Article

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy

August 1990
Biophysical Journal 58(1):1-12

August 1990
58(1):1-12

DOI:10.1016/S0006-3495(90)82348-1

Source
PubMed

Authors:

Donald C Chang

The Hong Kong University of Science and Technology

Thomas S Reese

National Institutes of Health

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.

Study on preservation of mango nectar using electric current and UV rays

Article

Full-text available

Jun 2016

Hasnaa Mahmoud Abo Taleb

The use of continues time of electric current field had more effect on preservation of mango nectar (by decreasing the microorganisms) than non-continues time. It was found that continues current field (5 minutes) exhibited a change in the color of mango nectar to become more browning than non-continues time of electric current field. By increasing electric current strength and the volt to be 220 it lead to more preservation. A clear decrease in the microorganisms was observed by increasing the exposure time of electric current field. Also the exposing of mango nectar by UV ray with high stirring led to a pronounced decrease in the total count of microorganisms (bacteria, molds and yeasts) without any change in the temperature degrees. studying the rheological properties of mango nectar led to a decrease in viscosity with increasing shear stress in different times resulting in a change consistency index and Flow behavior index of mango nectar. Keywords-Electric current field for food preservation, preservation of mango nectar by UV ray, rheological properties.

Study on preservation of mango nectar using electric current and UV rays

Article

Full-text available

Jun 2016

Acute safety and efficacy of pulse field ablation for atrial fibrillation in a Polish cohort of patients

Article

Jun 2024
KARDIOL POL

A perspective on modeling pore energy and pulsed electromagnetic field induced cell membrane perforation

Article

Mar 2023

Pulsed electric field-induced electroporation has been widely used, but its specific perforation theory has not been fully elucidated. Therefore, this Perspective paper takes as a clue the dynamic development relationship between the pore energy and the pore state in cell membranes. First, based on the contribution of line tension, surface tension, steric repulsion, and applied electric field to pore energy, the theoretical models of reversible electroporation of a microsecond pulsed electric field and irreversible electroporation of a high-frequency nanosecond pulsed electric field are reviewed. Then, the contribution of elastic strain energy to pore energy is increased, and the theoretical model of pulsed electric field electroporation considering the mechanical properties of cell membranes is further reviewed. Based on the contribution of magnetic stress generated by the magnetic field and the gradient magnetic field to pore energy, a theoretical model of cell membrane magnetoporation under the action of a pulsed magnetic field is proposed, which lays a theoretical foundation for the popularization and application of non-contact cell membrane perforation technology.

Tese Rodolfo Lauro Weinert

Thesis

Full-text available

Sep 2020

Rodolfo Lauro Weinert

Biological electropermeabilization is a phenomenon of opening pores in the cell membrane when exposed to intense electric fields. Although mathematical models have been exposed since the last, they are still necessary to use a model that represents the mathematical and physical point of view, with intense variation in the conductivity of the tissue during the occurrence of this phenomenon. This thesis validates a dynamic model for electroporation of biological tissues, where computer simulations were performed compared to experimental results. For the simulation, two calculation methods were used: Equivalent Circuit Method -ECM and Finite Element Method -FEM. In the ECM, the dielectric dispersion present in biological tissues was included. An electropermeabilizer capable of reaching 800 V was developed and a data acquisition system to save the voltage and current data that circulates through the sample during the application of an intense electric field. For obtaining the experimental data, the following biological tissues were used: heart, kidney and rabbit liver and rat liver. Voltage pulse protocols and voltage ramps were used. The properties of the intact tissue were obtained using an Agilent ® model 4294A impedance analyzer. Using the genetic algorithm, the dispersion parameters of the � band of the Cole-Cole model were obtained. There was good agreement between the simulated and experimental results, with the simulated results within the experimental standard deviation. Only for the protocol with fundamental frequency of 50 kHz, the simulation performed by the FEM using the commercial software COMSOL Multiphysics ® did not correctly represent the current reaching errors of 50 %. The justification for the error found is due to the dielectric dispersion that was not included in the current calculation in this simulator. Experiments to obtain the recovery time of the membrane pores were performed, with values between 1 s and 10 s being obtained. It is concluded that the dynamic model of electropermeabilization for biological tissues has characteristics that allow the correct computational simulation of the phenomenon in the tissues used. Thus, the next step is to use the present model in clinical applications.

Maxwell's equations explain why irreversible electroporation will not heat up a metal stent

Article

Full-text available

Apr 2021
INT J HEAT MASS TRAN

Irreversible Electroporation (IRE) is a promising clinical ablation therapy for the treatment of cancer, but issues with the generation of heat must be solved before safe and effective clinical results can be obtained. In the present study, we show that a metal stent will not be noticeably heated up by IRE pulses under typical clinical conditions. Derivation of this non-intuitive result required the application of Maxwell's equations to the tissue-stent configuration. Subsequently, straightforward and arguably accurate simplifications of the electric field generated by two needles in tissue surrounding a metal stent have enabled the modeling of the heat generation and the transport of heat in IRE procedures. Close to a stent that is positioned in between two needles, temperatures in a typical run of 100 s, 1 Hz pulses, may remain notably lower than without the stent. This is the explanation of the experimentally observed low temperature rim of viable tissue around the stent, whereas all tissue was non-viable without stent, found in tissue model experiments.

A Circular Multielectrode Pulsed-Field Ablation Catheter "Lasso PFA": Lesion Characteristics, Durability and Effect on Neighboring Structures

Article

Jan 2021

Background - Pulsed field ablation (PFA) is a nonthermal energy with potential safety advantages over radiofrequency ablation (RFA). This study investigated a novel PFA system- a circular multielectrode catheter ("PFA lasso") and a multichannel generator designed to work with Carto 3® mapping system. Methods - A 7.5F bidirectional circular catheter with 10 electrodes and variable expansion was designed for PFA (biphasic, 1800 Volts). This study included a total of 16 swine utilized to investigate the following 3 experimental aims: Aim 1 examined the feasibility to create a right atrial ablation line of block from the superior vena cava (SVC) to the inferior vena cava (IVC). Aim 2 examined the effect of PFA on lesion maturation including durability after a 30-day survival period. Aim 3 examined the effect of high intensity PFA (10 applications) on esophageal and phrenic nerve tissue in comparison to normal intensity RFA (1-2 applications). Histopathological analysis of all cardiac, esophageal and phrenic nerve tissue was performed. Results - Acute line of block was achieved in 12/12 swine (100%) and required a total PFA time of 14 sec (IQR:9-24.5) per line. Ablation line durability after 28&3 days was maintained in 11/12 (91.7%) swine. PFA resulted in transmural lesions in 179/183 (97.8%) sections and a median lesion width of 14.2mm. High intensity PFA (9 [IQR:8-14] application) had no effect on the esophagus while standard intensity RFA (1.5 [IQR:1-2] applications) resulted in deep esophageal tissue injury involving the muscularis propria and adventitia layers. High intensity PFA (16 [IQR:10-28] applications) has no effect on phrenic nerve function and structure while standard dose RFA (1.5 [IQR:1-2] applications) resulted in acute phrenic nerve paralysis. Conclusions - In this preclinical model, a multielectrode circular catheter and multichannel generator produced durable atrial lesions with lower vulnerability to esophageal or phrenic nerve damage.

Influences of Pulsed Electric Field Parameters on Cell Electroporation and Electrofusion Events: Comprehensive Understanding by Experiments and Molecular Dynamics Simulations

Preprint

Full-text available

Mar 2024

Electroporation and electrofusion are efficient methods, which have been widely used in different areas of biotechnology and medicine. Pulse strength and width, as an external condition, play an important role in the process of these methods. However, comparatively little work has been done to explore the effects of pulsed electric field parameters on electroporation and electrofusion. Herein, influences of pulse strength and width on the electroporation and electrofusion of phospholipid bilayers were systematically investigated by using experiments combined with molecular dynamics simulations. Experimental results and machine learning-based regression analysis showed that the number of pores is mainly determined by pulse strength, while the sizes of pores were enlarged by increasing the pulse widths. In addition, the formation of large-size pores is the most crucial factor that affects the fusion rate of myeloma cells. The same trend has taken place on coarse-grained and all-atom MD simulations. The result suggested that electroporation events occur only in an electric field exceeding the strength of threshold, and the unbalanced degree of electric potential between two membranes leads to pores formation during the process of electroporation. Generally, this work provides a comprehensive understanding of how pulse strength and width govern the poration event of bilayer lipid membranes, as well as guidance on the experimental design of electrofusion.

Modeling of the process of pore formation during electrical breakdown of bilayer lipid membranes

Article

Full-text available

Aug 2023

Targeted drug delivery is one of the most important areas in pharmacology. The drug can be placed in a liposomal shell and destroyed it in a specific location in the body using electroporation. In many experiments on the study of electroporation, the bilayer lipid membrane BLM (black film) is used as a model for the action of an electric field. The characteristic pore size during electroporation is several nanometers, which corresponds to the membrane thickness. These pores can be visualized using cryoelectron microscopy or atomic force microscopy. However, we cannot observe with the help of these methods the dynamics of pores: changes in their number in the membrane and changes in their size over time. We suggest using 100-500 nm color film until it has turned into a black BLM film as the BLM model. Metastable pore-defects about 6 µm in size were registered in the color film, which were observed with a light microscope (video is attached, pore-defects appear at the very end of the recording). The temporal characteristics of the registered pore-defects were considered: the lifetime of pores before membrane rupture, the rate of increase in the number of pores in the membrane. The results obtained show that a thick colored film can be used as a BLM model for studying the process of pore formation during electroporation.

A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes

Article

Full-text available

Feb 2023

CRISPR/Cas-based genome editing has dramatically improved genetic modification technology. In situ electroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need for ex vivo embryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise including micromanipulation techniques. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infected cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals and in situ electroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.

A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes

Preprint

Full-text available

Sep 2022

CRISPR/Cas-based genome editing has dramatically improved genetic modification technology. In-situ electroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need for ex vivo embryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infectious cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals and in-situ electroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.

TEMPERATURE DISTRIBUTION ANALYSIS IN PARALLEL PLATE TREATMENT CHAMBER FOR PULSED ELECTRIC FIELD PROCESSING: NUMERICAL STUDY

Article

Full-text available

Aug 2022

Studies on temperature distribution in parallel plate treatment chambers are limited due to its design which is more prone to arcing, thus, neglecting its use in continuous processing. Therefore, this study discusses the temperature distribution due to Joule heating in a parallel plate treatment chamber acting in continuous mode. The numerical results predict that at a slow flow rate (i.e., 0.0234 cm3/s), the fluid flow near the chamber wall is in a static state (0 cm/s), thus, increasing its residence time and resulting in receiving more pulses. In this situation, the temperature increased dramatically from 25 °C (inlet temperature) to approximately 58 °C, i.e., 132 % increment. On the other hand, a slight increase in temperature (i.e., < 27 °C) is predicted by numerical simulation at a higher flow rate (i.e., 0.138 cm3/s) at the same location (near the chamber wall). This less rise is due to the low residence time which causes the liquid to quickly leave the treatment area, thus, getting less pulse. The temperature soar in this condition is very low which is approximately 8 % of the inlet temperature. From the results obtained, flow rate control helps to reduce the temperature rise, thus, keeping the temperature at ambient temperature or slightly above the ambient and at the same time reducing the risk of the treated media from experiencing adverse effects on its physical attributes as a result of high temperatures.

Activation of Tissue Reparative Processes by Glow-Type Plasma Discharges as an Integral Part of the Therapy of Decubital Ulcers

Article

Full-text available

Aug 2022

The results of a clinical study of the complex treatment of pressure ulcers using the method of activation of reparative processes in tissues by cold plasma discharges initiated by high-frequency current are presented. Activation was carried out with a specialized device generating cold plasma discharges at frequencies of 0.11, 2.64, and 6.78 MHz. It was shown that the process of activation in the skin and muscle tissues of the bedsore zone proceeds most efficiently when using a current with a frequency of 6.78 MHz as compared to currents with a frequency of 2.64 and 0.11 MHz. For a needle electrode with a diameter of 0.3 mm, the optimal exposure parameters were power—(5.0 ± 1.5) W and time—(2.0–3.0) s. The results of the analysis of histological samples, histochemical, and bacteriological analysis confirmed the effect and showed the dynamics of the process of activation of reparative processes in the tissues of the bedsore wound under the influence of cold plasma discharges and a decrease in microbial contamination. The most pronounced effect of activation was formed during the period from 14 to 21 days. The effectiveness of therapy by the method of activation of reparative processes with cold plasma discharges, according to the criterion of the rate of wound healing, ranged from 14 to 16%, depending on the etiology of the decubitus wound. It is concluded that the activation of tissue reparative processes by glow-type plasma discharges as an integral part of the treatment of decubital ulcers is an effective link in the complex treatment of pressure sores.

In-vivo egfp expression in the honeybee Apis mellifera induced by electroporation and viral expression vector

Article

Full-text available

Jun 2022
PLOS ONE

In this study we describe egfp expression induced by two techniques: in vivo electroporation and viral transduction in several cell types of the adult honeybee brain. Non-neuronal and neuronal cell types were identified and the expression persisted at least during three days. Kenyon cells, optic lobe neurons and protocerebral lobe neurons were electroporated. Astrocyte-like glia cells, fibrous lamellar glia cells and cortex glia cells were identified. Viral transduction targeted one specific type of glia cells that could not be identified. EGFP positive cells types were rather variable after electroporation, and viral transduction resulted in more homogenous groups of positive cells. We propose that these techniques remain a good alternative to transgenic animals because they potentially target only somatic cells.

In‐vivo porcine characterization of atrial lesion safety and efficacy utilizing a circular pulsed‐field ablation catheter including assessment of collateral damage to adjacent tissue in supratherapeutic ablation applications

Article

Full-text available

May 2022

Introduction: Pulsed field ablation (PFA), an ablative method that causes cell death by irreversible electroporation, has potential safety advantages over radiofrequency ablation and cryoablation. Pulmonary vein (PV) isolation was performed in a porcine model to characterize safety and performance of a novel, fully-integrated biphasic PFA system comprising a multi-channel generator, variable loop circular catheter, and integrated PFA mapping software module. Methods: Eight healthy porcine subjects were included. To evaluate safety, multiple ablations were performed, including sites not generally targeted for therapeutic ablation, such as the right inferior PV lumen, right superior PV ostium, and adjacent to the esophagus and phrenic nerve. To evaluate efficacy, animals were recovered, followed for 30(±3) days, then re-mapped. Gross pathological and histopathological examinations assessed procedural injuries, chronic thrombosis, tissue ablation, penetration depth, healing, and inflammatory response. Results: All 8 animals survived follow-up. PV narrowing was not observed acutely nor at follow-up, even when ablation was performed deep to the PV ostium. No injury was seen grossly or histologically in adjacent structures. All PVs were durably isolated, confirmed by bidirectional block at re-map procedure. Histological examination showed complete, transmural necrosis around the circumference of the ablated section of right PVs. Conclusion: This pre-clinical evaluation of a fully-integrated PFA system demonstrated effective and durable ablation of cardiac tissue and PV isolation without collateral damage to adjacent structures, even when ablation was performed in more extreme settings than those used therapeutically. Histological staining confirmed complete transmural cell necrosis around the circumference of the PV ostium at 30 days. This article is protected by copyright. All rights reserved.

Identification of electroporation sites in the complex lipid organization of the plasma membrane

Article

Full-text available

Feb 2022
eLife

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon termed electroporation is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. Additionally, by analysing poration kinetics with Bayesian survival analysis we show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

Cell Membrane Permeabilization by Pulsed Electric Fields for Efficient Extraction of Intercellular Components from Foods

Chapter

Jan 2022

The chapter reviews applications of pulsed electric fields (PEF) for the efficient extraction of intercellular components from food plants. Mechanisms of cell membrane permeabilization by PEF including electroporation of plane membranes, spherical cells, cells with different shapes and sizes, and ensembles of cells and plant tissues are discussed. Different techniques to detect electroporation, PEF protocols, treatment chambers, and methods for optimization of PEF treatment are presented. Solid/liquid expression and solvent extraction assisted by PEF are described in detail. Numerous practical examples of PEF-enhanced extraction of intracellular compounds from foods (potatoes, apples, sugar crops, citruses, grapes, etc.) are presented.

The Good and the Bad of Cell Membrane Electroporation

Article

Full-text available

Dec 2021
ACTA CHIM SLOV

Electroporation is used to increase the permeability of the cell membrane through high-voltage electric pulses. Nowadays, it is widely used in different areas, such as medicine, biotechnology, and the food industry. Electroporation induces the formation of hydrophilic pores in the lipid bilayer of cell membranes, to allow the entry or exit of molecules that cannot otherwise cross this hydrophobic barrier. In this article, we critically review the basic principles of electroporation, along with the advantages and drawbacks of this method. We discuss the effects of electroporation on the key components of biological membranes, as well as the main applications of this procedure in medicine, such as electrochemotherapy, gene electrotransfer, and tissue ablation. Finally, we define the most relevant challenges of this romising area of research.

Making Waves: Pathogen Inactivation by Electric Field Treatment: From Liquid Food to Drinking Water

Article

Oct 2021
WATER RES

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Identification of electroporation sites in the complex lipid organization of the plasma membrane

Preprint

Full-text available

Oct 2021

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to a strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon, termed electroporation, is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. However, by analysing poration kinetics with Bayesian survival analysis we then show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

Recent electroporation-based systems for intracellular molecule delivery

Article

Full-text available

May 2021

Intracellular delivery of functional molecules, such as DNA probes and plasmids, is an important method for investigating cellular mechanisms and changing cell fates in biomedicine. Among various delivery methods, recent years have seen the emergence of electroporation-based techniques that provide versatile platforms for molecule delivery, with high efficiency and controlled dosage. In this Review, we describe recent electroporation-based systems for intracellular molecule delivery. The principles of electroporation for cell membrane perforation and cargo delivery are briefly summarized. Focusing on various scenarios for the application of electroporation, we review electroporation devices that variously employ structures based on nanochannels, nanostraws, and flow-through microfluidic channels for in vitro intracellular molecule delivery. We also consider in vivo targeted therapies based on delivery of active molecules by electroporation according to the lesion locations. Finally, we discuss the current challenges facing electroporation-based techniques, as well as opportunities for their future development, which may lead to innovations in intracellular molecule delivery both for cellular analysis in the laboratory and treatment in the clinic.

Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells

Article

Full-text available

Sep 2021

Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.

Pulsed-Field Ablation in Ventricular Myocardium Using a Focal Catheter: The Impact of Application Repetition on Lesion Dimensions

Article

Full-text available

Aug 2021

Background - Pulsed-field ablation (PFA) is a rapid and nonthermal energy with higher selectivity to myocardial tissue in comparison to radiofrequency ablation (RFA). However, its effects on ventricular myocardium, and titration of lesion dimensions have not been well studied. This study examined the effect of PFA on ventricular myocardium, and the influence of application repetition on lesion dimensions. Methods - An 8Fr lattice catheter with a compressible 9 mm nitinol tip was used with a PFA generator (Affera Inc) applying a bipolar and biphasic waveform (±1.3-2.0 kV, 4 sec per application). We examined the impact of single applications (1x) vs four repetitive applications (4x) interspaced by 10 seconds. The latter was performed to determine whether repetitions of a similar electrical field can increase lesion dimensions. Experiments were performed in an in-vivo porcine model and a survival period of 24 hours (n=6) or 23±5.4 days (n=6) for evaluation of early and late histopathological effects. Results - PFA in ventricular myocardium showed repetition dependency. Acute lesion depth and volume increased from 5.6±1.43mm and 860±362mm ³ to 8.8±0.74mm and 2383±548mm ³ for 1x and 4x applications, respectively (P<0.001 for both comparisons). This effect was maintained in the chronic lesion phase with lesion depth and volume of 3.9±0.92mm and 655±286mm ³ compared with 7.3±0.83mm and 2170±488mm ³ , respectively (P<0.001 for both comparisons). Acute lesions showed well demarcated necrotic core without coagulation necrosis while chronic lesions showed tissue thinning with fibro-fatty replacement. Conclusions - PFA in ventricular myocardium exhibits repetition dependency as lesion dimension increases with consecutive applications of a similar electrical field.

Electroporation-Based Therapy for Brain Tumors: A Review

Article

May 2021

Electroporation-based therapy (EBT), as a high-voltage-pulse technology has been prevalent with favorable clinical outcomes in the treatment of various solid tumors. The aim of this review paper is to promote the clinical translation of EBT for brain tumors. First, we briefly introduced the mechanism of pore formation in a cell membrane activated by external electric fields using a single cell model. Then, we summarized and discussed the current in vitro and in vivo preclinical studies, in terms of (1) the safety and effectiveness of EBT for brain tumors in animal models, and (2) the blood-brain barrier (BBB) disruption induced by EBT. Two therapeutic effects could be achieved in EBT for brain tumors simultaneously, i.e., the tumor ablation induced by irreversible electroporation (IRE) and transient blood-brain barrier (BBB) disruption induced by reversible electroporation (RE). The BBB disruption could potentially improve the uptake of anti-tumor drugs thereby enhancing brain tumor treatment. The challenges that hinder the application of EBT in the treatment of human brain tumors are discussed in the review paper as well.

The Defibrillation Conundrum: New Insights into the Mechanisms of Shock-Related Myocardial Injury Sustained from a Life-Saving Therapy

Article

Full-text available

May 2021
INT J MOL SCI

Implantable cardiac defibrillators (ICDs) are recommended to prevent the risk of sudden cardiac death. However, shocks are associated with an increased mortality with a dose response effect, and a strategy of reducing electrical therapy burden improves the prognosis of implanted patients. We review the mechanisms of defibrillation and its consequences, including cell damage, metabolic remodeling, calcium metabolism anomalies, and inflammatory and pro-fibrotic remodeling. Electrical shocks do save lives, but also promote myocardial stunning, heart failure, and pro-arrhythmic effects as seen in electrical storms. Limiting unnecessary implantations and therapies and proposing new methods of defibrillation in the future are recommended.

Irreversible Electroporation (IRE) in Locally Advanced Pancreatic Cancer: A Review of Current Clinical Outcomes, Mechanism of Action and Opportunities for Synergistic Therapy

Article

Full-text available

Apr 2021

Locally advanced pancreatic cancer (LAPC) accounts for 30% of patients with pancreatic cancer. Irreversible electroporation (IRE) is a novel cancer treatment that may improve survival and quality of life in LAPC. This narrative review will provide a perspective on the clinical experience of pancreas IRE therapy, explore the evidence for the mode of action, assess treatment complications, and propose strategies for augmenting IRE response. A systematic search was performed using PubMed regarding the clinical use and safety profile of IRE on pancreatic cancer, post-IRE sequential histological changes, associated immune response, and synergistic therapies. Animal data demonstrate that IRE induces both apoptosis and necrosis followed by fibrosis. Major complications may result from IRE; procedure related mortality is up to 2%, with an average morbidity as high as 36%. Nevertheless, prospective and retrospective studies suggest that IRE treatment may increase median overall survival of LAPC to as much as 30 months and provide preliminary data justifying the well-designed trials currently underway, comparing IRE to the standard of care treatment. The mechanism of action of IRE remains unknown, and there is a lack of data on treatment variables and efficiency in humans. There is emerging data suggesting that IRE can be augmented with synergistic therapies such as immunotherapy.

Mitral insufficiency following electrical cardioversion in pediatric patients

Article

Jan 2020

Understanding In Vivo Fate of Nucleic Acid and Gene Medicines for the Rational Design of Drugs

Article

Full-text available

Jan 2021

Nucleic acid and genetic medicines are increasingly being developed, owing to their potential to treat a variety of intractable diseases. A comprehensive understanding of the in vivo fate of these agents is vital for the rational design, discovery, and fast and straightforward development of the drugs. In case of intravascular administration of nucleic acids and genetic medicines, interaction with blood components, especially plasma proteins, is unavoidable. However, on the flip side, such interaction can be utilized wisely to manipulate the pharmacokinetics of the agents. In other words, plasma protein binding can help in suppressing the elimination of nucleic acids from the blood stream and deliver naked oligonucleotides and gene carriers into target cells. To control the distribution of these agents in the body, the ligand conjugation method is widely applied. It is also important to understand intracellular localization. In this context, endocytosis pathway, endosomal escape, and nuclear transport should be considered and discussed. Encapsulated nucleic acids and genes must be dissociated from the carriers to exert their activity. In this review, we summarize the in vivo fate of nucleic acid and gene medicines and provide guidelines for the rational design of drugs.

Analysis on reversible/irreversible electroporation region in lung adenocarcinoma cell model in vitro with electric pulses delivered by needle electrodes

Article

Full-text available

Nov 2020
PHYS MED BIOL

Irreversible electroporation (IRE) is a minimally invasive tumor therapy using pulsed electric field with high intensity while the important tissues such as blood vessel, bile duct, and nerve are preserved. In addition to ablation area, reversible electroporation (RE) region is also generated using needle electrodes for pulse delivery. The goal of this work is to study the generation of RE region and ablation region on a 2D lung adenocarcinoma cell model in vitro. The tumor model is exposed to electric pulses with various number. The calcium AM and propidium iodide (PI) are examined to detect the ablation area and electroporation area, respectively. The results show that electroporation area firstly tends to plateau after approximately 50 pulses, while the ablation area continues to increase. The percentage of IRE area in total electroporation area increases with additional pulses, which means that RE region could be gradually turned into ablation area with increased pulse number. However, the percentage of IRE area only achieves to 54% for 200 pulses, which indicates that RE region still cannot be completely removed. RE and IRE thresholds appear to converge as the number of pulses increases. An equation between pulse number and the electric field threshold of ablation including the electric field threshold of RE is also provided for lung adenocarcinoma cell ablation. This work may have the value for the optimization of IRE protocols on tumor ablation.

In vivo analysis of the origin and characteristics of gaseous microemboli during catheter-mediated irreversible electroporation

Article

Full-text available

Oct 2020
EUROPACE

Aims : Irreversible electroporation (IRE) ablation is a non-thermal ablation method based on the application of direct current between a multi-electrode catheter and skin electrode. The delivery of current through blood leads to electrolysis. Some studies suggest that gaseous (micro)emboli might be associated with myocardial damage and/or (a)symptomatic cerebral ischaemic events. The aim of this study was to compare the amount of gas generated during IRE ablation and during radiofrequency (RF) ablation. Methods and results: In six 60-75 kg pigs, an extracorporeal femoral shunt was outfitted with a bubble-counter to detect the size and total volume of gas bubbles. Anodal and cathodal 200 J IRE applications were delivered in the left atrium (LA) using a 14-electrode circular catheter. The 30 and 60 s 40 W RF point-by-point ablations were performed. Using transoesophageal echocardiography (TOE), gas formation was visualized. Average gas volumes were 0.6 ± 0.6 and 56.9 ± 19.1 μL (P < 0.01) for each anodal and cathodal IRE application, respectively. Also, qualitative TOE imaging showed significantly less LA bubble contrast with anodal than with cathodal applications. Radiofrequency ablations produced 1.7 ± 2.9 and 6.7 ± 7.4 μL of gas, for 30 and 60 s ablation time, respectively. Conclusion : Anodal IRE applications result in significantly less gas formation than both cathodal IRE applications and RF applications. This finding is supported by TOE observations.

The estimation of pore size distribution of electroporated MCF-7 cell membrane

Article

Jun 2024
ELECTROMAGN BIOL MED

In-silico research on the effects of variable voltage pulse protocols on the cell survival and permeabilization fractions in electroporated cancerous tissues

Article

May 2024
MICROCHEM J

A Brief Sketch of the History of EMB: Where Good Ideas Come From

Chapter

Sep 2023

C. Brosseau

Catheter-Based Electroporation

Article

Jun 2023

Recent Advances in Electrochemical Sterilization

Article

Apr 2023

Optoelectronic cell lysis

Conference Paper

Sep 2014

Structural dynamics: review of time-resolved cryo-EM

Article

Full-text available

Jul 2022

The structural determination of biological macromolecules has been transformative for understanding biochemical mechanisms and developing therapeutics. However, the ultimate goal of characterizing how structural dynamics underpin biochemical processes has been difficult. This is largely due to significant technical challenges that hinder data collection and analysis on the native timescales of macromolecular dynamics. Single-particle cryo-EM provides a powerful platform to approach this challenge, since samples can be frozen faster than the single-turnover timescales of most biochemical reactions. In order to enable time-resolved analysis, significant innovations in the handling and preparation of cryo-EM samples have been implemented, bringing us closer to the goal of the direct observation of protein dynamics in the milliseconds to seconds range. Here, the current state of time-resolved cryo-EM is reviewed and the most promising future research directions are discussed.

The Enlargement of Ablation Area by Electrolytic Irreversible Electroporation (E-IRE) Using Pulsed Field with Bias DC Field

Article

Jul 2022

Irreversible electroporation (IRE) by high-strength electric pulses is a biomedical technique that has been effectively used for minimally invasive tumor therapy while maintaining the functionality of adjacent important tissues, such as blood vessels and nerves. In general, pulse delivery using needle electrodes can create a reversible electroporation region beyond both the ablation area and the vicinity of the needle electrodes, limiting enlargement of the ablation area. Electrochemical therapy (EChT) can also be used to ablate a tumor near electrodes by electrolysis using a direct field with a constant current or voltage (DC field). Recently, reversible electroporated cells have been shown to be susceptible to electrolysis at relatively low doses. Reversible electroporation can also be combined with electrolysis for tissue ablation. Therefore, the objective of this study is to use electrolysis to remove the reversible electroporation area and thereby enlarge the ablation area in potato slices in vitro using a pulsed field with a bias DC field (constant voltage). We call this protocol electrolytic irreversible electroporation (E-IRE). The area over which the electrolytic effect induced a pH change was also measured. The results show that decreasing the pulse frequency using IRE alone is found to enlarge the ablation area. The ablation area generated by E-IRE is significantly larger than that generated by using IRE or EChT alone. The ablation area generated by E-IRE at 1 Hz is 109.5% larger than that generated by IRE, showing that the reversible electroporation region is transformed into an ablation region by electrolysis. The area with a pH change produced by E-IRE is larger than that produced by EChT alone. Decreasing the pulse frequency in the E-IRE protocol can further enlarge the ablation area. The results of this study are a preliminary indication that the E-IRE protocol can effectively enlarge the ablation area and enhance the efficacy of traditional IRE for use in ablating large tumors.

Cerebral safety after pulsed field ablation for paroxysmal atrial fibrillation

Article

Jun 2022
HEART RHYTHM

Background Pulsed field ablation (PFA) is a novel, nonthermal ablation modality that can ablate myocardial tissue with minimal effect on surrounding tissue. Preclinical data show absence of cerebral emboli after extensive PFA. However, clinical data SCL /SCE after PFA are lacking. Objectives This study investigates the occurrence of neurological deficits and silent cerebral lesions (SCL) and/or events (SCE) after PFA in paroxysmal AF (PAF) using National Institutes of Health Stroke Scale (NIHSS) scores and magnetic resonance imaging (MRI). Methods In patients with symptomatic paroxysmal AF, pulmonary vein isolation (PVI) using PFA was performed. NIHSS scores were assessed before, 2 and 30 days after PVI. One day after PVI, patients underwent cerebral 1.5 Tesla MRI-scanning using diffusion-weighted imaging (DWI) and FLAIR sequences to document the occurrence of SCL/SCE. Results In 30 patients (age 63±10 years), PFA was performed. No patient showed neurological deficits. All obtained NIHSS scores showed the minimum value of 0. Cerebral MRI scans were normal in 29/30 (97%) patients. In 1 (3%) patient, a single 7-mm cerebellar lesion was observed. Forty days after the procedure, a follow-up cerebral MRI scan showed complete regression of the lesion. Conclusions In patients treated with PFA for symptomatic paroxysmal AF, the incidence of MRI-detected asymptomatic thrombo-embolic cerebral events or lesions was as low as 3%. There were no neurological deficits in any of the patients.

Pore-Opening Dynamics of Single Nanometer Biovesicles at an Electrified Interface

Article

Full-text available

Jun 2022

Release from nanobiovesicles via a pore generated by membrane electroporation at an electrified interface can be monitored by vesicle impact electrochemical cytometry (VIEC) and provides rich information about the various vesicular content transfer processes, including content homeostasis, intraphase content transfer, or the transient fusion of vesicles. These processes are primarily influenced by the vesicular pore-opening dynamics at the electrified interface which has not been disclosed at the single nanobiovesicle level yet. In this work, after simultaneously measuring the size and release dynamics of individual vesicles, we employed a moving mesh-finite element simulation algorithm to reconstruct the accurate pore-opening dynamics of individual vesicles with different sizes during VIEC. We investigated the expansion times and maximal pore sizes as two characteristics of different vesicles. The pore expansion times between nanobiovesicles and pure lipid liposomes were compared, and that of the nanobiovesicles is much longer than that for the liposomes, 2.1 ms vs 0.18 ms, respectively, which reflects the membrane proteins limiting the electroporation process. For the vesicles with different sizes, a positive relationship of pore size (Rp,max) with the vesicle size (Rves) and also their ratio (Rp,max/Rves) versus the vesicle sizes is observed. The mechanism of the pore size determination is discussed and related to the membrane proteins and the vesicle size. This work accurately describes the dynamic pore-opening process of individual vesicles which discloses the heterogeneity in electroporation of different sized vesicles. This should allow us to examine the more complicated vesicular content transfer process between intravesicular compartments.

Is Irreversible electroporation (IRE) an effective and safe ablation method for local advanced pancreatic cancer: a meta-analysis

Article

May 2022

Background/Objectives : To evaluate the safety and efficacy of irreversible electroporation (IRE) for treating local advanced pancreatic carcinoma. Methods : Relevant literature published from January 1, 2010, to July 1, 2021, was retrieved from PubMed and EMBASE databases. The following keywords were applied: " IRE, "Irreversible electrophoresis, "pancreatic cancer, "ablation, "and " ablation therapy. "The primary outcomes was IRE related significant complications rate. Results : 19 studies were included, a total of 984 patients had undergone IRE. The probability of IRE-related major (Grade 3-5) complications rate was 17% (95% CI: 10% - 28%). 2 deaths related to IRE procedure were reported. The median overall survival time since IRE ranged from 6.1 to 27 months. Patients treated with IRE combined with surgical resection or standard chemotherapy showed a longer overall survival. Conclusions : IRE can benefit the survival of patients with locally advanced pancreatic cancer, but related adverse events should be treated with caution.

Microfluidics-based Fabrication of Flexible Ionic Hydrogel Batteries Inspired by Electric Eels

Article

Apr 2022

Electric eels in nature can generate high voltage with hundreds of volts based on the mechanism of gradient-induced ion flux, which provides an excellent prototype to inspire the exploration of more efficient and green energy generation strategies in artificial systems. Here we developed a novel microfluidics-based strategy to efficiently fabricate flexible ionic hydrogel batteries by mimicking ion-concentration gradients in biological organs. Microfluidic networks were designed to simultaneously transport four types of ionic hydrogel solutions into pre-defined well arrays and enable the perfusion of 801 hydrogel precursor wells within 1.53 minutes. A specifically-designed photomask was used to selectively solidify these precursors into separate hydrogel particles, which were further assembled into a flexible ionic hydrogel battery pack by using negative pressure. The voltage output of the resultant ionic hydrogel battery pack was found to increase linearly with the increase of battery unit number, and a maximum voltage of 73.33 V can be achieved by connecting eight ionic hydrogel battery packs in series. The resultant ionic hydrogel battery exhibited unique capability in stably working in wide temperature ranges (0-90°C) and large deformation conditions, and can be further preserved for a long period after dehydration and instantly recover its original discharge capacity once rehydrated. PEDOT:PSS was introduced to the ionic hydrogel battery for reduced internal resistance, resulting in a 1.5-fold improvement in electrical current output. The presented microfluidics-based and high-efficient fabrication strategy exhibits great promise to translate the biomimetic flexible power-generating systems into a viable soft power source for various electrically-driven applications.

Induction and Suppression of Cell Lysis in an Electrokinetic Microfluidic System

Article

Mar 2022

The ability to strategically induce or suppress cell lysis is critical for many cellular‐level diagnostic and therapeutic applications conducted within electrokinetic microfluidic platforms. The chemical and structural integrity of sub‐cellular components is important when inducing cell lysis. However, metal electrodes and electrolytes participate in undesirable electrochemical reactions that alter solution composition and potentially damage protein, RNA, and DNA integrity within device microenvironments. For many biomedical applications, cell viability must be maintained even when device‐imposed cell‐stressing stimuli (e.g., electrochemical reaction byproducts) are present. In this work, we explored a novel and tunable method to accurately induce or suppress device‐imposed artifacts on human red blood cell (RBC) lysis in non‐uniform AC electric fields. For precise tunability, a dielectric hafnium oxide (HfO2) layer was used to prevent electron transfer between the electrodes and the electric double layer and thus reduce harmful electrochemical reactions. Additionally, a low concentration of Triton X‐100 surfactant was explored as a tool to stabilize cell membrane integrity. The extent of hemolysis was studied as a function of time, electrode configuration (T‐shaped and star‐shaped), cell position, applied non‐uniform AC electric field, with uncoated and HfO2 coated electrodes (50 nm), and absence and presence of Triton X‐100 (70 μM). Tangible outcomes include a parametric analysis relying upon literature and this work to design, tune, and operate electrokinetic microdevices to intentionally induce or suppress cellular lysis without altering intracellular components. Implications are that devices can be engineered to leverage or minimize device‐imposed biological artefacts extending the versatility and utility of electrokinetic diagnostics. This article is protected by copyright. All rights reserved

In-vivo egfp expression in the honeybee Apis mellifera induced by electroporation and viral expression vector

Preprint

Full-text available

Jan 2022

In this study we describe egfp expression induced by two techniques: in vivo electroporation and viral transduction in several cell types of the adult honeybee brain. Non-neuronal and neuronal cell types were identified and the expression persisted at least during three days. Kenyon cells, optic lobe neurons and protocerebral lobe neurons were electroporated. Astrocyte-like glia cells, fibrous lamellar glia cells and cortex glia cells were identified. Viral transduction targeted one specific type of glia cells that could not be identified. EGFP positive cells were rather variable after electroporation, and viral transduction resulted in more homogenous groups of positive cells. We propose that these techniques remain a good alternative to transgenic animals because they potentially target only somatic cells.

Irreversible Electroporation Ablation for Atrial Fibrillation: Status and Challenges

Article

Jan 2022

Pulsed electric field (PEF) is a novel energy source by which high-voltage electric pulses are used to create irreversible electroporation. PEF is non-thermal and highly tissue-dependent in which specific targeting of the atrial myocardium is achieved and sparing of adjacent tissues is feasible, theoretically increasing the safety of the procedure, which could potentially break the trade-off between effective lesions and collateral damage and substantially improve risk-benefit ratios in atrial fibrillation (AF) ablation. Although recently published trials have shown a clear effect of AF ablation, large-scale clinical trials are lacking. Current clinical evidence has demonstrated significant efficacy in achieving durable pulmonary vein isolation without ablation-related adverse events. However, their putative benefits regarding efficacy, efficiency, and safety remain to be proven in randomized controlled trials.

Enhanced transmembrane transport of reactive oxygen species by electroporation effect of plasma

Article

Aug 2021

Recently, plasma has been proposed as a new efficient tool for various biological and medical applications. The function of plasma is mainly realized through two aspects: Electric field and reactive species. However, because the interactions between plasma and cells are quite complicated, the synergistic effect of electric field and reactive species has not been studied in depth. In this paper, the synergetic effects of electric field and transportation of reactive species on cells are investigated by a two-dimensional finite element model. The exposure of cells to plasma results in the sufficiently high transmembrane voltage (1 V) and electroporation. When the electric field of plasma is less than 10⁴ V/cm, the radius of nanopores generated by plasma increases with the electric field. The further increase of electric field to 10⁵ V/cm does not cause a significant increase of nanopore radius, whereas the pore density increases by five times. The increase of discharge frequency significantly decreases the time needed to generate stable nanopores. The nanopores enhance the diffusion of H2O2 generated by plasma through the cell membrane significantly. Compared with the reality that H2O2 does not freely diffuse across cell membranes, the “generation and enhanced transportation” effect will further expand the application range of plasma medicine.

Dynamic Electroporation Model Evaluation on Rabbit Tissues

Article

Full-text available

Jun 2021

Biological electroporation is a process of opening pores in the cell membrane when exposed to intense electric fields. This work provides results for validation of a dynamic model of electroporation on biological tissues. Computational simulations were carried out and results for the electrical current through the tissue and increase of the tissue temperature were compared to experimental results. Two calculation methods were used: Equivalent Circuit Method and Finite Element Method. With Equivalent Circuit Method the dielectric dispersion present in biological tissues was included. Liver, kidney and heart of rabbit were used in the experiments. Voltage pulse protocols and voltage ramps were applied using stainless steel needles electrodes. There is good agreement between the simulated and experimental results with mean errors below 15%, with the simulated results within the experimental standard deviation. Only for the protocol with fundamental frequency of 50 kHz, the simulation performed by the Finite Element Method using a commercial software did not correctly represent the current, with errors reaching 50%. The justification for the error found is due to the dielectric dispersion that was not included in this simulator.

Pulmonalvenenisolation bei Vorhofflimmern mittels „pulsed field ablation“Pulmonary vein isolation in atrial fibrillation using pulsed field ablation

Article

Jun 2021

Atrial fibrillation is the most common supraventricular arrhythmia with increasing incidence and prevalence. Until now, thermal energy sources such as radiofrequency or cryoablation have been used for pulmonary vein isolation of atrial fibrillation but these have led to indiscriminate tissue destruction in the target area. Pulsed field ablation (PFA) is an energy modality that does not utilize thermal effects. An ultrarapid electric field produces irreversible changes in cell membrane pores (irreversible electroporation) culminating in cell death. The myocardium is very sensitive to PFA compared to the esophagus, the pulmonary veins or the phrenic nerve. Consequently, it is possible to perform effective ablation of the pulmonary veins in a very short time and to make the treatment time more effective without causing relevant collateral damage. The treatment offers a potential paradigm shift from catheter ablation of cardiac arrhythmia.

Efficient encapsulation of functional proteins into erythrocytes by controlled shear- mediated membrane deformation

Article

May 2021

Red blood cells (RBCs) are attractive carriers of biomolecular payloads due to their biocompatibility and the ability to shelter their encapsulated cargo. Commonly employed strategies to encapsulate payloads into RBCs, such as hypotonic shock, membrane fusion or electroporation, often suffer from low throughput and unrecoverable membrane impairment. This work describes an investigation of a method to encapsulate protein payloads into RBCs by controlling membrane deformation either transiently or extendedly in a microfluidic channel. Under the optimized conditions, the loading efficiency of enhanced green fluorescent protein into mouse RBCs increased was about 2.5- and 4-fold compared to that with osmotic entrapment using transient and extended deformation, respectively. Significantly, mouse RBCs loaded with human arginase exhibit higher enzymatic activity and membrane integrity compared to their counterparts loaded by osmotic entrapment. These features together with the fact that this shear-mediated encapsulation strategy allows loading with physiological buffers highlight the key advantages of this approach compared to traditional osmotic entrapment.

2. The “Focal Membrane Fusion” Model Revisited: Toward a Unifying Structural Concept of Biological Membrane Fusion

Chapter

Dec 1993

Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release

Article

Full-text available

Jun 1979

We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.

Visualization of the hexagonal lattice of the membrane skeleton

Article

Full-text available

Apr 1987

The isolated membrane skeleton of human erythrocytes was studied by high resolution negative staining electron microscopy. When the skeletal meshwork is spread onto a thin carbon film, clear images of a primarily hexagonal lattice of junctional F-actin complexes crosslinked by spectrin filaments are obtained. The regularly ordered network extends over the entire membrane skeleton. Some of the junctional complexes are arranged in the form of pentagons and septagons, approximately 3 and 8%, respectively. At least five forms of spectrin crosslinks are detected in the spread skeleton including a single spectrin tetramer linking two junctional complexes, three-armed Y-shaped spectrin molecules linking three junctional complexes, three-armed spectrin molecules connecting two junctional complexes with two arms bound to one complex and the third arm bound to the adjacent complex, double spectrin filaments linking two junctional complexes, and four-armed spectrin molecules linking two junctional complexes. Of these, the crosslinks of single spectrin tetramers and three-armed molecules are the most abundant and represent 84 and 11% of the total crosslinks, respectively. These observations are compatible with the presence of spectrin tetramers and oligomers in the erythrocyte membrane skeleton. Globular structures (9-12 nm in diameter) are attached to the majority of the spectrin tetramers or higher order oligomer-like molecules, approximately 80 nm from the distal ends of the spectrin tetramers. These globular structures are ankyrinor ankyrin/band 3-containing complexes, since they are absent when ankyrin and residual band 3 are extracted from the skeleton under hypertonic conditions.

Electroportion for the efficient transfection of mammalian cells with DNA

Article

Full-text available

Mar 1987

A simple and reproducible procedure for the introduction of DNA into mammalian cells by electroporation is described. The parameters involving the cells, the DNA, and the electric field are investigated. The procedure has been applied to a broad range of animal cells. It is capable of transforming more than 1% of the viable cells to the stable expression of a selectable marker.

Translational mobility of the membrane intercalated particles of human erythrocyte ghosts. pH-dependent, reversible aggregation

Article

Full-text available

Jun 1972
J CELL BIOL

Pedro Pinto da Silva

This paper demonstrates the translational movement along the plane of the human erythrocyte ghost of the membrane particles exposed by freeze-fracture. The membrane particles can be aggregated by incubation of the ghosts in media with a pH in the vicinity of 5 5 or 3 5. The particles are disaggregated in neutral and alkaline media (pH 9 5) and also at pH 4.5 Aggregation of the particles at pH 5.5 is reversible, prevented by prefixation in glutaraldehyde and by media of high ionic strength. Particle aggregation occurs within 2-4 min. These results are consistent with the concept that the erythrocyte ghost membrane is a planar fluid domain formed by a bilayer membrane continuum which is interrupted by localized, yet mobile, proteic intercalations.

Permeability Changes Induced by Electric Impulses in Vesicular Membranes

Article

Full-text available

Jan 1973

Summary Electric impulses were found to cause transient permeability changes in the membranes of vesicles storing biogenic amines. Release of catecholamines induced by electric fields (of the order of 20 kV/cm and decaying exponentially with a decay time of about 150 μsec) was studied, using the chromaffin granules of bovine adrenomedullary cells as a vesicular model system. Far-UV-absorption spectroscopy was applied to determine the amount of catecholamines released from suspended vesicles. A polarization mechanism is suggested for the induction of short-lived permeability changes caused by electric fields. Such transient changes in permeability may possibly represent a part of the sequence of events leading to stimulated neurohumoral secretion.

A New Technique for the Assay of Infectivity of Human Adenovirus 5 DNA

Article

Full-text available

May 1973

A new technique for assaying infectivity of adenovirus 5 DNA has been developed. Viral DNA was diluted in isotonic saline containing phosphate at a low concentration, and calcium chloride was added, resulting in the formation of a calcium phosphate precipitate. DNA coprecipitated with the calcium phosphate and, when the resulting suspension was added to human KB cell monolayers, became adsorbed to the cells. Following adsorption, uptake of DNA into the cells occurred during an incubation in liquid medium at 37 ° in the continued presence of extra calcium chloride.For adenovirus 5 DNA the assay resulted in up to 100-fold more plaques than could be obtained using DEAE-dextran. Furthermore a reproducible relationship between amounts of DNA inoculated per culture and numbers of plaques produced was demonstrated. The assay was most efficient at high DNA concentrations (10–30 μg/ml); below this range the addition of carrier DNA was necessary for optimum results.In addition to adenovirus 5 DNA, the technique has been used successfully to assay infectivity of DNA from adenovirus 1 and simian virus 40.

Gene-Transfer into Mouse Lyoma Cells by Electroporation in High Electric-Fields

Article

Full-text available

Feb 1982

Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.

Characterization of electric field-induced fusion in erythrocyte ghost membranes

Article

Full-text available

Jan 1985

Arthur Edward Sowers

Fusion has been reported to occur in a variety of membrane systems in response to the application of certain electric currents to the medium (Zimmermann, U., 1982, Biochim. Biophys. Acta., 694:227-277). The application of a weak but continuous alternating current causes the membranes in suspension to become rearranged into the "pearl-chain" formation. Fusion can then be induced by one or more strong direct current pulses that cause pore formation. This results in the conversion of individual membranes in the "pearl-chain" formation to a single membrane with one or more hourglass constrictions that form lumens which connect the cytoplasmic compartments. As the diameter of the lumens increases, the overall membrane shape grows to one large sphere. To further characterize electric field-induced fusion, experiments were conducted using the erythrocyte ghost as a model membrane, and a new combination of electrical circuit and fusion chamber that is simpler and improved over previous systems. All odd-shaped ghosts (collapsed or partly collapsed spherical shapes, echinocytes, discocytes, and stomatocytes) in 30 mM phosphate buffer was first converted to spherocytes and then fused with increasing yields by increasing the number of pulses. After fusion, the lateral diffusion of a fluorescent lipid soluble label (Dil) from labeled to unlabeled membranes was observed to occur both with and without the appearance in phase-contrast optics of distinct communication (lumens) between cytoplasmic compartments of the fused membranes. Connections between cytoplasmic compartments, however, were unmistakable with the instant transfer of a fluorescent water-soluble label (fluorescein isothiocyanate-dextran) from labeled to unlabeled cytoplasmic compartments upon fusion. Although pulses still resulted in the lateral diffusion of Dil to unlabeled membranes, the presence of glycerol in the medium strongly reduced the yield of lumens observable by phase-contrast optics in fusion events. The presence of glycerol also inhibited the conversion of membranes to spherocytes, but did not inhibit the lateral diffusion of Dil from labeled to unlabeled membranes.

Beginning of exocytosis captured by rapid-freezing of Limulus Amebocytes

Article

Full-text available

Aug 1981

Structural changes underlying exocytosis evoked by the application of endotoxin to Limulus amebocytes were studied at the level of detail afforded by freeze-fracture and freeze-substitution techniques combined with the time resolution of direct rapid-freezing. The results with amebocytes prepared in this manner differed from those with other secretory cells prepared by conventional means. Exocytosis begins within seconds of endotoxin treatment when the plasmalemma invaginates to form pedestallike appositions with peripheral secretory granules. The juxtaposed membranes at these pedestal appositions form several punctate pentalaminar contacts, but examination of freeze-fractured pedestals failed to reveal any corresponding changes in the intramembrane particle distribution. Small secretory granule openings or pores, which are very infrequent, appear within the first 5 s after endotoxin treatment. These pores rapidly widen and this widening is immediately followed by the sequential dissolution of the granule contents, which then move into the surrounding extracellular space. Cytoplasmic filaments connecting the plasmalemma with the granule membrane are suitably deployed to be responsible for the plasmalemma invaginations. How pores begin is not certain, but the appearance of clear spaces between the granule core and the granule membrane at this point in exocytosis supports the possibility of a role of osmotic forces.

TRANSLATIONAL MOBILITY OF MEMBRANE INTERCALATED PARTICLES OF HUMAN ERYTHROCYTE GHOSTS - PH-DEPENDENT, REVERSIBLE AGGREGATION

Article

Jan 1972

PINTODAS.P

The Morphology of Adult Red Cells

Article

Dec 1974

Ronald S Weinstein

Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release

Article

May 1979

J. E. Heuser

The Relaxation Hysteresis of Membrane Electroporation

Article

Jan 1989

Eberhard Neumann

External electric fields have traditionally been applied in physical chemistry and biophysics to probe the ionic—electric properties and reactivities of molecules and molecular organizations such as biological membranes (Eigen and DeMaeyer, 1963; Neumann, 1986a). In recent years, electric field pulse techniques have also gained increasing importance in cellular and molecular biology, in gene technology, and in medicine. In particular, the methods of electroporation (Neumann et al., 1982) and electrofusion (Senda et al., 1979; Neumann et al., 1980; Zimmermann and Scheurich, 1981; Weber et al., 1981) have become powerful tools for cell manipulations (for reviews see Zimmermann, 1986; Berg, 1987; Sowers, 1987) and for the physical chemical study of electrically induced structural rearrangements in membranes (for review see Neumann, 1986a).

Development of drug carrier systems: Electrical field induced effects in cell membranes

Article

Sep 1980
Bioelectrochem Bioenerg

Mouse thymocytes and erythrocytes are loaded electrically with drugs and dyes in isotonic solution. The loaded cells are used for targeting the drugs to specific sites in the organism in order to achieve a controlled drug release in time and space. Erythrocytes are directed to the liver by changing the volume and the shape. Using erythrocytes as drug carrier systems, methotrexate, the most widely studied agent in chemotherapeutic cancer treatment, could be directed exclusively to the liver.Directing to other organs is obtained by either using electrically fused cells or by loading cells with magnetic particles (about 10 nm in diameter) and the drug simultaneously and by guiding the cells to any selected site of the organism. The field technique used for the loading of the cells is based on the electrical breakdown of the cell membrane which is observed when cell suspensions are subjected to external field pulses of 2 to 20 kV/cm for short time intervals (ns to ms). When an apparent membrane potential of about 1 V (pulse length in the range of μs) is reached in response to the external field the membrane breaks down reversibly. The breakdown of the membrane is associated with a remarkable and reversible permeability increase of the cell membrane. The increase in permeability depends on the strength and the duration of the field pulse. The duration of the high conductance state of the cell membrane induced by the electric field is sufficiently long to entrap large quantities of drugs or dyes inside the cells.Electron-micrographs of thymocytes subjected to field pulses of various strengths and durations show that the pulse length is a critical factor for the reversibility of the field induced effects in the cell. When loading thymocytes the field pulse length has to be less than 1 μs to avoid irreversible changes in the ultrastructure of the cell. On the other hand, erythrocytes can be subjected to field pulses of 40 μs duration without any irreversible changes in the membrane structure.The life-span of the loaded erythrocytes in the blood circulation can be considerably prolonged if loss of haemoglobin and intracellular enzymes is avoided during field application and the resealing process. The loss of intracellular proteins can be minimized if the erythrocytes are immobilized in a polymeric network of calcium-alginate during the field application. The matrix is permeable to low molecular weight compounds but impermeable towards larger molecules such as proteins. Chelation of the Ca2+-ions in the alginate network by addition of sodium citrate leads to the release of the loaded erythrocytes from the calcium-alginate matrix.

Third colloquium in biological sciences: Cellular signal transduction

Conference Paper

Jan 1987

F.L. Strand

This book contains over 100 papers. Some of the paper titles are: Characterization of Antibodies to DNA by Immunoadsorption on Cation-Complexed DNA; Simultaneous Evaluation of Radiochromatography Strips Using Gamma Camera Interfaced to a Computer; Simultaneous Quantification of Procine Myocardial Adenine Nucleotides and Creatine Phosphate by Ion-Pair Reverse-Phase High-Performance Liquid Chromatography; HPLC Analysis of Proteins from Alzheimer Paired Helical Filaments; and Internal Dosimetry Evaluations of Various Organs in Humans Using a Computerized Model.

Ion Channels of Excitable Membranes

Book

Jan 2001

Bertil Hille

Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts

Article

Oct 1986

Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field-induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent-tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius of at least 8.4 nm the electropores resealed within 200 ms to a radius of about 0.5 nm and stayed at that radius thereafter. Video sequences clearly show that pores are induced preferentially in the cathodal hemisphere. Pores induced in the hemisphere facing the positive electrode were either (i) never greater than 0.5 nm in radius, (ii) much smaller in number if they were greater than 0.5 nm in radius, or (iii) shorter lived. Calculations indicated that an upper limit of 700 electropores were induced per membrane.

Breakdown of lipid bilayer membranes in an electric field

Article

Dec 1983
BBA-BIOMEMBRANES

The behaviour of lipid bilayer membranes, made of oxidized cholesterol, and UO22+-modified azolectin membranes in a high electric field has been investigated using the voltage clamp method. When a voltage pulse is applied to the membrane of these compositions, the mechanical rupture of the membranes is preceded by a gradual conductance increase which remains quite reversible till a certain moment. The voltage drop at this reversible stage of breakdown leads to a very rapid (characteristic time of less than 5 μs) decrease in the membrane conductance. At repeated voltage pulses of the same amplitude with sufficient intervals between them (approx. 10 s), the current oscillograms reflecting the reversible resistance decrease are well reproduced on the same membrane. The time of attainment of the predetermined level of the membrane conductance is strongly dependent on voltage. At different stages of breakdown we have investigated changes in the conductance of UO22+-modified membrane after the application of two-step voltage pulses, the kinetics of development of the reversible decrease in the membrane resistance in solutions of univalent and divalent ions, and also the influence of sucrose and hemoglobin on the current evolution. The relationship between the reversible conductance increase, the reversible electrical breakdown [15] and the rupture of membrane in an electric field is discussed. We propose the general interpretation of these phenomena, based on the representation of the potential-dependent appearance in the membrane of pores, the development of which is promoted by an electric field.

Electroporation: The population distribution of macromolecular uptake and shape changes in red blood cells following a single 50 μs square wave pulse

Article

Dec 1988
Bioelectrochem Bioenerg

We describe a method of using flow cytometry for determining the distribution of electroporation effects within a statistically significant cell population. Here we illustrate basic aspects of the method by investigating the electroporation of red blood cells, which have been widely used by others in previous investigations of electroporation, including studies of reversible electrical breakdown, and molecular uptake or release associated with a transient high permeability state. We make two measurements on each cell in a population of 10,000 or more cells: (1) light scatter to indicate changes in cell morphology, and (2) fluorescence to determine the uptake of a fluorescence-labeled macromolecule (FITC-dextran; 70,000 dalton). Computer processing of the single cell data allows construction of statistical distributions which reveal how electroporation occurs within a large cell population. Using this method we find that after a single 50 μs square pulse of optimal magnitude about 30% have formed spherical ghosts because of electroporation, and that two distinct ghost subpopulations occur. One subpopulation (about 10% of analyzed cells) has negligible uptake, while the second subpopulation (about 20%) consists of ghosts which have taken up significant amounts of the test macromolecule. Two interesting findings are the high frequency-of-occurrence of electroporation due to a single, optimal pulse, and the implication, because of the two distinct ghost subpopulations, that there is a significant variation in pore sizes.

Antibodies introduced into living cells by red cell ghosts are functionally stable in the cytoplasm of the cells

Article

Jan 1980
CELL

The function and fate of antibodies introduced into living cells by red cell ghosts were studied using CRM 176 (a mutant diphtheria toxin having lower toxicity than the wild-type) and antibody against fragment A of diphtheria toxin. IgG labeled with iodine and FITC was found in the cytoplasm of the recipient cells. When about 1500 molecules of anti-fragment A antibody (rabbit IgG) were introduced into diphtheria toxin-sensitive Vero cells or FL cells, these cells became resistant to the toxin and formed normal colonies. It was calculated from the survival of cells without anti-fragment A IgG under these conditions that about 300 molecules of fragment A-176 were transferred to the cells. These results showed that the antigen-antibody reaction took place in living cells as effectively as in a cell-free system. The functional stability of antibody IgG in cells was examined by exposing Vero cells containing a subminimal amount of anti-fragment A IgG (about 1000 molecules) to the toxin for 2 hr at various times after the introduction of anti-fragment A IgG. More than 50% of the initial activity of the antibody to neutralize toxin still remained even after incubation of the cells at 37°C for 20 hr. The same degree of stability was also demonstrated using iodine-labeled specific anti-fragment A IgG. The IgG recovered from the recipient cells after various times of incubation at 37°C retained its full ability to bind to fragment A-conjugated Sepharose 4B, although the total amount of IgG associated with the cells decreased about 50% in 24 hr.

The role of spectrin in erythrocyte membrane-stimulated actin polymerisation

Article

Jun 1979

PROTEINS on the cytoplasmic surface of the erythrocyte membrane, including spectrin and actin, are postulated to comprise the red cell cytoskeleton1-3, but little is known about the role of actin or its association with the membrane. We have reported that monomeric (G) actin added to erythrocyte ghosts selectively associates with a component at the cytoplasmic surface of the membrane4,5. We now show that this component is unlikely to be spectrin and that actin binding occurs by stimulated actin polymerisation.

Voltage-induced conductance in human erythrocyte membranes

Article

Aug 1979
Biochim Biophys Acta

Isotonic suspensions of erythrocytes were exposed to intense electric fields for a duration in microseconds. Time-dependent increase in the conductivity of the suspension was observed under fields greater than a threshold of about 1.5 kV/cm. The threshold was independent of the ionic strength of the medium, and changed little with temperature or with the rise time of the applied field. Under fields greater than 3 kV/cm, the time course of the conductivity increase consisted of a rapid (approx. 1 μs) and a slow (approx. 100 μs) phases. The increase is attributed primarily to large membrane conductance induced by the applied field. The membrane conductance is in the order of in the rapid phase and in the slow phase. Comparison with previous results indicates that this induced membrane conductance corresponds to the formation of aqueous pores in the cell membrane. After the applied field was removed, the conductivity of the suspension returned nearly to its initial value, indicating that the induced membrane conductance is strongly dependent on the membrane potential. The conductivity then increased again in the time range of 10 s. This is attributed to the diffusional efflux of intracellular ions through the voltage-induced pores. From the rate of the efflux, number of the pores/cell is estimated to be in the order of 102. Final stage of the conductivity change was a slow decrease, corresponding to the colloid osmotic swelling of the perforated cells.

The direct measurement of temperature changes within freeze-fracture specimens during quenching in liquid coolants

Article

Feb 1978

We have evaluated the cooling rates of specimens mounted in a variety of freeze-fracture holders when plunged into a series of liquid coolants. These rates were measured using miniature thermocouples placed within the mounted specimens. The most rapid cooling rates were obtained using propane at 83 K as the coolant. When mounted on a newly devised ‘copper sandwich’ holder, specimen cooling rates in excess of 4500 K/s have been recorded. A simple guillotine-like device for quenching freeze-fracture specimens under reproducible conditions is presented.

Formation and resealing of pores of controlled sizes in human erythrocyte membrane

Article

Sep 1977

APPLICATION of an electric pulse, at field intensities of a few kV cm-1 and of duration in the µs range, to an isotonic suspension of erythrocytes is known to cause haemolysis of the red cells1-4. Studies from different laboratories suggest that the haemolysis is due to the field-induced transmembrane potential1,3,4. Our recent experiments5 indicate that once the transmembrane potential reaches a threshold of approximately 1 V, which corresponds to an applied field of 2.2 kV cm-1, the erythrocyte membrane becomes leaky to normally impermeant ions or molecules. The permeation of solutes leads to the swelling and eventual lysis of the red cells. This type of haemolysis is known as colloid osmotic haemolysis6,7. The voltage-induced permeability change is consistent with the formation of pores in the membrane. We show here that the size of these pores can be varied in a controlled manner, and that the leaky membrane can be resealed while the haemolysis is prevented. Foreign molecules have successfully been incorporated into the resealed, but otherwise intact, erythrocytes.

The Ionic Channels in Excitable Membranes

Article

Jan 1975
Ciba Found Symp

R D Keynes

The ionic channels in excitable membranes are of two classes: those that open and close when the membrane potential alters and those that respond to the release of an appropriate chemical transmitter. The former are responsible for the conduction of impulses in nerve and muscle fibres and the latter for synaptic transmission. It is now clear that the sodium and potassium channels in electrically excitable membranes are functionally distinct, since each can be blocked without affecting the behaviour of the other. It has recently proved possible to study, in the voltage-clamped squid giant axon, the movements of the mobile charges or dipoles that form the voltage-sensitive portion of the sodium channels, which give rise to the so-called 'gating' current. Detailed comparisons can now be made between the kinetics of the ionic conductances as described by Hodgkin & Huxley, and the steady-state distribution and kinetics of the charged controlling particles, which should lead to useful conclusions about the intramolecular organization of the sodium channels and the conformational changes that take place under the influence of the electric field. There is as yet little information about the chemical nature of the electrically excitable channels, but significant progress has been made towards the isolation and characterization of the acetylcholine receptors in muscle and electric organ.

Reversible plasma membrane ultrastructural changes correlated with electropermeabilization in CHO cells

Article

May 1988
Biochim Biophys Acta

Chinese hamster ovary cells (CHO) grown in monolayers were permeabilized to molecules with molecular weight up to 1000 by high intensity 100 mus square wave electric field pulses. This permeability was transient and the cell viability was not affected. It was not possible for molecules with molecular weight larger than 1500 to penetrate inside the cytoplasm if lytic pulsing conditions were not used. In order to investigate the ultrastructural changes associated with this transient and limited permeabilization, cells were chemically fixed a few seconds after their pulsation and observed by electron microscopy. By scanning electron microscopy, numerous microvilli and blebs were observed almost immediately after application of the field. No other membrane changes were observed. Permeabilization of the membrane was visualized at the electron microscopic level by penetration of Ruthenium red. The appearance of osmotic pressure-dependent 'blebs' was indicative of local weakening of the plasma membrane. Most of these effects were fully reversible and disappeared within 30 min at 37 degrees C with the formation of huge polykaryons when cells were in contact before pulsing.

Reversible electrical breakdown of lipid layers: Formation and evolution of pores

Article

Jun 1988
Biochim Biophys Acta

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.

Cell poration and cell fusion using an oscillating electric held

Article

Nov 1989

Donald C Chang

It has been shown in previous studies that cell poration (i.e., reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field. Recently we suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. This study is an experimental test of such an idea. Here, pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce cell poration and cell fusion. The fusion experiments were done on human red blood cells. The poration experiments were done on a fibroblast cell line using a molecular probe (which is a DNA plasmid containing the marker gene chloramphenicol acetyltransferase, CAT) and assayed by a gene transfection technique. It was found that the pulsed RF field is highly efficient in both cell fusion and cell poration. Also, in comparison with electro-poration using a DC field, the RF field results in a higher percentage of cells surviving the exposure to the electric field.

Electroporation in biology: Methods, applications, and instrumentation

Article

Dec 1988

Huntington Potter

Il apparait que l'electroporation (qui permet de realiser des trous dans des membranes cellulaires grâce a un choc electrique de haut voltage) est une methode simple et efficace pour introduire des gene dans des cellules animales, vegetales, des organismes unicellulaires et des microorganismes. Cette methode permet egalement un traitement des maladies genetiques chez l'homme

The Membrane Skeleton of Human Erythrocytes and its Implications for More Complex Cells

Article

Feb 1985

Vann Bennett

[Submicrosecond imaging of membrane potential under a pulsed-laser fluorescence microscope--electroporation of cell membrane visualized]

Article

Jan 1989
Seikagaku

Electroporation of cell membrane visualized under pulsed-laser fluorescence microscope

Article

Jul 1988

Controlled permeability can be conferred to cell membranes by exposing cells to a microsecond electric pulse of sufficient intensity (electroporation). By constructing a fluorescence microimaging system with a submicrosecond time resolution we have been able to resolve temporally and spatially the events in a single cell under a microsecond electric pulse. An enormous membrane conductance, corresponding to a loss of 0.01-0.1% of the membrane area, was observed in those membrane regions where the transmembrane potential induced by the electric pulse exceeded a critical value. The conductance decreased to a low level in a submillisecond after the pulse, leaving a moderately electroporated cell.

Electrical Breakdown, Electropermeabilization and Electrofusion

Article

Feb 1986
REV PHYSIOL BIOCH P

Ulrich Zimmermann

The considerable amount of activity in the field of electrofusion and electropermeabilization is very promising from the point of view of new insights into biomembranes and new technologies in the future for the production of new compounds and modification of cell systems for nutrition, energy production and the removal of waste products. It is particularly gratifying to see how basic science has provided the foundation for a useful technology, although in some cases the time needed to develop an application is very long. In other cases, it is necessary to overcome the difficulties posed by existing schools of thought which have been shown to be wrong. It is fascinating to observe the many developments and discoveries in the areas of physics, material science, space technology and electronics which are just waiting to be applied to biological systems. An increased interdisciplinary collaboration between physicists and biologists could provide considerable impetus to biology and its application in technology. However, this can only be achieved if basic research into biological membranes is accelerated. The techniques for electrical breakdown, electropermeabilization and electrofusion could be an important tool in this process, since we cannot rule out the possibility that the high electrical fields occurring naturally in the membrane play an important role in the selective transport of substances across the membrane as well as in natural regulatory processes.

Kinetics of ultrastructural changes during electrically induced fusion of human erythrocytes

Article

Feb 1986

The sequence of events during the electrically induced fusion of human erythrocytes was studied by rapid quench freeze-fracture electron microscopy. A single electric field pulse was used to induce fusion of human erythrocytes treated with pronase and closely positioned by dielectrophoresis. The electronic circuit was coupled to a rapid freezing mechanism so that ultrastructural changes of the membrane could be preserved at given time points. Pronase treatment enabled adjacent cells to approach each other within 15 nm during dielectrophoresis. The pulse caused a brief disruption of the aqueous boundaries which separated the cells. Within 100 msec following pulse application, the fracture faces exhibited discontinuous areas which were predominantly free of intramembranous particles. At 2 sec after the pulse, transient point defects attributed to intercellular contact appeared in the same membrane areas and replaced the discontinuous areas as the predominant membrane perturbation. At 10 sec after the pulse, the majority of the discontinuous areas and point defects disappeared as the intercellular distance returned to approximately 15 to 25 nm, except at sites of cytoplasmic bridge formation. Intramembranous particle clearing was observed at 60 sec following pulse application in discrete zones of membrane fusion.

Introduction of new genetic material into pluripotent stem cells of the mouse

Article

Aug 1984

An infectious retrovirus vector has been used to transfer a bacterial gene encoding resistance to the neomycin analogue G418 into pluripotent haematopoietic stem cells present in explanted murine bone marrow tissue. Subsequent transplantation of the cells into lethally irradiated mice results in engraftment of the animals with donor haematopoietic tissue containing the bacterial gene. This approach affords an efficient and rapid means of re-introducing genetically modified tissue into intact organisms and provides a system whereby the expression and regulation of cloned genes can be followed within the context of a well characterized developmental programme.

[30] Microinjection of tissue culture cells

Article

Feb 1983
METHOD ENZYMOL

The microinjection technique is based on the use of small glass capillaries; this allows the transfer of almost any type of molecules into either the cytoplasma or nuclei of the recipient cells. This technique has no cell type restriction, and also suspension culture cells (e.g., lymphocytes) are accessible for microinjection after they are bound to a substrate by suitable linkers. Among these techniques, microinjection is by far the most efficient. Up to 100% of the recipient cells allow expression of the transferred material, and stable transformed cell lines can be isolated with a frequency of 20-30% after intranuclear injection of DNA. Biochemical studies can be performed on 50 injected cells, and the transferred material can be reisolated and further analyzed from only 100-200 recipient cells.

Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis

Article

Apr 1983

D H Russell

This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [alpha-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, alpha-difluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [32P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 microM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32P]guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis.

Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields

Article

Feb 1982

By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to a field of 2 kV/cm, τ=200 μsec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2 nm. At 37°C these ‘equivalent pores’ remain patent for up to 1 hr. The formation and stability of these ‘pores’ is not affected by the Ca content of the bathing solution. The ‘pores’ permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water. Cells rendered ‘leaky’ in K glutamate medium containing 5mm Mg-ATP and EGTA to give an ionized Ca close to 10−8m release less than 1% of their total catecholamine. These same cells can release up to 30% of their catecholamine when exposed to 10−5m Ca. This Ca-dependent release is unaffected by Ca-channel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends on the final Ca concentration achieved. Half-maximal release occurs at about 1 μm Ca, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions. Calcium-evoked release of catecholamine is associated with the release of dopamine-β-hydroxylase (DβH) but not lactate dehydrogenase. The ratio DβH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of DβH and catecholamine is identical. Transmission electron microscopy of ‘leaky’ cells exposed to 10−8m Ca reveals no marked differences from unstimulated intact cells. The cytoplasm of ‘leaky’ cells exposed to 10−5m Ca contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles. Ca-dependent release of both catecholamine and DβH requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca. Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and DβH. Chloride causes a small increase in Ca-independent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate−>acetate−>Cl−>Br−>SCN−. Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the range pH 6.6 to 7.8. Raising the free Mg concentration reduces the extent of Ca-dependent exocytosis and also its apparent affinity for calcium. Calcium-dependent exocytosis in ‘leaky’ cells is largely unaffected by (i) a variety of agonists and antagonists of the nicotinic receptor; (ii) agents that disrupt microtubules and microfilaments; (iii) phalloidin; (iv) vanadate; (v) inhibitors of anion permeability; (vi) protease inhibitors; and (vii) agents that dissipate the vesicle pH gradient and potential. It is partially inhibited by (i) certain antipsychotic drugs; (ii) a rise in osmotic pressure, (iii) lowering the temperature below 20°C, and (iv) N-ethyl maleimide.

Electric field-induced cell-to-cell fusion

Article

Feb 1982

The sealing process of lipid bilayer after reversible electrical breakdown

Article

Feb 1981
Biochim Biophys Acta

The resealing process of lipid bilayer membranes after reversible electrical breakdown was investigated using two voltage pulses switched on together. Electrical breakdown of the membranes was induced with a voltage pulse of high intensity and short duration. The time course of the change in membrane conductance after the application of the high (short) voltage pulse was measured with a longer voltage pulse of low amplitude. The decrease in membrane conductance during the resealing process could be fitted to a single exponential curve with a time constant of 10--2 micros in the temperature range between 2 and 20 degrees C. The activation energy for this exponential decay process was found to be about 50 kJ/mol, which might indicate a diffusion process. Above 25 degrees C the resealing process is controlled by two exponential processes. The data obtained for the time course of the resealing process can be explained in terms of pore formation in the membrane in response to the high electrical field strength. A radius of about 4 nm is calculated for the initial pore size. From the assumed exponential change of the pore area with progressive resealing time a diffusion constant of 10(-8) cm2/s for lateral lipid diffusion can be estimated.

Erythrocyte entrapment of daunomycin by amphotericin B without hemolysis

Article

May 1980

Amphotericin B is a polyene that binds to sterols and perforates cell membranes. An antileukemic drug such as daunomycin added exogenously is impermeable to the red cell membrane. However, when the cells are incubated with a low concentration of amphotericin B, daunomycin is entrapped in the red cells without hemolysis or alteration in the chemical parameter of the erythrocytes. The erythrocyte has been used as a carrier vehicle to enhance the cytotoxic activity of daunomycin against L1210 leukemic cells. In comparison to control preparations, the greatest increase in survival was obtained in vivo when the erythrocytes with entrapped daunomycin were given to C57BL X DBA/2 F1 mice bearing L1210 cells.

bilayers: formation and evolution of pores

275-287

R W Glaser
S L Leikin
L V Chernomordik
V L Pastushenko

Glaser, R. W., S. L. Leikin, L. V. Chernomordik, V. L. Pastushenko, and A. bilayers: formation and evolution of pores. Biochim. Biophys. Acta. 940:275-287

Third Colloquium in Biological Sciences: Cellular Signal Transduction Red cell shape

Jan 1987
205-246

F L Strand
T L Steck

Strand, F. L. 1987. Third Colloquium in Biological Sciences: Cellular Signal Transduction. Ann. N.Y. Acad. Sci. 494 pp. Steck, T. L. 1989. Red cell shape. In Cell Shape: Determinants, Regulation and Regulatory Role. W. D. Stein and F. Bonner, editors. Academic Press, New York. 205-246.

Beginning of exocytosis captured by rapid freezing of Limulus amebocytes Translational mobility of membrane intercal-ated particles of human erythrocyte ghosts-pH-dependent, revers-ible aggregation

Jan 1972
40-54777

R L Ornberg
T S Reese
Pinto
P Silva

Ornberg, R. L., and T. S. Reese. 1981. Beginning of exocytosis captured by rapid freezing of Limulus amebocytes. J. Cell Biol. 90:40-54. Pinto da Silva, P. 1972. Translational mobility of membrane intercal-ated particles of human erythrocyte ghosts-pH-dependent, revers-ible aggregation. J. Cell. Biol. 53:777-787.

Basic Methods in Molecular Biology Reversible plasma membrane ultrastructural changes correlated with electropermeabilization in Chinese hamster ovary cells

Jan 1986
247-259

L G Davis
M D Dibner
J F Battey

Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic Methods in Molecular Biology. Elsevier, New York. 388 pp. Escande-Geraud, M. L., M. P. Rols, M. A. Dupont, N. Gas, and J. Teissie. 1988. Reversible plasma membrane ultrastructural changes correlated with electropermeabilization in Chinese hamster ovary cells. Biochim. Biophys. Acta. 939:247-259.

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy

Abstract

No full-text available

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