Modeling electroporation of the non-treated and vacuum impregnated heterogeneous tissue of spinach leaves

Uniform electroporation of the heterogeneous structure of spinach leaf cross section is a technological challenge that is addressed in this investigation. Three dimensional models were created with cells arranged in specific tissue types, considering a leaf with its air fraction and a leaf where the air fraction was replaced by a solution of known properties using vacuum impregnation. The models were validated before electroporation, in the frequency domain, where alternating voltage and current signal at frequencies from 20 Hz to I MHz were used to measure conductivity of the tissue. They were also validated through measurements of current during electroporation when a single 250 mu s rectangular pulse with amplitudes ranging from 50 to 500 V was applied. Model validations show that both the frequency dependent conductivity and electroporation are well predicted. The importance of the wax layer and stomata in the model is thoroughly discussed. Industrial relevance: Our aim was to investigate electroporation of the spinach leaf by developing a model which would enable us to meet the technological challenge of achieving uniform electroporation in a highly heterogeneous structure in the context of a process aimed at improving freezing stability of plant foods. Pulsed electric field treatment may be used to introduce the cryoprotectant molecules into the cells, and hence improve the structure and properties of frozen food plants. (C) 2014 Elsevier Ltd. All rights reserved

Modeling electroporation of the non-treated and vacuum impregnated heterogeneous tissue of spinach leaves

Uniform electroporation of the heterogeneous structure of spinach leaf cross section is a technological challenge that is addressed in this investigation. Three dimensional models were created with cells arranged in specific tissue types, considering a leaf with its air fraction and a leaf where the air fraction was replaced by a solution of known properties using vacuum impregnation. The models were validated before electroporation, in the frequency domain, where alternating voltage and current signal at frequencies from 20 Hz to 1 MHz were used to measure conductivity of the tissue. They were also validated through measurements of current during electroporation when a single 250 μs rectangular pulse with amplitudes ranging from 50 to 500 V was applied. Model validations show that both the frequency dependent conductivity and electroporation are well predicted. The importance of the wax layer and stomata in the model is thoroughly discussed. Industrial relevance: Our aim was to investigate electroporation of the spinach leaf by developing a model which would enable us to meet the technological challenge of achieving uniform electroporation in a highly heterogeneous structure in the context of a process aimed at improving freezing stability of plant foods. Pulsed electric field treatment may be used to introduce the cryoprotectant molecules into the cells, and hence improve the structure and properties of frozen food plants

APPLICATIONS OF THEORETICAL MODELS OF LIPID MEMBRANE ELECTROPORATION

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.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

Revisiting the role of pulsed electric fields in overcoming the barriers to in vivo gene electrotransfer

Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo

Membrane Electroporation and Electropermeabilization: Mechanisms and Models

International audienc

Cellular excitability and ns-pulsed electric fields

Recent studies showed that nanosecond pulsed electric fields (nsPEFs) can activate voltage-gated ion channels (VGICs) and trigger action potentials (APs) in excitable cells. Under physiological conditions, VGICs’ activation takes place on time scales of the order 10–100 μs. These time scales are considerably longer than the applied pulse duration, thus activation of VGICs by nsPEFs remains puzzling and there is no clear consensus on the mechanisms involved. Here we propose that changes in local electrical properties of the cell membrane due to lipid oxidation might be implicated in AP activation. We first use MD simulations of model lipid bilayers with increasing concentration of primary and secondary lipid oxidation products and demonstrate that oxidation not only increases the bilayer conductance, but also the bilayer capacitance. Equipped with MD-based characterization of electrical properties of oxidized bilayers, we then resort to AP modelling at the cell level with Hodgkin-Huxley-type models. We confirm that a local change in membrane properties, particularly the increase in membrane conductance, due to formation of oxidized membrane lesions can be high enough to trigger an AP, even when no external stimulus is applied. However, excessive accumulation of oxidized lesions (or other conductive defects) can lead to altered cell excitability

Gene electrotransfer into mammalian cells using commercial cell culture inserts with porous substrate

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we expand the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. We first use multiscale numerical modeling to determine the pulse parameters, substrate pore size, and other factors that are expected to result in successful gene electrotransfer. Based on the numerical results, we design a simple device combining an insert with substrate containing pores with 0.4 µm or 1.0 µm diameter, a multiwell plate, and a pair of wire electrodes. We test the device in three mammalian cell lines and obtain transfection efficiencies similar to those achieved with conventional bulk electroporation, but at better cell viability and with low-voltage pulses that do not require the use of expensive electroporators. Our combined theoretical and experimental analysis calls for further systematic studies that will investigate the influence of substrate pore size and porosity on gene electrotransfer efficiency and cell viability