Permeabilizing Phospholipid Bilayers with Non-normal Electric Fields.

Since 2003, molecular dynamics simulations of lipid bilayers have provided valuable insights into the mechanisms underlying electropermeabilization (electroporation)-an electric field-induced increase in the permeability of biological membranes. The convention in these studies has been to apply the electric field normal to the plane of the membrane. In a typical electroporation application, however, where the electric field is reasonably uniform and unidirectional, the field is perpendicular to the membrane only at a few locations-for spherical cells only at the poles of the cells along the axis defined by the direction of the electric field. Everywhere else on the cell surface the field is applied at an angle that is oblique to the plane of the membrane. On a microscopic level, the invaginations and protrusions that characterize a living cell membrane also present many angles to the applied electric field. Here we report the results of molecular dynamics simulations of lipid electropore formation when the electric field is not normal to the membrane surface, which show that the tangential component of the field has a small but non-zero effect.

A double-pulse approach for electrotransfection.

Gene transfer and expression can be obtained by delivering calibrated electric pulses on cells in the presence of plasmids coding for the activity of interest. The electric treatment affects the plasma membrane and induces the formation of a transient complex between nucleic acids and the plasma membrane. It results in a delivery of the plasmid in the cytoplasm. Expression is only obtained if the plasmid is translocated inside the nucleus. This is a key limit in the process. We previously showed that delivery of a high-field short-duration electric pulse was inducing a structural alteration of the nuclear envelope. This study investigates if the double-pulse approach (first pulse to transfer the plasmid to the cytoplasm, and second pulse to induce the structural alteration of the envelope) was a way to enhance the protein expression using the green fluorescent protein as a reporter. We observed that not only the double-pulse approach induced the transfection of a lower number of cells but moreover, these transfected cells were less fluorescent than the cells treated only with the first pulse.

Effect of nanosecond pulsed electric field on Escherichia coli in water: inactivation and impact on protein changes.

AIMS: This article shows the effect of nanosecond pulsed electric field (nsPEF) on Escherichia coli, which could imply a durable change in protein expressions and then impacted the phenotype of surviving bacteria that might lead to increase pathogenicity. METHODS AND RESULTS: The effects of nsPEF on E. coli viability and membrane permeabilization were investigated. One log10 reduction in bacterial counts was achieved at field strength of 10(7) V m(-1) with a train of 500 successive pulses of 60 × 10(-9) s. Incubation of germs after treatment with propidium iodide showed that membrane permeabilization was reversible. Possible protein changes of surviving bacteria were checked to assess potential phenotypical changes using two-dimensional electrophoresis. In our study, after 40 generations, only UniProt #P39187 was up-regulated with P ≤ 0·05 compared with the control and corresponded to the uncharacterized protein YtfJ. Antibiograms were used to check whether or not the pattern of cultivable bacteria after nsPEF deliveries changed. CONCLUSIONS: The results tend to show that nsPEFs are able to inactivate bacteria and have probably no serious impact in E. coli protein patterns. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of nsPEF is a safe promising new nonthermal method for bacterial inactivation in the food processing and environmental industry.

Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells.

The RNA interference-mediated gene silencing approach is promising for therapies based on the targeted inhibition of disease-relevant genes. Electropermeabilization is one of the nonviral methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is effective in the field of gene silencing by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time lapse fluorescence microscopy and was correlated with the silencing of egfp expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. When added after electropulsation, the siRNA was inefficient for gene silencing because it did not penetrate the cells. Therefore, we report that an electric field acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The transfer kinetics of siRNA are compatible with the creation of nanopores, which are described with the technique of synthetic nanopores. The mechanism involved was clearly specific for the physico-chemical properties of the electrotransferred molecule and was different from that observed with small molecules or plasmid DNA.

Minicircle DNA electrotransfer for efficient tissue-targeted gene delivery.

A major issue for successful human gene therapy or genetic vaccination is a safe high-transgene expression level. Plasmid-based (non-viral) physical methods of gene transfer offered attracting approaches but their low efficiencies have limited their use in human pre-clinical trials. One of the limits appears to be the size of the plasmid that must be transferred across the cell membrane to the nucleus for its processing. In the present work to enhance gene transfer and expression, we evaluated a new generation of DNA vector; the minicircle, combined with the electropulsation technique. Minicircle is a doubled-stranded circular DNA with reduced size as it is devoid of bacterial sequences. We showed that electrotransferred minicircle encoding green fluorescent protein had higher in vitro transfection level compared with full-length plasmid. We demonstrated that minicircle great efficiency was not because of cellular toxicity decrease but was correlated to more efficient vector uptake by cells. Vector electrotransfection was operated in vivo and, using fluorescence imaging, minicircle electrotransfer was shown to enhance the efficiency and duration of tissue-targeted gene delivery and expression. By combining powerful expression and delivery systems, we have provided a valuable method for new approaches in gene therapy and genetic vaccination.

Flow process for electroextraction of total proteins from microalgae.

Classical methods for protein extraction from microorganisms, used for large-scale treatments such as mechanical or chemical processes, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow-process yeast electroextraction proved that pulsed electric field technology allows us to preserve the integrity of released cytosolic proteins by keeping intact vacuole membranes. Furthermore, large volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electroextract total cytoplasmic proteins from microalgae (Nannochloropsis salina and Chlorella vulgaris). Given that induction of electropermeabilization is under the control of the target cell size, as the mean diameter for N. salina is only 2.5 µm, we used repetitive 2-ms-long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24-h incubation period in a salty buffer. The amount of total protein released was evaluated by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris under milder electrical conditions, as expected from their larger size. This innovative technology designed in our group should become familiar in the field of microalgae biotechnology.

Muscle gene electrotransfer is increased by the antioxidant tempol in mice.

Electropermeabilization (EP) is an effective method of gene transfer into different tissues. During EP, reactive oxygen species (ROS) are formed, which could affect transfection efficiency. The role of generated ROS and the role of antioxidants in electrotransfer in myoblasts in vitro and in Musculus tibialis cranialis in mice were, therefore, investigated. We demonstrate in the study that during EP of C2C12 myoblasts, ROS are generated on the surface of the cells, which do not induce long-term genomic DNA damage. Plasmid DNA for transfection (pEGFP-N1), which is present outside the cells during EP, neutralizes the generated ROS. The ROS generation is proportional to the amplitude of the electric pulses and can be scavenged by antioxidants, such as vitamin C or tempol. When antioxidants were used during gene electrotransfer, the transfection efficiency of C2C12 myoblasts was statistically significantly increased 1.6-fold with tempol. Also in vivo, the transfection efficiency of M. tibialis cranialis in mice was statistically significantly increased 1.4-fold by tempol. The study indicates that ROS are generated on cells during EP and can be scavenged by antioxidants. Specifically, tempol can be used to improve gene electrotransfer into the muscle and possibly also to other tissues.

Electric field orientation for gene delivery using high-voltage and low-voltage pulses.

Electropermeabilization is a biological physical process in response to the presence of an applied electric field that is used for the transfer of hydrophilic molecules such as anticancer drugs or DNA across the plasma membranes of living cells. The molecular processes that support the transfer are poorly known. The aim of our study was to investigate the effect of high-voltage and low-voltage (HVLV) pulses in vitro with different orientations on cell permeabilization, viability and gene transfection. We monitored the permeabilization with unipolar and bipolar HVLV pulses with different train repetition pulses, showing that HVLV pulses increase cell permeabilization and cell viability. Gene transfer was also observed by measuring green fluorescent protein (GFP) expression. The expression was the same for HVLV pulses and electrogenotherapy pulses for in vitro experimentation. As the viability was better preserved for HVLV-pulsed cells, we managed to increase the number of GFP-expressing cells by up to 65% under this condition. The use of bipolar HVLV train pulses increased gene expression to a higher extent, probably by affecting a larger part of the cell surface.

Electrotransformation of Saccharomyces cerevisiae.

Intact yeast cell transformation is easily achieved by gene electrotransfer (GET). The procedure is fast and efficient in terms of transformants/µg DNA. Yeast cells in exponential growth phase are washed, treated for a short period with dithiothreitol (DTT) and then mixed with the plasmid DNA in a buffer with a low conductivity. A single well defined electric pulsed is delivered. After a 1 h incubation in the growth medium without selection, transformants are obtained on a selective plate medium. After a short description of the present knowledge on the events affecting the yeast cell as a consequence of the pulsed electric field, a step-by-step protocol is reported for Saccharomyces cerevisiae.

Control by pulse parameters of DNA electrotransfer into solid tumors in mice.

Electrotransfer (electroporation) is recognized as one of the most promising alternatives to viral vectors for transfection of different tissues in vivo for therapeutic purposes. We evaluated the transfection efficiency of reporter genes (green fluorescent protein and luciferase) in murine subcutaneous tumors using different combinations of high-field (HV) (600-1400 V cm(-1), 100 mus, 8 pulses) and low-field (LV) (80-160 V cm(-1), 50-400 ms, 1-8 pulses) pulses and compared it to protocol using eight identical pulses of 600 V cm(-1) and 5 ms duration (electro-gene therapy, EGT). Expression of GFP was determined using a fluorescent microscope and flow cytometry and expression of luciferase by measuring its activity using a luminometer. The EGT protocol yielded the highest expression of both reporter genes. However, a careful optimization of combinations of HV and LV pulses may result in similar transfection as EGT pulses. With the combination protocol, relatively high fields of LV pulses were necessary to obtain comparable transfection to the EGT protocol. Expression of reporter genes was higher in B16 melanoma than in SA-1 fibrosarcoma. Our data support the hypothesis that both electropermeabilization and electrophoresis are involved in electrotransfer of plasmid DNA, but demonstrate that these components have to happen at the same time to obtain significant expression of the target gene in tumors.

Electric pulse-induced fusion of 3T3 cells in monolayer culture.

Swiss mouse 3T3-C2 fibroblasts, grown to confluence in monolayer culture, are shown to fuse when exposed to electric fields. Exposure to five repetitive electric pulses of about 1 kilovolt per centimeter with a duration of 50 microseconds caused approximately 20 percent of the cells to become fused (multinucleate) when 1 millimolar magnesium was present in the medium. The effects of minimum thresholds of field strength, pulse duration, and number of pulses were determined. Cell disruption was observed when the electric field exceeded 2.0 kilovolts per centimeter or the pulse was of longer duration than 120 microseconds.

Lateral communication by fast proton conduction: a model membrane study.

Lateral communication of information along biological membranes is thought to be a key process for many cellular activities. Support for this hypothesis comes from physicochemical experiments that show that an efficient facilitated lateral proton conduction exists along lipid-water interfaces. The existence of a local two-dimensional hydrogen bond network between the lipid headgroups and their associated water molecules would explain this phenomenon.

Electrotransfer of CpG free plasmids enhances gene expression in skin.

Skin is a very suitable target for gene therapy and DNA vaccination due to its accessibility, its surface and its ability to produce transgenes. Gene electrotransfer (GET) to the skin is under development for clinical applications for DNA vaccine or local treatment such as wound healing. Local treatments are effective if the expression of the plasmid affects only the local environment (skin) by inducing an efficient concentration over a prolonged period. In this study, we evaluate the control of expression in the skin of a plasmid coding a fluorescent protein by its CpG (cytosine-phosphate-guanine motif) content. Two fluorescent reporter genes are evaluated: tdTomato and GFP. The expression is followed on the long term by in vivo fluorescence imaging. Our results show that GET mediated expression in the skin can be controlled by the CpG content of the plasmid. Long term expression (>120 days) can be obtained at high level with CpG-free constructs associated with a proper design of the electrodes where the field distribution mediating the gene electrotransfer is present deep in the skin.

Electrochemotherapy in veterinary oncology.

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.

Recommendations and requirements for reporting on applications of electric pulse delivery for electroporation of biological samples.

Electric field-induced membrane changes are an important approach in the life sciences. However, the developments in knowledge and translational applications face problems of reproducibility. Indeed, a quick survey of the literature reveals a lack of transparent and comprehensive reporting of essential technical information in many papers. Too many of the published scientific papers do not contain sufficient information for proper assessment of the presented results. The general rule/guidance in reporting experimental data should require details on exposure conditions such that other researchers are able to evaluate, judge and reproduce the experiments and data obtained. To enhance dissemination of information and reproducibility of protocols, it is important to agree upon nomenclature and reach a consensus on documentation of experimental methods and procedures. This paper offers recommendations and requirements for reporting on applications of electric pulse delivery for electroporation of biological samples in life science.

In vivo electrically mediated protein and gene transfer in murine melanoma.

We show that efficient permeabilization of murine melanoma can be obtained in vivo by applying electric pulses. More than 80% of the cell population is affected as shown by the penetration of propidium iodide. A protein, beta-galactosidase, can be transferred and expressed into the cells by incorporating either the protein or a plasmid carrying the reporter gene with respective efficiencies of 20% and 4%. This is obtained by a direct injection of either the protein or the plasmid in the tumor, followed by the application of electric pulses with surface electrodes in contact with the skin. This approach is simple and safe to use, reproducible, and specific; moreover, it is potentially applicable to a wide variety of tissues, cell types, and animals.

Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of ?) knowledge.

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.

Irreversibly electropermeabilized yeast retains the capability for ATP synthesis via oxidative phosphorylation.

ATP synthesis in irreversibly electropermeabilized yeast Kluyveromyces lactis was studied by using different respiratory substrates. The permeabilization itself provoked a dramatic decrease of the total ATP level and the cells lost their ability to synthesize ATP via glycolysis. The addition of exogenous NADH supported ATP synthesis in irreversibly permeabilized cells for up to 4-6 h after substrate addition when the total ATP level became twice that of intact cells incubated for the same period with lactose.

Post-pulse addition of trans-cyclohexane-1,2-diol improves electrotransfer mediated gene expression in mammalian cells.

Electric field mediated gene transfer is facing a problem in expression yield due to the poor transfer across the nuclear envelope. Trans-cyclohexane-1,2-diol (TCHD) was shown to significantly increase chemically mediated transfection by collapsing the permeability barrier of the nuclear pore complex. We indeed observed a significant increase in expression by electrotransfer when cells are treated post pulse by a low non toxic concentration of TCHD. This was obtained for different pulsing conditions, cell strains and plasmid constructs. An interesting improvement in cell viability can be obtained. This can significantly enhance the non-viral gene electrical delivery.

Electrochemotherapy guided by intraoperative fluorescence imaging for the treatment of inoperable peritoneal micro-metastases.

Surgery is often the first therapeutic indication in cancer. Patient survival essentially depends on the completeness of tumor resection. This is a major challenge, particularly in patients with peritoneal carcinomatosis (PC), where tumors are widely disseminated in the large peritoneal cavity. These small tumors can be difficult to visualize and are often positioned in delicate locations, further increasing the risk of producing serious tissue/organ damage during their ablation. We propose an innovative therapeutic approach based on intraoperative fluorescence (IF) guided electrochemotherapy (ECT) for the treatment of peritoneal micro-metastases. ECT combines the effects of tissue electro-permeabilization (EP) with the administration of an antimitotic agent (bleomycin) that has poor permeability across intact membranes. IF significantly improves the detection of small tumor lesions. ECT is clinically validated for the treatment of cutaneous tumors in animals and humans, but this is the first time that it has been used along with IF imaging for the targeted treatment of peritoneal metastases in a preclinical model. We set up a murine model of PC that develops secondarily to the resection of a distant primary tumor. Tumor growth and metastasis were finely monitored by non-invasive multimodal imaging (bioluminescence and 3D fluorescence/microCT). Once metastases were detected, mice were randomized into three groups: the ECT group (bleomycin injected intravenously followed by EP) and 2 control groups (bleomycin alone and EP alone). Twenty four hours after the intravenous injection of the tumor targeting agent Angiostamp™700, mice in all groups underwent an abdominal surgery for metastases exploration assisted by fluorescence imaging with the Fluobeam®700 portative device. EP was applied to every nodule detected by IF, except in the bleomycin control group. After surgery, the metastatic invasion was tracked by bioluminescence imaging. In mice treated with bleomycin or EP alone, the metastatic load progressed very rapidly and mice showed no significant difference in lifespan compared to non-operated mice (median lifespan: 27days vs. 25days, respectively). In contrast, the mice treated with ECT displayed a decreased metastatic load and an increased survival rate (median lifespan: 34days). These results provide evidence that IF guided ECT is an effective approach for the treatment of inoperable intraperitoneal micro-metastases.