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Objective: Pelvic exenteration in women with recurrent vulvar carcinoma is associated with high morbidity and mortality and substantial treatment costs. Because pelvic exenteration severely affects the quality of life and can lead to significant complications, other treatment modalities, such as electrochemotherapy, have been proposed. The aim of this study was to evaluate the feasibility and suitability of electrochemotherapy in the treatment of recurrent vulvar cancer. We aimed to analyze the treatment options, treatment outcomes, and complications in patients with recurrent vulvar cancer of the perineum. Methods: A retrospective analysis of patients who had undergone pelvic exenteration for vulvar cancer at the Institute of Oncology Ljubljana over a 16-year period was performed. As an experimental, less mutilating treatment, electrochemotherapy was performed on one patient with recurrent vulvar cancer involving the perineum. Comparative data analysis was performed between the group with pelvic exenteration and the patient with electrochemotherapy, comparing hospital stay, disease recurrence after treatment, survival after treatment in months, and quality of life after treatment. Results: We observed recurrence of disease in 2 patients with initial FIGO stage IIIC disease 3 months and 32 months after pelvic exenteration, and they died of the disease 15 and 38 months after pelvic exenteration. Two patients with FIGO stage IB were alive at 74 and 88 months after pelvic exenteration. One patient with initial FIGO stage IIIC was alive 12 months after treatment with electrochemotherapy with no visible signs of disease progression in the vulvar region, and the lesions had a complete response. The patient treated with electrochemotherapy was hospitalized for 4 days compared with the patients with pelvic exenteration, in whom the average hospital stay was 19.75 (± 1.68) days. Conclusion: Our experience has shown that electrochemotherapy might be a less radical alternative to pelvic exenteration, especially for patients with initially higher FIGO stages.

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.

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.

Electrochemotherapy (ECT) is an effective locoregional therapy for cutaneous melanoma metastases and has been safely combined with immune checkpoint inhibitors in preliminary experiences. Since ECT is known to induce immunogenic cell death, its combination with immune checkpoint inhibitors might be beneficial. In this study, we aimed to investigate the effectiveness of ECT on cutaneous melanoma metastases in combination with pembrolizumab. We undertook a retrospective matched cohort analysis of stage IIIC%IV melanoma patients, included in the International Network for sharing practices of ECT (InspECT) and the Slovenian Cancer Registry. We compared the outcome of patients who received the following treatments: (a) pembrolizumab alone, (b) pembrolizumab plus ECT, and (c) ECT. The groups were matched for age, sex, performance status, and size of skin metastases. The local objective response rate (ORR) was higher in the pembrolizumab-ECT group than in the pembrolizumab group (78% and 39%, p < 0.001). The 1 year local progression-free survival (LPFS) rates were 86% and 51% (p < 0.001), and the 1 year systemic PFS rates were 64% and 39%, respectively (p = 0.034). The 1 year overall survival (OS) rates were 88% and 64%, respectively (p = 0.006). Our results suggest that skin-directed therapy with ECT improves superficial tumor control in melanoma patients treated with pembrolizumab. Interestingly, we observed longer PFS and OS in the pembrolizumab-ECT group than in the pembrolizumab group. These findings warrant prospective confirmation. Nasl. z nasl. zaslona. Opis vira z dne 31. 8. 2021. Bibliografija na koncu prispevka. Abstract.

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.

Endometrial carcinoma is the most common gynecological malignancy and the fifth most common malignancy in women. The worldwide incidence is 15.9 new cases per 100,000 women per year, and the incidence in Europe is 22.7 new cases. Minority of cases are diagnosed at an advanced stage of the disease. Cutaneous metastases are very rare with a prevalence of 0.8%. If cutaneous metastases are present, the prognosis is poor with an overall survival of up to 12 months. In this review, we presented clinical data on treatment of gynecological cancers with electrochemotherapy, with focus on treatment of cutaneous vulvar metastases from endometrial cancer. Further, we present our data on the case of a 64-year-old woman with recurrent endometrial adenocarcinoma with vulvar skin metastases. Treatment of endometrial carcinoma metastases is multimodal with surgery, chemotherapy, radiotherapy and hormone treatment. There is still no consensus about the specific treatment of cutaneous metastases from endometrial cancer, in particular in order to release symptoms. Electrochemotherapy may be a treatment option to reduce pain and bleeding and a safe option to treat multiple skin metastases.

Electrochemotherapy (ECT) is a local ablative treatment that is based on the reversible electroporation and intracellular accumulation of hydrophilic drug molecules, which greatly increases their cytotoxicity. In mucosal head and neck cancer (HNC), experience with ECT is limited due to the poor accessibility of tumors. In order to review the experience with ECT in mucosal HNC, we undertook a systematic review of the literature. In 22 articles, published between 1998 and 2020, 16 studies with 164 patients were described. Curative and palliative intent treatment were given to 36 (22%) and 128 patients (78%), respectively. The majority of tumors were squamous cell carcinomas (79.3%) and located in the oral cavity (62.8%). In the curative intent group, complete response after one ECT treatment was achieved in 80.5% of the patients, and in the palliative intent group, the objective (complete and partial) response rate was 73.1% (31.2% and 41.9%). No serious adverse events were reported during or soon after ECT and late effects were rare (19 events in 17 patients). The quality-of-life assessments did not show a significant deterioration at 12 months post-ECT. Provided these preliminary data are confirmed in randomized controlled trials, ECT may be an interesting treatment option in selected patients with HNC not amenable to standard local treatment. Nasl. z nasl. zaslona. Opis vira z dne 3. 5. 2021. Bibliografija na koncu prispevka. Abstract.

The beneficial effects of electrochemotherapy (ECT) for superficial tumours and, more recently, deepseated malignancies in terms of local control and quality of life are widely accepted. However, the variability in responses across histotypes needs to be explored. Currently, patient selection for ECT is based on clinical factors (tumour size, histotype, and exposure to previous oncological treatments), whereas there are no biomarkers to predict the response to treatment. In this field, two major areas of investigation can be identified, i.e., tumour cell characteristics and the tumour microenvironment (vasculature, extracellular matrix, and immune infiltrate). For each of these areas, we describe the current knowledge and discuss how to foster further investigation. This review aims to provide a summary of the currently used guiding clinical factors and delineates a research roadmap for future studies to identify putative biomarkers of response to ECT. These biomarkers may allow researchers to improve ECT practice by customising treatment parameters, manipulating the tumour and its microenvironment, and exploring novel therapeutic combinations.

The aim of this clinical study was to investigate the effectiveness and long-term safety of electrochemotherapy as an emerging treatment for HCC in patients not suitable for other treatment options. A prospective phase II clinical study was conducted in patients with primary HCC who were not suitable for other treatment options according to the Barcelona Clinic Liver Cancer classification. A total of 24 patients with 32 tumors were treated by electrochemotherapy. The procedure was effective, feasible, and safe with some procedure-related side effects. The responses of the 32 treated nodules were: 84.4% complete response (CR), 12.5% partial response (PR), and 3.1% stable disease (SD). The treatment was equally effective for nodules located centrally and peripherally. Electrochemotherapy provided a durable response with local tumor control over 50 months of observation in 78.0% of nodules. The patient responses were: 79.2% CR and 16.6% PR. The median progression-free survival was 12 months (range 2.7&ndash, 50), and the overall survival over 5 years of observation was 72.0%. This prospective phase II clinical study showed that electrochemotherapy was an effective, feasible, and safe option for treating HCC in patients not suitable for other treatment options.

The aim of this study was to evaluate the safety and efficacy of the combination of electrochemotherapy (ECT) with bleomycin and gene electrotransfer (GET) of plasmid encoding canine interleukin 12 (IL-12) for the treatment of canine oral malignant melanoma (OMM). Our focus was to determine the effect of the treatment on achieving local tumor control and stimulation of an antitumor immune response. Nine dogs with histologically confirmed OMM stage I to III were included in a prospective, non-randomized study. The dogs were treated with a combination of cytoreductive surgery, ECT and IL-12 GET, which was repeated up to five times, depending on the clinical response to the treatment, evaluated according to the follow-up protocol (7, 14 and 28 days after, the last treatment). One month after treatment, the objective response (OR) rate was 67% (6/9). Median survival time (MST) was 6 months and, even though the disease progressed in 8/9 patients at the end of the observation period (2 to 22 months), four animals were euthanized due to tumor-unrelated reasons. In addition, we observed a decline in the percentage of regulatory T cells (Treg) in the peripheral blood in the course of the treatment, which could be attributed to a systemic antitumor response to IL-12 GET. The results of this study suggest that a combination of ECT and IL-12 GET may be beneficial for dogs with OMM, especially when other treatment approaches are not acceptable due to their invasiveness or cost.

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.