Mechanisms of electroporation and physicochemical factors of importance to electroporation-based therapiesŠmerc, Rok (Avtor) Mahnič-Kalamiza, Samo (Mentor) electroporationelectroporation-based treatmentspulsed electric field (PEF) treatmentpulsed field ablation (PFA)numerical modellingskeletal muscle anisotropyelectrochemistrypH changeselectroosmosisElectroporation 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.20252025-07-08 08:40:01Doktorsko delo/naloga170512sl