Aim: Electrochemotherapy is a local drug delivery approach aimed at treatment with palliative intent of cutaneous and subcutaneous tumour nodules of different histologies. Electrochemotherapy, via cell membrane permeabilising electric pulses, potentiates the cytotoxicity of nonpermeant or poorly permeant anticancer drugs with high intrinsic cytotoxicity, such as bleomycin or cisplatin, at the site of electric pulse application. Methods: An overview of preclinical and clinical studies is presented, and the treatment procedure is further critically evaluated. Results: In clinical studies electrochemotherapy has proved to be a highly efficient and safe approach for treating cutaneous and subcutaneous tumour nodules. The treatment response for various tumours (predominantly melanoma) was w75% complete and 10% partial response of the treated nodules. Conclusions: Electrochemotherapy is a new, clinically acknowledged method for the treatment of cutaneous and subcutaneous tumours. Its advantages are high effectiveness on tumours with different histologies, simple application, minimal side effects and the possibility of effective repetitive treatment.

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An increased permeability of a cell membrane during the application of high-voltage pulses results in increased transmembrane transport of molecules that otherwise cannot enter the cell. Increased permeability of a cell membrane is accompanied by increased membrane conductivity; thus, by measuring electric conductivity the extent of permeabilized tissue could be monitored in real time. In this article the effect of cell electroporation caused by high-voltage pulses on the conductivity of a cell suspension was studied by current-voltage measurements during and impedance measurement before and after the pulse application. At the same time the percentage of permeabilized and survived cells was determined and the extent of osmotic swelling measured. For a train of eight pulses a transient increase in conductivity of a cell suspension was obtained above permeabilization threshold in low- and high-conductive medium with complete relaxation in <1 s. Total conductivity changes and impedance measurements showed substantial changes in conductivity due to the ion efflux in low-conductive medium and colloid-osmotic swelling in both media. Our results show that by measuring electric conductivity during the pulses we can detect limit permeabilization threshold but not directly permeabilization level, whereas impedance measurements in seconds after the pulse application are not suitable.

The paper presents an approach that reduces several difficulties related to the determination of induced transmembrane voltage (ITV) on irregularly shaped cells. We first describe a method for constructing realistic models of irregularly shaped cells based on microscopic imaging. This provides a possibility to determine the ITV on the same cells on which an experiment is carried out, and can be of considerable importance in understanding and interpretation of the data. We also show how the finite-thickness, nonzero-conductivity membrane can be replaced by a boundary condition in which a specific surface conductivity is assigned to the interface between the cell interior (the cytoplasm) and the exterior. We verify the results obtained using this method by a comparison with the analytical solution for an isolated spherical cell and a tilted oblate spheroidal cell, obtaining a very good agreement in both cases. In addition, we compare the ITV computed for a model of two irregularly shaped CHO cells with the ITV measured on the same two cells by means of a potentiometric fluorescent dye, and also with the ITV computed for a simplified model of these two cells.

Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, DeltaPsi (m)) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of DeltaPsi (m) and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between DeltaPsi (m) and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of DeltaPsi (m) are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.

Abstract-Electroporation-based applications require the use of specific pulse parameters for a successful outcome. When recommended values of pulse parameters cannot be set, similar outcomes can be obtained by using equivalent pulse parameters. We determined the relations between the amplitude and duration/number of pulses resulting in the same fraction of electroporated cells. Pulse duration was varied from 150 ns to 100 ms, and the number of pulses from 1 to 128. Fura 2-AM was used to determine electroporation of cells to Ca 2+ . With longer pulses or higher number of pulses, lower amplitudes are needed for the same fraction of electroporated cells. The expression derived from the model of electroporation could describe the measured data on the whole interval of pulse durations. In a narrower range (0.1-100 ms), less complex, logarithmic or power functions could be used instead. The relation between amplitude and number of pulses could best be described with a power function or an exponential function. We show that relatively simple two-parameter power or logarithmic functions are useful when equivalent pulse parameters for electroporation are sought. Such mathematical relations between pulse parameters can be important in planning of electroporationbased treatments, such as electrochemotherapy and nonthermal irreversible electroporation.

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The transport of propidium iodide into electropermeabilized Chinese hamster ovary cells was monitored with a photomultiplier tube during and after the electric pulse. The influence of pulse amplitude and duration on the transport kinetics was investigated with time resolutions from 200 ns to 4 ms in intervals from 400 micros to 8 s. The transport became detectable as early as 60 micros after the start of the pulse, continued for tens of seconds after the pulse, and was faster and larger for higher pulse amplitudes and/or longer pulse durations. With fixed pulse parameters, transport into confluent monolayers of cells was slower than transport into suspended cells. Different time courses of fluorescence increase were observed during and at various times after the pulse, reflecting different transport mechanisms and ongoing membrane resealing. The data were compared to theoretical predictions of the Nernst-Planck equation. After a delay of 60 micros, the time course of fluorescence during the pulse was approximately linear, supporting a mainly electrophoretic solution of the Nernst-Planck equation. The time course after the pulse agreed with diffusional solution of the Nernst-Planck equation if the membrane resealing was assumed to consist of three distinct components, with time constants in the range of tens of microseconds, hundreds of microseconds, and tens of seconds, respectively.