| Popis: |
The aim of this study was to compare standard eight 100 µs pulses with alternative pulse waveforms (namely nanosecond and short high-frequency biphasic (H-FIRE) pulses) at different levels important for electroporation-based applications (extent of electrochemical reactions, time required for cell membrane resealing, decrease of cell survival and intracellular accumulation of chemotherapeutic agent after electrochemotherapy). By measuring the release of metal ions from the electrodes after electroporation, we confirmed the hypothesis that the use of H-FIRE and nanosecond pulses reduces electrochemical reactions even when the parameters of the electric field (amplitude, pause between pulses, ...) are adjusted to have the same biological effect as with longer monophasic pulses. Our second hypothesis, that electroporation of cells with different pulse shapes leading to comparable permeabilization of the cell membrane results in comparable oxidation of cellular components, could neither be confirmed nor refuted because it was shown that Click-iT Lipid Peroxidation Imaging Kit - Alexa Fluor 488 is not a suitable method for measuring lipid peroxidation in the first hours after electroporation. Our results show that nanosecond pulses with appropriately chosen parameters are suitable for use in electrochemotherapy with cisplatin and bleomycin, as they cause the same decrease in cell survival as with the standard eight 100 μs pulses. We confirmed the hypothesis that electroporation with nanosecond pulses increases the amount of chemotherapeutic agent in the cell and consequently increases its cytotoxic effect. Cilj študije je bil primerjati standardnih osem 100 µs pulzov z alternativnimi oblikami pulzov (in sicer nanosekundnimi in kratkimi visokofrekvenčnimi bipolarnimi (H-FIRE) pulzi) na različnih ravneh pomembnih za aplikacije, ki temeljijo na elektroporaciji (obseg elektrokemijskih reakcij, čas za zaceljenje celične membrane, zmanjšanje preživetja celic in znotrajcelično kopičenje kemoterapevtika po elektrokemoterapiji). Z merjenjem sproščanja kovinskih ionov z elektrod po elektroporaciji smo potrdili hipotezo, da uporaba H-FIRE in nanosekundnih pulzov zmanjšuje elektrokemijske reakcije tudi, ko so parametri električnega polja (amplituda, pavza med pulzi, …) prilagojeni tako, da imajo enak biološki učinek kot daljši monopolarni pulzi. Naše druge hipoteze, da elektroporacija celic z različnimi oblikami pulzov, ki vodijo v primerljivo permeabilizacijo celične membrane, povzroči primerljivo oksidacijo celičnih komponent, nismo mogli niti potrditi niti ovreči, ker se je izkazalo, da Click-iT Lipid Peroxidation Imaging Kit - Alexa Fluor 488 ni ustrezna metoda za merjenje lipidne peroksidacije v prvih urah po elektroporaciji. Naši rezultati kažejo, da so nanosekundni pulzi s primerno izbranimi parametri primerni za uporabo v elektrokemoterapiji s cisplatinom in bleomicinom, saj povzročijo enako zmanjšanje celičnega preživetja kot z uporabo standardnih osmih 100 μs pulzov. Potrdili smo hipotezo, da elektroporacija z nanosekundnimi pulzi poveča količino kemoterapevtika v celici in posledično poveča tudi njegov citotoksični učinek. |
