To minimize neuromuscular electrical stimulation during electroporation-based treatments, the replacement of long monophasic pulses with bursts of biphasic high-frequency pulses in the range of microseconds was suggested in order to reduce muscle contraction and pain sensation due to pulse application. This treatment modality appeared under the term high-frequency electroporation (HF-EP), which can be potentially used for some clinical applications of electroporation such as electrochemotherapy, gene electrotransfer, and tissue ablation. In cardiac tissue ablation, which utilizes irreversible electroporation, the treatment is being established as Pulsed Field Ablation. While the reduction of muscle contractions was confirmed in multiple in vivo studies, the reduction of pain sensation in humans was not confirmed yet, nor was the relationship between muscle contraction and pain sensation investigated. This is the first study in humans examining pain sensation using biphasic high-frequency electroporation pulses. Twenty-five healthy individuals were subjected to electrical stimulation of the tibialis anterior muscle with biphasic high-frequency pulses in the range of few microseconds and both, symmetric and asymmetric interphase and interpulse delays. Our results confirm that biphasic high-frequency pulses with a pulse width of 1 or 2 µs reduce muscle contraction and pain sensation as opposed to currently used longer monophasic pulses. In addition, interphase and interpulse delays play a significant role in reducing the muscle contraction and/or pain sensation. The study shows that the range of the optimal pulse parameters may be increased depending on the prerequisites of the therapy. However, further evaluation of the biphasic pulse protocols presented herein is necessary to confirm the efficiency of the newly proposed HF-EP.

Electroporator is a generator of electric pulses that is used for permeabilization of cells. There are five major concepts of electroporation design. Capacitor discharge, square wave generator, and analog generator are used to generate classical electroporation pulses that are longer than microsecond and pulse forming network, and resonant charging generator that are used to generate nanosecond electroporation pulses. The choice of an electroporator design is always driven by the biotechnological or biomedical application. Electroporators can be used for introduction of small (electrochemotherapy) and large molecules (gene electrotransfer), cell fusion, insertion of proteins into cell membrane, electroporation of organelles, pasteurization, tissue ablation etc. Basic concepts and foreseeable future developments in electroporator design are presented in this article.

Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses. For this aim we used electroporator (EP-GMS 7.1) and developed new electrodes. We used finite-elements method to calculate and evaluate the electric field homogeneity between these new electrodes. Quick practical test was performed on confluent cell culture, to confirm and demonstrate electric field distribution. Then we experimentally evaluated the effectiveness of the new system and protocols on CHO cells. Gene transfection and cell survival were evaluated for different electric field protocols. The results of in-vitro gene electrotransfer experiments show that the fraction of transfected cells increases by changing the electric field direction between electrical pulses. The fluorescence intensity of transfected cells and cell survival does not depend on electric field protocol. Moreover, a new effect a shading effect was observed during our research. Namely, shading effect is observed during gene electrotransfer when cells are in clusters, where only cells facing negative electro-potential in clusters become transfected and other ones which are hidden behind these cells do not become transfected. On the basis of our results we can conclude that the new system can be used in in-vitro gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival.

In this Letter, we explore how cell electro-deformation and electro-poration are connected. We build a time-domain model of layered concentric shells (a model of biological cells) including their dielectric and elastic properties. We simulate delivery of one trapezoidal voltage pulse to either a single spherical cell or an assembly of three neighboring cells in a specific configuration and calculate cell deformation and pore formation. We describe the qualitative features of the electric field, surface charge density, transmembrane voltage, cell elongation, and pore density distribution at specific times i.e., before, during and after the application of the electric pulse and explore the correlations between them. Our results show that (1) the polarization charge redistribution plays a significant role in the spatial distribution of electrical stresses at μs time scales and (2) the cell deformation and pore density can be correlated with regions of high surface charge density. In future work, our model could be used for understanding basic mechanisms of electro-deformation and electro-poration with high-frequency short bipolar pulses of biological cells in suspension or tissues.