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Znanstveno izhodišče: Karcinom trebušne slinavke je četrti vodilni vzrok z rakom povezane smrti v zahodnem svetu. Ob postavitvi diagnoze pri približno 40 % bolnikov kirurško zdravljenje zaradi lokalno napredovale bolezni ni mogoče. Uspešnost zdravljenja s kemoterapijo in radioterapijo je pri teh bolnikih omejena, saj je preživetje kljub terapiji kratko (< 12 mesecev). Lokalne ablacijske metode bi lahko predstavljale novo možnost zdravljenja teh bolnikov. Elektrokemoterapija (ECT) je ablacijski poseg, ki temelji na lokalni uporabi reverzibilne elektroporacije. Poseg omogoča transport i. v. apliciranega kemoterapevtika, ki sicer ne prehaja celične membrane, neposredno v celico. Metoda je že uveljavljena pri zdravljenju kožnih in jetrnih tumorjev. Do sedaj varnost in učinkovitost ECT pankreasa še nista bili sistematično proučevani. Namen dela in hipoteza: Namen doktorskega dela je raziskati varnost ECT trebušne slinavke z bleomicinom na preživetvenem modelu pujsa. Naša hipoteza je bila, da ECT normalne trebušne slinavke ne povzroča klinično pomembnih lokalnih ali sistemskih neželenih učinkov in da ECT normalne trebušne slinavke povzroči nekrozo celic v ablacijskem območju. Metode: Dovoljenje za izvajanje poskusov je izdala Etična komisija za poskuse na živalih Uprave Republike Slovenije za varno hrano, veterinarstvo in varstvo rastlin (številka dovoljenja U34401-1/2017/4). Vsi postopki so bili skladni z nacionalno in evropsko zakonodajo. Raziskava je potekala na osmih (8) prašičih, ki so bili razdeljeni v dve skupini. V prvi skupini je bil poseg ECT izveden perkutano, pod kontrolo računalniške tomografije (CT), tretiran je bil rep trebušne slinavke. V drugi skupini je bil poseg ECT izveden ob kirurški laparotomiji, tretirana sta bila glava in rep trebušne slinavke. Elektroporacijo smo izvedli z uporabo dveh igelnih elektrod (z debelino 1,2 mm) s 3 cm dolgim aktivnim delom. Obe skupini smo po posegu sledili sedem dni. Za oceno morebitnih zapletov smo spremljali klinično stanje prašičev in laboratorijske parametre. Takoj po posegu in sedmi dan po posegu smo opravili preiskavo s CT. Za oceno histoloških sprememb smo prašiče sedmi dan po posegu evtanazirali in opravili patološki pregled trebušne slinavke. Rezultati: Poseg je bil uspešno opravljen pri vseh prašičih in vsi so preživeli načrtovano opazovalno obdobje. CT-preiskava in histološki pregled nista razkrila pomembnih lokalnih neželenih učinkov. Noben od prašičev ni razvil kliničnih znakov pankreatitisa. Pri dveh prašičih (št. 6 in 8) je prišlo do prehodnega povišanja koncentracije serumske amilaze in lipaze. Histološka analiza je pri teh prašičih pokazala iatrogeno poškodbo glavnega pankreatičnega voda zaradi vstavitve igelne elektrode. Histološka analiza je v ablacijskem območju pokazala prisotnost fibroze in nekroze tik ob vstavitvi igelnih elektrod ter prisotnost apoptoze v perifernih delih ablacijskega območja. Zaključek: Raziskava je pokazala, da ECT povzroči nekrozo in apoptozo v ablacijskem območju, ki pa se ne odraža v lokalnih ali sistemskih stranskih učinkih. Zaključimo lahko, da je ECT parenhima trebušne slinavke varna ablacijska metoda. Rezultati te študije bodo prispevali k nadaljnjemu razvoju ECT trebušne slinavke. Scientific background: Pancreatic cancer is the fourth leading cause of cancer-related death in the Western world. At the time of diagnosis approximately 40% of patients present with locally advanced disease , unsuitable for surgical treatment. The success of chemotherapy and radiotherapy therapy in these patients is limited, with a short survival (

Elektroporacija je pojav, pri katerem s kratkimi visokonapetostnimi električnimi pulzi spremenimo strukturo celične membrane in s tem povečamo njeno prepustnost. Z ustrezno izbiro parametrov pulzov je pojav lahko reverzibilen ali ireverzibilen. Obe vrsti elektroporacije se uporabljata v različnih medicinskih aplikacijah. Reverzibilna elektroporacija se uporablja pri elektrokemoterapiji in genski elektrotransfekciji, ireverzibilna elektroporacija pa se uporablja za ablacijo tumorjev ter ablacijo v srcu pri zdravljenju atrijske fibrilacije. Numerične metode so pomembno orodje za analizo kompleksnih dogajanj v biološkem tkivu med elektroporacijo. Številni vidiki klinične uporabe elektroporacije so še vedno neraziskani. Numerično modeliranje nam omogoča raziskovanje novih pristopov k zdravljenju, preizkušanje novih oblik elektrod ter analizo različnih kliničnih scenarijev z vidika izvedljivosti in varnosti. Uporaba numeričnih modelov tudi zmanjša število predkliničnih in kliničnih študij, potrebnih za razvoj zdravljenja na podlagi elektroporacije. Vendar je treba modele pred vključitvijo v klinični delovni proces, npr. za načrtovanje zdravljenja, potrditi s poskusi in (pred)kliničnimi študijami. Ena najpomembnejših aplikacij numeričnega modeliranja je računalniško podprto načrtovanje zdravljenja. Predpogoj za uspeh vseh zdravljenj, ki temeljijo na elektroporaciji, je popolna pokritost kliničnega ciljnega volumna z dovolj visokim električnim poljem. Razporeditev električnega polja v tkivu je odvisna predvsem od postavitve elektrod in parametrov dovedenih pulzov. Natančno določanje porazdelitve električnega polja v tkivu, zlasti pri globoko ležečih tarčah, ni enostavna naloga. Za zagotovitev uspešnega izida zdravljenja je priporočljivo načrtovanje zdravljenja, ki temelji na numeričnih modelih, prilagojenih bolniku, in optimizacija parametrov zdravljenja. Kljub tehnološkemu napredku načrtovanje zdravljenja pri terapijah z elektroporacijo še vedno ni del standardne klinične prakse glavna omejitev izhaja iz dejstva, da se načrti trenutno pripravljajo pred posegom. Razvoj načrtovanja zdravljenja v realnem času z uporabo dejanskih položajev elektrod bo omogočil nadzor parametrov zdravljenja in izida v realnem času in je ključni korak pri uvajanju računalniško podprtega načrtovanja zdravljenja v rutinsko klinično prakso. Namen disertacije je z uporabo numeričnega modeliranja in računalniško podprtega načrtovanja zdravljenja izboljšati zdravljenje globje ležečih tumorjev z elektroporacijo. V uvodnem delu je podan pregled kliničnih aplikacij elektroporacije, sledijo osnove načrtovanja zdravljenja, ter pregled metod, ki se uporabljajo pri modeliranju elektroporacije. Glavnino disertacije sestavlja šest izvirnih znanstvenih člankov, objavljenih v mednarodnih revijah, ki zajemajo delo, opravljeno v disertaciji. Metodologija in rezultati so v člankih podrobno obravnavani, zato so na koncu disertacije povzete le razprave in zaključki posameznih člankov. V disertacijo so vključeni trije izvirni znanstveni prispevki. Prvi prispevek obsega validacijo numeričnega modela elektroporacije v jetrih z uporabo kliničnih in predkliničnih podatkov. Drugi prispevek obsega vidike varnosti in učinkovitosti zdravljenja z elektroporacijo v tveganih kliničnih scenarijih, na primer v bližini srčnih spodbujevalnikov ali ob prisotnosti kovinskih kirurških sponk v območju zdravljenja. Numerično sta bila ovrednoteni tudi izvedljivost in varnost novega pristopa k zdravljenju metastaz v hrbtenici, kar dokazuje uporabnost numeričnega modeliranja pri razvoju novih načinov zdravljenja. Tretji prispevek zajema razvoj optimizacijskega algoritma za postavitev elektrod brez uporabe računsko zahtevnih metod. Algoritem bistveno skrajša čas in strokovno znanje, ki sta potrebna za razvoj načrta zdravljenja, in predstavlja prvi korak k načrtovanju zdravljenja v realnem času. Electroporation is a phenomenon in which short high-voltage electric pulses are used to change the structural integrity of the cell membrane, thereby increasing membrane permeability. With appropriate choice of pulse parameters, the phenomenon can be reversible or irreversible. Both types of electroporation are used in various medical applications. Reversible electroporation is used in electrochemotherapy and gene electrotransfer, while irreversible electroporation is used for ablation of tumors and ablation in the heart to treat atrial fibrillation. Numerical methods are an important tool for analyzing complex interactions in biological tissue during electroporation. Many aspects of the clinical applications of electroporation are still unresolved. Numerical modeling allows us to investigate new treatment approaches, test new electrode designs, and analyze different clinical scenarios for the feasibility and safety aspects. The use of numerical models also reduces the number of preclinical and clinical studies required to develop treatments based on electroporation. However, before the models can be integrated into the clinical workflow, e.g., for treatment planning, they need to be validated by experiments and (pre)clinical studies. One of the most important applications of numerical modeling is computer-assisted treatment planning. A prerequisite for the success of all electroporation-based treatments is the complete coverage of the clinical target volume with a sufficiently high electric field. The distribution of the electric field in the tissue depends mainly on the electrode configuration and the parameters of the delivered pulse. Accurately determining the distribution of the electric field in the tissue, especially for deep-seated targets, is not a trivial task. Treatment planning based on patient-specific numerical models and optimization of treatment parameters, is advisable to ensure a successful treatment outcome. Despite the technological advances, treatment planning is still not part of the standard clinical practice for electroporation-based treatments the major limitation stems from the fact that plans are currently generated prior to the procedure. The development of real-time treatment planning using actual electrode positions will allow real-time control of treatment parameters and outcome, and is a critical step toward introducing computer-assisted treatment planning into routine clinical practice. The aim of this dissertation is to improve future electroporation-based treatments through numerical modeling and computer-assisted treatment planning. First, a brief introduction to the clinical application of electroporation is given then, the fundamentals of treatment planning are described, followed by an overview of the numerical approaches used in modeling the electroporation phenomenon. The main body of the dissertation consists of six original research papers published in international journals that comprise the work performed in the dissertation. The methodology and results are discussed in detail in the papers, therefore only the discussion and conclusions of the presented papers are reproduced at the end of the dissertation. Three original contributions to science are included within this dissertation. First, the numerical model of electroporation in the liver was validated using preclinical and clinical data. Second, the safety and efficacy aspects of electroporation-based treatments were numerically evaluated in risky clinical scenarios, such as near implanted pacemakers or in the presence of multiple metallic implants within the treatment zone. The feasibility and safety of a new treatment approach for spinal metastases was also numerically evaluated, demonstrating the utility of numerical modeling in the development of new treatments. Finally, an optimization algorithm for electrode placement was developed without using computationally intensive methods. The algorithm significantly reduces the time and expertise required to develop a treatment plan and is a step toward real-time treatment planning.

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.

Nanosecond electric pulses have several potential advantages in electroporation-based procedures over the conventional micro- and millisecond pulses including low level of heating, reduced electrochemical reactions and reduced muscle contractions making them alluring for use in biomedicine and food industry. The aim of this study was to evaluate if nanosecond pulses can enhance the cytotoxicity of chemotherapeutics bleomycin and cisplatin in vitro and to quantify metal release from electrodes in comparison to 100 μs pulses commonly used in electrochemotherapy. The effects of nanosecond pulse parameters (voltage, pulse duration, number of pulses) on cell membrane permeabilization, resealing and on cell survival after electroporation only and after electrochemotherapy with bleomycin and cisplatin were evaluated on Chinese hamster ovary cells. Application of permeabilizing nanosecond pulses in combination with chemotherapeutics resulted in successful cell kill. Higher extracellular concentrations of bleomycin – but not cisplatin – were needed to achieve the same decrease in cell survival with nanosecond pulses as with eight 100 μs pulses, however, the tested bleomycin concentrations were still considerably lower compared to doses used in clinical practice. Decreasing the pulse duration from microseconds to nanoseconds and concomitantly increasing the amplitude to achieve the same biological effect resulted in reduced release of aluminum ions from electroporation cuvettes.

Objectives: The use of thermal ablative therapies in the pancreatic tumors is limited because of the risk of the vessel injury and potential pancreatitis or fistula formation. Electrochemotherapy (ECT) is an ablative therapy with established role in the treatment of cutaneous and liver tumors. This study was designed to evaluate the safety and feasibility of ECT of the pancreas in a porcine survival model. Methods: In the first group, 4 animals underwent computed tomography (CT)%guided percutaneous ECT with bleomycin of the pancreatic tail. In the second group (4 animals), the intraoperative ECTwith bleomycin of pancreatic tail and head was performed. Animals were followed for 7 days and then killed. Clinical parameters, CT imaging, laboratory, and histologic analysis were performed. Results: All pigs survived the ECT procedure and none of them developed clinical signs of acute pancreatitis or related complications. There were no signs of acute pancreatitis or damage to the large vessels present in the follow-up CT scans. No significant change in laboratory parameters was obtained after procedure. Conclusions: This study shows that ECT with bleomycin is feasible and safe in the pancreatic parenchyma. Clinical studies are needed to evaluate the efficacy of ECT in pancreatic cancer.

Currently, in high-frequency electroporation, much progress has been made but limited to research groups with custom-made laboratory prototype electroporators. According to the review of electroporators and economic evaluations, there is still an area of pulse parameters that needs to be investigated. The development of an asymmetric bipolar pulse generator with a maximum voltage of 4 kV and minimum duration time of a few hundred nanoseconds, would enable in vivo evaluation of biological effects of high-frequency electroporation pulses. Herein, from a series of most commonly used drivers and optical isolations in high-voltage pulse generators the one with optimal characteristics was used. In addition, the circuit topology of the developed device is described in detail. The developed device is able to generate 4 kV pulses, with theoretical 131 A maximal current and 200 ns minimal pulse duration, the maximal pulse repetition rate is 2 MHz and the burst maximal repetition rate is 1 MHz. The device was tested in vivo. The effectiveness of electrochemotherapy of high-frequency electroporation pulses is compared to “classical” electrochemotherapy pulses. In vivo electrochemotherapy with high-frequency electroporation pulses was at least as effective as with “classical” well-established electric pulses, resulting in 86% and 50% complete responses, respectively. In contrast to previous reports, however, muscle contractions were comparable between the two protocols.

Namen doktorske disertacije je bil ovrednotenje rezultatov slikovno-diagnostičnih metod po elektroporaciji (EP) in elektrokemoterapiji (EKT) jeter zdravih prašičev za preučitev varnostnega vidika EKT globoko ležečih jetrnih tumorjev ter rezultatov ultrazvočnega pregleda s kontrastnim sredstvom (UZ KS) po EKT in genskem elektroprenosu (GEP) mišjih in pasjih tumorjev kot prediktivnih dejavnikov zdravljenja. Slikovno-diagnostične spremembe po EP in EKT z bleomicinom (BLM) v jetrnem parenhimu in velikih krvnih žilah jeter zdravega prašiča s potencialno nevarno vstavitvijo linearnih in heksagonalnih elektrod v velike jetrne žile smo preučili z ultrazvočno preiskavo takoj po EP/EKT ter z računalniško tomografijo od 60 do 90 minut in en teden po EP/EKT. Rezultati slikovno-diagnostičnih metod jeter prašičev po EK so pokazali zmanjšano perfuzijo tretiranega področja. Spremembe niso bile posledica uporabe BLM, saj so bile enake pri poskusni in kontrolni skupini, kjer smo aplicirali samo električne pulze. Namerna vstavitev elektrod in aplikacija električnih pulzov neposredno v svetlino velikih žil ni povzročila tromboz, krvavitev ali drugih hemodinamskih motenj. Kot model preiskovanja uporabnosti UZ-KS kot prediktivnega dejavnika zdravljenja s terapevtskimi metodami, ki temeljijo na EP, smo uporabili miši z induciranimi melanomi. V prvi raziskavi smo na mišjem modelu, ki smo ga zdravili z obsevanjem in GEP plazmidne DNA, ki utiša adhezijsko molekulo celic melanoma, opazili značilno zmanjšanje perfuzije tumorjev v terapevtskih skupinah v primerjavi s kontrolnimi skupinami, rezultati UZ-KS pa so korelirali z zmanjšano gostoto žil. Povprečne vrednosti parametra, ki opisuje volumen pretoka, so bile značilno nižje pri tumorjih, ki so se odzvali na terapijo s popolnim odgovorom, in vrednosti PE so pokazale trend korelacije s protitumorsko učinkovitostjo. V drugi raziskavi na mišjih melanomih, zdravljenih z EKT z BLM v kombinaciji z GEP plazmida za interlevkin 12 (pIL-12), je UZ-KS pokazal zmanjšano perfuzijo v skupinah z daljšim časom podvojitve volumna tumorja in prediktivno vrednost UZ KS smo potrdili s statistično značilnimi Pearsonovimi koeficienti. S krajšim časom podvojitve volumna tumorja je bila povezana tudi večja heterogenost perfuzije tumorja. V klinični del raziskave smo vključili pse s kožnimi in podkožnimi tumorji, ki so bili zdravljeni s standardnim postopkom kombinacije EKT in GEP pIL-12. Rezultate UZ-KS smo primerjali z izidom zdravljenja. Tudi v klinični raziskavi pri psih smo ugotovili številne razlike v parametrih UZ KS med tumorji s popolnim odgovorom in tumorji z nepopolnim odgovorom: perfuzija in heterogenost perfuzije sta bili manjši pri tumorjih s popolnim odgovorom. The aim of this dissertation was to evaluate the results of diagnostic imaging after electroporation (EP) and electrochemotherapy (ECT) of porcine liver to investigate the safety aspect of ECT of deep-seated liver tumors and the results of dynamic contrast-enhanced ultrasound (DCE US) after ECT and gene electrotransfer (GET) of murine and canine tumors as potential predictive factors in treatment. Radiological findings after EP and ECT with bleomycin (BLM) on the hepatic parenchyma and major blood vessels of the liver of a healthy pig with potentially dangerous insertion of the electrodes into the major hepatic vessels were examined by ultrasound immediately after ECT and by computed tomography from 60 to 90 minutes and one week after ECT with linear and hexagonal electrodes. The results of diagnostic imaging of the porcine liver after ECT showed decreased blood flow in the treated area. The changes were not due to the use of BLM, as they were the same in the experimental and control groups, where only electrical pulses were administered. Intentional insertion of electrodes and application of electrical pulses directly into the lumen of large vessels did not result in thrombosis, haemorrhage, or other hemodynamically significant injury. Mice with induced melanoma were used as a model to investigate the utility of DCE-US as a prognostic factor for treatment with EP-based therapies. In the first study in a mouse model treated with irradiation and GET of plasmid DNA that silences the melanoma cell adhesion molecule, we observed a decrease in tumor perfusion in the treatment groups compared with the control groups, and the results of DCE US correlated with decreased vessel density detected histologically. Furthermore, mean values of parameters describing flow volume were significantly lower in tumors with complete response, and they showed a trend toward correlation with antitumor efficacy. In the second study, mouse melanomas were treated with ECT in combination with GET of plasmid DNA encoding interleukin-12 (pIL-12). DCE US showed reduced perfusion in groups with longer tumor volume doubling time, and the predicted DCE US value was confirmed by statistically significant Pearson coefficients. In addition, greater tumor perfusion heterogeneity was associated with shorter tumor doubling time. The clinical study included dogs with cutaneous and subcutaneous neoplasms treated with a standard procedure combining ECT and GET pIL-12. We also found a number of differences in DCE-US parameters between tumors with complete response and tumors with incomplete response in the clinical study: Perfusion and perfusion heterogeneity were lower in tumors with complete response.

Elektroporacija je fizikalna metoda, ki omogoča prehajanje številnih molekul skozi celično membrano, ki jih celica v normalnih pogojih ne prepušča. Z uporabo kratkih, visokonapetostnih električnih pulzov v plazemski membrani povzročimo nastanek hidrofilnih por. Ločimo reverzibilno elektroporacijo, po kateri se celična membrana zaceli in ireverzibilno elektroporacijo, po kateri se celična membrana ne more regenerirati ter posledično pride do celične smrti. Elektroporacija ima veliko možnih aplikacij na več področjih. Uporablja se jo za zlivanje celic, transfekcijo ali transformacijo celic, sterilizacijo vode, pasterizacijo hrane, ekstrakcijo snovi. Elektroporacija je dobila tudi mesto v medicini, na področju zdravljenja raka. Za ta namen lahko uporabimo reverzibilno elektroporacijo, s katero omogočimo, da v rakaste celice prodrejo citostatiki in tako povečamo njihov učinek. Pri uporabi citostatika cisplatin z elektroporacijo povečamo njegovo učinkovitost za 70-krat, pri bleomicinu pa kar do 700-krat. Možna je tudi uporaba ireverzibilne elektroporacije, kjer rakaste celice ubijemo in jo uporabljamo za netermično ablacijo tkiv in tumorjev. Electroporation is a physical method that allows the passage of many molecules through the cell membrane that is impermeable to this very molecules. Using short, high-voltage electrical pulses in the plasma membrane causes the formation of hydrophilic pores. Membrane electroporation can either be reversible (cell membrane reseal) or irreversible (cell membrane cannot return to its primary state and cell death occurs). Electroporation has many possible applications in several areas. It is used for cell fusion, water sterilization, food pasteurization, substance extraction, transfection, transformation, etc. Electroporation has also gained a place in medicine, especially in the field of cancer treatment. For this purpose, reversible electroporation is used to introduce cytostatic into cancer cells. When electroporation is used, the efficiency of cisplatin increases 70 times and the efficiency of bleomycin up to 700 times. We can also use irreversible electroporation, where we kill cancer cells, for non-thermal ablation of tissues and tumors.

The plasma concentration profile of bleomycin in the distribution phase of patients younger than 65 years is needed to determine the suitable time interval for efficient application of electric pulses during electrochemotherapy. Additionally, bleomycin concentrations in the treated tumors for effective tumor response are not known. In this study, the pharmacokinetic profile of bleomycin in the distribution phase in 12 patients younger than 65 years was determined. In 17 patients, the intratumoral bleomycin concentration was determined before the application of electric pulses. In younger patients, the pharmacokinetics of intravenously injected bleomycin demonstrated a faster plasma clearance rate than that in patients older than 65 years. This outcome might indicate that the lowering of the standard bleomycin dose of 15,000 IU/m$^2$ with intravenous bleomycin injection for electrochemotherapy is not recommended in younger patients. Based on the plasma concentration data gathered, a time interval for electrochemotherapy of 5–15 min after bleomycin injection was determined. The median bleomycin concentration in tumors 8 min after bleomycin injection, at the time of electroporation, was 170 ng/g. Based on collected data, the reduction of the bleomycin dose is not recommended in younger patients however, a shortened time interval for application of electric pulses in electrochemotherapy to 5–15 min after intravenous bleomycin injection should be considered.

Elektroporacija (EP) je fizikalna metoda s katero z dovajanjem električnih pulzov v celični membrani za kratek čas ustvarimo pore, kar olajša prehod v celice določenim molekulam. Za zdravljenje rakavih obolenj se vse bolj uporablja EP v kombinaciji s cisplatinom (CDDP) in bleomicinom, čemur rečemo elektrokemoterapija (EKT). Cisplatin je platinova (II) spojina, ki s kovalentnimi povezavami povzroči spremembe v strukturi DNA in vodi v smrt rakave celice. Kljub temu, da je CDDP eden najpogosteje uporabljenih kemoterapevtikov, pa povzroča hude stranske učinke in tudi odpornost rakavih celic. Zaradi tega se raziskave usmerjajo v razvoj novih platinovih (II) analogov s protitumorskim delovanjem, kamor spadata tudi [PtCl (5,7-dibromo-8-hidroksikinolinato)(S-dmso)] (spojina A) in njen analog, [PtCl (5,7-dibromo-8-hidroksikinolinato)(pta)] (spojina B). Citotoksičnost obeh spojin v kombinaciji z EP smo preverili na humani celični liniji raka debelega črevesja (HT29) in raka dojke (MCF7). S testom klonogenosti smo izračunali vrednosti IC50 in jih primerjali z vrednostmi IC50 CDDP, s pripravo citospinov in barvanjem po Giemsi pa smo na podlagi morfoloških značilnosti ugotovili delež apoptotičnih in nekrotičnih celic. Ugotovili smo, da spojini A in B delujeta citotoksično na tumorske celice in povzročata predvsem apoptozo ter deloma nekrozo. Elektroporacija statistično značilno poveča citotoksično delovanje vseh treh platinovih (II) spojin in je učinkovita metoda za izboljšanje vnosa ne samo CDDP, pač pa tudi spojine A in B. Spojina B se je v kombinaciji z in brez EP izkazala za bolj toksično od spojine A in hkrati tudi enako ali celo bolj citotoksično kot CDDP pri obeh celičnih linijah in je zato primerna za nadaljne testiranje, kot potencialni kemoterapevtik. Electroporation (EP) is a physical technique in which an electric field is applied to cells in order to increase the permeability of cell membrane for poorly permeating molecules. Electroporation in combination with cisplatin (CDDP), also called electrochemotherapy (ECT), is used for local treatment of several malignant tumors. Cisplatin is a well-known platinum (II) antitumor drug which antitumor activity has been linked to its ability to form DNA adducts, which subsequently lead to apoptosis of cancer cells. Although CDDP is broadly used, its main limitations are severe side effects and tumor cells’ resistance. Because of this, researchers are focusing on developing new platinum (II) complexes with antitumor activity, including [PtCl (5,7-dibromo-8-hydroxyquinolinato)(S-dmso)] (compound A) and it's analog, [PtCl (5,7-dibromo-8-hydroxyquinolinato)(pta)] (compound B). In the thesis, we analysed the cytotoxicity of compound A and B in combination with EP in human colon cancer (HT29) and human breast cancer cell line (MCF7). We determined IC50 values with clonogenic assay and compared it to IC50 values of CDDP. We used Giemsa staining to determine the percentage of apoptotic and necrotic cells. Results show that all compounds, A, B and CDDP have a cytotoxic effect on both tumor cell lines and cause primarily apoptotic cell death. Furthermore, EP has proved to be a very effective method for drug uptake as it has significantly improved cytotoxicity of all three platinum (II) complexes. Compound B has turned out to be more cytotoxic with and without EP than compound A and showed similar or better cytotoxicity than CDDP in both cell lines. Based on our results, compound B is very promising potential therapeutic agent.

Electroporation is a method of inducing an increase in permeability of the cell membrane through the application of an electric field and can be used as a delivery method for introducing molecules of interest (e.g., chemotherapeutics or plasmid DNA) into cells. Electroporation-based treatments (i.e., electrochemotherapy, gene electrotransfer, and their combinations) have been shown to be safe and effective in veterinary oncology, but they are currently mostly recommended for the treatment of those solid tumors for which clients have declined surgery and/or radiotherapy. Published data show that electroporation-based treatments are also safe, simple, fast and cost-effective treatment alternatives for selected oral and maxillofacial tumors, especially small squamous cell carcinoma and malignant melanoma tumors not involving the bone in dogs. In these patients, a good local response to treatment is expected to result in increased survival time with good quality of life. Despite emerging evidence of the clinical efficacy of electroporation-based treatments for oral and maxillofacial tumors, further investigation is needed to optimize treatment protocols, improve clinical data reporting and better understand the mechanisms of patients' response to the treatment.

Rak je druga po smrtnosti najučestalija bolest na svijetu. Zbog svoje kompleksnosti, liječenje još uvijek nije efikasno u mnogim slučajevima. Zbog raznolikih vrsta tumora i mutacija koje se pojavljuju, veoma je teško pronaći standardiziranu terapiju. Korištenje električnih polja spada u novije metode pri liječenju tumora i još je relativno neistraženo, ali obećavajuće područje. Poboljšava unos lijekova u stanicu, koristi se kod genske terapije i u ablaciji tumorskog tkiva. Terapija je veoma sigurna, nuspojave nisu izražene i postotak oporavka pacijenata je veoma visok. Dva su glavna ishoda djelovanja električnog polja na membranu. Ireverzibilna i reverzibilna elektroporacija. Kod ireverzibilne elektroporacije, struktura membrane je trajno narušena i ona se neće vratiti u prvotno stanje, te se time postiže smrt stanice. Kod reverzibilne elektroporacije, membrana se vraća u prvobitno stanje, te ne dolazi do smrti stanice. Elektrokemoterapija je poboljšan način dostave lijeka u tumorsku stanicu. Pomoću električnog polja se povećava propusnost membrane, te lijek lakše ulazi u tumorsku stanicu. Primjenom elektrokemoterapije, smanjuje se potreba doza kemoterapeutike za liječenje, broj nuspojava je reduciran, profil liječenja je poboljšan. Bleomicin i cisplatin su indentificirani kao lijekovi koji bi mogli imat poboljšanu terapiju sa elektrokemoterapijom. Istraživanja sa bleomicinom su pokazala da ima 50% poboljšano djelovanje kada se primjenjuje sa elektrokemoterapijom, u odnos na primjenjivanje njega samog. Genska terapija je nov pristup liječenju tumora. Može se koristiti da se ispravi gubitak funkcije uzrokovan mutacijom ili da se eksprimira genski proizvod s nedostatkom na fiziološkoj razini. Cancer is the second most common disease in the world. Due to its complexity, treatment is still not effective in many cases. Due to the various types of tumors and mutations that occur, it is very difficult to find standardized therapy. The use of electric fields is one of the newer methods in the treatment of tumors and is still a relatively unexplored but promising area. It improves the uptake of drugs into the cell, is used in gene therapy and in the ablation of tumor tissue. The therapy is very safe, the side effects are not pronounced and the recovery rate of the patients is very high. There are two main outcomes of the action of an electric field on a membrane. Irreversible and reversible electroporation. In irreversible electroporation, the structure of the membrane is permanently disrupted and it will not return to its original state, thus achieving cell death. With reversible electroporation, the membrane returns to its original state, and cell death does not occur. Electrochemotherapy is an improved way of delivering a drug to a tumor cell. The electric field increases the permeability of the membrane, and the drug enters the tumor cell more easily. With the application of electrochemotherapy, the need for doses of chemotherapy for treatment is reduced, the number of side effects is reduced, the treatment profile is improved. Bleomycin and cisplatin have been identified as drugs that could have improved electrochemotherapy. Studies with bleomycin have shown that it has a 50% improved effect when administered with electrochemotherapy, compared to self-administration. Gene therapy is a new approach to treating tumors. It can be used to correct a loss of function caused by a mutation or to express a deficient gene product at the physiological level.

Izhodišče in namen: Incidenca primarnih jetrnih tumorjev v svetu se povečuje. Najpogostejši med njimi (90 %) je hepatocelični karcinom (HCC) in skupaj z intrahepatičnim holangiokarcinom (CHC) predstavlja 98,5 % primarnih jetrnih tumorjev. Ne glede na dejstvo, da je to eden najpogostejših vzrokov smrti zaradi raka v svetu, je manj kot 20 % bolnikov s HCC kandidatov za kurativno zdravljenje – kirurško resekcijo ali presaditev jeter. Samo kurativno zdravljenje omogoča bolnikom možnost za dolgoletno preživetje s petletnim preživetjem do 70 % pri kirurških resekcijah in presaditvi jeter. Ko radikalni/kurativni tretmaji zaradi splošnega stanja telesne zmogljivosti, pridružene okvare jeter ali velikosti in razširjenosti tumorja ne pridejo v poštev, uporabljamo druge metode, kot sta transarterijska kemoembolizacija (TACE) in transarterijska radioembolizacija (TARE), ali lokalne ablativne tehnike, kot so radiofrekvenčna ablacija (RFA), mikrovalovna ablacija (MWA), krioterapija in druge metode za zdravljenje primarnih jetrnih tumorjev, ki podaljšajo preživetje teh bolnikov. Elektrokemoterapija (ECT) omogoča netoplotno ablacijo kožnih in tudi globlje ležečih tumorjev. Na podlagi dobrih rezultatov pri zdravljenju jetrnih zasevkov raka debelega črevesa in danke smo opravili prospektivno raziskavo zdravljenja hepatoceličnega karcinoma z namenom ugotavljanja izvedljivosti, varnosti in učinkovitosti elektrokemoterapije pri primarnih jetrnih tumorjih. Metode: Elektrokemoterapijo z bleomicinom smo izvedli pri 25 bolnikih s hepatoceličnim karcinomom, skupno je bilo tretiranih 33 lezij po predvidenem protokolu. Zdravljenje je bilo izvedeno med odprto laparotomijo pri 24 od 25 bolnikov, pri enem bolniku je bila elektrokemoterapija izvedena perkutano pod kontrolo računalniške tomografije. Ob zaključku spremljanja bolnikov je bila mediana opazovanja 23,5 meseca. Rezultati: Elektrokemoterapija je bila izvedljiva pri vseh 33 hepatoceličnih karcinomih. Pri nobenem bolniku ni prišlo do neželenih stranskih učinkov zdravljenja ali večjih pooperativnih zapletov. Do popolnega odgovora na zdravljenje je po 3 do 6 mesecih prišlo pri 92 % bolnikov in 94 % zdravljenih lezij. Ob zaključku spremljanja, tj. po medianem času spremljanja 23,5 meseca, je pri 31 od 33 lezij prišlo do popolnega odgovora. Zaključki: Elektrokemoterapija primarnih jetrnih tumorjev se je izkazala kot izvedljiv in varen način zdravljenja pri vseh 25 bolnikih. Za dokončno oceno učinkovitosti zdravljenja je potreben daljši interval sledenja bolnikov. Elektrokemoterapija je predvsem primerna pri bolnikih z zmanjšano jetrno funkcijo zaradi jetrne ciroze, pri bolnikih z zmerno do hudo portalno hipertenzijo in/ali pri zdravljenju lezij, pri katerih bi lahko zgolj operacija z velikim tveganjem za zaplete ponudila možnost popolne ozdravitve, saj elektrokemoterapija ponuja možnost popolnega odgovora lezije na zdravljenje. Background and objectives: The incidence of primary liver cancer (PLC) is rising across the world. Hepatocellular carcinoma (HCC) is the most common (90%) and it and intrahepatic cholangiocarcinoma represent more than 98.5% of all primary liver tumors. Despite being one of the most common causes of cancer-related death around the world, fewer than 20%of patients with HCC are eligible for curative treatment—that is, surgical resection or liver transplantation. Only curative treatment offers the patient a chance for long-term survival, with 5-year survival rates of 70% for liver resection and liver transplantation. When radical or curative treatment is not indicated due to the patient’s condition, underlying liver disease, or tumor extent, other methods such as transarterial chemoembolization (TACE), transarterial radioembolization (TARE), and local ablative treatments such as radiofrequency ablation (RFA), microwave ablation (MWA), cryotherapy, and other methods help in longer survival of those patients. Electrochemotherapy provides non-thermal ablation of cutaneous and deep-seated tumors. Based on positive results of the treatment of colorectal liver metastases, we conducted a prospective pilot study on hepatocellular carcinomas with the aim of testing the feasibility, safety, and effectiveness of electrochemotherapy. Patients and methods: Electrochemotherapy with bleomycin was performed on 33 hepatocellular carcinomas in 25 patients using a previously established protocol. The procedure was performed during open laparotomy for 24/25 patients, and percutaneous electrochemotherapy with computed tomography control was performed for one patient. At the end of the observation period, the median follow-up was 23.5 months. Results: Electrochemotherapy was feasible for all 33 lesions, and no treatment-related adverse events or major postoperative complications were observed. The complete response rate at 3 to 6 months was 92% per patient and 94% per treated lesion. At the end of the observation period, after a median follow-up time of 23.5 months, complete response was obtained in 31 out of 33 lesions. Conclusions: Electrochemotherapy of hepatocellular carcinoma proved to be a feasible and safe treatment in all 25 patients. To evaluate the effectiveness of this method, a longer observation period is needed. Electrochemotherapy is predominantly applicable for patients with impaired liver function due to liver cirrhosis, in patients with mild to severe portal hypertension, and/or for lesions where a high-risk operation is needed to achieve curative intent because ECT offers the possibility for complete response of the lesion to treatment.

Znanstveno izhodišče: Elektrokemoterapija je inovativen način zdravljenja raka, vendar je malo raziskav, ki bi bile osredotočene na njeno vlogo v zdravljenju sluzničnega in kožnega raka glave in vratu. Za ugotavljanje učinkovitosti in varnosti zdravljenja tudi pri tej skupini bolnikov so nujne pravilno zasnovane in izvedene prospektivne klinične raziskave. Z izboljšanjem tehnoloških postopkov zdravljenja z elektrokemoterapijo lahko zdravimo tudi globoko ležeče tumorje glave in vratu. Pri zdravljenju z elektrokemoterapijo se najpogosteje uporablja bleomicin. Vendar lahko sistemsko zdravljenje z bleomicinom izzove stranske učinke, predvsem pljučno fibrozo in razjede na mestih zdravljenja. Da bi bilo zdravljenje z elektrokemoterapijo kar najbolj učinkovito in varno, je poleg ostalega potrebno določiti tudi parametre farmakokinetike bleomicina. Prav tako je potrebno razjasniti vpliv tumorskega mikrookolja na porazdelitev bleomicina v tumorju in posledično na učinkovitost zdravljenja z elektrokemoterapijo. Namen dela: Namen doktorskega dela je bil ovrednotiti učinkovitost in varnost elektrokemoterapije z bleomicinom v zdravljenju raka glave in vratu. Specifični cilji so bili: (i) določitev optimalnega časovnega okvirja od časa injiciranja bleomicina do elektroporacije in optimalne koncentracije bleomicina, ki je potrebna za učinkovito elektrokemoterapijo, (ii) izboljšanje tehnološkega postopka zdravljenja globoko ležečih tumorjev v področju glave in vratu, (iii) določitev napovednih dejavnikov za uspešnost elektrokemoterapije. Hipoteza: Elektrokemoterapija je učinkovita in varna metoda zdravljenja raka v področju glave in vratu. Metode: V doktorski disertaciji smo spremljali učinek in varnost elektrokemoterapije pri bolnikih s kožnim in sluzničnim rakom glave in vratu. S sklopitvijo načrta zdravljenja in navigacijskega sistema smo poskušali izboljšati postopek zdravljenja globoko ležečih tumorjev v področju glave in vratu. Parametre farmakokinetike bleomicina smo določili tako pri bolnikih kot in vivo pri miših s tekočinsko kromatografijo sklopljeno z masno spektrometrijo. Tumorsko mikrookolje smo preučevali z imunohistokemično analizo na karcinomskem in melanomskem tumorskem modelu. Rezultati: S spremljanjem odgovora na zdravljenje smo dokazali, da je elektrokemoterapija varno in učinkovito zdravljenje raka glave in vratu. Pokazali smo, da je z optimizacijo tehnoloških postopkov elektrokemoterapije možno učinkovito in varno zdraviti tudi v globini ležeče tumorje glave in vratu. Pri starejših bolnikih je terapevtsko okno za aplikacijo električnih pulzov po intravenoznem injiciranju bleomicina daljše kot pri mlajših. Hkrati je pri njih nižji odmerek bleomicina enako učinkovit kot standardni odmerek. Odgovor na zdravljenje z elekokemoterapijo je odvisen od ožiljenosti tumorja, ki vpliva na razporeditev bleomicina v tumorju. Zaključki: Elektrokemoterapija se je izkazala za učinkovit in varen način zdravljenje raka v različnih predelih glave in vratu. Rezultati raziskave pomembno prispevajo k širjenju terapevtskih možnosti pri tem raku in k prilagoditvi elektrokemoterapevtskih protokolov posameznemu bolniku. Značilnosti tumorskega mikrookolja so pomemben dejavnik za napoved uspešnosti elektrokemoterapije. Scientific background: Electrochemotherapy is an inventive approach to cancer treatment. Howevwer, only a few studies so far have focused on its role in the treatment of skin and mucosal head and neck cancers. Properly designed and well-conducted prospective clinical trials are required to determine the efficiency and safety of electrochemotherapy in this group of patients. Technological improvements implement electrochemotherapy as a feasible treatment also for deep-seated head and neck tumors. Nevertheless, intravenously administrated bleomycin is currently the most utilized drug for electrochemotherapy, but it might have some specific side effects, predominantly lung fibrosis and possible ulceration in the treated areas. The main parameters of bleomycin pharmacokinetics need to be determined in order to provide optimal effectiveness and safety of electrochemotherapy. Furthermore, the role of tumor microenvironment in bleomycin distribution and its consequential impact on tumor response to electrochemotherapy has to be explained. Aim: The aim of the study was to evaluate the effectiveness and safety of bleomycin-based electrochemotherapy in the treatment of head and neck cancer. Specific objectives were: (i) to determine an optimal time frame between bleomycin injection and electroporation and optimal bleomycin concentration required for effective electrochemotherapy, (ii) the improvement of technological procedure in the treatment of deep-seated head and neck tumors and (iii) determination of predictive factors for effective electrochemotherapy. Hypothesis: Electrochemotherapy is an effective and safe treatment modality for the treatment of head and neck cancer. Methods: In the scope of the study, the local anti-cancer effectiveness and safety were followed on the skin and mucosal head and neck cancers treated with electrochemotherapy. The efficiency of electrochemotherapy as a treatment option of deep-seated head and neck tumors was evaluated by coupling treatment planning and navigation system. The parameters of bleomycin pharmacokinetics were determined in patients and in vivo in mice by liquid chromatography coupled with high-resolution mass spectrometry. Tumor microenvironment on carcinoma and melanoma tumor models was additionaly investigated with immunohistochemical analysis. Results: The evaluation of treatment outcome in patients with head and neck cancer treated with electrochemotherapy confirms the efficacy and safety of electrochemotherapy in this group of patients. The optimized technological approach enables the treatment of deep-seated tumors in the head and neck region. In the group of elderly patients, therapeutic window is prolonged compared to younger patients and a reduced dose of bleomycin is equally effective as a standard dose. The tumor response to electrochemotherapy is influenced by tumor vascularization, which affects bleomycin distribution in the tumor. Conclusion: Electrochemotherapy proved to be an efficient and safe treatment of cancers in different head and neck regions. The results of the study broaden therapeutic options in the treatment of head and neck cancer and contribute to the adaptation of the electrochemotherapeutic protocols to each individual patient. Characteristics of tumor microenvironment are an important predictive factor to the tumor response to electrochemotherapy.

Purpose Electrochemotherapy is increasingly entering into national and international guidelines, requiring formal evaluation of treatment costs and cost-effectiveness to ensure that its uptake provides value to budget-constrained health care systems. This study analyzed the early cost-effectiveness of electrochemotherapy in patients with Stage IIIc/IV skin melanoma in clinical practice in Slovenia. The costs of electrochemotherapy were compared to those of the standard of care, consisting of palliative treatment and therapy for symptoms. Methods wThe study enrolled 23 patients treated with electrochemotherapy at the Institute of Oncology (Ljubljana, Slovenia). The mean cost of electrochemotherapy was estimated using patient-specific cost data on electrochemotherapy procedures and subsequent follow-up. Quality-adjusted life-years (QALYs) were estimated by collecting EQ-5D-3L questionnaires at baseline, after complete or partial response following the treatment, and after a relapse of skin lesions. A discrete-time Markov model was built to estimate the lifetime costs and consequences of using electrochemotherapy compared to standard of care, from the perspective of the Slovenian health care system. The analysis was conducted separately in the whole patient sample and in the subset of patients with bleeding lesions. Deterministic and probabilistic sensitivity analyses were conducted to test model assumptions and to characterize the uncertainty around model parameters. Findings In the whole patient population, electrochemotherapy for skin melanoma Stage IIIc/IV was expected to increase QALYs by 0.29 (95% credible interval [CrI], 0.10–0.50), at the higher cost of 6568 EUR (95% CrI, 4593–8928) in comparison to the standard of care. At the cost-effectiveness threshold of 20,000 EUR/QALY, the estimated probabilities of electrochemotherapy being cost-effective compared to standard of care were 0.30 and 0.91 in the whole patient sample and in patients with bleeding lesions, respectively. In the whole sample population, a 50% reduction in the price of the electrodes was expected to increase the probability of electrochemotherapy being cost-effective from 0.30 to ~0.64. Implications The findings from this cost-effectiveness analysis of data from clinical practice were based on a small sample size (ie, 23 patents), which made the subgroup of patients with bleeding lesions very small. Therefore, the findings in this patient population should be carefully interpreted.

In the master's thesis, we investigated the possible effects of high-voltage electroporation pulses on the functioning of the pacemaker, under various conditions and methods of pulse delivery. We also developed simulation models that would allow us a more systematic study and understanding of physical phenomena and facilitate further research We were interested in how the influence of the pulses depends on the horizontal distance of the electrodes of the Cliniporator to the electrodes of the pacemaker, what are the maximum currents and voltages that can occur on the pacemaker electrodes and what are the effects when the Cliniporator pulses are delivered at different stages of pacing of the pacemaker. Measurements were carried out in a glass container, with a medium for two different values of electrical conductivity. The results obtained can be compared with voltages and currents that are caused in the body by other medical devices, for which it was proven that they cannot damage the pacemaker or cause its malfunction. While carrying out the measurements, we also came to a realization, that during the delivery of the Cliniporator pulses, the property of the media changed locally as evidenced by the sparking observed at the electrodes of the Cliniporator. Using the results of experimental measurements, two simulation models were constructed. The first model was constructed using linear electrical lumped elements, which were calculated by making the assumptions about the physical properties of the experimental setup during the measurements and using the results of these measurements. The second model was a numerical model using the finite element method, which required the proper composition of 3D geometry and the use of the output voltage of the Cliniporator. The Cliniporator pulse can affect the shape and amplitude of the pacemaker pulse, but the pacemaker pulse returns to its normal form in the next delivery cycle. In clinical circumstances, this should not present a problem, as the delivery of Clinipator pulses during electrochemotherapy are synchronized with the heart cycle and as such the Cliniporator pulses can never coincide with the pacemaker pulses. After all experimentation there was no change in the functioning of the pacemaker. The energy of the Cliniporator pulses delivered into the system is comparable to the energy delivered during defibrillation. Therefore, electrochemotherapy should be safe for patients with a pacemaker, taking similar precautions as for defibrillation. In order to confirm this, further examination of standards concerning cardiac pacemaker devices is needed. The simulation models achieved satisfactory results when computed signals were compared with the measured current and voltage signals. However, we should be aware of the inaccuracies introduced into the model with all the assumptions and simplifications used in building the models. V magistrski nalogi smo raziskovali možne vplive visoko napetostnih elektroporacijskih pulzov na delovanje srčnega spodbujevalnika, pod različnimi pogoji in načini dovajanja pulzov. Razvili smo tudi simulacijske modele, ki bi omogočili bolj sistematično preučevanje in razumevanje fizikalnih pojavov in olajšali nadaljnje raziskovanje. Zanimalo nas je, kakšen je vpliv pulzov v odvisnosti od horizontalne razdalje Cliniporatorjevih elektrod do elektrod srčnega spodbujevalnika, kakšni so maksimalni tokovi in napetosti, ki se lahko pojavijo na elektrodah spodbujevalnika, in kakšni so vplivi, kadar so Cliniporatorjevi pulzi dovedeni ob različnih fazah delovanja spodbujevalnika. Meritve smo izvajali v stekleni kadi z medijem pri dveh različnih vrednostih električne prevodnosti. Pridobljene rezultate lahko primerjamo z napetostmi in tokovi, ki jih v telesu povzročijo naprave, za katere je dokazano, da ne morejo drastično škodovati srčnemu spodbujevalniku. Pri izvajanju meritev smo prišli tudi do spoznanja, da se med dovajanjem Cliniporatorjevih pulzov, lokalno spreminja lastnost uporabljenega medija, zaradi pojava iskrenja ob Cliniporatorjevih elektrodah. S pomočjo rezultatov eksperimentalnih meritev smo sestavili dva simulacijska modela. Prvi model je sestavljen iz koncentriranih linearnih električnih elementov. Koncentrirane elemente smo poračunali s pomočjo predpostavk o fizikalnih lastnosti merilne postavitve in rezultatov meritev. Drugi model je bil numerični model z uporabo metode končnih elementov, ki je upošteval pravilno 3D geometrijo in izhodno napetost Cliniporatorja. Cliniporatorjevi pulzi lahko pri delujočem srčnem spodbujevalniku vplivajo na obliko in amplitudo dovedenega pulza spodbujevalnika, vendar se ta povrne nazaj na svojo normalno oblika že v naslednjemu ciklu delovanja. V klinični razmerah to ne bi smelo biti problematično, saj je dovajanje Cliniporatorjevih pulzov med elektrokemoterapijo natančno sinhronizirano z delovanjem srca in zato pri pravilni sinhronizaciji Cliniporatorjevi pulzi ne bi bili nikoli dovedeni med stimulacijskim pulzom spodbujevalnika. Po končanih eksperimentalnih meritvah nismo opazili nobenih sprememb v delovanju srčnega spodbujevalnika Dovedene energije Cliniporatorjevih pulzov v sistem so primerljive oziroma manjše kot so energije pri defibrilaciiji. Torej bi elektrokemoterapija s tega vidika morala varna za paciente z vgrajenim spodbujevalnikom ob upoštevanju podobnih previdnostnih ukrepov kot pri defibrilaciji. Za potrditev tega bo potrebno še podrobno preučiti standarde, ki se nanašajo na srčne spodbujevalnike. Simulacijski modeli, so dosegli zadovoljive rezultate, kadar smo izračunane signale primerjali s izmerjenimi signali tokov in napetosti. Vendar se je treba zavedati, kakšne napake smo vnesli v model z vsemi predpostavkami in poenostavitvami.

Electroporation is a phenomenon, which occurs when short high voltage pulses are applied to cells and tissues resulting in a transient increase in membrane permeability or cell death, presumably due to pore formation. If cells recover after pulse application, this is reversible electroporation. If cells die, this is irreversible electroporation. Electroporation is used in biotechnology for biocompound extraction and cryopreservation, in food processing for sterilization and pasteurization of liquid food and in medicine for treating tumors by electrochemotherapy or irreversible electroporation as an ablation technique, for gene electrotransfer, transdermal drug delivery, DNA vaccination, and cell fusion. In electroporation-based medical treatments, we can treat tumors with predefined electrode geometry and parameters of electric pulses. When we treat larger tumors of irregular shape treatment plan of the position of the electrodes and parameters of the electric pulses has to be calculated before each treatment to assure coverage of the tumor with a sufficient electric field. In treatment plans, currently, 1) we assume that above an experimentally determined critical electric field all cells are affected and below not, although, in reality, the transition between non-electroporated and electroporated state is continuous. 2) We do not take into account the excitability of some tissues. 3) The increase in tissues’ conductivity is described phenomenologically and does not include mechanisms of electroporation. 4) Transport of chemotherapeutics into the tumor cells in electrochemotherapy treatments is not included in the treatment plan although it is vital for a successful treatment. We focused on the mathematical and numerical models of electroporation with the aim of including them in the treatment planning of electroporation-based medical treatments. We aimed to model processes happening during electroporation of tissues, relevant in the clinical procedures, by taking into account processes happening at the single cell level. First, we used mathematical models of cell membrane permeability and cell death which are phenomenological descriptions of experimental data. The models were chosen on the basis of the best fit with the experimental data. However, they did not include mechanisms of electroporation, and their transferability to tissues was questionable. We modeled time dynamics of dye uptake due to increased cell membrane permeability in several electroporation buffers with regard to the electrosensitization, i.e., delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. We also modeled the strength-duration depolarization curve and cell membrane permeability curve of excitable and non-excitable cell lines which could be used to optimize pulse parameters to achieve maximal drug uptake at minimal tissue excitation. Second, we modeled change in dielectric properties of tissues during electroporation. Model of change in dielectric properties of tissues was built for skin and validated with current-voltage measurements. Dielectric properties of separate layers of skin before electroporation were determined by taking into account geometric and dielectric properties of single cells, i.e., keratinocytes, corneocytes. Dielectric properties of separate layers during electroporation were obtained from cell-level models of pore formation on single cells of lower skin layers (keratinocytes in epidermis and lipid spheres in papillary dermis) and local transport region formation in the stratum corneum. Current-voltage measurements of long low-voltage pulses were accurately described taking into account local transport region formation, pore formation in the cells of lower layers and electrode polarization. Voltage measurements of short high-voltage pulses were also accurately described in a similar way as with long low-voltage pulses however, the model underestimated the current, probably due to electrochemical reactions taking place at the electrode-electrolyte interface. Third, we modeled the transport of chemotherapeutics during electrochemotherapy in vivo. In electrochemotherapy treatments, transport of chemotherapeutics in sufficient amounts into the cell is vital for a successful treatment. We performed experiments in vitro and measured the intracellular platinum mass as a function of pulse number and electric field by inductively coupled plasma – mass spectrometry. Using the dualporosity model, we calculated the in vitro permeability coefficient as a function of electric field and number of applied pulses. The in vitro determined permeability coefficient was then used in the numerical model of mouse melanoma tumor to describe the transport of cisplatin to the tumor cells. We took into account the differences in the transport of cisplatin in vitro and in vivo caused by the decreased mobility of molecules and decreased membrane area available for the uptake in vivo due to the high volume fraction of cells, the presence of cell matrix and close cell connections. Our model accurately described the experimental results obtained in electrochemotherapy of tumors and could be used to predict the efficiency of electrochemotherapy in vitro thus reducing the number of needed animal experiments. In the thesis, we connected the models at the cell level to the models at the tissue level with respect to cell membrane permeability and depolarization, cell death, change in dielectric properties and transport. Our models offer a step forward in modeling and understanding electroporation at the tissue level. In future, our models could be used to improve treatment planning of electroporation-based medical treatments. Visokonapetostni električni pulzi povečajo prepustnost celične membrane (Tsong 1991 Weaver 1993 Kotnik et al. 2012) skozi pore (Abidor et al. 1979), ki nastanejo na tistih njenih delih, kjer vsiljena transmembranska napetost preseže kritično vrednost (Towhidi et al. 2008 Kotnik et al. 2010). Elektroporacija je reverzibilna, če si celica po pulzih opomore, in ireverzibilna, če je škoda preobsežna in celica odmre (Pakhomova et al. 2013b Jiang et al. 2015a). Trenutne optične metode por ne morejo zaznati, zato njihov nastanek zaznavamo posredno, bodisi z meritvami vnosa različnih molekul v celice ali z meritvami električnih lastnosti celic (Napotnik in Miklavčič 2017). Uporaba elektroporacije V živilski industriji (Toepfl 2012 Toepfl et al. 2014) uporabljamo elektroporacijo oziroma pulzirajoča električna polja (angl. pulsed electric fields), kar je uveljavljen izraz v tej industriji, za uničevanje patogenih organizmov in njihovih produktov (encimov in toksinov). V nasprotju s termično obdelavo hrane električni pulzi ne vplivajo na okus, barvo ali hranilno vrednost. V biotehnologiji uporabljamo elektroporacijo za ekstrakcijo molekul iz mikroorganizmov in rastlin, s čimer se izognemo uporabi kemičnih sredstev in ne uničimo celičnih organelov, torej se izognemo tudi dodatnemu čiščenju končnega produkta (Sack et al. 2010 Haberl et al. 2013a Mahnič-Kalamiza et al. 2014b Kotnik et al. 2015). Primeri: ekstrakcija DNK iz bakterij sladkorja iz sladkorne pese (Haberl et al. 2013b), sokov iz sadja polifenolov iz grozdja za izboljšanje kvalitete vina (Puértolas et al. 2010) vode pri sušenju zelene biomase, ki služi kot vir za biogorivo (Golberg et al. 2016). Elektroporacija je tudi nova metoda pri zamrzovanju celic in tkiv, angl. cryopreservation (Galindo in Dymek 2016 Dovgan et al. 2017). Elektroporacijo uporabljamo tudi v medicini (Miklavčič et al. 2010 Yarmush et al. 2014), in sicer pri elektrokemoterapiji (Miklavčič et al. 2012 Mali et al. 2013 Cadossi et al. 2014 Miklavčič et al. 2014 Campana et al. 2014 Serša et al. 2015), netermičnem odstranjevanju tkiva z ireverzibilno elektroporacijo (Davalos et al. 2005 Garcia et al. 2010 José et al. 2012 Cannon et al. 2013 Scheffer et al. 2014b Jiang et al. 2015a Rossmeisl et al. 2015), genski terapiji (Golzio et al. 2002 Vasan et al. 2011 Gothelf in Gehl 2012 Calvet et al. 2014 Heller in Heller 2015 Trimble et al. 2015) in vnosu učinkovin v kožo in skoznjo (Denet et al. 2004 Zorec et al. 2013b). Pri genski terapiji vnesemo v celice plazmide, v katerih je zapisana sinteza določenega proteina, ki lahko spremeni biološko funkcijo celice (Aihara in Miyazaki 1998 Heller in Heller 2015). Z elektroporacijo povišamo varnost genske terapije, saj se izognemo uporabi virusov in kemikalij. Mehanizmi genske terapije z elektroporacijo še niso popolnoma pojasnjeni, osnovni koraki so opisani v literaturi (Rosazza et al. 2016). Z elektroporacijo lahko zlivamo različne celice, s čimer pridobivamo celice, ki proizvajajo monoklonska protitelesa ali inzulin (Ramos in Teissié 2000 Trontelj et al. 2008 Rems et al. 2013). V doktorski disertaciji sem se osredotočila na uporabo elektroporacije v medicini, predvsem pri elektrokemoterapiji, netermičnem odstranjevanju tkiva z ireverzibilno elektroporacijo in pri vnosu učinkovin v kožo in skoznjo je, zato so ti trije posegi podrobneje opisani v naslednjem poglavju. Medicinski posegi z elektroporacijo – elektrokemoterapija, netermično odstranjevanje tkiva z ireverzibilno elektroporacijo in vnos učinkovin v kožo in skoznjo Elektrokemoterapija je kombinacija kemoterapije in električnih pulzov, dovedenih neposredno na tarčno tkivo. Električni pulzi povečajo prepustnost celične membrane za kemoterapevtike, zato povečamo učinkovitost zdravljenja, obenem pa zmanjšamo dovedeno dozo kemoterapevtika in omilimo stranske učinke. Celoten tumor mora biti pokrit z dovolj visokim električnim poljem, da povečamo prepustnost vseh tumorskih celic (Miklavčič et al. 2006a), zagotoviti pa moramo tudi dovolj visoko koncentracijo kemoterapevtika znotraj tumorja (Miklavčič et al. 2014). Okoliško tkivo ne sme biti uničeno, torej mora biti električno polje okoli tumorja pod mejo za ireverzibilno elektroporacijo. Pri elektrokemoterapiji običajno dovajamo osem pulzov dolžine 100 μs s ponavljalno frekvenco 1 Hz. S poskusi določena meja za povišanje prepustnosti tumorskega tkiva je 0,4 kV/cm (Miklavčič et al. 2010). Osem pulzov je bilo določenih kot optimalno število pulzov (Marty et al. 2006 Mir et al. 2006), večje število dovedenih pulzov namreč že zmanjšuje preživetje (Dermol in Miklavčič 2015). Za zdravljenje tumorjev z elektrokemoterapijo so bili definirani standardni postopki (angl. standard operating procedures) (Marty et al. 2006 Mir et al. 2006), kjer so glede na število tumorjev, njihovo velikost in lokacijo (na koži ali pod kožo) določeni tip elektrod, kemoterapevtik, anestezija in način dovajanja kemoterapevtika. Kemoterapevtik lahko dovedemo lokalno ali sistemsko. V elektrokemoterapiji oz. terapiji z električnimi pulzi sta najbolj razširjena kemoterapevtika cisplatin in bleomicin. Z elektrokemoterapijo je možno zdraviti tudi globlje ležeče tumorje (Miklavčič et al. 2010 Pavliha et al. 2013 Edhemović et al. 2014 Miklavčič in Davalos 2015). V zadnjem času se uveljavlja tudi uničevanje tumorskih celic z visokimi koncentracijami kalcija in električnimi pulzi (Frandsen et al. 2015 Frandsen et al. 2016 Frandsen et al. 2017). Pri elektrokemoterapiji se pojavijo še dodatni učinki, ki povišajo učinkovitost elektroporacije. Vazokonstrikcija zmanjša spiranje kemoterapevtika iz tumorja in s tem ohranja visoko koncentracijo kemoterapevtika v tumorju, obenem se zmanjša pretok krvi skozi tumor, kar povzroči hipoksijo in pomanjkanje hranilnih snovi (Mir 2006 Serša et al. 2008). Elektrokemoterapija sproži tudi odziv imunskega sistema, ki nato odstrani preostale tumorske celice (Serša et al. 2015). Z ireverzibilno elektroporacijo netermično odstranjujemo tumorje brez uporabe kemoterapevtika (Jiang et al. 2015a). Tako se popolnoma izognemo stranskim učinkom kemoterapevtikov, vendar na račun več dovedene energije in posledično Joulovega gretja. Pri ireverzibilni elektroporaciji dovajamo več (okoli 90) električnih pulzov, dolgih od 50 μs do 100 μs, s ponavljalno frekvenco 1 Hz. Dovedeno električno polje je v rangu nekaj kV/cm, kar je dosti več kot pri elektrokemoterapiji. Pri ireverzibilni elektroporaciji lahko z visoko natančnostjo odstranimo želeno tkivo – območje med uničenim in nepoškodovanim tkivom je široko le nekaj premerov celic (Rubinsky et al. 2007). Za odstranjevanje tumorjev tradicionalno uporabljamo termične metode (Hall et al. 2014) – radiofrekvenčno odstranjevanje in odstranjevanje s tekočim dušikom, kjer tkivo uničujemo z visoko oz. z nizko temperaturo. Prednost ireverzibilne elektroporacije pred uveljavljenimi termičnimi metodami je krajši čas zdravljenja, izognemo se učinkom hlajenja oz. gretja tkiva zaradi bližine žil (Golberg et al. 2015), pri čemer ostanejo okoliške pomembne strukture (žile, živci) nedotaknjene (Jiang et al. 2015a). Tudi pri ireverzibilni elektroporaciji je v dokončno odstranitev tumorskih celic vpleten imunski sistem (Neal et al. 2013). Pri elektrokemoterapiji in ireverzibilni elektroporaciji se zaradi daljših pulzov in ponavljalne frekvence 1 Hz pojavljajo težave zaradi krčenja mišic (Miklavčič et al. 2005), bolečine med dovajanjem pulzov, heterogenosti električnih lastnosti tkiv v tem frekvenčnem področju ter zaradi možnosti srčnih aritmij (Ball et al. 2010). Bolečini in krčenju mišic se lahko izognemo, če pulze dovajamo z višjo frekvenco, npr. 5 kHz (Županič et al. 2007 Serša et al. 2010). Srčnim aritmijam se izognemo tako, da s sinhroniziramo dovedene električne pulze z električno aktivnostjo srčne mišice (Mali et al. 2008 Deodhar et al. 2011a Mali et al. 2015). Bolečini, krčenju mišic in heterogenosti električnih lastnosti tkiv se lahko izognemo z dovajanjem 1 μs bipolarnih pulzov (Arena et al. 2011 Arena in Davalos 2012 Sano et al. 2015). V zadnjem času so se pojavile tudi metode, s katerimi so vnos barvil v celico dosegli brezkontaktno s t. i. magnetoporacijo (Chen et al. 2010 Towhidi et al. 2012 Kardos in Rabussay 2012 Novickij et al. 2015 Kranjc et al. 2016 Novickij et al. 2017b Novickij et al. 2017a). Elektroporacijo lahko uporabljamo ne le za zdravljenje tumorjev, temveč tudi za vnos učinkovin v kožo in skoznjo. Vnos učinkovin skozi kožo je neinvaziven, poleg tega pa se izognemo degradaciji učinkovin pri prehodu skozi prebavni trakt. Skozi kožo lahko preide le malo molekul, zato uporabljamo različne metode za povečanje prehoda učinkovin – iontoforezo, radiofrekvenčno mikroablacijo, laser, mikroigle, ultrazvok in elektroporacijo (Zorec et al. 2013b). Proces elektroporacije kože je slabo razumljen. Predpostavljamo, da pri dovajanju visokonapetostnih električnih pulzov v roženi plasti nastanejo lokalna transportna območja, kjer sta povišani električna prevodnost in prepustnost (Pliquett et al. 1996 Pliquett et al. 1998 Pliquett et al. 1998 Pavšelj in Miklavčič 2008a). Skozi lokalna transportna območja lahko nato učinkovine še nekaj ur po dovedenih pulzih vstopajo skozi kožo v krvni obtok (Zorec et al. 2013a). Gostota teh območij je odvisna od električnega polja v koži – višje električno polje jih povzroči več. Velikost lokalnih transportnih območij je odvisna od trajanja pulza. Med samim pulzom se zaradi Joulovega gretja topijo lipidi v roženi plasti, kar povzroči njihovo širjenje (Pliquett et al. 1996 Prausnitz et al. 1996 Pliquett et al. 1998 Weaver et al. 1999 Vanbever et al. 1999 Gowrishankar et al. 1999b). Načrtovanje posegov elektrokemoterapije in netermičnega odstranjevanja tkiva z ireverzibilno elektroporacijo Pri zdravljenju tumorjev z elektroporacijo lahko uporabimo standardne oblike in postavitve elektrod z že določenimi parametri električnih pulzov (Marty et al. 2006 Mir et al. 2006 Campana et al. 2014). Če zdravimo velike tumorje ali tumorje nepravilnih oblik, ki pogosto ležijo globlje, s standardno postavitvijo elektrod ne moremo zagotoviti ustrezne pokritosti tumorja z dovolj visokim električnim poljem. V tem primeru lahko elektrode med samim posegom večkrat premaknemo ali pa prilagodimo njihovo število in postavitev. Pri tem moramo prej pripraviti načrt posega (Kos et al. 2010 Miklavčič et al. 2010 Pavliha et al. 2012 Linnert et al. 2012 Edhemović et al. 2014). V njem zagotovimo, da bo cel tumor izpostavljen dovolj visokemu električnemu polju (Miklavčič et al. 2006a), obenem pa škoda na okoliškem tkivu minimalna. Načrtovanje posega poteka v več korakih: 1. zajem medicinskih slik (računalniška tomografija, magnetna resonanca) tumorja in okoliškega tkiva 2. obdelava slik 3. razgradnja slik in določitev geometrije tkiva 4. vzpostavitev tridimenzionalnega modela 5. optimizacija postavitve elektrod glede na obliko in velikost tumorja 6. izdelava modela elektroporacije (izračun električnega polja in spremembe električne prevodnosti tkiva) 7. optimizacija napetosti med elektrodami in položaja elektrod (Pavliha et al. 2012). Na sliki 1 lahko vidimo izračunano električno polje v tumorju in okoliškem tkivu pri eni izmed možnih postavitev elektrod.

In addition to several well-known drug delivery strategies developed to facilitate effective chemotherapy with anticancer agents, some new approaches have been recently established, based on specific effects arising from the applications of ultrasound, magnetic and electric fields on drug delivery systems. This paper gives an overview of newly developed methods of drug delivery to tumors and of the related anticancer therapies based on the combined use of different physical methods and specific drug carriers. The conventional strategies and new approaches have been put into perspective to revisit the existing and to propose new directions to overcome the threatening problem of cancer diseases., Osim dobro poznatih metoda prijenosa lijekova u kemoterapijskom pristupu liječenja tumora, nedavno su otkriveni novi načini prijenosa koji se zasnivaju na specifičnim mehanizmima uzrokovanim upotrebom ultrazvuka, magnetskih i električnih polja. Članak sadrži prikaz fizikalnih mehanizama na kojima se temelje ove nove metode, kao i pregled novootkrivenih prijenosnika lijekova (Pluronske micele, magnetoliposomi, magnetski fluidi), novih terapija tumora (magnetska hipertermija, elektrokemoterapija) i najnovijih istraživanja temeljenih na fizikalnom pristupu ovoj problematici.