of the plasma membrane of cells. Electrochemotherapy combines local or systemic administration of chemotherapeutic drugs such as bleomycin or cisplatin that have poor membrane permeability with electropermeabilization by direct application of electric pulses to the tumors. Preclinical studies have demonstrated excellent antitumor effectiveness of electrochemotherapy on different animal models and various tumor types, minimal toxicity, and safety of the procedure. Based on results of preclinical studies, clinical studies were conducted in human patients, which demonstrated pronounced antitumor effectiveness of electrochemotherapy with 80–85% objective responses of the treated cutaneous and SC tumors. Clinical studies in veterinary oncology have demonstrated that electrochemotherapy is very effective in the treatment of cutaneous and SC tumors of different histologic types in cats, dogs, and horses. The results of these studies have also demonstrated approximately 80% long-lasting objective responses of tumors treated by electrochemotherapy. Primary tumors of different histologic types were treated. Electrochemotherapy in veterinary oncology has future promise to be highly effective, and could be used to treat primary or recurrent solitary or multiple cutaneous and SC tumors of different histology or as an adjuvant treatment to surgery.
M Rols - One of the best experts on this subject based on the ideXlab platform.
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Pulsed Electric Field Treatment Enhances the Cytotoxicity of Plasma-Activated Liquids in a Three-Dimensional Human Colorectal Cancer Cell Model
Scientific Reports, 2019Co-Authors: Elena Griseti, M Rols, Jelena Kolosnjaj-tabi, Laure Gibot, Isabelle Fourquaux, Mohammed Yousfi, Nofel Merbahi, Muriel GolzioAbstract:Cold atmospheric plasma and more recently, plasma-activated liquids (culture media, water or buffered solutions previously exposed to plasma), are gathering momentum in cancer cells treatment. Nevertheless, in vitro tests show that this novel approach is sometimes less efficient than expected. We here evaluate the mechanisms of action of the plasma-activated PBS and suggest to use electropermeabilization (EP) in combination with the plasma-activated phosphate-buffered saline (PBS), in order to potentiate the cytotoxic effect of the plasma activated liquid. Human multicellular tumor spheroids (MCTS), a three-dimensional cell model, which resembles small avascular tumors, was used to define the optimal treatment conditions for single and dual-mode treatments. MCTS growth, viability, and global morphological changes were assessed by live cell video-microscopy. In addition, the induction of caspases activation, the appearance of DNA damages, and cell membrane permeabilization, as well as the early modifications in the cellular ultrastructure, were examined by immunofluorescence, propidium iodide staining, confocal fluorescence microscopy and transmission electron microscopy, respectively. Altogether, our results show that a combined treatment resulted in an earlier onset of DNA damage and caspases activation, which completely abolished MCTS growth. This report is a proof of concept study evidencing that electropermeabilization greatly potentiates the cytotoxic effect of plasma-activated PBS in vitro in a three-dimensional cancer cell model.
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new insights in the gene electrotransfer process evidence for the involvement of the plasmid dna topology
Current Gene Therapy, 2012Co-Authors: Jeanmichel Escoffre, Muriel Golzio, Justin Teissié, Cyril Favard, Biliana Nikolova, Laetitia Mallet, Julien Henri, Iana Tsoneva, M RolsAbstract:Electropermeabilization is a non-viral method that can be used to transfer plasmid DNA (pDNA) into cells and tissues. According the applications and considered tissues, this safe method can be less efficient than the viral approaches. Biophysical mechanisms of gene electrotransfer are not entirely known. Contrary to small molecules that have direct and fast access to the cytoplasm, pDNA is electrophoretically pushed towards the permeabilized membrane where it forms a complex before being transferred into the cytoplasm. In order to understand the biophysical mechanisms of gene electrotransfer and in this way to improve it, we investigated the dependence of the topology of pDNA i.e. linear versus supercoiled on both pDNA/membrane interaction and gene expression. Our results revealed that: i) even if pDNA/membrane interactions are only slightly affected by the topology of pDNA, ii) gene transfer and expression are strongly influenced by it. Indeed, the linearization of pDNA leads to a decrease in the transfection level.
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What is (Still not) Known of the Mechanism by Which Electroporation Mediates Gene Transfer and Expression in Cells and Tissues
Molecular Biotechnology, 2009Co-Authors: Jeanmichel Escoffre, David S Dean, Thomas Portet, Luc Wasungu, Teissié Justin, M RolsAbstract:Cell membranes can be transiently permeabilized under application of electric pulses. This treatment allows hydrophilic therapeutic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. This process, called electropermeabilization or electroporation, has been rapidly developed over the last decade to deliver genes to tissues and organs, but there is a general agreement that very little is known about what is really occurring during membrane electropermeabilization. It is well accepted that the entry of small molecules, such as anticancer drugs, occurs mostly through simple diffusion after the pulse while the entry of macromolecules, such as DNA, occurs through a multistep mechanism involving the electrophoretically driven interaction of the DNA molecule with the destabilized membrane during the pulse and then its passage across the membrane. Therefore, successful DNA electrotransfer into cells depends not only on cell permeabilization but also on the way plasmid DNA interacts with the plasma membrane and, once into the cytoplasm, migrates towards the nucleus. The focus of this review is to describe the different aspects of what is known of the mechanism of membrane permeabilization and associated gene transfer and, by doing so, what are the actual limits of the DNA delivery into cells.
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time dependence of electric field effects on cell membranes a review for a critical selection of pulse duration for therapeutical applications
Radiology and Oncology, 2008Co-Authors: Justin Teissié, M Rols, Jeanmichel Escoffre, Muriel GolzioAbstract:Background. Electropulsation is one of the non-viral methods successfully used to transfer drugs and genes into living cells in vitro as in vivo. This approach shows promise in field of gene and cellular therapies. This presentation first describes the temporal factors controlling electropermeabilization to small molecules (< 4kDa) and then the processes supporting DNA transfer in vitro. The description of in vitro events brings our attention on the processes occurring before (s), during (ms) and after electropulsation (ms to hours) of DNA and cells. They all appear to be multistep events with well defined kinetics. They cannot be described as just punching holes in a lipid matrix in a two states process. Conclusions. The faster events (may be starting on the ns time scale) appear to be under the control of the external field while the slower ones are linked to the cell metabolism. Investigating the associated collective molecular reorganization by fast kinetics methods and molecular dynamics simulation will help in their safe developments for the in vivo processes and their present and potential clinical applications.
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membrane perturbation by an external electric field a mechanism to permit molecular uptake
European Biophysics Journal, 2007Co-Authors: Jeanmichel Escoffre, M Rols, David S Dean, Marie Hubert, Cyril FavardAbstract:Electropermeabilisation is a well established physical method, based on the application of electric pulses, which induces the transient permeabilisation of the cell membrane. External molecules, otherwise nonpermeant, can enter the cell. Electropermeabilisation is now in use for the delivery of a large variety of molecules, as drugs and nucleic acids. Therefore, the method has great potential in the fields of cancer treatment and gene therapy. However many open questions about the underlying physical mechanisms involved remain to be answered or fully elucidated. In particular, the induced changes by the effects of the applied field on the membrane structure are still far from being fully understood. The present review focuses on questions related to the current theories, i.e. the basic physical processes responsible for the Electropermeabilisation of lipid membranes. It also addresses recent findings using molecular dynamics simulations as well as experimental studies of the effect of the field on membrane components.
Lluis M. Mir - One of the best experts on this subject based on the ideXlab platform.
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electropermeabilization of inner and outer cell membranes with microsecond pulsed electric fields quantitative study with calcium ions
Scientific Reports, 2017Co-Authors: Hanna Hanna, Micaela Liberti, Agnese Denzi, Franck M Andre, Lluis M. MirAbstract:Microsecond pulsed electric fields (μsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, μsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology.
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Sequential finite element model of tissue electropermeabilization.
IEEE Transactions on Biomedical Engineering, 2017Co-Authors: Davorka Sel, Danute Batiuskaite, David Cukjati, Lluis M. Mir, Tomaž Slivnik, Damijan MiklavčičAbstract:Permeabilization, when observed on a tissue level, is a dynamic process resulting from changes in membrane permeability when exposing biological cells to external electric field (E). In this paper we present a sequential finite element model of E distribution in tissue which considers local changes in tissue conductivity due to permeabilization. These changes affect the pattern of the field distribution during the high voltage pulse application. The presented model consists of a sequence of static models (steps), which describe E distribution at discrete time intervals during tissue permeabilization and in this way present the dynamics of electropermeabilization. The tissue conductivity for each static model in a sequence is determined based on E distribution from the previous step by considering a sigmoid dependency between specific conductivity and E intensity. Such a dependency was determined by parameter estimation on a set of current measurements, obtained by in vivo experiments. Another set of measurements was used for model validation. All experiments were performed on rabbit liver tissue with inserted needle electrodes. Model validation was carried out in four different ways: 1) by comparing reversibly permeabilized tissue computed by the model and the reversibly permeabilized area of tissue as obtained in the experiments; 2) by comparing the area of irreversibly permeabilized tissue computed by the model and the area where tissue necrosis was observed in experiments; 3) through the comparison of total current at the end of pulse and computed current in the last step of sequential electropermeabilization model; 4) by comparing total current during the first pulse and current computed in consecutive steps of a modeling sequence. The presented permeabilization model presents the first approach of describing the course of permeabilization on tissue level. Despite some approximations (ohmic tissue behavior) the model can predict the permeabilized volume of tissue, when exposed to electrical treatment. Therefore, the most important contribution and novelty of the model is its potentiality to be used as a tool for determining parameters for effective tissue permeabilization.
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Impact of external medium conductivity on cell membrane electropermeabilization by microsecond and nanosecond electric pulses
Scientific Reports, 2016Co-Authors: Aude Silve, Isabelle Leray, Clair Poignard, Lluis M. MirAbstract:The impact of external medium conductivity on the efficiency of the reversible permeabilisation caused by pulsed electric fields was investigated. Pulses of 12 ns, 102 ns or 100 μs were investigated. Whenever permeabilisation could be detected after the delivery of one single pulse, media of lower conductivity induced more efficient reversible permeabilisation and thus independently of the medium composition. Effect of medium conductivity can however be hidden by some saturation effects, for example when pulses are cumulated (use of trains of 8 pulses) or when the detection method is not sensitive enough. This explains the contradicting results that can be found in the literature. The new data are complementary to those of one of our previous study in which an opposite effect of the conductivity was highlighted. It stresses that the conductivity of the medium influences the reversible permeabilization by several ways. Moreover, these results clearly indicate that Electropermeabilisation does not linearly depend on the energy delivered to the cells.
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cell membrane permeabilization by 12 ns electric pulses not a purely dielectric but a charge dependent phenomenon
Bioelectrochemistry, 2015Co-Authors: Aude Silve, Isabelle Leray, Clair Poignard, Michael Leguebe, Lluis M. MirAbstract: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.
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comparison of the effects of the repetition rate between microsecond and nanosecond pulses electropermeabilization induced electro desensitization
Biochimica et Biophysica Acta, 2014Co-Authors: Aude Silve, Guimera A Brunet, B Alsakere, Antoni Ivorra, Lluis M. MirAbstract:Abstract Background Applications of cell electropermeabilization are rapidly growing but basic concepts are still unclear. In particular, the impact of electric pulse repetition rate in the efficiency of permeabilization has not yet been understood. Methods The impact of electric pulse repetition rate in the efficiency of permeabilization was analyzed in experiments performed on potato tissue and partially transposed on mice liver. On potato tissue, pulses with durations of 100 μs or 10 ns are applied. The intensity of permeabilization was quantified by means of bioimpedance changes and electric current measurements and a new index was defined. Results For the two pulse durations tested, very low repetition rates (below 0.1 Hz) are much more efficient to achieve cell permeabilization in potato tissue. In mice liver, using 100 μs pulses, the influence of the repetition rate is more complex. Indeed, repetition rates of 1 Hz and 10 Hz are more efficient than 100 Hz or 1 kHz, but not the repetition rate of 0.1 Hz for which there is an impact of the living mice organism response. Conclusions We propose that the effects reported here might be caused by an electroporation-induced cell membrane ‘electro-desensitization’ which requires seconds to dissipate due to membrane resealing. General significance This study not only reinforces previous observations, but moreover it sustains a new concept of ‘electro-desensitization’ which is the first unifying mechanism enabling to explain all the results obtained until now both in vitro and in vivo, with long and short pulses.
Muriel Golzio - One of the best experts on this subject based on the ideXlab platform.
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Pulsed Electric Field Treatment Enhances the Cytotoxicity of Plasma-Activated Liquids in a Three-Dimensional Human Colorectal Cancer Cell Model
Scientific Reports, 2019Co-Authors: Elena Griseti, M Rols, Jelena Kolosnjaj-tabi, Laure Gibot, Isabelle Fourquaux, Mohammed Yousfi, Nofel Merbahi, Muriel GolzioAbstract:Cold atmospheric plasma and more recently, plasma-activated liquids (culture media, water or buffered solutions previously exposed to plasma), are gathering momentum in cancer cells treatment. Nevertheless, in vitro tests show that this novel approach is sometimes less efficient than expected. We here evaluate the mechanisms of action of the plasma-activated PBS and suggest to use electropermeabilization (EP) in combination with the plasma-activated phosphate-buffered saline (PBS), in order to potentiate the cytotoxic effect of the plasma activated liquid. Human multicellular tumor spheroids (MCTS), a three-dimensional cell model, which resembles small avascular tumors, was used to define the optimal treatment conditions for single and dual-mode treatments. MCTS growth, viability, and global morphological changes were assessed by live cell video-microscopy. In addition, the induction of caspases activation, the appearance of DNA damages, and cell membrane permeabilization, as well as the early modifications in the cellular ultrastructure, were examined by immunofluorescence, propidium iodide staining, confocal fluorescence microscopy and transmission electron microscopy, respectively. Altogether, our results show that a combined treatment resulted in an earlier onset of DNA damage and caspases activation, which completely abolished MCTS growth. This report is a proof of concept study evidencing that electropermeabilization greatly potentiates the cytotoxic effect of plasma-activated PBS in vitro in a three-dimensional cancer cell model.
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new insights in the gene electrotransfer process evidence for the involvement of the plasmid dna topology
Current Gene Therapy, 2012Co-Authors: Jeanmichel Escoffre, Muriel Golzio, Justin Teissié, Cyril Favard, Biliana Nikolova, Laetitia Mallet, Julien Henri, Iana Tsoneva, M RolsAbstract:Electropermeabilization is a non-viral method that can be used to transfer plasmid DNA (pDNA) into cells and tissues. According the applications and considered tissues, this safe method can be less efficient than the viral approaches. Biophysical mechanisms of gene electrotransfer are not entirely known. Contrary to small molecules that have direct and fast access to the cytoplasm, pDNA is electrophoretically pushed towards the permeabilized membrane where it forms a complex before being transferred into the cytoplasm. In order to understand the biophysical mechanisms of gene electrotransfer and in this way to improve it, we investigated the dependence of the topology of pDNA i.e. linear versus supercoiled on both pDNA/membrane interaction and gene expression. Our results revealed that: i) even if pDNA/membrane interactions are only slightly affected by the topology of pDNA, ii) gene transfer and expression are strongly influenced by it. Indeed, the linearization of pDNA leads to a decrease in the transfection level.
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non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery
Journal of Controlled Release, 2009Co-Authors: Serge Mazeres, Muriel Golzio, Y Tamzali, Davorka Sel, Damijan Miklavčič, Gorazd Pucihar, Justin TeissiéAbstract:For an effective tissue controlled electropermeabilization as requested for electrochemotherapy and electrogenotherapy, it is very important to have informations about the electric field distribution provided by a defined set of electrodes. Computer simulations using the finite element models approach predicted the associated field distributions and currents. Phantoms made of gels with well-defined electrical conductance were used to measure the current responses of a new electrode geometry (wires), A good agreement between the measured and predicted currents was observed supporting the validity of the prediction for the field distribution. Field distribution was observed to be very localized and highly homogeneous with the new concept of contact wire electrodes. They allowed to focus the field effect along the surface of the tissue to induce a controlled release of drugs or plasmids. Non invasive (contact) electrodes can be moved rapidly on the body and avoid puncturing the skin and the tissue. They can be used for large surface effects, to treat the skin and subcutaneous tumors. The use of contact electrodes after drug or DNA intradermal injection were validated by clinical treatment of large surface skin tumors and by in vivo imaging of permeabilization or of gene expression.
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time dependence of electric field effects on cell membranes a review for a critical selection of pulse duration for therapeutical applications
Radiology and Oncology, 2008Co-Authors: Justin Teissié, M Rols, Jeanmichel Escoffre, Muriel GolzioAbstract:Background. Electropulsation is one of the non-viral methods successfully used to transfer drugs and genes into living cells in vitro as in vivo. This approach shows promise in field of gene and cellular therapies. This presentation first describes the temporal factors controlling electropermeabilization to small molecules (< 4kDa) and then the processes supporting DNA transfer in vitro. The description of in vitro events brings our attention on the processes occurring before (s), during (ms) and after electropulsation (ms to hours) of DNA and cells. They all appear to be multistep events with well defined kinetics. They cannot be described as just punching holes in a lipid matrix in a two states process. Conclusions. The faster events (may be starting on the ns time scale) appear to be under the control of the external field while the slower ones are linked to the cell metabolism. Investigating the associated collective molecular reorganization by fast kinetics methods and molecular dynamics simulation will help in their safe developments for the in vivo processes and their present and potential clinical applications.
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mechanisms of cell membrane electropermeabilization a minireview of our present lack of knowledge
Biochimica et Biophysica Acta, 2005Co-Authors: Justin Teissié, Muriel Golzio, M RolsAbstract:Abstract Cell electropulsation is routinely used in cell Biology for protein, RNA or DNA transfer. Its clinical applications are under development for targeted drug delivery and gene therapy. Nevertheless, the molecular mechanisms supporting the induction of permeabilizing defects in the membrane assemblies remain poorly understood. This minireview describes the present state of the investigations concerning the different steps in the reversible electropermeabilization process. The different hypotheses, which were proposed to give a molecular description of the membrane events, are critically discussed. Other possibilities are then given. The need for more basic research on the associated loss of cohesion of the membrane appears as a conclusion.