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Properties of lipid electropores I: Molecular dynamics simulations of stabilized pores by constant charge imbalance
No full textMolecular dynamics (MD) simulations have become a powerful tool to study electroporation (EP) in atomic detail. In the last decade, numerous MD studies have been conducted to model the effect of pulsed electric fields on membranes, providing molecular models of the EP process of lipid bilayers. Here we extend these investigations by modeling for the first time conditions comparable to experiments using long (mu s-ms) low intensity (similar to KV/cm) pulses, by studying the characteristics of pores formed in lipid bilayers maintained at a constant surface tension and subject to constant charge imbalance. This enables the evaluation of structural (size) and electrical (conductance) properties of the pores formed, providing information hardly accessible directly by experiments. Extensive simulations of EP of simple phosphatidylcholine bilayers in 1 M NaCl show that hydrophilic pores with stable radii (1-2.5 nm) form under transmembrane voltages between 420 and 630 mV, allowing for ionic conductance in the range of 6.4-29.5 nS. We discuss in particular these findings and characterize both convergence and size effects in the MD simulations. We further extend these studies in a follow-up paper (Rems et al., Bioelectrochemistry, Submitted), by proposing an improved continuum model of pore conductance consistent with the results from the MD simulations
Insights into cell membrane electroporation with molecular dynamics simulations
No full textIzpostavitev bioloških celic kratkim visokonapetostnim električnim pulzom povzroči strukturne spremembe v njihovih membranah. Te spremembe povečajo prepustnost celičnih membran in omogočijo transmembranski transport molekulam, za katere so membrane običajno neprepustne. Pojav se imenuje elektroporacija in se uporablja na raznih področjih medicine, biologije in biotehnologije. Razumevanje mehanizmov elektroporacije je močno napredovalo z uporabo simulacij molekularne dinamike (MD). Te omogočajo napoved gibanja atomov in molekul v preučevanem sistemu ter vizualizacijo procesov na molekularni ravni. Simulacije MD so razkrile, da lahko povečano prepustnost membran pripišemo vodnim poram, ki nastanejo pod vplivom električnega polja v lipidnih domenah membrane, ali pa celo v napetostnih senzorjih nekaterih napetostno-odvisnih ionskih kanalov. Slednji igrajo ključno vlogo pri proženju električnih signalov imenovanih akcijski potenciali pri mišičnih in nevronskih celicah. Odpira se vprašanje, kolikšna je verjetnost poškodb ionskih kanalov pri elektroporaciji oziroma ali je nastanek por ugodnejši v lipidnih domenah membrane ali v ionskih kanalih. Zato smo v diplomski nalogi z uporabo atomističnih simulacij MD primerjali hitrost s katero električno polje ustvarja pore v različnih lipidnih dvoslojih in ionskih kanalih. Začeli smo s simulacijami dvosloja zgrajenega iz lipidov 1-pamitoil 2-oleoil fosfatidilholin (POPC), ki predstavlja najbolj poenostavljen model celične membrane in se pogosto uporablja na področju raziskav elektroporacije s simulacijami MD. Simulirali smo dva dvosloja POPC različnih velikosti in ugotovili, da velikost dvosloja vpliva na hitrost elektroporacije, pri čemer se pore hitreje tvorijo v večjem dvosloju. To ugotovitev smo upoštevali v nadaljevanju, ko smo simulirali elektroporacijo dvosloja, sestavljenega iz 15 različnih vrst lipidov, ki je predstavljal realističen model lipidne domene v membrani sesalcev. Ta lipidni dvosloj smo zasnovali na podlagi rezultatov predhodne študije in je predstavljal lipidno domeno, ki je najbolj dovzetna za elektroporacijo. Čas, potreben za elektroporacijo lipidnih dvoslojev, smo nato primerjali s časom, potrebnim za nastanek por v dveh klinično relevantnih napetostno-odvisnih ionskih kanalih, NaV1.5 in CaV1.1. Natrijev kanal NaV1.5 je pretežno izražen v srčno-mišičnih celicah, kalcijev kanal CaV1.1 pa v skeletno-mišičnih celicah. Ugotovili smo, da elektroporacija ionskih kanalov v povprečju poteče pri nižjih vrednostih električnega polja kot elektroporacija lipidnih dvoslojev, saj je bil pri dani vrednosti električnega polja nastanek por skozi napetostne senzorje hitrejši v primerjavi z nastankom por v lipidnih domenah. To pomeni, da je elektroporacija napetostno-odvisnih ionskih kanalov NaV1.5 in CaV1.1 energetsko ugodnejša od elektroporacije lipidnih domen. Rezultati nakazujejo, da je verjetnost poškodb ionskih kanalov pri elektroporaciji mišičnih in nevronskih celic zelo visoka. Te poškodbe lahko vplivajo na sposobnost proženja akcijskih potencialov in obliko le-teh po elektroporaciji mišičnih in nevronskih celic. Ugotovitve študije so pomembne za optimizacijo zdravljenj temelječih na elektroporaciji, kot so ablacija srčne mišice za zdravljenje artimij, ablacija možganskih tumorjev in vnos nukleinskih kislin v skeletne in srčne mišične celice za gensko terapijo.Exposure of biological cells to short, high-voltage electrical pulses induces structural changes in their membranes, leading to an increase in membrane permeability and allowing transmembrane transport of molecules that are otherwise unable to cross the membrane. This phenomenon is termed electroporation and is used in various applications in medicine, biology and biotechnology. Our mechanistic understanding of electroporation has considerably improved through the use of molecular dynamics (MD) simulations. MD enables simulations of the movement of atoms and molecules within the studied system and offers a visualization of processes at the molecular level. MD simulations have revealed that the increase in membrane permeability can be attributed to the formation of aqueous pores formed under the influence of a strong electric field in the membrane lipid bilayer, or even in the voltage sensors of certain voltage-gated ion channels (VGICs). The latter play a crucial role in the generation of electrical signals called action potentials in muscle and neuronal cells. This opens the question on whether electroporation is favoured in lipid domains or VGICs and how susceptible VGICs are to damage due to electroporation. In this thesis we thus used atomistic MD simulations to compare how fast an electric field can form pores in different lipid bilayers and VGICs. We first simulated a 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) bilayer that represents the simplest model of a cell membrane and is commonly used for studying electroporation with MD simulations. We studied electroporation in two POPC bilayers of different sizes and found that the rate of pore formation depends on the bilayer size, with pores forming faster in the larger bilayer. This finding was considered in the following step, where we simulated electroporation of a bilayer composed of 15 different types of lipids, modeling a realistic lipid domain within a mammalian cell membrane. This bilayer was designed based on results from a previous study and represented a lipid domain that has the highest propensity for electroporation. The time required to form pores in these lipid bilayers was then compared with the time required for electroporation of two clinically relevant VGICs, NaV1.5 and CaV1.1. The sodium channel NaV1.5 is predominantly expressed in cardiac muscle cells, while the calcium channel CaV1.1 is predominantly expressed in skeletal muscles. Our results showed that electroporation of VGICs occurs on average at lower electric field strength than electroporation of lipid bilayers. At a given electric field strength, formation of pores within the voltage sensors of VGICs occurs faster compared to pores within lipid bilayers. This means that electroporation of the studied VGICs, NaV1.5 and CaV1.1, is energetically favorable compared to lipid bilayer electroporation. Consequently, our results suggest that the likeness of electroporation-induced damage to these VGICs is very high. Such damage may affect the ability of muscle and neuronal cells to trigger action potentials and alter the shape of these action potentials after electroporation. The findings of this study are particularly important for optimizing electroporation-based treatments, such as cardiac ablation for treatment of heart arrhythimias, ablation of brain tumours, and nucleic acid delivery into skeletal and cardiac muscle for gene therapy
Monitoring changes in transmembrane voltage after in vitro cell electroporation in the presence of ion channel inhibitors
No full textZ elektroporacijo povečamo prepustnost plazemske membrane, kar nam omogoči, da v celico vnesemo ali iz nje ekstrahiramo želene učinkovine, ki plazemske membrane sicer ne prehajajo. Ta tehnika se uporablja v različnih medicinskih aplikacijah, kot so gensko zdravljenje, zdravljenje tumorjev in dostava zdravil. Uspešnost elektroporacije temelji na doseganju ravnotežja med povišanjem prepustnosti plazemske membrane in preživetjem celic. Pomembno vlogo pri preživetju celic predstavlja sposobnost celice, da obnovi mirovno transmembransko napetost in vzpostavi celično homeostazo.
V nalogi smo raziskovali, kako elektroporacija vpliva na spremembe v transmembranski napetosti pri ovarijskih celicah kitajskega hrčka (CHO-K1) in kako na te spremembe vplivata zaviralec TRPM4 kanalov 9-hydroxyphenanthrene (9-phenantrol) in zaviralec kalijevih ionskih kanalov tetraetilamonij (TEA). Predpostavili smo, da bi zaviralec 9-phenantrol lahko imel vpliv na spremembe v TMN, saj so predhodne študije pokazale, da CHO celice izražajo endogene TRPM4 kanale. Zaviralec TEA pa je bil izbran kot negativna kontrola, saj CHO celice ne izražajo znatnega števila kalijevih kanalov.
Elektroporacijo smo dosegli z izpostavitvijo celic enemu električnemu pulzu dolžine 10 µs, 100 µs ali 1000 µs. Amplitudo pulza smo določili na podlagi meritev vnosa kalcija v celice s pomočjo fluorescenčnega barvila Fluo4-AM. Za vsako dolžino pulza smo določili najnižjo amplitudo, pri kateri smo zaznali znatni vnosa kalcija v celice. Poleg te najnižje amplitude, ki predstavlja prag za elektroporacijo, smo izbrali še približno 2-krat višjo amplitudo. Pulze izbranih amplitud smo nato uporabili pri nadaljnjih meritvah sprememb v transmembranski napetosti. Za slednje smo uporabili potenciometrično barvilo FLIPR, katerega fluorescenca se spreminja s spremembo v TMN.
Ugotovili smo, da se membrana po elektroporaciji depolarizira, vendar nobeden od testiranih zaviralcev ni imel signifikantnega vpliva na to depolarizacijo, ne glede na dolžino in amplitudo pulza. To nakazuje, da je bila depolarizacija v veliki meri povzročena zaradi neselektivnega povečanja prepustnosti membrane zaradi elektroporacije. Za lažjo interpretacijo eksperimentalnih rezultatov smo razvili teoretični model, s katerim smo preverili, v kolikšni meri bi TRPM4 kanali sploh lahko prispevali k spremembam TMN po elektroporaciji. Model je potrdil, da TRPM4 kanali nimajo bistvenega vpliva na depolarizacijo, saj se pri danih pogojih membrana povsem depolarizira že zaradi neselektivnega toka ionov prek por v elektroporirani membrani.
Poskusi so razkrili še, da nizkonapetostni pred-pulz dolžine 45 ms, ki ga proži elektroporator BTX Gemini za meritev upornosti vzorca pred proženjem elektroporacijskega pulza, zmanjša fluorescenco barvila FLIPR. Mehanizmi tega vpliva niso povezani s povečanjem prepustnosti membrane in ostajajo nepojasnjeni. V nadaljnjih poskusih bi bilo priporočljivo uporabljati elektroporator, ki pred dovajanjem pulza ne proži pred-pulza, tako da celice ne bi po nepotrebnem izpostavljali stresu, ki lahko vpliva na rezultat elektroporacije. Hkrati bi bilo smiselno testirati druga barvila za merjenje sprememb v transmembranski napetosti, ki bi lahko nadomestila barvilo FLIPR.Electroporation increases the permeability of the plasma membrane, allowing us to introduce or extract desired substances into/from the cell that would not normally pass through the plasma membrane. This technique has various medical applications such as gene therapy, tumor treatment, and drug delivery. The success of electroporation is based on achieving a balance between increasing the permeability of the plasma membrane and cell survival. A crucial role in cell survival is played by the cell\u27s ability to restore the resting transmembrane potential and establish cellular homeostasis.
In the thesis, we investigated how electroporation affects changes in transmembrane voltage in Chinese hamster ovary (CHO-K1) cells and how these changes are influenced by the TRPM4 channel inhibitor 9-hydroxyphenanthrene (9-Phenanthrene) and the potassium ion channel inhibitor tetraethylammonium (TEA). We hypothesized that the inhibitor 9-Phenanthrene might have an effect on the changes in TMN, as previous studies have shown that CHO cells express endogenous TRPM4 channels. However, the TEA inhibitor was chosen as a negative control as CHO cells do not express a significant number of potassium channels.
Electroporation was achieved by exposing the cells to a single electrical pulse of 10 µs, 100 µs or 1000 µs. The amplitude of the pulse was determined from measurements of calcium uptake into the cells using the fluorescent dye Fluo4-AM. For each pulse length, we determined the lowest amplitude at which significant calcium uptake into the cells was detected. In addition to this lowest amplitude, which represents the threshold for electroporation, we selected an amplitude approximately 2x higher. Pulses of the selected amplitudes were then used in further measurements of changes in transmembrane voltage. For the latter, we used the FLIPR potentiometric dye, whose fluorescence changes with the change in TMN.
We found that the membrane depolarizes after electroporation, but none of the inhibitors tested had a significant effect on this depolarization, regardless of the pulse length and amplitude. This suggests that the depolarization was largely caused by a non-selective increase in membrane permeability due to electroporation. To facilitate the interpretation of the experimental results, we developed a theoretical model to test the extent to which TRPM4 channels could contribute at all to the changes in TMN after electroporation. The model confirmed that TRPM4 channels do not have a significant effect on depolarization, as under the given conditions the membrane is already fully depolarized due to non-selective ion flux through the pores in the electroporated membrane.
Experiments further revealed that a low-voltage pre-pulse of 45 ms, delivered by a BTX Gemini electroporator to measure the sample resistance before delivering the electroporation pulse, reduces the fluorescence of the FLIPR dye. The mechanisms of this effect are unrelated to the increase in membrane permeability and remain unexplained. In further experiments, it would be advisable to use an electroporator that does not deliver a pre-pulse before the electroporation pulse, to avoid unnecessarily exposing the cell to stress that may affect the electroporation result. At the same time, it would be useful to test other dyes to measure changes in transmembrane voltage that could replace the FLIPR dye
Metoda za konstrukcijo in optimizacijo lipidnih nanodiskov za simulacije molekularne dinamike
No full textLipid nanodisks are lipid bilayer structures stabilized by a belt of alipoproteins.
They play a critical role in studying membrane proteins and developing drug
delivery systems. Mimetic peptides offer a simplified yet effective alternative to
full-length apolipoproteins in stabilizing these nanodisks, providing a platform for
understanding lipid-protein interactions and exploring therapeutic applications.
The objective of this thesis was to develop, implement, and test a computational
protocol for constructing and simulating nanodisks composed of lipid bilayers
and stabilized by mimetic peptides, using molecular dynamics all-atom simula
tions with the charmm36m forcefield. Five all-atom simulations were conducted
to study the structural dynamics of nanodisks formed by 14A peptide dimers and
DMPClipids. Anadditional simulation was done by replacing DMPC with POPC
and cholesterol, to study the effect of different lipids on nanodisk properties. The
presence of cholesterol in the POPC system led to slower stabilization and less
shrinkage, likely due to increased membrane rigidity. Coarse-grained simulations
using the Martini 3 forcefield were explored for their potential to accelerate sim
ulation times. Construction was successful, but the coarse-grain models failed
to maintain structural integrity during production runs, indicating that current
coarse-grain models may not accurately represent interactions between peptide
dimers. While coarse-grain simulations offer the potential for significant accelera
tion of the simulation speed, further refinement of the forcefield and stabilization
strategies are necessary for reliable nanodisk modeling.Lipoproteini so nanostrukture, bistvene za transport lipidov v krvnem obtoku
zaradi hidrofobnosti lipidov. Sestavljeni so iz apolipoproteinov in razliˇ cnih lipi
dov, vkljuˇcno s holesterolom in trigliceridi. Med ˇstirimi glavnimi vrstami lipopro
teinov—HDL, LDL, VLDL in IDL—je HDL najbolj znan po svoji sestavi in
funkciji [1]. HDL, ki je sestavljen predvsem apolipoproteina A1 (Apo-AI) in
fosfolipidov, je kljuˇcen za reverzni transport holesterola (angl. reverse cholesterol
transport), kar zmanjˇsuje aterosklerozo in tveganje za kardiovaskularne bolezni
Gene electrotransfer using porous membranes
No full textGenska elektrotransfekcija je ena izmed glavnih nevirusnih metod za dostavo plazmidne DNK
v notranjost bioloških celic. Ta metoda temelji na pojavu elektroporacije, pri katerem se ob
izpostavitvi celic pulzirajočemu električnemu polju začasno poveča prepustnost celičnih
membran, kar pa omogoča vnos terapevtskih molekul v celice. Uporaba naprav z
nanostrukturiranimi geometrijami, kot so nanoslamice, nanokanali in nanopore, omogoča
lokalizacijo električnega polja na majhnih delih celične membrane, s čimer je mogoče znatno
povečati učinkovitost genske elektrotransfekcije in izboljšati preživetje celic. Slabosti takšnih
nanostrukturiranih naprav so, da niso splošno dostopne in zahtevajo strokovno znanje
postopkov nanofabrikacije ter dostop do čistih prostorov. Nedavno pa je bilo dokazano, da je
mogoče doseči zelo učinkovito elektrotransfekcijo plazmidov tudi z uporabo komercialnih
polikarbonatnih membran z nanoporami polmera 0,05 μm. Zato v tej raziskovalni nalogi
preučujem in želim oblikovati pristop elektrotransfekcije, ki temelji na komercialno dostopnih
vstavkih s poroznimi membranami iz polietilentereftalata (PET), namenjenim za kulturo in
preučevanje biologije celic. Najprej z numeričnim modeliranjem na ravni ene celice določim
parametre električnega pulza, velikost por porozne membrane in druge dejavnike, za katere se
pričakuje, da bodo povzročili znotrajcelično dostavo plazmidov. Na podlagi numeričnih
rezultatov oblikujem preprosto napravo, ki vsebuje vstavek, porozno membrano s porami
polmera 0,2 m, 0,5 m ali 1,5 m, večjamično ploščo in par žičnih elektrod. Napravo nato
tudi numerično modeliram, da načrtam ustrezno postavitev in konfiguracijo elektrod, ki
omogoči homogeno porazdelitev električne napetosti vzdolž porozne membrane. Napravo nato
preizkusim na treh celičnih linijah sesalcev in ovrednotim učinkovitost genske transfekcije ter
preživetje celic. Pridobljeni rezultati so primerljivi z rezultati klasične genske
elektrotransfekcije, pri kateri suspenzijo celic ali celice, pritrjene na podlago, postavimo med
par vzporednih elektrod in jih izpostavimo homogenemu električnemu polju. Vendar uporaba
poroznih vstavkov omogoča dovajanje nizkonapetostnih pulzov, ki ne zahtevajo uporabe dragih
visokonapetostnih elektroporatorjev in je možno pulzni generator izdelati tudi doma. Na koncu
diskutiram še o možnostih nadaljnjih raziskav, vezanih na vpliv lastnosti porozne membrane,
kot so debelina, poroznost in polmer por, na učinkovitost transfekcije.Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid
DNA, wherein exposure of biological cells to pulsed electric field induces electroporation, that
is a transient increase in cell membrane permebility to exogenous molecules. By localizing the
electric field on small parts of the cell membrane using nanostructured geometries such as
nanostraws, nanochannels, and nanopores, the efficiency of gene electrotransfer and cell
viability can be significantly increased. Disadvantages of such nanofabricated devices are that
they are not widely available and require nanofabrication expertise and access to cleanroom. It
has recently been shown that highly efficient electrotransfer of plasmids can also be achieved
using commercial polycarbonate membranes with nanopores of 0.05 μm in radius. Therefore,
in this thesis I study and design an electrotransfer approach based on commercially available
cell culture inserts with porous membrane from polyethylene terephthalate (PET). First, I use
numerical modeling at the single cell level to determine electric pulse parameters, size of pores
in the porous membrane, and other factors expected to result in successful gene electrotransfer.
Based on the numerical results, I design a simple device that contains an insert with porous
membrane containing pores with radius of 0,2 µm, 0,5 µm or 1,5 µm, a multiwell plate, and a
pair of wire electrodes. I numerically model the device to improve the placement and
configuration of the electrodes, in order to achieve a homogenous voltage along the entire
porous membrane. I then test the device using three mammalian cell lines and assess the
transfection efficiency and cell viability. The results are comparable to those obtained in
previous studies using classical gene electrotransfer, where cells in suspension or adhered to
surface are placed between a pair of parallel electrodes and exposed to homogenous electric
field. However, the porous membranes enable application of low-voltage pulses, which do not
require the use of expensive high-voltage electroporators. Finally, I discuss further research
directions to assess the influence of porous membrane properties, such as thickness, porosity,
and pore radius on transfection efficiency and cell viability
Modelling the effect of electroporation on the triggering of action potentials in cardiomyocytes
No full textSrčne aritmije so nepravilnosti srčnega ritma, ki lahko segajo od blagih in asimptomatskih do
življenjsko ogrožajočih stanj. Zdravljenje srčnih aritmij je individualizirano in temelji na vrsti
aritmije, resnosti, zdravstvenih razmerah posameznika ter tudi na starosti in življenjskem slogu bolnika. Atrijska fibrilacija (AF) je najpogostejša motnja srčnega ritma, ki jo zaznamuje
neurejen in hiter srčni utrip. Pogosto povzroča simptome, kot so palpitacije, utrujenost in
zasoplost, ter povečuje tveganje za možgansko kap in srčno popuščanje.
V zadnjih letih je katetrska ablacija postala prednostna metoda za zdravljenje atrijske fibrilacije, kar odražajo tudi najnovejša priporočila. Postopek ablacije uniči določena območja srčnega tkiva, ki povzročajo aritmijo oz. ta območja električno izolira od preostalega dela srca. Klasični načini katetrske ablacije vključujejo radiofrekvenčno ablacijo (RFA) in krioablacijo, ki sta se izkazali za učinkoviti pri odpravljanju aritmičnih žarišč.
Ireverzibilna elektroporacija je inovativna netermična ablacijska metoda v srčni
elektrofiziologiji, ki temelji na uporabi visokonapetostnih električnih pulzov, ki povzročijo
povečanje prepustnosti celičnih membran. Povečana prepustnost poruši celično homeostazo,
kar lahko vodi v smrt tarčnih celic. Ta proces omogoča uničenje tarčnih celic, kar se je izkazalo učinkovito za zdravljenje srčnih aritmij. Ireverzibilna elektroporacija obeta manjše tveganje za poškodbe okoliških tkiv in boljšo varnost za bolnike v primerjavi s termičnimi metodami ablacije.
Znano pa je, da se okrog ireverzibilno elektroporiranega tkiva vedno nahaja tudi regija
reverzibilno elektroporiranega tkiva, v katerem celice preživijo izpostavitev električnim
pulzom. Reverzibilna elektroporacija kardiomiocitov bi lahko vplivala njihovo sposobnost
proženja akcijskih potencialov v teh regijah. Zato je potrebno bolje razumeti, kako
elektroporacija vpliva na funkcijo kardiomiocitov.
Cilj tega diplomskega dela je bil raziskati vpliv elektroporacije na funkcijo kardiomiocitov s
poudarkom na modeliranju vpliva elektroporacije na proženje akcijskih potencialov pri
izoliranih kardiomiocitih. Z modelom smo želeli razjasniti, kako povečanje prepustnosti
membrane in neselektivnega toka ionov prek membrane vpliva na delovanje ionskih kanalov in prenašalcev v celični membrani ter na dinamiko akcijskih potencialov in časovnega poteka
znotrajceličnega kalcija, ki je povezan s krčenjem kardiomiocita.
Za dosego cilja raziskave smo uporabili dva matematičnega modela, ki opisujeta dinamiko
akcijskih potencialov pri izoliranih kardiomiocitih s pomočjo nadomestnega električnega vezja.
Izbrana modela smo nadgradili z vključitvijo opisa neselektivnega toka ionov prek por v
membrani, nastalih zaradi elektroporacije. Model smo implementirali in simulirali v okolju
Matlab, sistem diferencialnih enačb pa rešili s funkcijo ode15s.
Naše simulacije so pokazale, da nastanek por v membrani močno vpliva na proženje akcijskih
potencialov. Z višanjem števila por se akcijski potencial najprej podaljšuje, dokler se pri
določenem številu por kardiomiociti ne morejo več repolarizirati in ostanejo depolarizirani ter niso več sposobni proženja akcijskih potencialov. Ko kardiomiociti postanejo depolarizirani, se možno poveča tudi koncentracija znotrajceličnega kalcija. Vse te spremembe opazimo že pri zelo majhnem številu ustvarjenih por (manj kot 20), kar pomeni, da že zelo šibka elektroporacija lahko močno vpliva na sposobnost proženja akcijskih potencialov. Rezultati raziskave bodo pripomogli k razumevanju sprememb v znotrajsrčnih elektrogramih, ki jih opazijo kardiologi po ablaciji srca z elektroporacijo, in potencialno k uporabi teh signalov za napovedovanje uspeha zdravljenja.Cardiac arrhythmias are irregularities in the heart\u27s rhythm that can range from mild and
asymptomatic to life-threatening conditions. The treatment of cardiac arrhythmias is
individualized and based on the type of arrhythmia, its severity, the individual\u27s health
conditions, as well as the patient\u27s age and lifestyle. Atrial fibrillation (AF) is the most common heart rhythm disorder, characterized by an irregular and rapid heartbeat. It often causes symptoms such as palpitations, fatigue, and shortness of breath and increases the risk of stroke and heart failure.
In recent years, catheter ablation has become the preferred method for treating atrial fibrillation, as reflected in the latest recommendations. The ablation procedure destroys specific areas of heart tissue that cause the arrhythmia or electrically isolates these areas from the rest of the heart. Traditional methods of catheter ablation include radiofrequency ablation (RFA) and cryoablation, which have proven effective in eliminating arrhythmic foci.
Irreversible electroporation is an innovative non-thermal ablation method in cardiac
electrophysiology that uses high-voltage electric pulses to increase the permeability of cell
membranes. The increased permeability disrupts cellular homeostasis, which can lead to the
death of target cells. This process allows the destruction of target cells and has been shown to be effective in treating cardiac arrhythmias. Irreversible electroporation promises a lower risk of damage to surrounding tissues and better safety for patients compared to thermal ablation methods.
However, it is known that around the irreversibly electroporated tissue, there is always a region of reversibly electroporated tissue, in which cells survive exposure to the electric pulses. Reversible electroporation of cardiomyocytes could affect their ability to trigger action potentials in these regions. Therefore, it is necessary to better understand how electroporation affects the function of cardiomyocytes.
The aim of this thesis was to investigate the impact of electroporation on the function of
cardiomyocytes, with a focus on modeling the impact of electroporation on the triggering of
action potentials in isolated cardiomyocytes. The model aimed to clarify how increased membrane permeability and non-selective ionic current across the membrane affect the
functioning of ion channels and transporters in the cell membrane, as well as the dynamics of action potentials and the time course of intracellular calcium associated with cardiomyocyte contraction.
To achieve the research goal, we used two mathematical models that describe the dynamics of action potentials in isolated cardiomyocytes using an equivalent electrical circuit. The selected models were upgraded by including a description of the non-selective ionic current through pores in the membrane created due to electroporation. The model was implemented and simulated in the Matlab environment, and the system of differential equations was solved using the ode15s function.
Our simulations showed that the formation of pores in the membrane significantly affects the triggering of action potentials. As the number of pores increases, the action potential starts to prolong until, at a certain number of pores, cardiomyocytes can no longer repolarize and remain depolarized, becoming incapable of triggering action potentials. When cardiomyocytes become depolarized, the concentration of intracellular calcium also increases. These changes are observed already with a very small number of created pores (less than 20), indicating that even very weak electroporation can significantly affect the ability of cardiomyocytes to trigger action potentials. The results of this research will contribute to better understanding of the changes in intracardiac
electrograms that are observed by cardiologists following cardiac ablation with electroporation,and potentially to the use of these signals for improving the treatment outcome
Identifikacija in opis mehanizmov molekularnega transporta preko celične membrane pri elektroporaciji
No full textElectroporation is a phenomenon which results in transient increase in cell membrane permeability for ions and molecules when exposing biological cells to short high-voltage electric pulses. If cells survive the exposure to electric pulses, electroporation is called reversibleotherwise, if cells die, electroporation is called irreversible. Electroporation is used in biomedicine for electrochemotherapy, gene electrotransfer, transdermal drug delivery, DNA vaccination, and as an ablation method to treat heart arrhythmia and tumors. It is also used for various purposes in biotechnology, food processing, and environmental applications, such as extraction of compounds from plant tissue, inactivation of bacteria, cell fusion, and genetic engineering of microorganisms.
Electrochemotherapy uses electroporation to enhance the delivery of chemotherapeutic drugs into tumor cells. It is successfully used in clinics to treat cutaneous and subcutaneous tumors with ongoing trials for the treatment of deep-seated tumors. However, the monopolar pulses with duration of 100 μs, used classically for electrochemotherapy, cause pain and muscle contractions. To overcome these drawbacks the use of bursts of high-frequency short bipolar pulses has been suggested. Furthermore, recent efforts have been focused on making electrochemotherapy a systemic treatment by combining it with gene electrotransfer for immunotherapy. Gene electrotransfer is also based on electroporation, where millisecond pulses are used for intracellular delivery of DNA molecules that code for proteins able to stimulate the immune response. Thus, using pulse types alternative to classical 100 μs pulses could be beneficial for improving the electrochemotherapy treatment. However, it is not well understood whether different types of pulses can be equally effective for electrochemotherapy. Therefore, the first aim of the dissertation was to investigate how different types of pulses affect cisplatin uptake and cytotoxicity. We performed in vitro experiments using cisplatin and three types of pulses: classical electrochemotherapy pulses, high-frequency bipolar pulses, and millisecond pulses. We demonstrated that all tested types of pulses can be considered equivalent in terms of cisplatin uptake and cytotoxicity and can potentially replace classical, i.e., monopolar 100 μs electrochemotherapy pulses.
For electrochemotherapy to be successful two main conditions need to be met: (i) the entire tumor must be exposed to a sufficiently high electric field that results in electroporation of the tumor cells and (ii) a sufficient amount of a chemotherapeutic drug (typically bleomycin or cisplatin) must enter the cells to bind to DNA and kill the tumor cells. The pulse parameters needed to successfully treat cutaneous tumors are provided in the standard operating procedures, whereas the treatment of deep-seated tumors is guided by a computational model that predicts the distribution of the electric field inside a tissue depending on the electrode configuration. To further improve such computational treatment planning, it would be useful to upgrade the model with a description of electroporation and the associated uptake of chemotherapeutic drugs into tumor cells. To enable the development of such models, it is necessary to determine the number of cisplatin molecules needed inside the cell to achieve a cytotoxic effect. Therefore, the second aim of the dissertation was to quantify the number of cisplatin molecules, delivered into cells by different types of pulses, and determine the lethal number that results in eradication of almost all treated cells. We found that the number of cisplatin molecules needed to achieve a cytotoxic effect is in the range of 2-7 ×107 molecules per cell, irrespective of the type of pulses used.
Mathematical models are also useful for understanding the phenomenon of electroporation. Many different models that describe electroporation and the associated transmembrane molecular transport are present in the literature. Whilst these models differ in their theoretical description, they typically show good agreement with a specific set of data. It is not clear if any of the models can be applied to describe the molecular transport for the broad range of pulse parameters and other experimental conditions used in electroporation research. Therefore, the third aim of the dissertation was to critically assess existing mechanistic models describing electroporation-mediated transmembrane transport of ions and molecules at the single-cell level. We confronted the models with a broad range of experimental measurements and observed that none of the models was reliable enough to predict molecular transport in all tested conditions. We underlined the limitations of the models and proposed further research to improve them. Nevertheless, the existing models can still help interpret certain experimental results, such as the influence of cardiomyocyte orientation on electroporation using pulses of different durations.Če biološko celico izpostavimo električnemu polju z dovolj visoko jakostjo, dosežemo zača-sno povečanje prevodnosti in prepustnosti celične membrane. Ta pojav se imenuje elektroporacija. Če celice preživijo izpostavljenost električnim pulzom, se elektroporacija imenuje reverzibilnače celice umrejo, se elektroporacija imenuje ireverzibilna. Elektroporacija se v biomedicini uporablja pri elektrokemoterapiji, genski terapiji, vnosu zdravilnih učinkovin skozi kožo, cepljenju z DNK ter kot metoda ablacije za zdravljenje srčnih aritmij ali tumorjev. Uporablja se tudi za različne namene v biotehnologiji in predelavi hrane, na primer za ekstrakcijo snovi iz rastlinskega tkiva, uničevanje bakterij, zlivanje celic in genski inženiring mikroorganizmov.
Pri elektrokemoterapiji z elektroporacijo izboljšamo vnos kemoterapevtskih učinkovin v tumorske celice, kar se v klinikah uspešno uporablja za zdravljenje kožnih in podkožnih tumorjev, v teku pa so tudi študije za zdravljenje globlje ležečih tumorjev. Monopolarni pulzi s trajanjem 100 μs, ki jih običajno dovajamo pri elektrokemoterapiji, povzročajo bolečine in krčenje mišic. Z dovajanjem vlakov visokofrekvenčnih kratkih bipolarnih pulzov lahko omilimo bolečine in krčenje mišic. Nedavno so se pojavile študije, kjer elektrokemoterapijo kombiniramo z imunsko gensko terapijo in dosežemo sistemsko zdravljenje. Pri genski terapiji, ki temelji na elektroporaciji, se uporabljajo milisekundni pulzi za znotrajcelični prenos molekul DNK, ki kodirajo beljakovine, sposobne spodbuditi imunski odziv. Z dovajanjem različnih pulzov, ki so alternativni klasičnim 100 μs pulzom, bi lahko izboljšali zdravljenje z elektrokemoterapijo. Ker še ni povsem jasno, ali so lahko različne vrste pulzov enako učinkovite pri elektrokemoterapiji, je bil prvi cilj disertacije raziskati, kako različne vrste pulzov vplivajo na vnos cisplatina in na citotoksičnost. Izvedli smo poskuse in vitro z dovajanjem cisplatina in treh vrst pulzov: klasičnih elektrokemoterapevtskih pulzov, visokofrekvenčnih bipolarnih pulzov in milisekundnih pulzov. Dokazali smo, da lahko vse preizkušene vrste pulzov štejemo za enakovredne v smislu vnosa cisplatina in citotoksičnosti.
Za uspešno elektrokemoterapijo morata biti izpolnjena dva glavna pogoja: i) celoten tumor mora biti izpostavljen dovolj visokemu električnemu polju, ki povzroči elektroporacijo tumorskih celic, in ii) zadostna količina kemoterapevtika (običajno bleomicina ali cisplatina) mora vstopiti v celice, da se veže na DNK in uniči tumorske celice.
Parametri pulzov, ki so potrebni za uspešno zdravljenje kožnih tumorjev, so določeni v standardnih operativnih postopkih, medtem ko je zdravljenje globokih tumorjev načrtovano z računalniškim modelom, ki predvideva porazdelitev električnega polja v tkivu glede na postavitev elektrod. Za nadaljnje izboljšanje takšnega računalniškega načrtovanja zdravljenja bi bilo koristno model nadgraditi z opisom elektroporacije in z njo povezanega vnosa kemoterapevtskih učinkovin v tumorske celice. Za razvoj takih modelov je treba določiti število molekul cisplatina, potrebnih v celici za citotoksični učinek. Drugi cilj disertacije je bil torej izmeriti število molekul cisplatina, ki jih v celice vnesemo z različnimi vrstami pulzov, in določiti zadostno število, ki povzroči celično smrt. Ugotovili smo, da je število molekul cisplatina, potrebnih za doseganje citotoksičnega učinka, v razponu 2-7 ×107 molekul na celico ne glede na vrsto dovedenih pulzov.
Za razumevanje pojava elektroporacije so koristni matematični modeli. V literaturi je veliko različnih modelov, ki opisujejo elektroporacijo in z njo povezan prenos molekul skozi celično membrano. Čeprav se ti modeli razlikujejo v svojem teoretičnem opisu, običajno kažejo dobro ujemanje z določenim nizom podatkov. Ni jasno, ali je mogoče katerega od modelov uporabiti za opis molekularnega transporta za širok razpon parametrov pulzov in drugih eksperimentalnih pogojev. Tretji cilj disertacije je bil kritično oceniti obstoječe mehanistične modele transporta ionov in molekul skozi elektroporirano celično membrano. Modele smo preverjali na širokem naboru eksperimentalnih meritev in ugotovili, da nobeden od modelov ni bil dovolj zanesljiv za napoved molekularnega transporta v vseh preizkušenih pogojih. Poudarili smo omejitve modelov in predlagali nadaljnje raziskave za izboljšanje in nadgradnjo obstoječih modelov. Kljub omejitvam lahko obstoječi modeli še vedno pomagajo pri razlagi nekaterih eksperimentalnih rezultatov, na primer vpliva orientacije kardiomiocitov v električnem polju in trajanja pulzov na učinkovitost elektroporacije
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
APPLICATIONS OF THEORETICAL MODELS OF LIPID MEMBRANE ELECTROPORATION
Full text linkV doktorski disertaciji smo pokazali, da lahko z numeričnimi modeli učinkovito prispevamo k eksperimentalnim raziskavam. Numerični modeli sicer ne morejo nadomestiti eksperimentov, lahko pa vodijo začetni razvoj eksperimentalnega protokola in njegovo nadaljnjo optimizacijo, ter pripomorejo k interpretaciji eksperimentalnih
rezultatov. Povezave med modeli na molekularni ravni, ravni celice in ravni tkiva so dosegljive in pomembne za napredovanje v razumevanju elektroporacije, s tem pa posledično razvoju učinkovitejših terapij in tehnologij. Eksperimentalni protokol za zlivanje različno velikih bioloških celic z električnimi pulzi trajanja največ nekaj sto nanosekund. Elektrozlivanje je vzpostavljena metoda za proizvodnjo hibridnih celic. Za razliko od kemičnih in virusnih metod, je elektrozlivanje fizikalna metoda, zato jo lahko varno uporabljamo tudi v kliniki. Pri elektrozlivanju je pomembno, da celice preživijo izpostavitev električnim pulzom, saj lahko le tako pridobimo žive zlite celice. Pri večini aplikacij si želimo doseči zlivanje med celicami različnega tipa. Če se celice močno razlikujejo v velikosti, lahko to predstavlja težavo pri elektrozlivanju, saj lahko večje celice postanejo poškodovane pri parametrih električnih pulzov, ki so potrebni za elektroporacijo manjših celic. Večje celice se namreč običajno elektroporirajo pri nižjih jakostih električnega polja kot manjše celice, posebej kadar dovajamo običajne pulze z dolžino nekaj 10 μs in več. Z uporabo numeričnega modeliranja smo pokazali, da lahko s pulzi dolžine v področju nanosekund dosežemo selektivno elektroporacijo stikov med celicami, ne glede na velikost celic.
Nanosekundni pulzi tako lahko omogočijo elektrozlivanje celic različnih velikosti, ne da bi pri tem poškodovali celice. Na podlagi predpostavk numeričnih izračunov smo razvili uspešen eksperimentalni protokol za elektrozlivanje celic z nanosekundnimi pulzi. Rezultati eksperimentov pa so potrdili napovedi numeričnega
modela. Numerični model biološkega tkiva z razločeno celično strukturo, ki omogoča načrtovanje elektroporacije tega tkiva Numerični modeli elektroporacije tkiva običajno obravnavajo tkivo kot homogeno strukturo s povprečnimi električnimi lastnostmi. Pri heterogenih tkivih je takšna obravnava preveč poenostavljena, saj heterogenost celične strukture povzroči nemohogeno porazdelitev električnega polja v tkivu. Modeliranje heterogenih tkiv kot homogeno strukturo tako lahko vodi do nepravilne interpretacije numeričnih rezultatov. Pri protokolih elektroporacije, kjer je preživetje celic ključnega pomena, je hkrati tudi pomembno, da pri načrtovanju protokolov upoštevamo heterogenost tkivne strukture. Napredek v razvoju modelov tkiv z razločeno celično
strukturo ima torej visok pomen. Krioprezervacija predstavlja primer aplikacije, kjer morajo električni pulzi zagotoviti elektroporacijo vseh celic v tkivu, kar omogoči da krioprotektant vstopi v vse celice in jih ščiti med zamrzovanjem. Hkrati pa električni pulzi tudi ne smejo poškodovati celic. V namen optimizacije krioprezervacije špinače smo razvili numerični model z razločeno celično strukturo špinačnega lista. Model je bil validiran na podlagi meritev električnih lastnosti špinačnih listov v frekvenčni domeni in z meritvami
električnega toka skozi list med dovajanjem elektroporacijskih pulzov. Ta model je prvi numerični model elektroporacije tkiva, ki upošteva celotno celično strukturo tkiva. Postopek, po katerem smo razvili model, pa lahko vodi nadaljnji razvoj podobnih modelov za druge tipe tkiva. Numerični model prevodnosti vodne pore v lipidnem dvosloju, validiran s simulacijami
molekularne dinamike Prevodnost pore je eden najpomebnejših parametrov, ki premošča teoretične in eksperimentalne študije vodnih por, ki nastanejo v lipidnem dvosloju pod vplivom vsiljene transmembranske napetosti (tj. elektroporacije). Simulacije molekularne dinamike namreč ponujajo možnost študije por na molekularnem nivoju, pri eksperimentih na ravninskih lipidnih dvoslojih pa raziskovanje por poteka preko meritev njihove prevodnosti. Most med eksperimentalnimi študijami in simulacijami molekularne dinamike ponujajo modeli, ki sistem opisujejo s strani zveznih teorij. V namen učinkovite premostitve smo razvili numerični Poisson-Nernst- Planckov model ionskega toka prek lipidne pore. Model je bil zgrajen neposredno po molekularnem sistemu, s katerim smo izmerili prevodnost por s pomočjo simulacij molekularne dinamike. S kvantitativno primerjavo med
napovedmi modela in rezultati, ki smo jih pridobili z analizo simulacij, smo numerični model validirali. Ta model predstavlja prvo direktno replikacijo molekularnega sistema z numeričnim modelom v smislu ionske prevodnosti lipidne pore. Pričakujemo lahko, da bo ta model prispeval k učinkovitejši karakterizaciji lipidnih por v eksperimentalnih študijah, hkrati pa lahko pričakujemo, da bo izboljšal tudi napovedi modelov, ki opisujejo elektroporacijo celičnih membran.Electroporation, electropermeabilization, or pulsed-electric-field (PEF) treatment, are all terms naming the treatment of cells with short (ns–ms) electric pulses, which induce an increase in cell membrane permeability. This technique is widely used in various medical and biotechnological applications, e.g. for increasing the uptake of drugs and genetic material into cells and tissues, for nonthermal tissue ablation, extraction of different components from plant tissues, food preservation, as well as inactivation of bacteria in food processing and environmental applications. Electroporation is generally achieved by placing the target cells or tissue between electrodes, to which electric pulses are delivered. During pulse application, the resulting electric field induces a transmembrane voltage across the cell membranes, which, when sufficiently high, leads to membrane structural rearrangement. At least part of these rearrangements are attributed to formation of aqueous pores in the membrane lipid domains, since similar phenomenon can also be observed in model lipid membranes, such as planar lipid bilayers and lipid vesicles. The induced transmembrane voltage is determined by the pulse parameters, electric field strength, cell size, geometry, orientation, and the proximity of other structures, which perturb the local electric field, such as neighboring cells. The most complex is thereby electroporation in tissues, which can be highly heterogeneous.
In many applications of electroporation, the protocol of applying electric pulses needs to be carefully tailored as to ensure that the cells are not damaged by excessive electric field, allowing them to survive the exposure after being electroporated. For such purpose, theoretical models of electroporation can be of great help, as they provide the means to probe the effects of different pulse parameters and can guide the optimization of experimental protocols. The first aim of the present thesis was thereby to use theoretical (numerical) modeling to complement and guide in vitro experimental work. We performed three studies, each addressing a different application of electroporation. In the first study we investigated the possibility of using nanosecond electric pulses for electroporating intracellular liposomes. Liposomes are drug delivery vehicles which have the advantage to protect the drug from the hostile environment, particularly in the blood plasma, as well as the organism itself from the toxic effects of the drug. But once the liposomes reach the target cells, their content needs to be released into the cytosol. Nanosecond electric pulses, which are able to electroporate intracellular organelles, could provide a method to control the release of the liposomal content. Our numerical results predicted that that nanosecond pulses can efficiently be used for electroporating the liposomes without affecting the cell viability, provided that the pulses are not much longer than 10 ns, if liposomes are ~100 nm large.
Our second study was oriented towards cell electrofusion and demonstrated the potential advantage of using nanosecond electric pulses for electrofusing cells with different size. Cell cultures characterized by a larger size are generally electroporated at lower electric field strength. When simultaneously electroporating two cell cultures with different size, which is performed in cell electrofusion protocols, the larger cells may become damaged when exposed to an electric field required to electroporate the smaller cells, in particular when conventional tens or hundreds of microseconds long pulses are applied. This is known to be an issue in electrofusion of lymphocytes with myeloma cells in hybridoma technology for monoclonal antibody production. Using numerical modeling, we demonstrated that when cells placed in a low conductive medium, typical for electrofusion protocols, are exposed to pulses with duration in the nanosecond range, the induced transmembrane voltage is the highest in the contact zone between cells, i.e., the target area for electrofusion. Amplification of the transmembrane voltage at the contact zone allows one to optimize the pulse parameters to specifically electroporate the contact zones and avoid problems due to cell size differences. We further developed an experimental protocol for fusing cells with nanosecond pulses, and confirmed our numerical predictions by experimental results.
The third study presents the development of an experimentally validated numerical model of a spinach leaf with resolved tissue structure in order to address the problems in cryopreservation of spinach leaves. In the latter, the cryoprotectant (e.g. trehalose) is first introduced into the extracellular space inside the leaf tissue by means of vacuum impregnation. Afterwards, the leaf is electroporated to allow the cryoprotectant to enter the cells, as the cryoprotectant needs to be present on both sides of the membrane in order to increase the freezing tolerance of the leaves. The leaf tissue is heterogeneous and it is difficult to achieve electroporation and survival of all cells in the tissue after exposure to electric pulses. In addition, the leaf is too thick to allow microscopic examination of all tissue layers. Consequently, the developed model allowed us to investigate electroporation of cells in different tissue layers and provided the possibility to further optimize the pulse parameters for reversible electroporation of all cells in the tissue.
Despite the general usefulness of numerical models of electroporation, the predictive power of the models relies on the proper description of the underlying electroporation process, which is not yet sufficiently well characterized on the molecular level. The possibility to progress towards improving the theoretical descriptions of electroporation, which are based on continuum theories, is offered by molecular dynamics simulations. The second aim of the thesis was thereby to compare the predictions arising from continuum electroporation models with results from molecular dynamics simulations. Our focus was the characterization of pore conductance, which is an important parameter in continuum electroporation models, and it can also be directly related to experimental measurements. We compared the results of pore conductance extracted from molecular dynamics simulations with the predictions of a continuum model based on the Poisson-Nernst-Planck theory. This theory is the origin of all theoretical descriptions of pore conductance, which are used in continuum electroporation models. Nevertheless, these descriptions contain many simplified assumptions. Our study demonstrated that the theory is able to describe the overall pore conductance to Na+ and Cl– ions very well, provided that we take into account the toroidal shape of the pore. In addition, we provided a continuum approach which allows to describe also the pore selectivity, i.e., higher conduction of Cl– than Na+ ions. We further compared our results to simplified theoretical expressions of pore conductance and demonstrated that the simplifications do indeed influence the overall predictions of continuum electroporation models.
In conclusion, theoretical models of electroporation provide a convenient way to complement experimental investigations by enhancing the understanding of the physics underlying the experimental data. Interconnections between molecular-scale, cell-scale, and tissue-scale models are feasible and important for progressing towards better understanding of the electroporation phenomenon and consequently developing more efficient therapies and technologies
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