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Background: Despite the numerous literature results about biological effects of electromagnetic field (EMF) exposure, the interaction mechanisms of these fields with organisms are still a matter of debate. Extremely low frequency (ELF) MFs can modulate redox homeostasis and we showed that 24 h exposure to 50 Hz–1 mT has a pro-oxidant effect and effects on the epigenome of SH-SY5Y cells, decreasing miR-34b/c expression through the hypermethylation of their promoter. Methods: Here, we investigated the role of the electromagnetic deposited energy density (ED) during exposures lasting 24 h to 1 mT amplitude MFs at a frequency of 50 Hz in inducing the above mentioned effects. To this end, we delivered ultrashort electric pulses, in the range of microsecond and nanosecond duration, with the same ED of the previously performed magnetic exposure to SH-SY5Y cells. Furthermore, we explored the effect of higher deposited energy densities. Analysis of i) gene and microRNA expression, ii) cell morphology, iii) reactive oxygen species (ROS) generation, and iv) apoptosis were carried out. Results: We observed significant changes in egr-1 and c-fos expression at very low deposited ED levels, but no change of the ROS production, miR-34b/c expression, nor the appearance of indicators of apoptosis. We thus sought investigating changes in egr-1 and c-fos expression caused by ultrashort electric pulses at increasing deposited ED levels. The pulses with the higher deposited ED caused cell electroporation and even other morphological changes such as cell fusion. The changes in egr-1 and c-fos expression were more intense, but, again, no change of the ROS production, miR-34b/c expression, nor apoptosis induction was observed. Conclusions: These results, showing that extremely low levels of electric stimulation (never investigated until now) can cause transcriptional changes, also reveal the safety of the electroporating pulses used in biomedical applications and open up the possibility to further therapeutic applications of this technology.

Mesenchymal Stem Cells or Multipotent Stromal Cells (MSCs) are adult stem cells able to differentiate into many cell types such as osteoblasts, adipocytes or chondrocytes. These cells have become of particular interest for cell-based therapy these last decades. In another respect, calcium is a ubiquitous secondary cell messenger, known to encode important information for the cells in the form of oscillations. It has been shown that MSCs naturally exhibit calcium oscillations, whose frequency is varying over the course of differentiation processes. The main question addressed in this work is to assess whether, by manipulating the frequency of calcium oscillations, we could influence proliferation or differentiation events in MSCs. The technology used for this purpose involves the use of short high voltage pulsed electric fields capable of permeabilizing the cell membrane to calcium ions presents in the surrounding medium. Long-term exposure of the cells to the pulsed electric fields was performed using a programmable generator along with an original custom device to expose the cells under regular attached cell culture conditions. We report the experimental constraints that were found and how they were solved.

[EN] The work here reported analyzes the effect of increased efficiency of brainderived neurotrophic factor (BDNF) production by electroporated Schwann cells (SCs) on the axonal extension in a coculture system on a biomaterial platform that can be of interest for the treatment of injuries of the nervous system, both central and peripheral. Rat SCs are electrotransfected with a plasmid coding for the BDNF protein in order to achieve an increased expression and release of this protein into the culture medium of the cells, performing the best balance between the level of transfection and the number of living cells. Gene-transfected SCs show an about 100-fold increase in the release of BDNF into the culture medium, compared to nonelectroporated SCs. Cocultivation of electroporated SCs with rat dorsal root ganglia (DRG) is performed on highly aligned substrates of polylactic acid (PLA) microfibers coated with the electroconductive polymer polypyrrol (PPy). The coculture of DRG with electrotransfected SCs increase both the axonal extension and the axonal sprouting from DRG neurons compared to the coculture of DRG with nonelectroporated SCs. Therefore, the use of PLA¿PPy highly aligned microfiber substrates preseeded with electrotransfected SCs with an increased BDNF secretion is capable of both guiding and accelerating axonal growth., The authors acknowledge financial support from the Spanish Government's State Research Agency (AEI) through projects DPI2015-72863-EXP and RTI2018-095872-B-C22/ERDF. F.G.R. acknowledges the scholarship FPU16/01833 and the short stay mobility aid EST18/00524 of the Spanish Ministry of Universities. F.G.R. also acknowledges the hosting at the Vectorology and Anti-cancer Therapies Centre (UMR 8203 CNRS). The authors thank the Electron Microscopy Service at the UPV, where the FESEM images were obtained.

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 (&micro, sPEFs) 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 &micro, sPEFs. 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 &micro, sPEFs 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.

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.

Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 μs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate.

Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltage-sensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is approximately 1nm. The ion-channel-like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable "conventional" pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.

The use of electric pulses to transfect all types of cells is well known and regularly used in vitro for bacteria and eukaryotic cells transformation. Electric pulses can also be delivered in vivo either transcutaneously or with electrodes in direct contact with the tissues. After injection of naked DNA in a tissue, appropriate local electric pulses can result in a very high expression of the transferred genes. This manuscript describes the evolution in the concepts and the various optimization steps that have led to the use of combinations of pulses that fit with the known roles of the electric pulses in DNA electrotransfer, namely cell electropermeabilization and DNA electrophoresis. A summary of the main applications published until now is also reported, restricted to the in vivo preclinical trials using therapeutic genes.

The membrane of cultured cells exposed in vitro to electric pulses of adequate field amplitude and pulse duration becomes permeable to otherwise “non permeant” molecules [1]. Indeed the delivery of these electric pulses results in changes in the structure of the cell membrane. This phenomenon, which affects the membranes of all live cells, is known as cell electroporation or cell electropermeabilization. It allows the introduction into the cells in culture of various types of molecules such as drugs, dyes, peptides, oligonucleotides, proteins, and large nucleic acids such as plasmids, mRNAs, minicircles, etc… In vivo as well, adequate electric pulses facilitate the uptake of molecules by the cells of a large number of tissues (tumors, muscle, skin, liver, etc. [2]). Nowadays, two applications are well developed: — The electrotransfer of cytotoxic drugs into tumor cells: the combination of electroporating electric pulses and drugs such as bleomycin, termed electrochemotherapy, results in very clear beneficial effects for the patients, with the complete destruction of about 80% of the treated nodules after one single treatment, with almost no side effect. After several years of development (technological improvements of the pulse generators, preclinical trials as well as clinical trials, preparation of Standard Operating Procedures [3, 4]), electrochemotherapy is now used in more than 140 cancer centres in Europe to treat skin tumors and cutaneous and subcutaneous metastasis of any origin. Electrochemotherapy is already reimbursed in 7 EU countries. The electrochemotherapy Users 2nd International Meeting, hold in 2013 in Italy, gathered 350 physicians using this antitumor approach, and more than 3 000 patients were treated in the EU in 2013. — The electrotransfer of genes into various kinds of target cells (tumor cells, muscle cells, skin cells, hepatocytes, …): described at the end of the last century, the mechanism of DNA electrotranfer in vivo, which includes cells permeabilization and DNA electrophoresis inside the tissue, was analysed in several papers from 2000 to 2008 [5]. The first molecular description of the crossing of a lipid bilayer by a siRNA under ultra-short (10 nanoseconds) and very intense (6 MV/m) electric pulses, by means of numerical simulations which were experimentally validated, was reported recently [6]. Several clinical trials are presently exploring the feasibility in humans of this method of gene transfer for non-viral gene therapy and DNA vaccination. Actually, in vivo, the electric pulses have consequences other than the increase of exogenous molecules uptake by the target cells as described here above. — The occurrence of a vascular lock has been extensively described. It affects all the tissues exposed to the electric pulses, but its intensity is very different on normal versus tumor tissues. In normal tissues, under the electric conditions corresponding to the applications mentioned here above, the vascular lock does not last for more than a couple of minutes [7]. On the contrary, in tumors, the blood flow recovers only after several hours [8]. This vascular lock, unpredictable from the in vitro experiments, has very positive therapeutic consequences. Indeed, it results in the absence of bleeding after the application of penetrating electrodes (like bundles of needles) rendering the electrochemotherapy procedures very easy for the professionals and very bearable by the patients. Moreover, in some cases (intratumor injection of drugs like the cisplatin [9] or just of a concentrated calcium solution [10]), it blocks the active agent in the tumor volume allowing a prolonged (and thus more efficient) antitumor effect. — The permeabilization of the membranes also allows the release of intracellular substances in the extracellular space. In the recent years, the immunogenic aspects of the cell death have been extensively revisited. It has been shown that in immunogenic cell death types, one of the DAMPs (Danger-Associated Molecular Patterns) is the ATP. Indeed, the ATP that can be released by the dying cell is a strong immunogenic signal because ATP plays a role of chemoattractant (“find me” signal) for the dendritic cells and their precursors [11] and favors their differentiation and maturation into dendritic cells with antigen-presenting capacity [12]. Thus it plays a pivotal role in raising an effective immune response. We have recently demonstrated that there is a very large release of ATP from the transiently electroporated cells that could add a new and important role for electroporation-based DNA vaccination [13]. Therefore, in vivo, cells electroporation is not merely a way to increase the uptake of drugs or nucleic acids by the cells. Electroporation-dependent vascular lock is certainly beneficial in electrochemotherapy protocols, and electroporation could be also a crucial adjuvant in vaccination protocols.

Isolated (SF 6 ) 55 clusters, as studied by computer simulation, exhibit a phase behavior which is essentially similar to the bulk material. Conversely, the molecular packing of (SF 6 ) 13 is close-to-icosahedral. No sign of a transition with coexistence of rigid and non-rigid states was found in (SF 6 ) 13 . We conclude that polyatomic van der Waals systems may exhibit quite different phase behaviors than the more commonly studied atomic ones.

Molecular dynamics (MD) simulations have been performed on free clusters of sulfur hexafluoride (SF 6 ) of various size: 229, 137, and 55 molecules. Some preliminary simulations of 13-molecule clusters have also been performed. A systematic study of the melting and solid-solid transitions and the effect of cluster size on their temperature spreads was undertaken. The stable structures of the 229-, 137-, and 55-molecule clusters are bcc at elevated temperatures and C2/m monoclinic at low temperature, as in the bulk crystal. The melting transition is rounded and shifted to low temperatures when decreasing the cluster size. In the whole size range, from the thermodynamic limit down to 55 molecules, melting initiates at the surface

The low temperature structures of sulfur hexafluoride have been investigated by molecular dynamics (MD) simulation, using the free cluster method. Clusters containing 229 molecules and having an initial BCC structure can transform either into a stable monoclinic structure identical to that of the bulk material or into an hexagonal structure, depending on the cooling procedure. The hexagonal phase is stable, in the MD time scale, below 65 K. In the range of 70–85 K it experiences a spontaneous transformation into the (monoclinic + cubic) intermediate structure which is known to be stable in such clusters. Essentially identical results have been obtained for a cluster containing 137 molecules. This, together with the fact that the parameters of the hexagonal structure obtained here are in good agreement with those obtained experimentally on a thin single crystal, favours the hypothesis of the existence of a metastable hexagonal phase in solid SF6.

It has been widely accepted that nanosecond electric pulses (nsEP) are distinguished from micro- and millisecond duration pulses by their ability to cause intracellular effects and cell death with reduced effects on the cell plasma membrane. However, we found that nsEP-induced cell death is most likely mediated by the plasma membrane disruption. We showed that nsEP can cause long-lasting (minutes) increase in plasma membrane electrical conductance and disrupt electrolyte balance, followed by water uptake, cell swelling and blebbing. These effects of plasma membrane permeabilization could be blocked by Gd(3+) in a dose-dependent manner, with a threshold at sub-micromolar concentrations. Consequently, Gd(3+) protected cells from nsEP-induced cell death, thereby pointing to plasma membrane permeabilization as a likely primary mechanism of lethal cell damage.

We found that ultra-short electric pulses (60 or 600 ns duration) applied to mammalian cells cause profound, dose-dependent increase of plasma membrane electrical conductance. This effect is detectable even minutes after the exposure and is explained by formation of long-lived, voltagesensitive, inward-rectifying pores of nanometer diameter (”nanopores”). The phenomenon of nanoelectroporation and the extended lifetime of nanopores can also be demonstrated by a surge of Tl + uptake in the presence of K + channel blockers, as well as in CHO cells that express no endogenous voltage-gated K + channels. Due to their long lifetime, nanopores can have significant impact on cell physiology.

International audience; Gene transfer by electroporation is gaining momentum now that high-level, long-term expression of transgenes is being obtained. Several different pulse regimens are efficient, yet little information is available about the physiological muscular response to gene electrotransfer. This paper provides a comprehensive evaluation of the physiological and molecular effects on host tissue after DNA electrotransfer. We have tested several pulse regimens with special emphasis on the pulse combination of a short (100 microsec) high-voltage (HV) pulse followed by a long low-voltage (LV) pulse used for DNA electrotransfer, comparing it with 8 HV pulses designed to ensure extensive permeabilization of the muscle membrane. Using both mouse and rat skeletal muscle tissue, we investigated cell permeabilization by the 51Cr-labeled EDTA assay, lactate dehydrogenase release, Na+ and Ca2+ influx, K+ efflux, ATP release, and water content, as well as muscle function both in vivo and ex vivo, Hsp70 induction, and histology. In all these assays, the HVLV pulse combination gave rise to minimal disturbance of cell function, in all cases significantly different from results when using 8 HV pulses. The evaluated parameters were normalized after 1 week. The addition of DNA caused significantly more transmembrane exchange, and this may be due to entrance of the DNA through the membrane. In conclusion, this study comprehensively documents the immediate effects of DNA electrotransfer and shows that only slight cell disturbances occur with the HVLV pulses used for gene transfer. This is highly important, as minimal perturbation of cell physiology is essential for efficient transgene expression.

We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 μs at 0:3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%), in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation. © 2007 Al-Sakere et al.

Nucleic acids electrotransfer has been shown to be a safe and efficient non-viral technique in a wide variety of cells (including primary cells) even with large nucleic acids. Using a small 3.5 kbp GFP reporter plasmid we previously obtained electrotransfer efficiency of up to 90%, with around 70% cell viability, in Mesenchymal Stem Cells (MSCs). However with larger plasmids (about 15 kbp), we observed highly decreased cell viability and electrotransfer efficacy in MSCs. Our results suggested that this could be the consequence of the increased time necessary for larger plasmids to cross the plasma membrane.Here we further studied the mechanisms of small and large plasmid electrotransfer. We investigated the roles of calcium, temperature and pH.Our results shed light on some of the mechanisms involved in gene electrotransfer as well as provide means to enhance electrotransfer efficacy and cell viability for small and large plasmids.

The simple injection of DNA into muscles is known to result in the expression of the injected genes, even though at low and variable levels. We report that this variability in DNA expression is partly dependent on the injection speed. The acceleration of the injection speed from values around 2 mul/s up to ones around 25 mul/s (depending on the tissue) results in a significant increase in gene expression in skeletal muscle (280 times on an average) and in liver (50 times) and a nonsignificant sevenfold increase in tumors. Heparin, which inhibits the spontaneous uptake of the injected DNA, also inhibits the increases related to the injection speed. However, at the highest injection speed, this inhibition is not total because very fast injections provoke a direct permeabilization of the cells. This 'hydroporation' could be similar to the permeabilization found in the hydrodynamics method based on the fast intravascular injection of a huge volume of DNA. Neither the 'hydroporation' nor the heparin-inhibitable uptake mechanism induces histologically detectable lesions. There is a limited muscle cell stress independent of the injection speed. Heterogeneity in the injection speed might thus be an explanation for the variability in DNA expression after simple injection.Gene Therapy advance online publication, 27 July 2006; doi:10.1038/sj.gt.3302827.

DNA vaccination consists of administering an antigen-coding nucleotide sequence. In order to improve the efficacy of DNA vaccines, electroporation is one of the most commonly used methods to enhance DNA uptake. Here, we discuss additional immunological effects of electroporation that are key aspects for inducing immunity in response to DNA vaccines.

Electrochemotherapy (ECT) is a local cancer treatment that has been used over the course of more than 2 decades for the removal of cutaneous and subcutaneous tumors. Several lines of evidence support the premise that the immune system is an important factor underlying anticancer treatment efficacy, potentially including patient responses to ECT. The concept of immunogenic cell death (ICD) arose a few years ago, stating that some cancer treatments generate danger-associated molecular patterns (DAMPs) that trigger an adaptive immune response against tumors. Hence, dying cancer cells behave as a therapeutic vaccine, eliciting a cytotoxic immune response against surviving malignant cells. In our study, we sought to evaluate the ability of ECT to generate cancer cell death encompassing the immunostimulatory characteristics of ICD. To this end, we assayed CT26 murine colon cancer cells in vitro in response to either electric pulses (EPs) application only or in combination with the anticancer drug bleomycin (that is ECT) by quantification of calreticulin (CRT) membrane externalization, as well as the liberation of adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1) protein. We show here that cell permeabilizing yet non-lethal electric pulses induce CRT exposure on the cell surface of EP-only treated cancer cells, as well as ATP release. However, the association of electric pulses along with the chemotherapeutic agent bleomycin was mandatory for HMGB1 release coincident with regimen-induced cell death. These data obtained in vitro were then substantiated by vaccination protocols performed in immunocompetent mice, showing that the injection of dying ECT-treated cells elicits an antitumor immune response that prevents the growth of a subsequent administration of viable cancer cells. We also confirmed previous results showing ECT treatment is much more efficient in immunocompetent animals than in immunodeficient ones, causing complete regressions in the former but not in the latter. This supports a central role for immunity in this beneficial outcome. In conclusion, we show that ECT not only possesses an intrinsic cytotoxic property toward cancer cells but also generates a systemic anticancer immune response via the activation of ICD. Hence, ECT may represent an interesting approach to treat solid tumors while preventing recurrence and metastasis, possibly in combination with immunostimulating agents.

The major limitation of gene therapy is the vector used for gene transfer. Viruses were used first and are still the most common approach. Due to adverse events in clinical trials, there was a need for non viral methods. Among them, DNA electrotransfer is very promising. In vivo DNA electrotransfer consist in the injection of a DNA plasmid, either locally or intravenously, followed by electric pulses directly applied to the targeted tissue. To obtain a good gene transfection in skeletal muscles, 8 short square Electric Pulses (EP) of 200 V/cm and 20ms duration delivered at a repetition frequency of 1Hz are often used. However, in our laboratory, a further increase in the efficacy of the gene transfer in skeletal muscles was obtained using combinations of short, High Voltage EP (HV; 800 V/cm, 100 |[mu]|s) and long, Low Voltage EP (LV; 80 V/cm, 100 ms) 1. The efficacy of the HV+LV combinations is due to the two different effects of the EP: the electropermeabilization of the target cells (mainly due to the HV) and the electrophoresis of the DNA (caused by the LV) towards or across the permeabilized plasma membranes.

Efficient DNA electrotransfer can be achieved with combinations of short high-voltage (HV) and long low voltage (LV) pulses that cover two effects of the pulses, namely, target cell electropermeabilization and DNA electrophoresis within the tissue. Because HV and LV can be delivered with a lag up to 3000 sec between them, we considered that it was possible to analyze separately the respective importance of the two types of effects of the electric fields on DNA electrotransfer efficiency. The tibialis cranialis muscles of C57BL/6 mice were injected with plasmid DNA encoding luciferase or green fluorescent protein and then exposed to various combinations of HV and LV pulses. DNA electrotransfer efficacy was determined by measuring luciferase activity in the treated muscles. We found that for effective DNA electrotransfer into skeletal muscles the HV pulse is prerequisite; however, its number and duration do not significantly affect electrotransfer efficacy. DNA electrotransfer efficacy is dependent mainly on the parameters of the LV pulse(s). We report that different LV number, LV individual duration, and LV strength can be used, provided the total duration and field strength result in convenient electrophoretic transport of DNA toward and/or across a permeabilized membrane.

Top of pageAbstract The simple injection of naked DNA into skeletal muscle is known to result in the expression of the injected gene even though at very low and variable levels. We report here that, with respect to the local (intratissular) slow injection of the DNA (in about 25 seconds), the local fast injection in 1 to 2 seconds results in an increase in gene expression from 10 times in tumors and 100 times in liver, up to 500 times in the skeletal muscle. We also show that the fast liquid injection affects cell membrane and we demonstrate that the main mechanism of the increase in DNA uptake and gene expression is the enhancement of the receptor-mediated endocytosis of the injected DNA. However, at the highest speed of injection, there is not a complete inhibition of this receptor-mediated endocytosis process by means of a huge excess of a competitive inhibitor (heparin), suggesting the existence of another minor process for DNA uptake. Using a classical marker of membrane permeability (propidium iodide) we show that this small amount of non heparin-inhibitable DNA uptake could result from the direct permeabilisation of the cells by the high speed/high pressure DNA injection. This hydroporation could be similar to the permeabilisation found in the hydrodynamics method based upon the fast intravascular DNA injection. Neither the hydroporation nor the increase in DNA receptor-mediated endocytosis induce histologically detectable lesions, and the level of cell stress in the muscle, as measured by Hsp70 gene transcription induction, is similar whatever the injection speed. Thus the fast intratissular injection of DNA is a safe, easy and quick way to achieve local gene expression using naked DNA.