Identification and description of molecular transport mechanisms across cell membrane due to electroporation

SCUDERI, MARIA (author), Rems, Lea (mentor), Dermol - Černe, Janja (co-mentor)

Electroporation is a phenomenon which results in transient increase in cell membrane permeability for ions and molecules when exposing biological cells to short high-voltage electric pulses. If cells survive the exposure to electric pulses, electroporation is called reversible; otherwise, if cells die, electroporation is called irreversible. Electroporation is used in biomedicine for electrochemotherapy, gene electrotransfer, transdermal drug delivery, DNA vaccination, and as an ablation method to treat heart arrhythmia and tumors. It is also used for various purposes in biotechnology, food processing, and environmental applications, such as extraction of compounds from plant tissue, inactivation of bacteria, cell fusion, and genetic engineering of microorganisms. Electrochemotherapy uses electroporation to enhance the delivery of chemotherapeutic drugs into tumor cells. It is successfully used in clinics to treat cutaneous and subcutaneous tumors with ongoing trials for the treatment of deep-seated tumors. However, the monopolar pulses with duration of 100 μs, used classically for electrochemotherapy, cause pain and muscle contractions. To overcome these drawbacks the use of bursts of high-frequency short bipolar pulses has been suggested. Furthermore, recent efforts have been focused on making electrochemotherapy a systemic treatment by combining it with gene electrotransfer for immunotherapy. Gene electrotransfer is also based on electroporation, where millisecond pulses are used for intracellular delivery of DNA molecules that code for proteins able to stimulate the immune response. Thus, using pulse types alternative to classical 100 μs pulses could be beneficial for improving the electrochemotherapy treatment. However, it is not well understood whether different types of pulses can be equally effective for electrochemotherapy. Therefore, the first aim of the dissertation was to investigate how different types of pulses affect cisplatin uptake and cytotoxicity. We performed in vitro experiments using cisplatin and three types of pulses: classical electrochemotherapy pulses, high-frequency bipolar pulses, and millisecond pulses. We demonstrated that all tested types of pulses can be considered equivalent in terms of cisplatin uptake and cytotoxicity and can potentially replace classical, i.e., monopolar 100 μs electrochemotherapy pulses. For electrochemotherapy to be successful two main conditions need to be met: (i) the entire tumor must be exposed to a sufficiently high electric field that results in electroporation of the tumor cells and (ii) a sufficient amount of a chemotherapeutic drug (typically bleomycin or cisplatin) must enter the cells to bind to DNA and kill the tumor cells. The pulse parameters needed to successfully treat cutaneous tumors are provided in the standard operating procedures, whereas the treatment of deep-seated tumors is guided by a computational model that predicts the distribution of the electric field inside a tissue depending on the electrode configuration. To further improve such computational treatment planning, it would be useful to upgrade the model with a description of electroporation and the associated uptake of chemotherapeutic drugs into tumor cells. To enable the development of such models, it is necessary to determine the number of cisplatin molecules needed inside the cell to achieve a cytotoxic effect. Therefore, the second aim of the dissertation was to quantify the number of cisplatin molecules, delivered into cells by different types of pulses, and determine the lethal number that results in eradication of almost all treated cells. We found that the number of cisplatin molecules needed to achieve a cytotoxic effect is in the range of 2-7 ×107 molecules per cell, irrespective of the type of pulses used. Mathematical models are also useful for understanding the phenomenon of electroporation. Many different models that describe electroporation and the associated transmembrane molecular transport are present in the literature. Whilst these models differ in their theoretical description, they typically show good agreement with a specific set of data. It is not clear if any of the models can be applied to describe the molecular transport for the broad range of pulse parameters and other experimental conditions used in electroporation research. Therefore, the third aim of the dissertation was to critically assess existing mechanistic models describing electroporation-mediated transmembrane transport of ions and molecules at the single-cell level. We confronted the models with a broad range of experimental measurements and observed that none of the models was reliable enough to predict molecular transport in all tested conditions. We underlined the limitations of the models and proposed further research to improve them. Nevertheless, the existing models can still help interpret certain experimental results, such as the influence of cardiomyocyte orientation on electroporation using pulses of different durations.

• Language: English
• Keywords: electroporation, electrochemotherapy, cisplatin, numerical modeling, molecu-lar transport
• Work type: Doctoral dissertation
• Organization: FE - Faculty of Electrical Engineering
• Year: 2024
• PID: 20.500.12556/RUL-155394
• COBISS.SI-ID: 190915587
• Publication date in RUL: 29.03.2024

Secondary language

• Language: Slovenian
• Title: Identifikacija in opis mehanizmov molekularnega transporta preko celične membrane pri elektroporaciji

Abstract

Če biološko celico izpostavimo električnemu polju z dovolj visoko jakostjo, dosežemo zača-sno povečanje prevodnosti in prepustnosti celične membrane. Ta pojav se imenuje elektroporacija. Če celice preživijo izpostavljenost električnim pulzom, se elektroporacija imenuje reverzibilna; če celice umrejo, se elektroporacija imenuje ireverzibilna. Elektroporacija se v biomedicini uporablja pri elektrokemoterapiji, genski terapiji, vnosu zdravilnih učinkovin skozi kožo, cepljenju z DNK ter kot metoda ablacije za zdravljenje srčnih aritmij ali tumorjev. Uporablja se tudi za različne namene v biotehnologiji in predelavi hrane, na primer za ekstrakcijo snovi iz rastlinskega tkiva, uničevanje bakterij, zlivanje celic in genski inženiring mikroorganizmov. Pri elektrokemoterapiji z elektroporacijo izboljšamo vnos kemoterapevtskih učinkovin v tumorske celice, kar se v klinikah uspešno uporablja za zdravljenje kožnih in podkožnih tumorjev, v teku pa so tudi študije za zdravljenje globlje ležečih tumorjev. Monopolarni pulzi s trajanjem 100 μs, ki jih običajno dovajamo pri elektrokemoterapiji, povzročajo bolečine in krčenje mišic. Z dovajanjem vlakov visokofrekvenčnih kratkih bipolarnih pulzov lahko omilimo bolečine in krčenje mišic. Nedavno so se pojavile študije, kjer elektrokemoterapijo kombiniramo z imunsko gensko terapijo in dosežemo sistemsko zdravljenje. Pri genski terapiji, ki temelji na elektroporaciji, se uporabljajo milisekundni pulzi za znotrajcelični prenos molekul DNK, ki kodirajo beljakovine, sposobne spodbuditi imunski odziv. Z dovajanjem različnih pulzov, ki so alternativni klasičnim 100 μs pulzom, bi lahko izboljšali zdravljenje z elektrokemoterapijo. Ker še ni povsem jasno, ali so lahko različne vrste pulzov enako učinkovite pri elektrokemoterapiji, je bil prvi cilj disertacije raziskati, kako različne vrste pulzov vplivajo na vnos cisplatina in na citotoksičnost. Izvedli smo poskuse in vitro z dovajanjem cisplatina in treh vrst pulzov: klasičnih elektrokemoterapevtskih pulzov, visokofrekvenčnih bipolarnih pulzov in milisekundnih pulzov. Dokazali smo, da lahko vse preizkušene vrste pulzov štejemo za enakovredne v smislu vnosa cisplatina in citotoksičnosti. Za uspešno elektrokemoterapijo morata biti izpolnjena dva glavna pogoja: i) celoten tumor mora biti izpostavljen dovolj visokemu električnemu polju, ki povzroči elektroporacijo tumorskih celic, in ii) zadostna količina kemoterapevtika (običajno bleomicina ali cisplatina) mora vstopiti v celice, da se veže na DNK in uniči tumorske celice. Parametri pulzov, ki so potrebni za uspešno zdravljenje kožnih tumorjev, so določeni v standardnih operativnih postopkih, medtem ko je zdravljenje globokih tumorjev načrtovano z računalniškim modelom, ki predvideva porazdelitev električnega polja v tkivu glede na postavitev elektrod. Za nadaljnje izboljšanje takšnega računalniškega načrtovanja zdravljenja bi bilo koristno model nadgraditi z opisom elektroporacije in z njo povezanega vnosa kemoterapevtskih učinkovin v tumorske celice. Za razvoj takih modelov je treba določiti število molekul cisplatina, potrebnih v celici za citotoksični učinek. Drugi cilj disertacije je bil torej izmeriti število molekul cisplatina, ki jih v celice vnesemo z različnimi vrstami pulzov, in določiti zadostno število, ki povzroči celično smrt. Ugotovili smo, da je število molekul cisplatina, potrebnih za doseganje citotoksičnega učinka, v razponu 2-7 ×107 molekul na celico ne glede na vrsto dovedenih pulzov. Za razumevanje pojava elektroporacije so koristni matematični modeli. V literaturi je veliko različnih modelov, ki opisujejo elektroporacijo in z njo povezan prenos molekul skozi celično membrano. Čeprav se ti modeli razlikujejo v svojem teoretičnem opisu, običajno kažejo dobro ujemanje z določenim nizom podatkov. Ni jasno, ali je mogoče katerega od modelov uporabiti za opis molekularnega transporta za širok razpon parametrov pulzov in drugih eksperimentalnih pogojev. Tretji cilj disertacije je bil kritično oceniti obstoječe mehanistične modele transporta ionov in molekul skozi elektroporirano celično membrano. Modele smo preverjali na širokem naboru eksperimentalnih meritev in ugotovili, da nobeden od modelov ni bil dovolj zanesljiv za napoved molekularnega transporta v vseh preizkušenih pogojih. Poudarili smo omejitve modelov in predlagali nadaljnje raziskave za izboljšanje in nadgradnjo obstoječih modelov. Kljub omejitvam lahko obstoječi modeli še vedno pomagajo pri razlagi nekaterih eksperimentalnih rezultatov, na primer vpliva orientacije kardiomiocitov v električnem polju in trajanja pulzov na učinkovitost elektroporacije.

• Keywords: elektroporacija, elektrokemoterapija, cisplatin, numerično modeliranje, molekularni transport