Alenka Maček Lebar

Feasibility of Employing Model-Based Optimization of Pulse Amplitude and Electrode Distance for Effective Tumor Electropermeabilization

In electrochemotherapy (ECT) electropermeabilization, parameters (pulse amplitude, electrode setup) need to be customized in order to expose the whole tumor to electric field intensities above permeabilizing threshold to achieve effective ECT. In this paper, we present a model-based optimization approach toward determination of optimal electropermeabilization parameters for effective ECT. The optimization is carried out by minimizing the difference between the permeabilization threshold and electric field intensities computed by finite element model in selected points of tumor. We examined the feasibility of model-based optimization of electropermeabilization parameters on a model geometry generated from computer tomography images, representing brain tissue with tumor. Continuous parameter subject to optimization was pulse amplitude. The distance between electrode pairs was optimized as a discrete parameter. Optimization also considered the pulse generator constraints on voltage and current. During optimization the two constraints were reached preventing the exposure of the entire volume of the tumor to electric field intensities above permeabilizing threshold. However, despite the fact that with the particular needle array holder and pulse generator the entire volume of the tumor was not permeabilized, the maximal extent of permeabilization for the particular case (electrodes, tissue) was determined with the proposed approach. Model-based optimization approach could also be used for electro-gene transfer, where electric field intensities should be distributed between permeabilizing threshold and irreversible threshold-the latter causing tissue necrosis. This can be obtained by adding constraints on maximum electric field intensity in optimization procedure

Chapter Seven Electroporation of Planar Lipid Bilayers and Membranes

Abstract Strong external electric field can destabilize membranes and induce formation of pores thus increasing membrane permeability. The phenomenon is known as membrane electroporation, sometimes referred to also as dielectric breakdown or electropermeabilization. The structural changes involving rearrangement of the phospholipid bilayer presumably lead to the formation of aqueous pores, which increases the conductivity of the membrane and its permeability to water-soluble molecules which otherwise are deprived of membrane transport mechanisms. This was shown in variety of experimental conditions, on artificial membranes such as planar lipid bilayers and vesicles, as well as on biological cells in vitro and in vivo. While studies of electroporation on artificial lipid bilayers enabled characterization of the biophysical processes, electroporation of biological cells led to the development of numerous biomedical applications. Namely, cell electroporation increases membrane permeability to otherwise nonpermeant molecules, which allows different biological and medical applications including transfer of genes (electrogene transfer), transdermal drug delivery and electrochemotherapy of tumors. In general, the key parameter for electroporation is the induced transmembrane voltage generated by an external electric field due to the difference in the electric properties of the membrane and the external medium, known as Maxwell–Wagner polarization. It was also shown that pore formation and the effectiveness of cell electroporation depend on parameters of electric pulses like number, duration, repetition frequency and electric field strength, where the later is the crucial parameter since increased transmembrane transport due to electroporation is only observed above a certain threshold field. Two main theoretical approaches were developed to describe electroporation. The electromechanical approach considers membranes as elastic or viscoelastic bodies, and applying principles of electrostatics and elasticity predict membrane rupture above critical membrane voltage. A conceptually different approach describing formation and expansion of pores is based on energy consideration; it is assumed that external electric field reduces the free energy barrier for formation of hydrophilic pores due to lower polarization energy of water in the pores compared to the membrane. Combined with stochastic mechanism of pores expansion it can describe experimental data of bilayer membranes. Still, the molecular mechanisms of pore formation and stabilization during electroporation are not fully understood and rigorous experimental conformation of different theories is still lacking. The focus of this chapter is to review experimental and theoretical data in the field of electroporation and to connect biophysical aspects of the process with the phenomenological experimental observations obtained on planar lipid bilayers, vesicles and cells.

A System for the Determination of Planar Lipid Bilayer Breakdown Voltage and Its Applications

In this paper, we focus on measurement principles used in electroporation studies on planar lipid bilayers. In particular, we point out the voltage-clamp measurement principle that has great importance when the breakdown voltage of a planar lipid bilayer is under consideration; however, it is also appropriate for the determination of other planar lipid bilayer electrical properties such as resistance and capacitance. A new experimental system that is based on the voltage-clamp measurement principle is described. With the use of a generator that can generate arbitrary-type signals, many specific shapes of a voltage signal could be generated, and therefore, the experimental system is appropriate for a broad spectrum of measurements.

Measurement protocol for planar lipid bilayer viscoelastic properties

This paper describes how to estimate planar lipid bilayers elasticity module E and surface tension sigma by means of measuring its breakdown voltage and using Dimitrovs viscoelastic model of electric field-induced breakdown of lipid bilayers. Planar lipid bilayers (BLMs) were made of two components: 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl 2-oleoyl phosphatidylserine (POPS) in five different compositions. Folding method for forming planar lipid bilayers in the salt solution of 100 mM KCl was used. Breakdown voltages Ubr and membrane life times tbr were measured by means of applying linear rising voltage signals of seven different slopes. Specific capacitances cBLM of bilayers were measured with charge pulse method. Then Dimitrovs viscoelastic model was fitted to measured data allowing for estimation of elasticity module and surface tension of the lipid bilayer. Our results show that onecomponent bilayers composed from POPS were more stable and thus having higher breakdown voltages and elasticity moduli then bilayers composed of POPC. Surface tension values were similar regardless of the membrane composition. Values of the elasticity (E) and surface tension (sigma) are comparable to those published in the literature. We conclude that the protocol used, though time consuming, is an alternative to other methods used for determination of bilayers mechanical properties.

Internal configuration and electric potential in planar negatively charged lipid head group region in contact with ionic solution.

The lipid bilayer composed of negatively charged lipid 1-palmitoyl-3-oleoyl-sn-glycero-3-phosphatidylserine (POPS) in contact with an aqueous solution of monovalent salt ions was studied theoretically by using the mean-field modified Langevin-Poisson-Boltzmann (MLPB) model. The MLPB results were tested by using molecular dynamic (MD) simulations. In the MLPB model the charge distribution of POPS head groups is theoretically described by the negatively charged surface which accounts for negatively charged phosphate groups, while the positively charged amino groups and negatively charged carboxylate groups are assumed to be fixed on the rod-like structures with rotational degree of freedom. The spatial variation of relative permittivity, which is not considered in the well-known Gouy-Chapman (GC) model or in MD simulations, is thoroughly derived within a strict statistical mechanical approach. Therefore, the spatial dependence and magnitude of electric potential within the lipid head group region and its close vicinity are considerably different in the MLPB model from the GC model. The influence of the bulk salt concentration and temperature on the number density profiles of counter-ions and co-ions in the lipid head group region and aqueous solution along with the probability density function for the lipid head group orientation angle was compared and found to be in qualitative agreement in the MLPB and MD models.

Specific electrical capacitance and voltage breakdown as a function of temperature for different planar lipid bilayers

The breakdown voltage and specific electrical capacitance of planar lipid bilayers formed from lipids isolated from the membrane of archaeon Aeropyrum pernix K1 as a function of temperature were studied and compared with data obtained previously in MD simulation studies. Temperature dependence of breakdown voltage and specific electrical capacitance was measured also for dipalmitoylphosphatidylcholine (DPPC) bilayers and bilayers formed from mixture of diphytanoylphosphocholine (DPhPC) and DPPC in ratio 80:20. The breakdown voltage of archaeal lipids planar lipid bilayers is more or less constant until 50°C, while at higher temperatures a considerable drop is observed, which is in line with the results from MD simulations. The breakdown voltage of DPPC planar lipid bilayer at melting temperature is considerably higher than in the gel phase. Specific electrical capacitance of planar lipid bilayers formed from archaeal lipids is approximately constant for temperatures up to 40°C and then gradually decreases. The difference with MD simulation predictions is discussed. Specific electrical capacitance of DPPC planar lipid bilayers in fluid phase is 1.75 times larger than that of the gel phase and it follows intermediated phases before phase transition. Increase in specific electrical capacitance while approaching melting point of DPPC is visible also for DPhPC:DPPC mixture.

Electrical Properties of Hydrogenated Planar Lipid Bilayers Formed from Archaea Lipids at Different Temperatures

This paper presents the study of electrical properties of hydrogenated planar lipid bilayers formed from thermophilic archaeal lipids extracted from Aeropyrum pernix K1. Study is focused on measuring electrical capacitance as parameter of the planar lipid bilayer thickness and breakdown voltage as parameter of planar lipid bilayer stability in electric field. The results show that both measured parameters of hydrogenated planar lipid bilayers formed from archeal lipids extracted from A. pernix K1 are not temperature dependant on measured temperature interval between 19 °C and 56 °C. Electrical capacitance is lower than in Molecular Dynamic simulations and in experiments using 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) lipids or 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphoserine (POPS) lipids. Breakdown voltage is higher than in experiments with POPC or POPS lipids.

Planar lipid bilayers: Observing pore creation and extinction

From an electrical point of view a planar lipid bilayer can be considered as a non-perfect capacitor; it can be presented as an ideal capacitor in parallel with resistor. In this study the whole measuring system including planar lipid bilayer was modeled by an equivalent electric circuit in Spiceopus software. Such a model gives additional information of experimentally obtained results. In this way we analyze measurements of transmembrane voltage that appears on planar lipid bilayer as consequence of linear rising current. Small voltage drops were obtained before the planar lipid bilayer breakdown. The model showed that effective current on planar lipid bilayer is actually much smaller than the current applied with current generator and should be used in calculations of a conductance related to voltage drops.