Gintautas Saulis

PORE DISAPPEARANCE IN A CELL AFTER ELECTROPORATION : THEORETICAL SIMULATION AND COMPARISON WITH EXPERIMENTS

The process of pore disappearance after cell electroporation is analyzed theoretically. On the basis of the kinetic model, in which the formation and annihilation of a metastable hydrophilic pore are considered as random one-step processes, a distribution function of cell resealing times, Fr(t), is derived. Two cases are studied: 1) the rate of pore resealing, k(r), is significantly greater than the rate of pore formation, k(f); and 2) the rate of pore formation, k(f), is comparable with k(r). It is determined that the shape of the distribution function depends on the initial number of pores in a cell, n(i). If in the absence of an external electric field the rate of pore formation, k(f), is significantly less than the rate of pore resealing, k(r) (case 1), pores disappear completely, whereas when k(f) approximately k(r) (case 2), the cell achieves a steady state in which the number of pores is equal to k(f)/k(r). In case 1, when n(i) = 1, the distribution function Fr(t) is exponential. The developed theory is compared with experimental data available in the literature. Increasing the time of incubation at elevated temperature increases the fraction of resealed cells. This indicates that the time necessary for the resealing varies from cell to cell. Although the shape of experimental relationships depends on the electroporation conditions they can be described by theoretical curves quite well. Thus it can be concluded that the disappearance of pores in the cell membrane after electroporation is a random process. It is shown that from the comparison of presented theory with experiments, the following parameters can be estimated: the average number of pores, n(i), that appeared in a cell during an electric pulse; the rate of pore disappearance, k(r); the ratio k(f)/k(r); and the energy barrier to pore disappearance deltaWr(0). Estimated numerical values of the parameters show that increasing the amplitude of an electric pulse increases either the apparent number of pores created during the pulse (the rate of pore resealing remains the same) or the rate of pore resealing (the average number of pores remains the same).

Energy-efficient biomass processing with pulsed electric fields for bioeconomy and sustainable development

Fossil resources-free sustainable development can be achieved through a transition to bioeconomy, an economy based on sustainable biomass-derived food, feed, chemicals, materials, and fuels. However, the transition to bioeconomy requires development of new energy-efficient technologies and processes to manipulate biomass feed stocks and their conversion into useful products, a collective term for which is biorefinery. One of the technological platforms that will enable various pathways of biomass conversion is based on pulsed electric fields applications (PEF). Energy efficiency of PEF treatment is achieved by specific increase of cell membrane permeability, a phenomenon known as membrane electroporation. Here, we review the opportunities that PEF and electroporation provide for the development of sustainable biorefineries. We describe the use of PEF treatment in biomass engineering, drying, deconstruction, extraction of phytochemicals, improvement of fermentations, and biogas production. These applications show the potential of PEF and consequent membrane electroporation to enable the bioeconomy and sustainable development.

Size of the pores created by an electric pulse: microsecond vs millisecond pulses.

Here, the sizes of the pores created by square-wave electric pulses with the duration of 100 μs and 2 ms are compared for pulses with the amplitudes close to the threshold of electroporation. Experiments were carried out with three types of cells: mouse hepatoma MH-22A cells, Chinese hamster ovary (CHO) cells, and human erythrocytes. In the case of a short pulse (square-wave with the duration of 100 μs or exponential with the time constant of 22 μs), in the large portion (30-60%) of electroporated (permeable to potassium ions) cells, an electric pulse created only the pores, which were smaller than the molecule of bleomycin (molecular mass of 1450 Da, r≈0.8 nm) or sucrose (molecular mass of 342.3 Da, radius-0.44-0.52 nm). In the case of a long 2-ms duration pulse, in almost all cells, which were electroporated, there were the pores larger than the molecules of bleomycin and/or sucrose. Kinetics of pore resealing depended on the pulse duration and was faster after the shorter pulse. After a short 100-μs duration pulse, the disappearance of the pores permeable to bleomycin was completed after 6-7 min at 24-26°C, while after a long 2-ms duration pulse, this process was slower and lasted 15-20 min. Thus, it can be concluded that a short 100-μs duration pulse created smaller pores than the longer 2-ms duration pulse. This could be attributed to the time inadequacy for pores to grow and expand during the pulse, in the case of short pulses.

Electric field-induced effects on yeast cell wall permeabilization.

The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPP(+) ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 µs and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPP(+) absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPP(+) by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPP(+) absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPP(+) , is due to the poration of the plasma membrane. The applicability of the TPP(+) absorption measurements for the analysis of yeast cells electroporation process is also discussed.

Release of Iron Ions From the Stainless Steel Anode Occurring During High-Voltage Pulses and Its Consequences for Cell Electroporation Technology

One of the plausible reactions occurring during high-voltage pulses, which are used to electroporate the cells, is the oxidation of the metal ions of the anode resulting in the dissolution of the anode. In the case of the anode made from stainless steel, which is one of the most popular electrode materials, iron ions ( Fe2+ and Fe3+) are released from the anode. Here, this process and its consequences have been studied. A single square-wave electric pulse with the duration of 2 ms and the amplitude of 1.2 kV/cm increased the concentration of iron ions in solution by over 0.5 mM. Iron ions released from the anode behave as a Lewis acid and hydrolyze the water molecules in the solution, reducing the pH of a solution and might play a role in the changes of the medium conductivity. In addition, the roughness of the stainless steel anode increased progressively, in proportion with the total amount of the electric charge that had passed through the unit area of the electrode. The reduction of the viability of cells by iron ions has been demonstrated. The iron ions quench the fluorescence of anticancer drugs, which are used when photodynamic tumor therapy is combined with electroporation, such as porphyrin sulfonate and Adriamycin.

Theoretical Analysis and Experimental Determination of the Relationships Between the Parameters of the Electric Field Pulse Required to Electroporate the Cells

Here, theoretical relationships between the parameters of the electric pulse, which is necessary to porate the cell by electric pulse of various shapes, have been obtained. The theoretical curves were compared with the experimental relationships. Experiments were carried out with human erythrocytes, Chinese hamster ovary and mouse hepatoma MH-22A cells. The fraction of electroporated MH-22A cells was determined from the extent of the release of intracellular potassium ions and erythrocytes-from the extent of their hemolysis after long (20-24 h) incubation in 0.63% NaCl solution at 4°C. The dependence of the fraction of electroporated cells on the amplitude of the electric field pulse was determined for pulses with the duration from 95 ns to 2 ms. The shapes of theoretical dependencies are in agreement with experimental ones. The cell poration time depended on the intensity of the pulse: the shorter the pulse duration, the higher the electric field strength has to be. This dependence is much more pronounced for pulses . For example, if the pulse amplitude required to electroporate 50% of human erythrocytes increased from 1.0 to 1.76 kV/cm, when the duration of a square-wave pulse was reduced from 2 ms to 20 μs, it increased from 3 to 12 kV/cm, when the pulse duration was reduced from 950 to 95 ns. The relationships between the electric field strength required for electroporation and the frequency of the applied ac field were calculated for different pulselengths. It has been obtained that although the electric field strength is constant for frequencies but its value depends on the pulselength decreasing with increasing pulse duration. At higher frequencies, electric field strength is dependent on the frequency of the ac field.

Cell Electromanipulation Procedures Change the pH of a Solution

Pulses of strong electric field (up to 30 kV/cm) are commonly utilized for cell electromanipulation (electroporation, electrofusion, electrotransformation etc.) (Chang et al., 1992; Neumann et al., 1989). Exposure of cell suspension to such strong electric fields leads to the formation of transient aqueous pores in the cell membrane (Chang et al., 1992; Neumann et al., 1989). However, an electrically induced cell membrane perturbation is not the only consequence of the exposure of cell suspension to a strong electric field. When an electric current passes through the aqueous solution, it causes heating (Joule heating) and at the same time various chemical reactions occur at the surface between the solution and the electrodes (electrolysis). These may include the evolution of gas, the separation of substances, the dissolution of the electrode or the appearance of new substances in the solution (Milazzo, 1963). The processes of electrolysis lead to the changes of the temperature, pH, and the chemical composition of the experimental medium. It must be stressed that these changes go along with the permeabilization of the cell membrane. Therefore any changes of the physical chemical properties of a solution during electroporation procedures are especially undesirable, as various substances may enter the cells through the pores created by electroporation (Saulis et al., 1991).

System for the Nanoporation of Biological Cells Based on an Optically-Triggered High-Voltage Spark-Gap Switch

This nanosecond electric pulse generator is designed for the electroporation of biological cells suspended in a liquid media. It is based on a spark-gap switch, which is optically triggered by a 0.45-ns duration and 1-mJ energy laser pulse (wavelength 1062 nm). This system can also be triggered manually by changing the distance between the spark-gap electrodes. It is able to generate in a 75- Ω impedance transmission line near-perfect square-shaped electric pulses (rise and fall times ) with durations of 10, 40, 60, or 92 ns. The maximal amplitude of such pulses is 12.5 kV. The main advantage of this system is its ability to generate single pulses, the amplitude and duration of which can be precisely set in advance. To treat the cells, a coaxial cuvette with a 0.03-mL active volume and a 1-mm distance between the 28.3- mm2 circular-shaped electrodes was used. The system was tested on human erythrocytes. It was demonstrated that for the 92- and 40-ns duration pulse, the amplitude required to electroporate 50% of the cells was 20 and 65 kV/cm, respectively.

Mechanism of Intermolecular Electron Transfer in Bionanostructures

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Most patients are inoperable and hepatoma cells are resistant to conventional chemotherapies. Thus, the development of novel therapies for HCC treatment is of paramount importance. Amongst different alimentary factors, vitamin C and vitamin K3 In the present work, it has been shown that the treatment of mouse hepatoma MH-22A cells by vitamin C and vitamin K3 at the ratio of 100:1 greatly enhanced their cytotoxicity. When cells were subjected to vitamin C at 200 μM or to vitamin K3 at 2 μM separately, their viability reduced by only about 10%. However, when vitamins C and K3 were combined at the same concentrations, they killed more than 90% of cells. To elucidate the mechanism of the synergistic cytotoxicity of the C&K3 mixture, theoretical quantum-chemical analysis of the dynamics of intermolecular electron transfer (IET) processes within the complexes containing C (five forms) and K3 (one form) has been carried out. Optimization of the ground state complex geometry has been provided by means of GAUSSIAN03 package. Simulation of the IET has been carried out using NUVOLA package, in the framework of molecular orbitals (MO). The rate of IET has been calculated using Fermi Golden rule. The results of simulations allow us to create the preliminary model of the reaction pathway.

Modeling of the Processes of Pore Creation and Disappearance in a Cell Under the Influence of Strong Electric Field as Random One-Step Processes

Electroporation of biological membranes has numerous applications in molecular biology, biotechnology and medicine1, but the mechanisms of pore formation under the influence of an electric field and their subsequent resealing are not fully understood. In most of theoretical papers the formation of pores in planar lipid bilayer membranes is investigated. Besides, in those studies where the electroporation of a cell or vesicle was analysed2–4, few characteristics of this process that can be determined experimentally are presented. In addition, so far theorists have not paid proper attention to the process of pore disappearance after the electric pulse.