doctoral dissertation
Lea Rems (Avtor), Damijan Miklavčič (Mentor), Mounir Tarek (Komentor)

Povzetek

V 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.

Ključne besede

elektroporacija;biološka celica;tkivo;liposom;zlivanje celic;krioprezervacija;pora v lipidnem dvosloju;prevodnost pore;numerično modeliranje;simulacije molekularne dinamike;

Podatki

Jezik: Angleški jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL FE - Fakulteta za elektrotehniko
Založnik: [L. Rems]
UDK: 602.621(043.3)
COBISS: 11300948 Povezava se bo odprla v novem oknu
Št. ogledov: 2043
Št. prenosov: 417
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Slovenski jezik
Sekundarni naslov: APPLICATIONS OF THEORETICAL MODELS OF LIPID MEMBRANE ELECTROPORATION
Sekundarni povzetek: 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.
Sekundarne ključne besede: lipidne membrane;elektroporacija;numerični modeli;Elektroporacija;Disertacije;Modeli;
Vrsta dela (COBISS): Doktorsko delo/naloga
Študijski program: 1000319
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za elektrotehniko
Strani: XLIV, 176 str.
ID: 9127560