Cyclin B1 knockdown mediated by clinically approved pulsed electric fields siRNA delivery induces tumor regression in murine melanoma.

RNA interference (RNAi) represents a promising therapy for the specific inhibition of gene expression in targeted tissues including tumors. To realize the therapeutic potential of RNAi drugs, non-immunogenic, efficient, and tissue-specific delivery technologies must be developed. We have previously shown that pulsed electric field (PEF) can deliver siRNAs into tumor cells thanks to long electrophoretic drift occurring during the use of millisecond duration pulses. Here, optical fluorescence imaging is used at first to evaluate the efficiency of microsecond-duration pulses for siRNA delivery. These pulsed electric fields (PEF) parameters, which are already used in clinics for electrochemotherapy (ECT) were compared to previous parameters optimized for electrogenotherapy (EGT) that use microsecond-duration pulses. Secondly, these PEF protocols were evaluated for the delivery of specific siRNAs targeting the cyclin B1 in subcutaneous tumors in mice. When a single treatment was performed, millisecond duration pulses led to a better efficiency. However, when multiple treatments were performed, both protocols were equally efficient and potentially silenced cyclin B1 endogenous gene, leading to a tumor growth reduction. Our findings provide insights into pulsed electric field-siRNA delivery that could benefit from existing clinical protocols for siRNA delivery to tumors.

Calcium electroporation: The bioelectrochemical treatment of spontaneous equine skin tumors results in a local necrosis.

Electrochemotherapy (ECT) is an anticancer bioelectrochemical therapy where electrical field pulses (electropermeabilization) increase intracellular concentration of antitumor drugs. The procedure is very effective against skin tumors. The restrictive regulations concerning anticancer drugs in veterinary medicine limit use of ECT. Electroporation with calcium (Electroporation Calcium Therapy)(ECaT) was proved to be effective in vivo on induced tumors in laboratory animals. This study evaluated the effects of ECaT in equine sarcoids (spontaneous skin tumors) on an animal cohort. Pulse parameters for ECaT were choosen for using skin contact electrodes. ECaT was applied under general anesthesia. The tumors were removed at different days after the treatment and analyzed by histology. The study assessed the volume fraction of necrosis that was >50% for 9 of 13 sarcoids. Sixteen sarcoids in 10 horses were treated with ECaT. Macroscopic changes (a crust) were observed in 14/16 tumors. The main microscopic changes were necrosis, ulceration,hemorrhages, calcifications and thrombosis. The adverse effect was an inflammatory local reaction. Surrounding tissues were not affected. This targeted effect can be explained by its control by the field distribution in the tissue and on the interstitial diffusion of the injected Ca2+.

Pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma.

BACKGROUND: Melanoma is a very aggressive skin tumor that can be cured when diagnosed and treated in its early stages. However, at the time of identification, the tumor is frequently in a metastatic stage. Intensive research is currently ongoing to improve the efficacy of the immune system in eliminating cancer cells. One approach is to boost the activation of cytotoxic T cells by IL-12 cytokine that plays a central role in the activation of the immune system. In parallel, physical methods such as electropermeabilization-based treatments are currently under investigation and show promising results. METHODS: In this study, we set electrical parameters to induce a partial-irreversible electropermeabilization (pIRE) of melanoma to induce a sufficient cell death and potential release of tumor antigens able to activate immune cells. This protocol mimics the situation where irreversible electropermeabilization is not fully completed. Then, a peritumoral plasmid IL-12 electrotransfer was combined with pIRE treatment. Evaluation of the tumor growth and survival was performed in mouse strains having a different immunological background (C57Bl/6 (WT), nude and C57Bl6 (TLR9-/-)). RESULTS: pIRE treatment induced apoptotic cell death and a temporary tumor growth delay in all mouse strains. In C57Bl/6 mice, we showed that peritumoral plasmid IL-12 electrotransfer combined with tumor pIRE treatment induced tumor regression correlating with a local secretion of IL-12 and IFN-γ. This combined treatment induced a growth delay of distant tumors and prevented the emergence of a second tumor in 50% of immunocompetent mice. CONCLUSIONS: The combination of pIL-12 GET and pIRE not only enhanced survival but could bring a curative effect in wild type mice. This two-step treatment, named Immune-Gene Electro-Therapy (IGET), led to a systemic activation of the adaptive immune system and the development of an anti-tumor immune memory.

A protein nanocontainer targeting epithelial cancers: rational engineering, biochemical characterization, drug loading and cell delivery.

The development of drug delivery and imaging tools is a major challenge in human health, in particular in cancer pathologies. This work describes the optimization of a protein nanocontainer, belonging to the lectin protein family, for its use in epithelial cancer diagnosis and treatment. Indeed, it specifically targets a glycosidic marker, the T antigen, which is known to be characteristic of epithelial cancers. Its quaternary structure reveals a large hydrated inner cavity able to transport small therapeutic molecules. Optimization of the nanocontainer by site directed mutagenesis allowed controlling loading and release of confined drugs. Doxorubicin confinement was followed, both theoretically and experimentally, and provided a proof of concept for the use of this nanocontainer as a vectorization system. In OVCAR-3 cells, a human ovarian adenocarcinoma cell line that expresses the T antigen, the drug was observed to be delivered inside late endosomes/lysosomes. These results show that this new type of vectorization and imaging device opens new exciting perspectives in nano-theranostic approaches.

Noninvasive Gene Electrotransfer in Skin.

The skin is considered as well suited for gene therapy and vaccination. DNA vaccines elicit both broad humoral and cellular immune responses when injected in the skin. Physical and chemical methods are needed to boost the expression. Gene electrotransfer (GET) is one of the most effective approaches. This step-by-step protocol describes the procedures to obtain an efficient GET targeted to the skin by using easy-to-use noninvasive electrodes after intradermal plasmid injection (i.d. GET). A specific pulse sequence is reported. Expression is observed by in vivo fluorescence imaging during >2 weeks as the plasmid was coding for tdTomato. The protocol is efficient for the transient expression of clinical proteins.

Safe and efficient novel approach for non-invasive gene electrotransfer to skin.

Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.

In Vivo Evaluation of a New Recombinant Hyaluronidase to Improve Gene Electro-Transfer Protocols for DNA-Based Drug Delivery against Cancer.

Cancer vaccines based on plasmid DNA represent a good therapeutic perspective, despite their low potency. Animal-derived hyaluronidases (Hyals) are employed in oncological clinical practice. Hyal has been also demonstrated to be a good enhancer of intramuscular Gene Electro-Transfer (GET) efficiency in anti-cancer preclinical protocols, with increased transfected cells and higher expression of the encoded genes. Nevertheless, the use of animal-derived Hyals results limited respect to their potentialities, since such preparations could be affected by low purity, variable potency and uncertain safety. To improve the delivery of intramuscular GET-based protocols in mouse, we investigated a new recombinant Hyal, the rHyal-sk, to assess in vivo safety and activity of this treatment at cellular and biochemical levels. We evaluated the cellular events and the inflammation chemical mediators involved at different time points after rHyal-sk administration plus GET. Our results demonstrated the in vivo safety and efficacy of rHyal-sk when injected once intramuscularly in association with GET, with no toxicity, good plasmid in-take ability, useful inflammatory response activation, and low immunogenicity. Following these findings, we would recommend the use of the new rHyal-sk for the delivery of DNA-based vaccines and immunotherapy, as well as into clinical practice, for tumor disease treatments.

Spatio-temporal dynamics of calcium electrotransfer during cell membrane permeabilization.

Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10 µs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80 kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45 kV/cm and 100 pulses at 1 kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.

Increased permeability of blood vessels after reversible electroporation is facilitated by alterations in endothelial cell-to-cell junctions.

Delivery of electric field pulses, i.e. electroporation (EP), to tissues has been shown to have a blood flow modifying effect. Indeed, the diameter of blood vessels exposed to EP is immediately reduced resulting in blood flow abrogation, followed by an increase in vascular permeability. The main cause of the increased permeability remains unknown. The aim of this study was to determine whether the in vivo effects of EP on permeability of blood vessels are linked to the permeabilization of endothelial cells' membrane (EC) and/or disruption of cell-to-cell junctions. We used a dorsal window chamber model in C57Bl/6 mice coupled with multiphoton microscopy and fluorescently labelled antibodies against PECAM-1 (CD31) to visualize endothelial cell-to-cell junctions. Clinically validated EP parameters were used and behavior of cell-to-cell junctions, in combination with leakage of 70 kDa fluorescein isothiocyanate labelled dextran (FD), was followed in time. After EP, a constriction of blood vessels was observed and correlated with the change in the shape of ECs. This was followed by an increase in permeability of blood vessels for 70 kDa FD and a decrease in the volume of labelled cell-to-cell junctions. Both parameters returned to pre-treatment values in 50% of mice. For the remaining 50%, we hypothesize that disruption of cell-to-cell junctions after EP may trigger the platelet activation cascade. Our findings show for the first time in vivo that alterations in cell-to-cell junctions play an important role in the response of blood vessels to EP and explain their efficient permeabilization.

Control by Low Levels of Calcium of Mammalian Cell Membrane Electropermeabilization.

Electric pulses, when applied to a cell suspension, induce a reversible permeabilization of the plasma membrane. This permeabilized state is a long-lived process (minutes). The biophysical molecular mechanisms supporting the membrane reorganization associated to its permeabilization remain poorly understood. Modeling the transmembrane structures as toroidal lipidic pores cannot explain why they are long-lived and why their resealing is under the control of the ATP level. Our results describe the effect of the level of free Calcium ions. Permeabilization induces a Ca2+ burst as previously shown by imaging of cells loaded with Fluo-3. But this sharp increase is reversible even when Calcium is present at a millimolar concentration. Viability is preserved to a larger extent when submillimolar concentrations are used. The effect of calcium ions is occurring during the resealing step not during the creation of permeabilization as the same effect is observed if Ca2+ is added in the few seconds following the pulses. The resealing time is faster when Ca2+ is present in a dose-dependent manner. Mg2+ is observed to play a competitive role. These observations suggest that Ca2+ is acting not on the external leaflet of the plasma membrane but due to its increased concentration in the cytoplasm. Exocytosis will be enhanced by this Ca2+ burst (but hindered by Mg2+) and occurs in the electropermeabilized part of the cell surface. This description is supported by previous theoretical and experimental results. The associated fusion of vesicles will be the support of resealing.

A new mechanism for efficient hydrocarbon electro-extraction from Botryococcus braunii.

BACKGROUND: Recent understanding that specific algae have high hydrocarbon production potential has attracted considerable attention. Botryococcus braunii is a microalga with an extracellular hydrocarbon matrix, which makes it an appropriate green energy source. RESULTS: This study focuses on extracting oil from the microalgae matrix rather than the cells, eliminating the need for an excessive electric field to create electro-permeabilization. In such a way, technical limitations due to high extraction energy and cost can be overcome. Here, nanosecond pulsed electric fields (nsPEF) with 80 ns duration and 20-65 kV/cm electric fields were applied. To understand the extraction mechanism, the structure of the algae was accurately studied under fluorescence microscope; extraction was quantified using image analysis; quality of extraction was examined by thin-layer chromatography (TLC); and the cell/matrix separation was observed real-time under a microscope during nsPEF application. Furthermore, optimization was carried out by screening values of electric fields, pulse repetition frequencies, and energy spent. CONCLUSIONS: The results offer a novel method applicable for fast and continues hydrocarbon extraction process at low energy cost.

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats.

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane permeabilization by means of "electroporation". To achieve the "electroporation" of the cells, electric pulses are generated by a generator and delivered to the target tissue by the use of electrodes. Electroporation is a physical method which is used to introduce molecules, like cytostatic drugs, into the cells that could not pass the cell membrane on their own. In electrochemotherapy, currently, cisplatin and bleomycin are clinically used. Electrochemotherapy antitumor effectiveness is high, for example up to 100% complete response of canine mast cell tumors smaller than 2 cm3 was achieved. Additionally, electrochemotherapy can be used for the treatment of inoperable tumors. One of the important characteristics of electrochemotherapy is that it can be effective as a one-time treatment only. However, in the case of failure or partial tumor response it can be repeated several times with equal or improved effectiveness. Electrochemotherapy is already a standard treatment for cutaneous and subcutaneous tumors of various histologies in human and veterinary oncology. Furthermore, several clinical studies exploiting electrochemotherapy for deep-seated tumors are on-going.

Energy-efficient biomass processing with pulsed electric fields for bioeconomy and sustainable development.

Fossil resources-free sustainable development can be achieved through a transition to bioeconomy, an economy based on sustainable biomass-derived food, feed, chemicals, materials, and fuels. However, the transition to bioeconomy requires development of new energy-efficient technologies and processes to manipulate biomass feed stocks and their conversion into useful products, a collective term for which is biorefinery. One of the technological platforms that will enable various pathways of biomass conversion is based on pulsed electric fields applications (PEF). Energy efficiency of PEF treatment is achieved by specific increase of cell membrane permeability, a phenomenon known as membrane electroporation. Here, we review the opportunities that PEF and electroporation provide for the development of sustainable biorefineries. We describe the use of PEF treatment in biomass engineering, drying, deconstruction, extraction of phytochemicals, improvement of fermentations, and biogas production. These applications show the potential of PEF and consequent membrane electroporation to enable the bioeconomy and sustainable development.

Imaging of Electrotransferred siRNA.

SiRNA delivery to the cytoplasm can be obtained through the application of calibrated electric field pulses to a mixture of cells and oligonucleotides. To investigate the uptake pathway, time lapse confocal fluorescence microscopy provides a direct visualization of the transfer. SiRNA is electrophoretically drifted directly to the cytoplasm during the pulse. No post pulse transfer is observed. The uploaded siRNA then freely diffuse in the cytoplasm with no access to the nuclei.

Electric Destabilization of Supramolecular Lipid Vesicles Subjected to Fast Electric Pulses.

Biological membranes are weakly permeable to hydrophilic molecules and ions and electric pulses can induce their transient permeabilization, but this process is not well characterized. We directly assay the electropermeabilization process, on the minimum model of lipid vesicles, by using a highly sensitive fluorescence method based on manganese ion transport. The approach gives access, at the single-lipid self-assembly level, to the transmembrane potential needed to detect divalent ion permeabilization on supramolecular giant unilamellar lipid vesicles. The critical values are strongly dependent on the lipid composition and are observed to vary from 10 to 150 mV. These values appear to be much lower than those previously reported in the literature for cells and vesicles. The detection method appears to be a decisive parameter as it is controlled by the transport of the reporter dye. We also provide evidence that the electropermeabilization process is a transient transition of the lipid self-organization due to the loss of assembly cohesion induced by bioelectrochemical perturbations of the zwitterionic interface with the solution.

Electrochemotherapy of tumors as in situ vaccination boosted by immunogene electrotransfer.

Electroporation is a platform technology for drug and gene delivery. When applied to cell in vitro or tissues in vivo, it leads to an increase in membrane permeability for molecules which otherwise cannot enter the cell (e.g., siRNA, plasmid DNA, and some chemotherapeutic drugs). The therapeutic effectiveness of delivered chemotherapeutics or nucleic acids depends greatly on their successful and efficient delivery to the target tissue. Therefore, the understanding of different principles of drug and gene delivery is necessary and needs to be taken into account according to the specificity of their delivery to tumors and/or normal tissues. Based on the current knowledge, electrochemotherapy (a combination of drug and electric pulses) is used for tumor treatment and has shown great potential. Its local effectiveness is up to 80 % of local tumor control, however, without noticeable effect on metastases. In an attempt to increase systemic antitumor effectiveness of electrochemotherapy, electrotransfer of genes with immunomodulatory effect (immunogene electrotransfer) could be used as adjuvant treatment. Since electrochemotherapy can induce immunogenic cell death, adjuvant immunogene electrotransfer to peritumoral tissue could lead to locoregional effect as well as the abscopal effect on distant untreated metastases. Therefore, we propose a combination of electrochemotherapy with peritumoral IL-12 electrotransfer, as a proof of principle, using electrochemotherapy boosted with immunogene electrotransfer as in situ vaccination for successful tumor treatment.

Content Delivery of Lipidic Nanovesicles in Electropermeabilized Cells.

Lipidic nanovesicles (the so-called liposomes) were among the one of the earliest forms of nanovectors. One of their limits was our lack of knowledge on the delivery pathway of their content to the target cell cytoplasm. In most models, it appears to be linked to endocytotic transfer. Their direct content delivery can be enhanced by electric field pulses applied to a cell liposomes mixture. The optimal form for liposomes was shown to be large unilamellar vesicles (LUV). The present communication describes an optimization to enhance the delivery. When lipidic nanovesicles (LUVs) are electrostatically brought in contact with electropermeabilized cells by a salt bridge, their content is delivered into the cytoplasm of electropermeabilized cells. The PEF parameters are selected to affect specifically the cells leaving the vesicles unaffected. Cell viability is positively affected by the treatment. High-field short pulses are more efficient than low-field long pulses. A homogeneous cytoplasm labeling is observed under digitized videomicroscopy. The process is a content mixing, not an endocytotic pathway. The lipidic composition of the LUV should contain charged lipids (phosphatidylserine), fusion promoting lipids (phosphatidylethanolamine), and cholesterol.

A Comparative Study on the Effects of Millisecond- and Microsecond-Pulsed Electric Field Treatments on the Permeabilization and Extraction of Pigments from Chlorella vulgaris.

The interdependencies of the two main processing parameters affecting "electroporation" (electric field strength and pulse duration) while using pulse duration in the range of milliseconds and microseconds on the permeabilization, inactivation, and extraction of pigments from Chlorella vulgaris was compared. While irreversible "electroporation" was observed above 4 kV/cm in the millisecond range, electric field strengths of ≥10 kV/cm were required in the microseconds range. However, to cause the electroporation of most of the 90 % of the population of C. vulgaris in the millisecond (5 kV/cm, 20 pulses) or microsecond (15 kV/cm, 25 pulses) range, the specific energy that was delivered was lower for microsecond treatments (16.87 kJ/L) than in millisecond treatments (150 kJ/L). In terms of the specific energy required to cause microalgae inactivation, treatments in the microsecond range also resulted in greater energy efficiency. The comparison of extraction yields in the range of milliseconds (5 kV, 20 ms) and microseconds (20, 25 pulses) under the conditions in which the maximum extraction was observed revealed that the improvement in the carotenoid extraction was similar and chlorophyll a and b extraction was slightly higher for treatments in the microsecond range. The specific energy that was required for the treatment in the millisecond range (150 kJ/L) was much higher than those required in the microsecond range (30 kJ/L). The comparison of the efficacy of both types of pulses on the extraction enhancement just after the treatment and after a post-pulse incubation period seemed to indicate that PEF in the millisecond range created irreversible alterations while, in the microsecond range, the defects were a dynamic structure along the post-pulse time that caused a subsequent increment in the extraction yield.

Versatile cellular uptake mediated by catanionic vesicles: simultaneous spontaneous membrane fusion and endocytosis.

Lactose-derived catanionic vesicles offer unique opportunities to overcome cellular barriers. These potential nanovectors, very easy to formulate as drug delivery systems, are able to encapsulate drugs of various hydrophilicity. This article highlights versatile interaction mechanisms between these catanionic vesicles, labeled with hydrophilic and amphiphilic fluorescent probes, and a mammalian cell line, Chinese Hamster Ovary. Confocal microscopy and flow cytometry techniques show that these vesicles are internalized by cells through cellular energy dependent processes, as endocytosis, but are simultaneously able to spontaneously fuse with cell plasma membranes and release their hydrophilic content directly inside the cytosol. Such innovative and polyvalent nanovectors, able to deliver their content via different internalization pathways, would positively be a great progress for the coadministration of drugs of complementary efficiency.