EFFECTS OF ELECTRICAL AND THERMAL PRE-TREATMENT ON MASS TRANSPORT IN BIOLOGICAL TISSUE

Električno polje dovoljšne jakosti lahko povzroči znatno povečanje električne prevodnosti in prepustnosti celične membrane. Pojav je poznan kot elektroporacija in je pripisan vzpostavitvi vodnih kanalov v lipidnem dvosloju imenovanih pore. Elektroporacija je bila in je še vedno predmet intenzivnih raziskav na številnih področjih, kot so biomedicina (za gensko transfekcijo, elektrokemoterapijo, vnos zdravil preko kože, in ablacijo mehkih tkiv, npr. tumorjev)v predelavi hrane in kemijskem inženirstvu (za povečanje količine ekstrakta pri pridobivanju sokov ali dragocenih snovi, izboljšanju kakovosti pridobljenih snovi, ali konzervacijo hrane)prav tako pa tudi v okoljskih znanostih (pri predelavi odpadnih voda, pridobivanju lipidov iz mikroorganizmov, ali stimulaciji rasti rastlin). Skoraj na vseh teh področjih nas zanima izboljšanje vnosa snovi v biološke celice ali pa pridobivanja snovi iz bioloških celic. Razumevanje in kvantifikacija transporta snovi v povezavi z elektroporacijo sta pomembna cilja pri raziskavah elektroporacije. Sposobnost dobrega razumevanja procesov transporta snovi ima pomembne posledice za, naprimer, nadaljnje izboljšanje selektivnega pridobivanja snovi ali sokov iz rastlinskih celic, za izboljšanje dostave zdravil v ciljna tkiva oziroma celice, in za uspešno reševanje trenutnih okoljskih izzivov. Čeprav so elektroporacija in z njo povezani pojavi predmet intenzivnih raziskav, je občutiti pomanjkanje kompleksnejših oziroma popolnejših modelov, ki bi se lahko uporabljali za modeliranje transporta snovi v kompleksnih strukturah, kakršna so biološka tkiva, še posebej v povezavi z elektroporacijo. Pričujoča dizertacija predstavlja poskus izgradnje teoretičnega matematičnega opisa – t.j. modela – za proučevanje transporta snovi v elektroporiranem tkivu. Model, poimenovan model dvojne poroznosti, je osnovan na podlagi obstoječe teoretične analize tlačnih razmer (hidrodinamike) v prsteh, sedimentih, prodovnih nanosih ter lomljenih kamninah. Med temi sistemi ter strukturo bioloških tkiv obstaja analogija, ki je izkoriščena za vzpostavitev ekvivalentne matematične obravnave bioloških tkiv z že obstoječimi metodami in pristopi, ki so bili prvotno razviti v geoloških znanostih, so pa bili že uporabljeni tudi v povezavi z biološkimi materiali, denimo pri obravnavi procesa prešanja z oljem bogatih semen. Model dvojne poroznosti je bil razvit upoštevaje zakone o ohranitvi mase in zakone masnega transporta (termodinamika sistemov v neravnovesju), in omogoča sklapljanje efektov 9 elektroporacije na membrano posameznih celic z rezultirajočim transportom snovi preko membrane in v izvenceličnem prostoru. Slednje predstavlja tudi poglavitni izvirni prispevek znanosti, saj model dvojne poroznosti še ni bil zapisan za difuzijo in v preteklih delih ni predstavljen v povezavi z elektroporacijo. Predstavljena je tudi poučna analitična rešitev modela, ki dovoljuje fizikalno interpretacijo, model pa se lahko enostavno nadgradi z dodatnimi odvisnostmi in s tem modelira učinke elektroporacije, ter nato reši numerično. Dizertacijo sestavlja pet znanstvenih člankov. Prvi podaja pregled nad aplikacijami elektroporacije v industriji predelave hrane ter biorafineriji. Drugi znanstveni članek zajema kontrukcijo modela dvojne poroznosti za problem difuzije topljenca v elektroporiranem tkivu po elektroporaciji. V tretjem članku je zajeta prilagoditev oz. predelava modela dvojne poroznosti za difuzijski problem (koncentracijske razmere) v formo primerno za obravnav problema iztiskanja soka iz elektroporiranega tkiva oz. problem konsolidacije elektroporiranega vzorca rastlinskega tkiva (tlačne razmere). Četrti članek združuje oba modela, model difuzije ter konsolidacije, ter dodaja potrebno validacijo modela prek primerjave eksperimentalno pridobljenih podatkov z rezultati pridobljenimi prek simulacij na modelu. Sklepno poglavje podaja še kratek oris najnovejših rezultatov v smeri izboljšav modela z vključitvijo drugih pomembnih dejavnikov v model kot so izguba turgorskega tlaka in elektroosmoza, ter predloge za možne prihodnje nadgradnje modela, njegovo razširitev in posplošitev.An electric field of sufficient strength can cause a significant increase of conductivity and permeability of cell membrane. Effect is known as electroporation and is attributed to creation of aqueous pathways in the lipid bilayer. Understanding and quantifying mass transport in connection with electroporation of biological tissues is an important goal in research. The ability to fully comprehend transport processes has ramifications in improved juice extraction and improved selective extraction of compounds from plant cells, improved drug delivery, and solutions to current environmental challenges. While electroporation is intensively investigated, there is a lack of comprehensive models that can be used to model mass transport in complex structures such as biological tissues with relation to electroporation. This thesis presents an attempt at constructing a theoretical mathematical description – a model, for studying mass transport in electroporated tissue. The model was developed employing mass conservation and transport laws and enables coupling effects of electroporation to the membrane of individual cells with the resulting mass transport in tissue. An instructive analytical solution has been found via simplifications and the model can be extended with additional dependencies to account for the phenomenon of electroporation, and solved numerically. Thesis comprises five peer-reviewed papers describing electroporation in the food industry, model creation for the problem of diffusion, translation of the model to the mathematically-related case of juice expression, model validation, as well as suggestions for possible future development, extension, and generalization

Scratching the electrode surface

Electroporation is employed ever more frequently and broadly to deliver energy to tissues and liquid media in various applications, thus answering questions on the associated electrochemistry and electrode material alteration is becoming important. The aim of the present study is firstly to introduce and elucidate the basic relations between voltage, current, electrical impedance, and heat generation in the medium, and secondly, to characterize electrode material alteration due to pulse delivery, both by performing an in vitro and an in-silico study. Saline was used as a(n) (over)simplified model medium representing biological tissue, and exposed to high-amplitude, high-current electroporation pulses of varying duration, polarity, and pulse repetition rate. The controlled experiment was conducted by using seven different electrode metals of high purity, delivering pulses using three different protocols, and concurrently or sequentially measuring as many physical properties as available (electric current, voltage, electrode-electrolyte impedance, temperature). The intent is to present a multi-physics approach to what is occurring during procedures such as in vivo electrochemotherapy, gene delivery and in vitro gene transfection, intracardiac irreversible electroporation/pulsed-field ablation, or indeed electroporation in liquid food products such as juice. Modelling is also used to see whether it is possible to detect, via electrical measurements, any alterations in medium properties (e.g. composition) due to electrochemical effects, and if any such effects can be decoupled from the ohmic and thermal effects. Water electrolysis was observed indirectly (gas production), but not detected by electrical measurements during pulse application. Reactions at the electrodes alter the electrode electrical properties depending on the electrode material as expected, which might be important especially in applications where the same electrodes are used for delivery of electroporation pulses and also for sensing small electrical signals such as ECG for example. The demonstrated approach using saline as a model medium allows for rapid validation, and can more easily be developed further, as compared to experiments with more complex electrode materials (e.g. alloys), media (e.g. fluids, growth media, biological cell suspensions), or tissues

Dual-porosity model of solute diffusion in biological tissue modified by electroporation

AbstractIn many electroporation applications mass transport in biological tissue is of primary concern. This paper presents a theoretical advancement in the field and gives some examples of model use in electroporation applications. The study focuses on post-treatment solute diffusion.We use a dual-porosity approach to describe solute diffusion in electroporated biological tissue. The cellular membrane presents a hindrance to solute transport into the extracellular space and is modeled as electroporation-dependent porosity, assigned to the intracellular space (the finite rate of mass transfer within an individual cell is not accounted for, for reasons that we elaborate on). The second porosity is that of the extracellular space, through which solute vacates a block of tissue.The model can be used to study extraction out of or introduction of solutes into tissue, and we give three examples of application, a full account of model construction, validation with experiments, and a parametrical analysis. To facilitate easy implementation and experimentation by the reader, the complete derivation of the analytical solution for a simplified example is presented.Validation is done by comparing model results to experimentally-obtained data; we modeled kinetics of sucrose extraction by diffusion from sugar beet tissue in laboratory-scale experiments. The parametrical analysis demonstrates the importance of selected physicochemical and geometrical properties of the system, illustrating possible outcomes of applying the model to different electroporation applications. The proposed model is a new platform that supports rapid extension by state-of-the-art models of electroporation phenomena, developed as latest achievements in the field of electroporation

Educational application for visualization and analysis of electric field strength in multiple electrode electroporation

Abstract Background Electrochemotherapy is a local treatment that utilizes electric pulses in order to achieve local increase in cytotoxicity of some anticancer drugs. The success of this treatment is highly dependent on parameters such as tissue electrical properties, applied voltages and spatial relations in placement of electrodes that are used to establish a cell-permeabilizing electric field in target tissue. Non-thermal irreversible electroporation techniques for ablation of tissue depend similarly on these parameters. In the treatment planning stage, if oversimplified approximations for evaluation of electric field are used, such as U/d (voltage-to-distance ratio), sufficient field strength may not be reached within the entire target (tumor) area, potentially resulting in treatment failure. Results In order to provide an aid in education of medical personnel performing electrochemotherapy and non-thermal irreversible electroporation for tissue ablation, assist in visualizing the electric field in needle electrode electroporation and the effects of changes in electrode placement, an application has been developed both as a desktop- and a web-based solution. It enables users to position up to twelve electrodes in a plane of adjustable dimensions representing a two-dimensional slice of tissue. By means of manipulation of electrode placement, i.e. repositioning, and the changes in electrical parameters, the users interact with the system and observe the resulting electrical field strength established by the inserted electrodes in real time. The field strength is calculated and visualized online and instantaneously reflects the desired changes, dramatically improving the user friendliness and educational value, especially compared to approaches utilizing general-purpose numerical modeling software, such as finite element modeling packages. Conclusion In this paper we outline the need and offer a solution in medical education in the field of electroporation-based treatments, e.g. primarily electrochemotherapy and non-thermal irreversible tissue ablation. We present the background, the means of implementation and the fully functional application, which is the first of its kind. While the initial feedback from students that have evaluated this application as part of an e-learning course is positive, a formal study is planned to thoroughly evaluate the current version and identify possible future improvements and modifications.</p

Time-dependent model of temperature distribution in continuous flow pulsed electric field treatment chambers

A key component of a continuous flow pulsed electric field (PEF) system is the treatment chamber, where the product is exposed to electric pulses. Determination of the temperature distribution in the chamber during PEF treatment is important since high local increases in temperatures can affect the quality of the product. Coupled simulations of electric field, fluid flow, and heating in the existing literature do not model each individual electric pulse, but rather employ a “duty cycle” approach, which does not account for transient variations in treatment intensity and temperature changes in the medium. We present a time-dependent approach to modelling PEF treatment in continuous flow treatment chambers, which can model each pulse separately, and thus enables a more accurate study of temporal and spatial distributions of electric field and temperature. The model has been validated on laboratory scale treatment chambers of parallel plate or colinear design and using realistic protocols. Industrial relevance text: The paper is relevant to all pulsed electric field (PEF) applications either on laboratory or industrial scale that implement a continuous flow treatment chamber. It presents an improved modelling approach which allows for an analysis of the electrical current, electric field, and temperature distribution in the chamber during, at the end, and in between application of electrical pulses. The model can be used to predict the peak temperature at the end of each pulse in the hot spots, which if large enough could potentially lead to thermal damage of the product or in extreme cases even potential local boiling of the medium, resulting not only in degradation of the treated product, but also in accelerated electrode fouling, oxidation, and dissolution (etching), as well as arcing. This would not only affect the quality of the treated product but would also affect the wear and lifetime of electrodes/chambers, and of the pulse generator. The model can also be used to avoid expensive trial-and-error optimization of the PEF protocols and chamber geometries in situ

MOESM1 of Education on electrical phenomena involved in electroporation-based therapies and treatments: a blended learning approach

Additional file 1. Knowledge assessment test with the corresponding correct answers. The knowledge assessment test was composed of ten questions related to the educational content of the e-learning practical work. The questions and the corresponding correct answers to the questions

Electrical Impedance Spectroscopy insight into plant tissues treated by Pulsed Electric Fields

Electroporation or pulsed electric field (PEF) treatment is known to cause an increase of cell membrane permeability and consequently an increase of the cell membrane conductivity. This is explained by the creation of aqueous pathways in the lipid domain of the cell membrane exposed to the external electric field. Since the cell membrane exhibits relatively high impedance, any permeabilization will result in a drop in impedance of the single cell and consequently of the tissue. Hence, the electroporation effect on biological matrices can be assessed by measurements of their electrical properties. The electrical impedance spectroscopy (EIS) has been suggested as a reliable method to estimate the extent of tissue damage due to high voltage treatment. This study reports on results of the bioimpedance measurements performed on different PEF-treated plant tissues (i.e. apples and potatoes). Furthermore, since bioimpedance depends on several physiological parameters, and changes in electrical properties can be masked by other processes, EIS was performed on a model system, i.e. an agarose phantom, lacking any cell structures and constituents. As expected, no changes of the measured electrical parameters were detected in the agarose samples. On the contrary, plant tissues showed a pronounced drop of the normalized impedance proportional to the electric field amplitude applied to the tissue