When high-amplitude, short-duration pulsed electric fields are applied to cells and tissues, the permeability of the cell membranes and tissue is increased. This increase in permeability is currently ...explained by the temporary appearance of aqueous pores within the cell membrane, a phenomenon termed electroporation. During the past four decades, advances in fundamental and experimental electroporation research have allowed for the translation of electroporation-based technologies to the clinic. In this review, we describe the theory and current applications of electroporation in medicine and then discuss current challenges in electroporation research and barriers to a more extensive spread of these clinical applications.
Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. ...Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.
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•IL-12 GET locally potentiates the antitumor response of ECT.•Treatment of tumors with ECT + IL-12 GET does not cause unwanted severe side effects.•ECT + GET i.t. therapy induces an ...antitumor immune response at the systemic level.•Higher expression of PDL-1 is associated with poorer response after ECT + IL-12 GET.•Treatment response should be assessed at least 2 months after treatment.
The combined treatment of electrochemotherapy (ECT) and interleukin-12 (IL-12) gene electrotransfer (GET) has already been used in clinical studies in dogs to treat various histological types of spontaneous tumors. The results of these studies show that the treatment is safe and effective. However, in these clinical studies, the routes of administration of IL-12 GET were either intratumoral (i.t.) or peritumoral (peri.t.). Therefore, the objective of this clinical trial was to compare the two IL-12 GET routes of administration in combination with ECT and their contribution to the enhanced ECT response. Seventy-seven dogs with spontaneous mast cell tumors (MCTs) were divided into three groups: one treated with a combination of ECT + GET peri. t. (29 dogs), the second with the combination of ECT + GET i.t. (30 dogs), and the third with ECT alone (18 dogs). In addition, immunohistochemical studies of tumor samples before treatment and flow cytometry of peripheral blood mononuclear cells (PBMCs) before and after treatment were performed to determine any immunological aspects of the treatment. The results showed that local tumor control was significantly better in the ECT + GET i.t. group (p < 0.050) than in the ECT + GET peri.t. or ECT groups. In addition, disease-free interval (DFI) and progression-free survival (PFS) were significantly longer in the ECT + GET i.t. group than in the other two groups (p < 0.050). The data on local tumor response, DFI, and PFS were consistent with immunological tests, as we detected an increased percentage of antitumor immune cells in the blood after treatment in the ECT + GET i.t. group, which also indicated the induction of a systemic immune response. In addition, we did not observe any unwanted severe or long-lasting side effects. Finally, due to the more pronounced local response after ECT + GET i.t., we suggest that treatment response assessment should be performed at least two months after treatment, which meets the iRECIST criteria.
Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination ...therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4 T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status.
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•Combination of electrochemotherapy and gene electrotransfer of IL-12 was tested.•Electrochemotherapy is more effective in more immunogenic tumors.•Gene electrotransfer exhibits greater contribution in less immunogenic tumors.•Therapy selection should be predominantly based on tumor immune status.
Wound healing is a complex biological response to injury characterized by a sequence of interdependent and overlapping physiological actions. To study wound healing and cutaneous regeneration ...processes, the complexity of wound healing requires the use of animal models. In this chapter, we describe the protocol to generate skin wounds in a mouse model. In the mouse splinted excisional wound model, two full-thickness wounds are firstly created on the mouse dorsum, which is followed by application of silicone splint around wounded area. A splinting ring tightly adheres to the skin around full-thickness wound, preventing wound contraction and replicating human processes of re-epithelialization and new tissue formation. The wound is easily accessible for treatment as well as for daily monitoring and quantifying the wound closure.This technique represents valuable approach for the study of wound healing mechanisms and for evaluation of new therapeutic modalities. In this protocol, we describe how to utilize the model to study the effect of gene electrotransfer of plasmid DNA coding for antiangiogenic molecules. Additionally, we also present how to precisely regulate electrical parameters and modify electrode composition to reach optimal therapeutic effectiveness of gene electrotransfer into skin around wounded area.
Introduction: Electroporation allows efficient delivery of DNA into cells and tissues, thereby improving the expression of therapeutic or immunogenic proteins that are encoded by plasmid DNA. This ...simple and versatile method holds a great potential and could address unmet medical needs such as the prevention or treatment of many cancers or infectious diseases.
Areas covered: This review explores the electroporation mechanism and the parameters affecting its efficacy. An analysis of past and current clinical trials focused on DNA electroporation is presented. The pathologies addressed, the protocol used, the treatment outcome and the tolerability are highlighted. In addition, several of the possible optimization strategies for improving patient compliance and therapeutic efficacy are discussed such as plasmid design, use of genetic adjuvants for DNA vaccines, choice of appropriate delivery site and electrodes as well as pulse parameters.
Expert opinion: The growing number of clinical trials and the results already available underline the strong potential of DNA electroporation which combines both safety and efficiency. Nevertheless, it remains critical to further increase fundamental knowledge to refine future strategies, to develop concerted and common DNA electroporation protocols and to continue exploring new electroporation-based therapeutic options.
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.
High-frequency bipolar electric pulses have been shown to mitigate undesirable muscle contraction during irreversible electroporation (IRE) therapy. Here, we evaluate the potential applicability of ...such pulses for introducing exogenous molecules into cells, such as in electrochemotherapy (ECT). For this purpose we develop a method for calculating the time course of the effective permeability of an electroporated cell membrane based on real-time imaging of propidium transport into single cells that allows a quantitative comparison between different pulsing schemes. We calculate the effective permeability for several pulsed electric field treatments including trains of 100μs monopolar pulses, conventionally used in IRE and ECT, and pulse trains containing bursts or evenly-spaced 1μs bipolar pulses. We show that shorter bipolar pulses induce lower effective membrane permeability than longer monopolar pulses with equivalent treatment times. This lower efficiency can be attributed to incomplete membrane charging. Nevertheless, bipolar pulses could be used for increasing the uptake of small molecules into cells more symmetrically, but at the expense of higher applied voltages. These data indicate that high-frequency bipolar bursts of electrical pulses may be designed to electroporate cells as effectively as and more homogeneously than conventional monopolar pulses.
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•Real-time imaging and permeability analysis were performed post-electroporation treatment.•Rapid bipolar electrical pulses induce lower membrane permeability than longer monopolar pulses.•Transmembrane transfer of molecules may be achieved using trains of short pulse widths.•Rapid bipolar pulses need larger amplitudes to disrupt cells similar to longer monopolar pulses.
Electrochemotherapy is a local ablative therapy that increases the cytotoxicity of either bleomycin or cisplatin by applying electric pulses (electroporation) to tumors. It has already been widely ...used throughout Europe for the treatment of various types of human and veterinary cutaneous tumors, with an objective response rate ranging from 70%-90%, depending on the tumor histotype. Recently, electrochemotherapy was introduced for the treatment of primary liver tumors, such as hepatocellular carcinoma (HCC). The complete response rate was 85% per treated lesion, with a durable response. Therefore, electrochemotherapy could become a treatment of choice for HCC, especially after achieving a transition from an open surgery approach to a percutaneous approach that uses dedicated electrodes. Electrochemotherapy elicits a local immune response and can be considered an in situ vaccination. HCC, among others, is a potentially immunogenic tumor; thus, electrochemotherapy could boost adjuvant immunotherapy to achieve a better and longer-lasting antitumor response. Therefore, therapeutic strategies that combine electrochemotherapy with immune checkpoint inhibitors or adjuvant treatment with cytokines are indicated for HCC. Immunogene therapy using electroporation as a delivery system for plasmid DNA coding for interleukin-12 is a highly promising approach. This electroporation approach has shown efficacy in preclinical settings and veterinary oncology and is awaiting translation for the treatment of liver tumors,
HCC.
The gene delivery to skeletal muscles is a promising strategy for the treatment of both muscular disorders (by silencing or overexpression of specific gene) and systemic secretion of therapeutic ...proteins. The use of a physical method like electroporation with plate or needle electrodes facilitates long-lasting gene silencing in situ. It has been reported that electroporation enhances the expression of the naked DNA gene in the skeletal muscle up to 100 times and decreases the changeability of the intramuscular expression. Coelectransfer of reporter genes such as green fluorescent protein (GFP), luciferase or beta-galactosidase allows the observation of correctly performed silencing in the muscles. Appropriate selection of plasmid injection volume and concentration, as well as electrotransfer parameters, such as the voltage, the length and the number of electrical pulses do not cause long-term damage to myocytes. In this review, we summarized the electroporation methodology as well as the procedure of electrotransfer to the gastrocnemius, tibialis, soleus and foot muscles and compare their advantages and disadvantages.
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