Selective susceptibility to nanosecond pulsed electric field (nsPEF) across different human cell types.

Tumor ablation by nanosecond pulsed electric fields (nsPEF) is an emerging therapeutic modality. We compared nsPEF cytotoxicity for human cell lines of cancerous (IMR-32, Hep G2, HT-1080, and HPAF-II) and non-cancerous origin (BJ and MRC-5) under strictly controlled and identical conditions. Adherent cells were uniformly treated by 300-ns PEF (0-2000 pulses, 1.8 kV/cm, 50 Hz) on indium tin oxide-covered glass coverslips, using the same media and serum. Cell survival plotted against the number of pulses displayed three distinct regions (initial resistivity, logarithmic survival decline, and residual resistivity) for all tested cell types, but with differences in LD50 spanning as much as nearly 80-fold. The non-cancerous cells were less sensitive than IMR-32 neuroblastoma cells but more vulnerable than the other cancers tested. The cytotoxic efficiency showed no apparent correlation with cell or nuclear size, cell morphology, metabolism level, or the extent of membrane disruption by nsPEF. Increasing pulse duration to 9 µs (0.75 kV/cm, 5 Hz) produced a different selectivity pattern, suggesting that manipulation of PEF parameters can, at least for certain cancers, overcome their resistance to nsPEF ablation. Identifying mechanisms and cell markers of differential nsPEF susceptibility will critically contribute to the proper choice and outcome of nsPEF ablation therapies.

The Electroporation as a Tool for Studying the Role of Plasma Membrane in the Mechanism of Cytotoxicity of Bisphosphonates and Menadione.

In this study, the role of the cell plasma membrane as a barrier in the mechanism of the cytotoxicity of nitrogen-containing bisphosphonates and menadione was studied, and the possibility of increasing the efficiency of bisphosphonates and menadione (vitamin K3) as chemotherapeutic agents by permeabilizing the cell plasma membrane has been investigated in vitro. The plasma membrane barrier was reduced by electropermeabilization with the pulse of strong electric field. Two membrane-impermeant bisphosphonates with different hydrophilicities were chosen as study objects: ibandronate and pamidronate. For the comparison, an amphiphilic vitamin K3, which is able to cross the cell membrane, was studied as well. The impact of nitrogen-containing bisphosphonates and vitamin K3 on MH-22A cells viability was evaluated for the case of long (9 days) and short (20 min) exposure. When cells were cultured in the medium with vitamin K3 for 9-10 days, it exhibited toxicity of 50 % over the control at 6.2 µM for mouse hepatoma MH-22A cells. Ibandronate and pamidronate were capable of reducing drastically the cell viability only in the case of long 9-days incubation and at high concentrations (~20 µM for pamidronate and over 100 µM for ibandronate). Single, square-wave electric pulse with the duration of 100 µs and the field strength of 2 kV/cm was used to electroporate mouse hepatoma MH-22A cells in vitro. The results obtained here showed that the combination of the exposure of cells to membrane-impermeable bisphosphonates pamidronate and ibandronate with electropermeabilization of the cell plasma membrane did not increase their cytotoxicity. In the case of membrane-permeable vitamin K3, cell electropermeabilization did increase vitamin K3 killing efficiency. However, this increase was not substantial, within the range of 20-30 % depending on the duration of the exposure. Electropermeabilization improved cytotoxic effect of vitamin K3 but not of pamidronate and ibandronate.

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.

Electroporation-Based Biopsy Treatment Planning with Numerical Models and Tissue Phantoms.

Molecular sampling with vacuum-assisted tissue electroporation is a novel, minimally invasive method for molecular profiling of solid lesions. In this paper, we report on the design of the battery-powered pulsed electric field generator and electrode configuration for an electroporation-based molecular sampling device for skin cancer diagnostics. Using numerical models of skin electroporation corroborated by the potato tissue phantom model, we show that the electroporated tissue volume, which is the maximum volume for biomarker sampling, strongly depends on the electrode's geometry, needle electrode skin penetration depths, and the applied pulsed electric field protocol. In addition, using excised human basal cell carcinoma (BCC) tissues, we show that diffusion of proteins out of human BCC tissues into water strongly depends on the strength of the applied electric field and on the time after the field application. The developed numerical simulations, confirmed by experiments in potato tissue phantoms and excised human cancer lesions, provide essential tools for the development of electroporation-based molecular markers sampling devices for personalized skin cancer diagnostics.

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.

Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media.

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1-13 kV/cm) was assessed by oxidation of 2',7'-dichlorodihydrofluoresein (H(2)DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H(2)DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2',7'-dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H(2)DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H(2)O(2) in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H(2)O(2) and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage.

Cytotoxicity of a Cell Culture Medium Treated with a High-Voltage Pulse Using Stainless Steel Electrodes and the Role of Iron Ions.

High-voltage pulses applied to a cell suspension cause not only cell membrane permeabilization, but a variety of electrolysis reactions to also occur at the electrode-solution interfaces. Here, the cytotoxicity of a culture medium treated by a single electric pulse and the role of the iron ions in this cytotoxicity were studied in vitro. The experiments were carried out on mouse hepatoma MH-22A, rat glioma C6, and Chinese hamster ovary cells. The cell culture medium treated with a high-voltage pulse was highly cytotoxic. All cells died in the medium treated by a single electric pulse with a duration of 2 ms and an amplitude of just 0.2 kV/cm. The medium treated with a shorter pulse was less cytotoxic. The cell viability was inversely proportional to the amount of electric charge that flowed through the solution. The amount of iron ions released from the stainless steel anode (>0.5 mM) was enough to reduce cell viability. However, iron ions were not the sole reason of cell death. To kill all MH-22A and CHO cells, the concentration of Fe3+ ions in a medium of more than 2 mM was required.

To breathe or not to breathe? Hypoxia after pulsed-electric field treatment reduces the effectiveness of electrochemotherapy in vitro.

Bleomycin, which is the most widely used drugs in electrochemotherapy, requires oxygen to be able to make single- or double-strand brakes in DNA. However, the concentration of oxygen in tumours can be lower than 1%. The aim of this study was to find out whether oxygen concentration in the medium in which cells loaded with bleomycin are incubated, affects the effectiveness of electrochemotherapy in vitro. Experiments were carried out on mouse hepatoma MH-22A cells. Cells were loaded with bleomycin by using a single square-wave electric pulse (2 kV/cm, 100 µs) under normoxic conditions, seeded into Petri dishes, and grown under normoxic and hypoxic conditions. Cell viability was determined by means of a colony-forming assay. We demonstrated that when cells loaded with bleomycin were incubated in hypoxia (0.2% O2), up to 5.3-fold higher concentrations of bleomycin were needed to kill them in comparison with cells grown in normoxia (18.7% O2).

In vitro cytotoxicity studies of industrial Eucalyptus kraft lignins on mouse hepatoma, melanoma and Chinese hamster ovary cells.

Kraft lignin is a polyphenolic compound generated as a by-product from the kraft pulping process in large quantities annually worldwide. In addition to its commercial availability, its structural features make it worth to be considered in the pharmaceutical area. The present study was carried out to evaluate in vitro antioxidant and cytotoxic properties of kraft lignin on mouse hepatoma MH-22A, melanoma B16 (tumor cells) and Chinese hamster ovary (CHO, non-cancerous) cells. Moreover, several analytical techniques were used in order to elucidate the chemical structure of isolated industrial lignins (FT-IR, GPC, Py-GC-MS, 2D HSQC NMR). Results revealed high phenolic content in their composition, high-condensed structure and high phenolic hydroxyls group content. DPPH and ABTS⁎+ radical scavenging assays demonstrated their strong antioxidant activity, which was higher than found for commercial antioxidant (BHT). Kraft lignins act cytotoxically inducing apoptosis- and necrosis-like processes on both on tumor and normal cells. However, the results evidenced that MH-22A cells showed greater sensitive behavior than B16 and non-cancerous CHO cells, which were more tolerant of kraft lignin.

Physicochemical and in vitro cytotoxic properties of chitosan from mushroom species (Boletus bovinus and Laccaria laccata).

Chitosan samples from two mushroom species (Boletus bovinus, Laccaria laccata) were obtained and characterized by viscosimetry, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), elemental analyses (EA), nuclear magnetic resonance spectroscopy (13C NMR), X-ray diffraction (XRD) and thermogravimetric (TGA) analyses. Properties of the fungal chitosan samples were compared to commercial low-molecular weight chitosan, crustacean chitosan (Cervimunida johni) and chitosan obtained from an insect (Hilobius abietis). Additionally, the cytotoxic properties of chitosan in vitro on cancerous hepatoma and non-cancerous ovary cells cultivated on films with different chitosan concentrations was evaluated. As a conclusion, this study clearly revealed that low-molecular weight chitosan films and solutions with high degree of deacetylation can act cytotoxically on both tumor MH-22A and normal CHO cells in vitro. Consequently, this work may be useful for further investigations of natural anticancer products in medical areas.

Increase of the roughness of the stainless-steel anode surface due to the exposure to high-voltage electric pulses as revealed by atomic force microscopy.

The changes of the stainless-steel electrode surface morphology occurring due to dissolution of the anode under the action of electric pulses which are commonly utilized in cell electromanipulation procedures, have been studied by using atomic force microscopy. The surface of the polished electrode was rather smooth--the average roughness was 13-17 nm and the total roughness 140-180 nm. After the treatment of the chamber filled with 154 mM NaCl solution to a series of short (about 20 mus), high-voltage (4 kV) pulses, the roughness of the surface of the anode has increased, depending on the total amount of the electric charge that has passed through the unit area of the electrode, and exceeded 400 nm for the dissolution charge of 0.24 A s/cm(2). No changes of the cathode surface were detected. Well-defined peaks with the width of 1-2 mum and the height of over 400 nm have appeared. These peaks create local enhancements of the electric field at the interface between the solution and the electrode surface which can lead to the non-homogeneity treatment of cells by electric pulses and can facilitate the occurrence of the electrical breakdown of the liquid samples.

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.

Changes of the solution pH due to exposure by high-voltage electric pulses.

The change of the pH of a NaCl solution (139-149 mM NaCl) buffered with 5-15 mM sodium phosphates (pH 7.4) during electromanipulation was studied. It has been determined that an increase in the pH value of electroporation solution of a whole chamber volume, caused by the application of electric field pulses, commonly used in cell electromanipulation procedures, can exceed 1-2 pH units. Several materials for the cathode were tested. In all cases a stainless steel anode was utilized. The aluminum cathode gave a two-fold greater DeltapH in comparison with platinum, copper or stainless steel cathodes. In addition, a substantial release of aluminum (up to 1 mg/l) from the cathode was observed. It has also been found that the shift in pH depended on the medium conductivity: DeltapH of the solution, in which sucrose was substituted for NaCl, was about 5 times less. On the basis of the results obtained here, to avoid the plausible undesirable consequences of the cathodic electrolysis processes, in particular under the conditions of strong electric treatment, it could be recommended that chambers with aluminum electrodes not be utilized and one should use strongly buffered solutions of low conductivity and alternating current (sine or square wave) bipolar electric pulses.

Determination of cell electroporation in small-volume samples.

Expose of cells to electric field pulses increases the cell membrane permeability. Intracellular potassium ions leak out of the cells through aqueous pores created in the membrane. This release is used here for the determination of the fraction of electroporated cells. To determine cell membrane electroporation in small-volume samples (40-50 miacrol), mini both potassium ion-selective and reference electrodes, with tip diameters of 1-1.5 mm and minimum immersion depths of 1 mm, were utilized. The obtained calibration graph was linear within the concentration range 0.2-100 mM. The slope was 50-51 and 53-56 mV per concentration order at 10-11 and 19-21 degrees C, respectively. Detection limit of the electrode was determined to be 0.05-0.08 mM, however, it was possible to work down to concentrations in the range of 0.01 mM. Experiments have been carried out on human erythrocytes exposed to a square-wave electric pulse with the duration of 0.1-2 ms. The extracellular potassium concentrations were in the range between 0.04-0.08 mM (intact cells) and 3-5 mM (100% electroporation). The obtained dependences of the fraction of electroporated cells on the pulse intensity were of a sigmoid shape. The dependence of the pulse amplitude required to electroporate 50% of cells on the pulse duration, obtained from the release of intracellular potassium ions, coincided with the one determined from the extent of hemolysis after 24 h-incubation at low temperature.

Determination of cell electroporation from the release of intracellular potassium ions.

When cells are exposed to a strong enough external electric field, transient aqueous pores are formed in the membrane. The fraction of electroporated cells can be determined by measuring the release of intracellular potassium ions. The current work is the first study where such a method was employed successfully not only with cells suspended in the medium with a rather high concentration of potassium (4-5 mM) but also with cells that release some part of intracellular potassium responding, in this way, to the stress caused by manipulation procedures during the preparation of the cell suspension. Experiments were carried out on mouse hepatoma MH-A22 cells exposed to a square-wave electric pulse. The curves showing the dependence of the fraction of the cells that have become permeable to bleomycin, a membrane-impermeable cytotoxic drug, are close to the ones showing the release of intracellular potassium ions.

The loading of human erythrocytes with small molecules by electroporation.

The technology for loading the cell with membrane-impermeable substances by means of electroporation consists of the following three stages: (i) the creation of pores permeable for the desired substance; (ii) the introduction of a substance into the cell cytosol; and (iii) the restoration of the membrane barrier function. In this paper, the experimental data on the loading of human erythrocytes with small molecules (molecular weight below 500 Da) is presented. The results obtained show that increasing the intensity of the electric field pulse increases the fraction of electroporated cells. The pores through which the molecules of ascorbic acid and mannitol (radius below 0.5 nm) can enter the erythrocytes appear when the field strength exceeds 2.5 kV/cm. The concentration of ascorbic acid inside the cells increases linearly. At 4 degrees C, the rate of ascorbic acid influx was constant for at least 4 hours. The original permeability of most of the cells towards ascorbic acid and mannitol was restored after about 6-7 min at 37 degrees C, and the characteristic time for complete resealing was about 20-40 min. The procedure described here can be used for loading cells with membrane-impermeable substances.