ID: 2202

• Title:
  Subnanosecond Pulsed Electric Fields For Biological Cell Electropermeabilization
  
  Authors:
  

  Vallet Leslie A, Université Paris-Saclay, CNRS, METSY, Villejuif, France (affiliation 1)
  Ibrahimi Njomza, Université de Pau et des Pays de l’Adour/E2S UPPA, SIAME, Pau, France (affiliation 2)
  Ariztia Laurent, (affiliation 2)
  Rivaletto Marc, (2)
  Silvestre de Ferron Antoine, (affiliation 2)
  Novac Bucur M., Loughborough University, United Kingdom & (affiliation 2)
  Andre franck M, (affiliation 1) 
  Pecastaing laurent, (affiliation 2)
  Mir Lluis M., (affiliation 1)

  
  
  Abstract:
  The understanding of the effects of ultrashort electric pulses on the cell membrane is an important issue. Indeed, the ability of short pulsed electric fields (PEFs) to generate cell electroporation (reversible and irreversible) has been extensively studied for decades [1, 2]. From this knowledge has emerged a wide scope of applications in various domains. In the biomedical field, electroporation can be used to vectorize molecules inside the cells, which is the principle of therapeutic approaches such as electrochemotherapy [3] or electro-gene-transfer [4]. Electroporation can also be used as an ablative method, performing cell killing in itself [5]. In the food process industry, electroporation can be used to enhance extraction of cellular compounds [6], or can also be used for decontamination purposes [1, 7]. In the perspective of optimizing these applications, and to develop new ones, it is important to describe all the mechanisms involved in the electroporation phenomenon, taking advantage of the progress in the generation of transient voltage pulses of shorter and shorter durations. If the action of µsPEFs and nsPEFs on the cell membranes have been already well characterized, studies investigating the ability of subnanosecond pulsed electric fields to electroporate cells are still relatively scarce, mainly as a consequence of the lack of reliable subnanosecond cells exposure pulsed power systems [8]. The present work focuses on the effects of ca. 910 ps duration PEFs on the level of electroporation of bacteria (E. coli DH5α strain) to YO-PRO-I iodide, a cell-impermeant DNA binding dye. While some results are those expected (for example, the permeability level as a function of the number of pulses applied), other results are different from those expected. They have led to a careful examination of all the experimental conditions and to the elaboration of new mechanistic hypothesis that are being experimentally validated.
  
  
  
  METHODS
  
  The present study was performed using a pulsed power picosecond generator based on a pulse forming line having a peak voltage impulse of 20 kV, a duration varying between 910 and 925 ps full width at half maximum amplitude and a maximum pulse repetition frequency (PRF) of 200 Hz. The electric field intensity applied to the samples subjected to PEFs exposure was determined using detailed CST simulations, with real input data provided by V-dot sensors. The electroporation level of the DH5α strain of the E. coli bacteria. was assessed by flow cytometry analysis using the YO-PRO-1 iodide cell-impermeant DNA binding dye.
  
  
  
  RESULTS
  
  The results reveal an efficient electropermeabilization of E. coli to YO-PRO-1 iodide with an electric field amplitude in the order of tens of kV/cm. The evolution in the percentage of electroporated bacteria, and in the type of electroporation, with respect to the number of pulses applied was found as expected: the higher the number of pulses delivered, the higher the percentage of reversibly permeabilized bacteria. Similarly, more irreversible electropermeabilization was observed with the application of a higher number of pulses. The influence of the PRF was limited in the PRF range tested (0,1 to 200 Hz). On the contrary, the influence of the temperature during exposures was found important. Finally, the influence of the amplitude of the electric field was striking, pointing out mechanistic differences when reaching this timescale. In this context, it has been possible to exclude a number of artifacts, which demonstrates the strength of the results achieved. Therefore, further mechanistic investigations, involving novel explanations, are in progress.
  
  CONCLUSIONS
  
  The achievement of efficient electroporation (both reversible and irreversible) of E. coli using subnanosecond pulses of relatively low electric field amplitude is a step forward in the perspective of development of novel technologies of PEF delivery.
  
  
  
  Keywords:
  
  
  Refs:
  [1] Guionet, A., Joubert?Durigneux, V., Packan, D., Cheype, C., Garnier, J. P., David, F., ... & Blanckaert, V. (2014). Effect of nanosecond pulsed electric field on Escherichia coli in water: inactivation and impact on protein changes. Journal of applied microbiology, 117(3), 721-728. [2] Silve, A., Leray, I., Poignard, C., & Mir, L. M. (2016). Impact of external medium conductivity on cell membrane electropermeabilization by microsecond and nanosecond electric pulses. Scientific reports, 6, 19957. [3] Mir, L. M., Orlowski, S., Belehradek, J., Jr, & Paoletti, C. (1991). Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. European journal of cancer (Oxford, England: 1990), 27(1), 68–72. [4] Mir, L. M., Moller, P. H., André, F., & Gehl, J. (2005). Electric pulse-mediated gene delivery to various animal tissues. Advances in genetics, 54, 83–114. [5] Savic, L. J., Chapiro, J., Hamm, B., Gebauer, B., & Collettini, F. (2016). Irreversible Electroporation in Interventional Oncology: Where We Stand and Where We Go. RoFo : Fortschritte auf dem Gebiete der Rontgenstrahlen und der Nuklearmedizin, 188(8), 735–745. [6] Carpentieri, S., Režek Jambrak, A., Ferrari, G., & Pataro, G. (2022). Pulsed Electric Field-Assisted Extraction of Aroma and Bioactive Compounds From Aromatic Plants and Food By-Products. Frontiers in nutrition, 8, 792203. [7] Puligundla, P., Pyun, Y. R., & Mok, C. (2018). Pulsed electric field (PEF) technology for microbial inactivation in low-alcohol red wine. Food science and biotechnology, 27(6), 1691–1696. [8] Xiao, S., Semenov, I., Petrella, R., Pakhomov, A. G., & Schoenbach, K. H. (2017). A subnanosecond electric pulse exposure system for biological cells. Medical & biological engineering & computing, 55(7), 1063-1072. [9] Schoenbach, K. H., Xiao, S., Joshi, R. P., Camp, J. T., Heeren, T., Kolb, J. F., & Beebe, S. J. (2008). The effect of intense subnanosecond electrical pulses on biological cells. IEEE Transactions on plasma science, 36(2), 414-422. [10] Semenov, I., Xiao, S., & Pakhomov, A. G. (2016). Electroporation by subnanosecond pulses. Biochemistry and biophysics reports, 6, 253-259. [11] Gao, M., Xie, Y., Wang, S., Shang, S., Zhao, J., & Lu, X. (2021). A wideband picosecond pulsed electric fields (psPEF) exposure system for the nanoporation of biological cells. Bioelectrochemistry, 140, 107790.
  
  Topic 1:
  6. Cancer treatment and tumor ablation
  
  Topic 2:
  13. Pulsed power devices and methods