Mechanisms of electroporation and physicochemical factors of importance to electroporation-based therapies

Šmerc, Rok (author), Mahnič-Kalamiza, Samo (mentor)

Electroporation is a biophysical phenomenon induced by the application of short electrical high-voltage pulses to biological tissue. These pulses temporarily increase the permeability of the cell membrane, allowing ions and molecules to which the membrane is otherwise impermeable to pass through, thus entering or leaving the cells. Depending on the pulse parameters, electroporation can either be reversible, allowing the cells to recover, or irreversible, leading to cell death. Thanks to its precise control over membrane permeability, electroporation is widely used in biomedicine, biotechnology, and food processing. Its applications range from tumour ablation, gene therapy, electrochemotherapy, treatment of cardiac arrhythmias to various applications in the food industry. Despite the widespread use of electroporation, several important factors influencing its efficacy remain poorly understood. This doctoral dissertation investigates three such factors: skeletal muscle anisotropy, electrochemically induced pH changes, and electroosmotic flow, and explores their implications for electroporation-based treatments. Firstly, the anisotropic electrical behaviour of skeletal muscle tissue was investigated in detail. Multiscale numerical models were developed to represent skeletal muscle from the level of individual cells up to the tissue level, capturing how the microscale structure influences the macroscopic electrical properties. The models and findings were validated by in vivo ablation experiments as well as ex vivo MRI-based current density imaging and impedance spectroscopy. Together, these approaches have demonstrated that the anisotropy of skeletal muscle significantly influences the electric field distribution, lesion geometry, and current pathways. Experimental and numerical results have shown that the anisotropic electrical properties originate primarily from the cell plasma membrane. Secondly, a mechanistic numerical model was constructed to predict the electrochemically induced pH dynamics associated with electroporation pulses, particularly relevant for gene electrotransfer protocols. This model accounted for the electrochemical reactions at the electrodes and was experimentally validated using agarose gels with embedded pH indicators. A calibrated imaging system was developed to enable real-time tracking of pH fronts. The developed model reliably captured the spatial and temporal evolution of acidic and alkaline zones, providing quantitative insights into the extent and implications of pH variations during electroporation treatments. Thirdly, electroosmotic flow, a relatively understudied phenomenon of electrically induced fluid transport, was experimentally characterised in plant tissue (potato), animal skeletal muscle, and agarose phantoms under pulsed electric field (PEF) and direct current (DC) conditions. The experimental results demonstrated a significant electroosmotically driven contribution to tissue deformation and fluid movement, revealing synergistic effects between electroporation-induced membrane permeabilisation and subsequent electroosmotic fluid transport, especially at lower current amplitudes. This systematic characterisation highlights the potential role of electroosmotic flow in influencing fluid motion during electroporation and supports its consideration in the development of future electroporation-based applications. The dissertation comprises five original scientific papers, each presenting experimental and theoretical advances. Collectively, the work advances our understanding of the mechanisms and physicochemical factors shaping electroporation-based treatments, offering new insights and practical contributions for future applications. Together, the findings and models provide a solid foundation for improving the design and optimisation of electroporation-based protocols across biomedical, biotechnological, and food-related applications.

• Language: English
• Keywords: electroporation, electroporation-based treatments, pulsed electric field (PEF) treatment, pulsed field ablation (PFA), numerical modelling, skeletal muscle anisotropy, electrochemistry, pH changes, electroosmosis
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FE - Faculty of Electrical Engineering
• Year: 2025
• PID: 20.500.12556/RUL-170512
• COBISS.SI-ID: 242086147
• Publication date in RUL: 08.07.2025

Secondary language

• Language: Slovenian
• Title: Mehanizmi elektroporacije in fizikalno-kemijski dejavniki pomembni pri terapijah z elektroporacijo

Abstract

Elektroporacija je biofizikalni pojav, ki ga povzroči dovajanje kratkih visokonapetostnih električnih pulzov biološkemu tkivu. Dovajanje pulzov povzroči začasno povečanje prepustnosti celične membrane, kar omogoči prehod ionov in molekul, ki sicer ne bi mogli skozi membrano, v celico ali iz nje. Odvisno od parametrov pulzov je elektroporacija lahko reverzibilna, pri kateri si celice po določenem času opomorejo, ali ireverzibilna, ki vodi v celično smrt. Zaradi možnosti selektivnega spreminjanja prepustnosti celične membrane se elektroporacija široko uporablja v biomedicini, biotehnologiji in živilski industriji. Njene aplikacije segajo od ablacije tumorjev, genskega zdravljenja, elektrokemoterapije in zdravljenja srčnih aritmij do različnih postopkov v živilski industriji. Kljub razširjeni uporabi elektroporacije ostaja več pomembnih dejavnikov, ki vplivajo na njeno učinkovitost, slabo raziskanih. Ta doktorska disertacija obravnava tri takšne dejavnike: anizotropijo skeletne mišice, elektrokemično povzročene spremembe pH, in elektroosmozni tok, ter raziskuje njihov pomen za terapije, temelječe na elektroporaciji. Najprej smo podrobno raziskali anizotropno električno obnašanje skeletne mišice. Razvili smo večnivojske numerične modele, ki predstavljajo skeletno mišico od ravni posameznih celic do ravni celotnega tkiva, pri čemer zajamejo vpliv mikroskopske strukture na makroskopske električne lastnosti. Modele in izsledke smo potrdili z ablacijskimi poskusi in vivo, in z MRI-slikanjem gostote toka ter impedančno spektroskopijo ex vivo. Skupaj so ti pristopi pokazali, da anizotropija skeletne mišice pomembno vpliva na porazdelitev električnega polja, geometrijo lezij, in poti električnega toka v tkivu. Eksperimentalni in numerični rezultati so pokazali, da anizotropne električne lastnosti mišice izvirajo predvsem iz neprepustnosti plazemske membrane celice. V drugem delu smo razvili mehanistični numerični model za napovedovanje elektrokemično povzročenih sprememb pH, povezanih z dovajanjem elektroporacijskih pulzov, zlasti pri protokolih, kot se uporabljajo pri genski elektrotransfekciji. V modelu smo upoštevali elektrokemične reakcije na elektrodah in ga eksperimentalno potrdili z uporabo agaroznih gelov, ki smo jim dodali pH indikatorje. Razvili smo kalibriran sistem slikanja, ki omogoča sledenje pH frontam v realnem času. Razviti model je zanesljivo zajel prostorsko in časovno evolucijo kislih in bazičnih območij ter podal vpogled v obseg in posledice pH sprememb med in po postopku elektroporacije. V tretjem delu raziskav smo obravnavali elektroosmozni tok, ki je sorazmerno slabo raziskan pojav električno induciranega gibanja tekočin. Eksperimentalno smo ga ovrednotili v rastlinskem tkivu (krompir), skeletni mišici živalskega izvora, in agaroznih fantomih pri obdelavi s pulzirajočim električnim poljem (PEF) ter enosmernim tokom (DC). Rezultati so pokazali pomemben prispevek elektroosmozno induciranega toka k deformaciji tkiva in gibanju tekočin. Zlasti pri nižjih jakostih toka je sinergijsko delovanje med povečanjem prepustnosti membran zaradi elektroporacije in kasnejšim elektroosmoznim tokom še posebej izrazito. Ta sistematična karakterizacija poudarja pomen elektroosmoze na vpliv gibanja tekočin med elektroporacijo ter podpira njeno vključitev v prihodnji razvoj aplikacij, temelječih na elektroporaciji. Disertacija vključuje pet izvirnih znanstvenih člankov, ki vsak zase predstavljajo nova eksperimentalna in teoretična dognanja. Skupaj prispevajo k boljšemu razumevanju mehanizmov in fizikalno-kemijskih dejavnikov, ki oblikujejo potek terapij, temelječih na elektroporaciji, ter ponujajo nova spoznanja in rešitve za prihodnjo rabo. Ugotovitve in razviti modeli skupaj predstavljajo osnovo za izboljšanje načrtovanja in optimizacije elektroporacijskih postopkov v biomedicini, biotehnologiji, in živilski industriji.

• Keywords: elektroporacija, elektroporacijske terapije, pulzirajoča električna polja (PEF), ablacija s pulzirajočim poljem (PFA), numerično modeliranje, anizotropija skeletne mišice, elektrokemija, pH spremembe, elektroosmoza