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1.O.2.  The Electroporation-induced Pore (EiP) Family 

Electroporation, the transient increase in the permeability of cell membranes when exposed to a high electric field, is an established in vitro technique and is used to introduce DNA or other molecules into cells. When the trans-membrane voltage induced by an external electric field exceeds a certain threshold (normally 0.2-1 V), a rearrangement of the molecular structure of the membrane occurs, leading to pore formation in the membrane and a considerable increase in the cell membrane permeability to ions, molecules and even macromolecules. This phenomenon is, potentially, the basis for many in vivo applications such as electrochemotherapy and gene therapy (Chen et al. 2006). Electrochemotherapy (ECT) can be used for the treatment for metastatic nodules of solid tumors on the skin or subcutaneous tissue. ECT is a combination of a physical effect, cell membrane poration, and cytotoxic drug administration (Giardino et al. 2006). Pulse duration dependent asymmetry in molecular transmembrane transport occurs during electroporation (Batista Napotnik and Miklavčič 2021). Electroporation sites in the complex lipid organization of the plasma membrane have been identified (Rems et al. 2022), and models of electroporation and the associated transmembrane molecular transport have been reviewed (Scuderi et al. 2022). Fluctuations in the electric parameters of the electroporated cells before and during electrofusion have been considered (Sherif et al. 2022). The complex interplay between pulse duration, electrical field strength and cell orientation on electroporation outcome have been considered using a time-dependent nonlinear numerical model (Scuderi et al. 2023).  Cell swelling giving rise to osmotic imbalance induced by electroporation has been examined (Qian et al. 2023). Genetically engineered HEK cells provide a valuable tool for studying electroporation in excitable cells (Batista Napotnik et al. 2024).

Single-cell electroporation (SCEP) has emerged for single-cell studies. When a large enough electric field is applied to a single cell, transient nano-pores form in the cell membrane allowing molecules to be transported into and out of the cell (Wang et al. 2010). Unlike bulk electroporation, in which a homogenous electric field is applied to a suspension of cells, in SCEP, an electric field is created locally near a single cell. Pore formation has been discussed from theoretical and experimental approaches. Current SCEP techniques using microelectrodes, micropipettes, electrolyte-filled capillaries, and microfabricated devices are all thoroughly discussed for adherent and suspended cells. SCEP has been applied in in-vivo and in-vitro studies for delivery of cell-impermeant molecules such as drugs, DNA, and siRNA, and for morphological observations (Wang et al. 2010). Lipid pores can be induced by external fields, stress, and peptides (Kirsch and Böckmann 2016). Electroporation induced by long monopolar and short bipolar pulses on pore formation in realistic 3D irregularly shaped cells has been studied (Chiapperino et al. 2020). During the application of unipolar pulse sequences, the pore radius and perforation area showed a step-like accumulation, and significant increases in the perforation area and intracellular ion concentration were observed with higher frequency pulse sequences and wider subpulse intervals (Guo et al. 2021). A bipolar cancellation effect was also observed in terms of membrane permeability and pore radius.

The applications of nanosecond, megavolt-per-meter electric field pulses to biological systems show cellular and subcellular electric field induced effects of electroporation. The absolute rate theory, with experimentally based parameter input, is consistent with membrane pore creation on a nanosecond time scale. Use of a Smoluchowski equation-based model for a planar cell membrane patch exposed to a 10 ns trapezoidal pulse with 1.5 ns rise and fall times, reversible supraelectroporation behavior in terms of transmembrane voltage, pore density, membrane conductance, fractional aqueous area, pore distribution, and average pore radius has been determined (Vasilkoski et al. 2006). Pore creation dominates the electrical response, and pore expansion proved to be negligible on this ns time scale.  The electric field induces lateral stress on the bilayer, manifested by surface tensions of magnitudes in the order of 1 mN.m-1 (Tarek 2005). The biphasic shape of DeltaVm at sites of shock-induced hyperpolarization is caused by membrane electroporation (Cheek and Fast 2004). Two methods, external fields and charge imbalance, are suitable for studying large-scale transitions of lipid membranes that involve changes of membrane capacitance (Kasparyan and Hub 2023).

Electroporation (EP) involves both nonlinear biophysical processes and complex geometries. When exposed to strong electric fields, the formation of pores within a cell membrane increases the membrane permeability. Discontinuous Galerkin (DG) finite element methods can directly enforce these flux jumps across the thin cell membrane interface. Sweeney and Davalos 2018 implemented a DG finite element method to model the electric field, pore formation, and transmembrane flux of charged solutes during EP.  Kotnik et al. 2019 reviewed the field of electroporation.  They first reviewed evidence for electrically induced membrane permeability, its correlation with transmembrane voltage, and continuum models of electropermeabilization that disregard the molecular-level structure and events. Second, they present insights from molecular-level modeling, particularly atomistic simulations that enhance an understanding of pore formation.  Third, they discuss evidence of chemical modifications of membrane lipids and functional modulation of membrane proteins affecting membrane permeability. Finally, they consider remaining challenges for a full understanding of electroporation and electropermeabilization (Kotnik et al. 2019). Flow Micropillar-array Electroporation (FME) systems have been developed to wisely regulate an important transmembrane-determining factor, namely cell size variations among individual cells, to achieve more effective transfection (Zu et al. 2019). Extracellular calcium ions inhibit the electrotransfer of small charged molecules, an effect that is related to an increased rate of membrane resealing (Navickaite et al. 2020). Pulsed electric fields can create pores in the voltage sensors of voltage-gated ion channels (Rems et al. 2020). Electroporation induced by long monopolar and short bipolar pulses has been studied (Chiapperino et al. 2020).

Cyclically alternating the membrane area leads to the generation of mechanoelectric current (El-Beyrouthy et al. 2022). This current is negligible without a transmembrane voltage until a composition mismatch between the membrane monolayers is produced, such as a one-sided accumulation of disruptive agents. The generated mechanoelectric current is eliminated when an applied electric field compensates for this asymmetry, enabling measurement of the transmembrane potential offset. Tracking the compensating voltage with respect to time then reveals the gradual accumulation of disruptive agents prior to membrane permeabilization (El-Beyrouthy et al. 2022). By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. Vindiš et al. 2022 expanded the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. The larger perforated area produced on cells in a microchip under a specific electric field allows a higher substance transfer efficiency, and the electroporation results are less affected by cell size. Furthermore, the relative perforation area increases with the decrease of the cell diameter in the microchip (Mou et al. 2023).

Nanochannel electroporation (NEP) is a new technology for cell transfection, which provides superior gene delivery and cell viability to conventional bulk electroporation (BEP). In NEP, the cells laid on a porous substrate are subjected to an asymmetric electric field which induces asymmetric membrane poration. The cell membrane near the channel outlet ('transfection membrane') is porated intensely, allowing direct delivery of genetic materials, while the rest of the cell membrane ('non-transfection membrane') remains much less perturbed for low cellular damage (Pan et al. 2023). Hydrophilic reactive oxygen species can not penetrate the membrane lipids through oxidation, but electroporation provided a new channel for reactive species to penetrate the membrane, and the oxidation effect reduced the electric field threshold of membrane electroporation (Cui et al. 2023).

References associated with 1.O.2 family:

Batista Napotnik, T. and D. Miklavčič. (2021). Pulse Duration Dependent Asymmetry in Molecular Transmembrane Transport Due to Electroporation in H9c2 Rat Cardiac Myoblast Cells In Vitro. Molecules 26:. 34770979
Batista Napotnik, T., B. Kos, T. Jarm, D. Miklavčič, R.P. O''Connor, and L. Rems. (2024). Genetically engineered HEK cells as a valuable tool for studying electroporation in excitable cells. Sci Rep 14: 720. 38184741
Cheek, E.R. and V.G. Fast. (2004). Nonlinear changes of transmembrane potential during electrical shocks: role of membrane electroporation. Circ Res 94: 208-214. 14670844
Chen, C., S.W. Smye, M.P. Robinson, and J.A. Evans. (2006). Membrane electroporation theories: a review. Med Biol Eng Comput 44: 5-14. 16929916
Chiapperino, M.A., L. Mescia, P. Bia, B. Staresinic, M. Cemazar, V. Novickij, A. Tabasnikov, S. Smith, J. Dermol-Cerne, and D. Miklavcic. (2020). Experimental and Numerical Study of Electroporation Induced by Long Monopolar and Short Bipolar Pulses on Realistic 3D Irregularly Shaped Cells. IEEE Trans Biomed Eng 67: 2781-2788. 32011999
Cui, Y., T. Zhao, H. Wang, X. Wang, D. Wang, and Y. Zhang. (2023). Molecular dynamics simulation of the transmembrane transport process of reactive species under the synergistic effect of plasma oxidation and an electric field. Free Radic Biol Med 208: 372-383. [Epub: Ahead of Print] 37657762
El-Beyrouthy, J., M.M. Makhoul-Mansour, and E.C. Freeman. (2022). Studying the Mechanics of Membrane Permeabilization through Mechanoelectricity. ACS Appl Mater Interfaces. [Epub: Ahead of Print] 35073482
Giardino, R., M. Fini, V. Bonazzi, R. Cadossi, A. Nicolini, and A. Carpi. (2006). Electrochemotherapy a novel approach to the treatment of metastatic nodules on the skin and subcutaneous tissues. Biomed Pharmacother 60: 458-462. 16930935
Guo, F., K. Qian, L. Zhang, X. Liu, and H. Peng. (2021). Multiphysics modelling of electroporation under uni- or bipolar nanosecond pulse sequences. Bioelectrochemistry 141: 107878. 34198114
Kasparyan, G. and J.S. Hub. (2023). Equivalence of Charge Imbalance and External Electric Fields during Free Energy Calculations of Membrane Electroporation. J Chem Theory Comput 19: 2676-2683. 37052575
Kirsch, S.A. and R.A. Böckmann. (2016). Membrane pore formation in atomistic and coarse-grained simulations. Biochim. Biophys. Acta. 1858: 2266-2277. 26748016
Kotnik, T., L. Rems, M. Tarek, and D. Miklavčič. (2019). Membrane Electroporation and Electropermeabilization: Mechanisms and Models. Annu Rev Biophys. [Epub: Ahead of Print] 30786231
Mou, Q., Y. Bai, M. Xu, D. Lv, J. Deng, N. Hu, and J. Yang. (2023). Microarray Chip and Method for Simultaneous and Highly Consistent Electroporation of Multiple Cells of Different Sizes. Anal Chem 95: 8533-8540. 37230941
Navickaite, D., P. Ruzgys, V. Novickij, M. Jakutaviciute, M. Maciulevicius, R. Sinceviciute, and S. Satkauskas. (2020). Extracellular-Ca-Induced Decrease in Small Molecule Electrotransfer Efficiency: Comparison between Microsecond and Nanosecond Electric Pulses. Pharmaceutics 12:. 32375426
Pan, J., C.L. Chiang, X. Wang, P. Bertani, Y. Ma, J. Cheng, V. Talesara, L.J. Lee, and W. Lu. (2023). Cell membrane damage and cargo delivery in nano-electroporation. Nanoscale 15: 4080-4089. 36744418
Qian, K., Y. Wang, Y. Lei, Q. Yang, and C. Yao. (2023). An experimental and theoretical study on cell swelling for osmotic imbalance induced by electroporation. Bioelectrochemistry 157: 108637. [Epub: Ahead of Print] 38215652
Rems, L., M.A. Kasimova, I. Testa, and L. Delemotte. (2020). Pulsed Electric Fields Can Create Pores in the Voltage Sensors of Voltage-Gated Ion Channels. Biophys. J. [Epub: Ahead of Print] 32559411
Rems, L., X. Tang, F. Zhao, S. Pérez-Conesa, I. Testa, and L. Delemotte. (2022). Identification of electroporation sites in the complex lipid organization of the plasma membrane. Elife 11:. 35195069
Scuderi, M., J. Dermol-Černe, C. Amaral da Silva, A. Muralidharan, P.E. Boukany, and L. Rems. (2022). Models of electroporation and the associated transmembrane molecular transport should be revisited. Bioelectrochemistry 147: 108216. 35932533
Scuderi, M., J. Dermol-Černe, T. Batista Napotnik, S. Chaigne, O. Bernus, D. Benoist, D.C. Sigg, L. Rems, and D. Miklavčič. (2023). Characterization of Experimentally Observed Complex Interplay between Pulse Duration, Electrical Field Strength, and Cell Orientation on Electroporation Outcome Using a Time-Dependent Nonlinear Numerical Model. Biomolecules 13:. 37238597
Sherif, S., Y.H. Ghallab, and Y. Ismail. (2022). Theoretical analysis for the fluctuation in the electric parameters of the electroporated cells before and during the electrofusion. Med Biol Eng Comput. [Epub: Ahead of Print] 36258107
Sweeney, D.C. and R.V. Davalos. (2018). Discontinuous Galerkin Model of Cellular Electroporation. Conf Proc IEEE Eng Med Biol Soc 2018: 5850-5853. 30441666
Tarek, M. (2005). Membrane electroporation: a molecular dynamics simulation. Biophys. J. 88: 4045-4053. 15764667
Vasilkoski, Z., A.T. Esser, T.R. Gowrishankar, and J.C. Weaver. (2006). Membrane electroporation: The absolute rate equation and nanosecond time scale pore creation. Phys Rev E Stat Nonlin Soft Matter Phys 74: 021904. 17025469
Vindiš, T., A. Blažič, D. Khayyat, T. Potočnik, S. Sachdev, and L. Rems. (2022). Gene Electrotransfer into Mammalian Cells Using Commercial Cell Culture Inserts with Porous Substrate. Pharmaceutics 14:. 36145709
Wang, M., O. Orwar, J. Olofsson, and S.G. Weber. (2010). Single-cell electroporation. Anal Bioanal Chem 397: 3235-3248. 20496058
Zu, Y., X. Liu, A.Y. Chang, and S. Wang. (2019). Flow micropillar array electroporation to enhance size specific transfection to a large population of cells. Bioelectrochemistry 132: 107417. [Epub: Ahead of Print] 31830670