Ultrashort electrical pulses open a new gateway into biological cells

An electrical model for biological cells predicts that for pulses with durations shorter than the charging time of the outer membrane, there is an increasing probability of electric field interactions with intracellular structures. Experimental studies in which human cells were exposed to pulsed electric fields of up to 300-kV/cm amplitude, with durations as short as 10 ns, have confirmed this hypothesis. The observed effects include the breaching of intracellular granule membranes without permanent damage to the cell membrane, abrupt rises in intracellular free calcium levels, and enhanced expression of genes. At increased electric fields, the application of submicrosecond pulses induces apoptosis (programmed cell death) in biological cells, an effect that has been shown to reduce the growth of tumors. Possible applications of the intracellular electroeffect are enhancing gene delivery to the nucleus, controlling cell functions that depend on calcium release (causing cell immobilization), and treating tumors.     

Theoretical and experimental analysis of electroporated membrane conductance in cell suspension

An intense electric field can be applied to increase the membrane conductance G(m) and consequently, the conductivity of cell suspension. This phenomenon is called electroporation. This mechanism is used in a wide range of medical applications, genetic engineering, and therapies. Conductivity measurements of cell suspensions were carried out during application of electric fields from 40 to 165 kV/m. Experimental results were analyzed with two electroporation models: the asymptotic electroporation model was used to estimate G(m) at the beginning and at the end of electric field pulse, and the extended Kinosita electroporation model to increase G(m) linearly in time. The maximum G(m) was 1-7 × 10(4) S/m(2), and the critical angle (when the G(m) is insignificant) was 50°-65°. In addition, the sensitivity of electroporated membrane conductance to extracellular and cytoplasmatic conductivity and cell radius has been studied. This study showed that external conductivity and cell radius are important parameters affecting the pore-opening phenomenon. However, if the cell radius is larger than 7 μm in low conductivity medium, the cell dimensions are not so important.     

Experimental characterization and numerical modeling of tissue electrical conductivity during pulsed electric fields for irreversible electroporation treatment planning

Irreversible electroporation is a new technique to kill cells in targeted tissue, such as tumors, through a nonthermal mechanism using electric pulses to irrecoverably disrupt the cell membrane. Treatment effects relate to the tissue electric field distribution, which can be predicted with numerical modeling for therapy planning. Pulse effects will change the cell and tissue properties through thermal and electroporation (EP)-based processes. This investigation characterizes these changes by measuring the electrical conductivity and temperature of ex vivo renal porcine tissue within a single pulse and for a 200 pulse protocol. These changes are incorporated into an equivalent circuit model for cells and tissue with a variable EP-based resistance, providing a potential method to estimate conductivity as a function of electric field and pulse length for other tissues. Finally, a numerical model using a human kidney volumetric mesh evaluated how treatment predictions vary when EP- and temperature-based electrical conductivity changes are incorporated. We conclude that significant changes in predicted outcomes will occur when the experimental results are applied to the numerical model, where the direction and degree of change varies with the electric field considered.     

Case for applying subnanosecond high-intensity, electrical pulses to biological cells

In this paper, model analysis into the time-dependent transmembrane potential at the outer cell membrane is presented, for applied high-intensity electric pulses having durations in the nanosecond range or smaller. It is argued that the frequency-dependent dielectric response of cell membranes could be used to advantage for stronger bioeffects by employing shorter pulses. Our model calculations predict faster transmembrane voltages and larger electroporation densities for a given external energy with pulse durations in the subnanosecond regime. This temporal regime would be used, for example, in the electrotherapy of mixed cell ensembles having different dielectric response properties.     

Second-order model of membrane electric field induced by alternating external electric fields

With biological cells exposed to ac electric fields below 100 kHz, external field is amplified in the cell membrane by a factor of several thousands (low-frequency plateau), while above 100 kHz, this amplification gradually decreases with frequency. Below 10 MHz, this situation is well described by the established first-order theory which treats the cytoplasm and the external medium as pure conductors. At higher frequencies, capacitive properties of the cytoplasm and the external medium become increasingly important and thus must be accounted for. This leads to a broader, second-order model, which is treated in detail in this paper. Unlike the first-order model, this model shows that above 10 MHz, the membrane field amplification stops decreasing and levels off again in the range of tens (high-frequency plateau). Existence of the high-frequency plateau could have an important impact on present theories of high-frequency electric fields effects on cells and their membranes.     

Potential distribution for a spheroidal cell having a conductivemembrane in an electric field

When a cell is situated in a uniform electric field, the field is modified due to the relatively low conductance of the cell membrane compared to that of the surrounding fluids. In certain cases, such as in the estimation of internal and external electrokinetic forces, one requires a means of estimating the magnitude of the electric field inside and outside the cell. Most treatments consider the case when the membrane has zero conductivity, or the case of only a spherical cell. The authors solve Laplace's equation for the electric potential distribution inside and outside a cell having a prolate spheroidal shape and having a membrane with a finite, nonzero conductivity     

The course of tissue permeabilization studied on a mathematical model of a subcutaneous tumor in small animals

One of the ways to potentiate antitumor effectiveness of chemotherapeutic drugs is by local application of short intense electric pulses. This causes an increase of the cell membrane permeability and is called electropermeabilization. In order to study the course of tissue permeabilization of a subcutaneous tumor in small animals, a mathematical model was built with the commercial program EMAS, which uses the finite element method. The model is based on the tissue specific conductivity values found in literature, experimentally determined electric field threshold values of reversible and irreversible tissue permeabilization, and conductivity changes in the tissues. The results obtained with the model were then compared to experimental results from the treatment of subcutaneous tumors in mice and a good agreement was obtained. Our results and the reversible and irreversible thresholds used coincide well with the effectiveness of the electrochemotherapy in real tumors where experiments show antitumor effectiveness for amplitudes higher than 900 V/cm ratio and pronounced antitumor effects at 1300 V/cm ratio.     

Aqueous Two‐Phase System Patterning of Microbubbles: Localized Induction of Apoptosis in Sonoporated Cells

Ultrasound‐driven microbubbles produce mechanical forces that can disrupt cell membranes (sonoporation). However, it is difficult to control microbubble location with respect to cells. This lack of control leads to low sonoporation efficiencies and variable outcomes. In this study, aqueous two‐phase system (ATPS) droplets are used to localize microbubbles in select micro‐regions at the surface of living cells. This is achieved by stably partitioning microbubbles in dextran (DEX) droplets, deposited on living adherent cells in medium containing polyethylene glycol (PEG). The interfacial energy at the PEG‐DEX interface overcomes microbubble buoyancy and prevents microbubbles from floating away from the cells. Spreading of the small DEX droplets retains microbubbles at the cell surface in defined lateral positions without the need for antibody or cell‐binding ligand conjugation. The patterned microbubbles are activated on a cell monolayer exposed to a broadly applied ultrasound field (center frequency 1.25 MHz, active element diameter 0.6 cm, pulse duration 8 μs or 30 s). This system enables efficient testing of different ultrasound conditions for their effects on sonoporation‐mediated membrane disruption and cell viability. Regions of cells without patterned microbubbles show no injury or membrane disruption. In microbubble patterned regions, 8 μs ultrasound pulses (0.2‐0.6 MPa) produce cell death that is primarily apoptotic. Ultrasound‐induced apoptosis increases with higher extracellular calcium concentrations, with cells displaying all of the hallmarks of apoptosis including annexinV labeling, loss of mitochondrial membrane potential, caspase activation and changes in nuclear morphology.     

A numerical model of permeabilized skin with local transport regions

The protective function of skin and hence its low permeability presents a formidable obstacle in therapeutical applications such as transdermal drug delivery and gene delivery in skin. One of the methods to temporarily increase skin permeability is electroporation, creating aqueous pathways across lipid-based structures by means of electric pulses. Also, the application of electric pulses to biological cells causes increased permeability of cell membrane, thus enabling the uptake of larger molecules that otherwise cannot cross the membrane, such as drug molecules or DNA, into the cell. The creation of localized sites of increased molecular transport termed local transport regions (LTRs) can be observed during electroporation, as well as changes in the bulk electric properties of skin layers. We modeled these phenomena with a numerical model and compared the output of the model with our own in vivo experiments and previously published results of skin electroporation and a good agreement was obtained. With the model presented, we used the available data to describe the nonlinear process of skin electropermeabilization from the aspect of tissue conductivity changes and the presence of local transport regions in the permeabilized stratum corneum. The observations derived from various in vivo experiments by different authors were thus confirmed theoretically.     

Magnetic resonance electrical impedance tomography for monitoring electric field distribution during tissue electroporation

Electroporation is a phenomenon caused by externally applied electric field of an adequate strength and duration to cells that results in the increase of cell membrane permeability to various molecules, which otherwise are deprived of transport mechanism. As accurate coverage of the tissue with a sufficiently large electric field presents one of the most important conditions for successful electroporation, applications based on electroporation would greatly benefit with a method of monitoring the electric field, especially if it could be done during the treatment. As the membrane electroporation is a consequence of an induced transmembrane potential which is directly proportional to the local electric field, we propose current density imaging (CDI) and magnetic resonance electrical impedance tomography (MREIT) techniques to measure the electric field distribution during electroporation. The experimental part of the study employs CDI with short high-voltage pulses, while the theoretical part of the study is based on numerical simulations of MREIT. A good agreement between experimental and numerical results was obtained, suggesting that CDI and MREIT can be used to determine the electric field during electric pulse delivery and that both of the methods can be of significant help in planning and monitoring of future electroporation based clinical applications.     

Dependence of induced transmembrane potential on cell density,arrangement, and cell position inside a cell system

A nonuniform transmembrane potential (TMP) is induced on a cell membrane exposed to external electric field. If the induced TMP is above the threshold value, cell membrane becomes permeabilized in a reversible process called electropermeabilization. Studying electric potential distribution on the cell membrane gives us an insight into the effects of the electric field on cells and tissues. Since cells are always surrounded by other cells, we studied how their interactions influence the induced TMP. In the first part of our study, we studied dependence of potential distribution on cell arrangement and density in infinite cell suspensions where cells were organized into simple-cubic, body-centered cubic, and face-centered cubic lattice. In the second part of the study, we examined how induced TMP on a cell membrane is dependent on its position inside a three-dimensional cell cluster. Finally, the results for cells inside the cluster were compared to those in infinite lattice. We used numerical analysis for the study, specifically the finite-element method (FEM). The results for infinite cell suspensions show that the induced TMP depends on both: cell volume fraction and cell arrangement. We established from the results for finite volume cell clusters and layers, that there is no radial dependence of induced TMP for cells inside the cluster.     

扫描离子电导显微镜及其在医学研究中的初步应用

扫描离子电导显微镜(scanning ion conductance microscopy,SICM)是一种非接触式的扫描探针显微技术(scanning probe microscopy,SPM),可以实现生物样品在近生理条件下的成像.随着技术发展,目前广泛应用于生物医学领域的SICM主要包括两种:跳跃式离子电导显微技术(hopping probe ion conductance microscopy,HPICM)和外加压力模式的SICM.前者可以应用于软的、黏的、对外力或其它机械信号敏感的样品的高分辨成像;后者可以通过探针微管对样品局部施加外力刺激或化学、电学、光学或生物分子等信号,实现对样品动力学性质或相关生理过程局部的原位研究.此外,SICM技术具有良好的开放性,能够越来越多地与其它技术手段联用,极大地丰富了其在生物医学领域的应用,可用于疾病发病机理、药物作用以及临床诊断等的研究.但是,目前SICM时间分辨率较低,这制约了它在生物体系动力学行为方面的研究.     

Murine cardiac catheterizations and hemodynamics: on the issue of parallel conductance

Catheter-based measurements are extensively used nowadays in animal models to quantify global left ventricular (LV) cardiac function and hemodynamics. Conductance catheter measurements yield estimates of LV volumes. Such estimates, however, are confounded by the catheter's nonhomogeneous emission field and the contribution to the total conductance of surrounding tissue or blood conductance values (other than LV blood), a term often known as parallel conductance. In practice, in most studies, volume estimates are based on the assumptions that the catheter's electric field is homogeneous and that parallel conductance is constant, despite prior results showing that these assumptions are incorrect. This study challenges the assumption for spatial homogeneity of electric field excitation of miniature catheters and investigated the electric field distribution of miniature catheters in the murine heart, based on cardiac model-driven (geometric, lump component) simulations and noninvasive imaging, at both systolic and diastolic cardiac phases. Results confirm the nonuniform catheter emission field, confined spatially within the LV cavity and myocardium, falling to 10% of its peak value at the ring electrode surface, within 1.1-2.0?mm, given a relative tissue permittivity of 33,615. Additionally, <1% of power leaks were observed into surrounding cavities or organs at end-diastole. Temporally varying parallel conductance effects are also confirmed, becoming more prominent at end-systole.     

Electric-field-induced volume and membrane ionic permeabilitychanges of red blood cells

When an external electric field (EF) is applied to red blood cells (RBCs), the RBCs are observed to undergo a swelling action. The swelling may or may not lead to hemolysis, depending on the EF strength. An objective verification of this swelling is by measuring the RBC mean corpuscular volume (MCV). In this study, the RBC's were exposed to the appropriate EF strength to induce swelling, but caused minimal hemolysis. The MCV was measured. The change in the erythrocyte membrane ionic permeability as a result of the EF exposure was also determined, as an objective verification of presumed membrane conductance change concomitant with the swelling. The fluxes of cations K⁺, Na ⁺, and Ca⁺⁺ and anion Cl^- were measured. The results showed that red cell MCV was indeed increased after EF application. The EF also altered the membrane ionic conductance to allow ions to flow down their respective concentration gradient across the membrane. Without a counterbalancing ionic pressure gradient, hemoglobin colloidal pressure inevitably drew H₂O in, thus producing the observed swelling     

Electrical impedance tomography for imaging tissue electroporation

Electroporation is a method to introduce molecules, such as gene constructs or small drugs, into cells by temporarily permeating the cell membrane with electric pulses. In molecular medicine and biotechnology, tissue electroporation is performed with electrodes placed in the target area of the body. Currently, tissue electroporation, as with all other methods of molecular medicine, is performed without real-time control or near-term information regarding the extent and degree of electroporation. This paper expands the work from our previous study by implementing new ex vivo experimental data with "front-tracking" analysis for the image reconstruction algorithm. The experimental data is incorporated into numerical simulations of electroporation procedures and images are generated using the new reconstruction algorithm to demonstrate that electrical impedance tomography (EIT) can produce an image of the electroporated area. Combining EIT with electroporation could become an important biotechnological and medical technique to introduce therapeutic molecules into cells in tissue at predetermined areas of the body.     

Microdosimetry for nanosecond pulsed electric field applications: a parametric study for a single cell

A microdosimetric study of nanosecond pulsed electric fields, including dielectric dispersivity of cell compartments, is proposed in our paper. A quasi-static solution based on the Laplace equation was adapted to wideband signals and used to address the problem of electric field estimation at cellular level. The electric solution was coupled with an asymptotic electroporation model able to predict membrane pore density. An initial result of our paper is the relevance of the dielectric dispersivity, providing evidence that both the transmembrane potential and the pore density are strongly influenced by the choice of modeling used. We note the crucial role played by the dielectric properties of the membrane that can greatly impact on the poration of the cell. This can partly explain the selective action reported on cancerous cells in mixed populations, if one considers that tumor cells may present different dielectric responses. Moreover, these kinds of studies can be useful to determine the appropriate setting of nsPEF generators as well as for the design and optimization of new-generation devices.     

Accuracy of the conductance catheter for measurement of ventricularvolumes seen clinically: effects of electric field homogeneity andparallel conductance

The conductance-volume method is an important clinical tool which allows the assessment of left ventricular function in vivo. However, the accuracy of this method is limited by the homogeneity of electric field the conductance catheter produces and the parallel conductance of surrounding structures. This paper examines these sources of error in volumes seen clinically, The characteristics of electric field within a chamber were examined using computer simulation. Nonconductive and conductive models were constructed and experimental measurements obtained using both single-field (SF) and dual-field (DF) excitation. Results from computer simulations and in vitro measurements were compared to validate the proposed theoretical model of conductance-volume method. The effects of field homogeneity and significance of parallel conductance in volume measurement were then determined. The results of this study show that DF provide a more accurate measure of intraventricular volume than SF, especially at larger volumes. However, both significantly underestimate true volume at larger volumes. In addition, the parallel conductance due to the chamber wall is significant at small volumes, but diminishes at larger volumes. Furthermore, the effect of parallel conductance beyond the chamber wall may be negligible. This study demonstrates the limitations in applying current conductance technology to patients with dilated hearts     

A model study of electric field interactions between cardiacmyocytes

The transmission of excitation via electric field coupling was studied in a model comprising two myocytes abutted end-to-end and placed in an unbounded volume conductor. Each myocyte was modeled as a small cylinder of membrane (10 microns in diameter and 100 microns in length) capped at both ends. A Beeler-Reuter model modified for the Na+ current dynamics served to simulate the membrane ionic current. There was no resistive coupling between the myocytes and the intercellular junction consisted of closely apposed pre- and post-junctional membranes, separated by a uniform cleft distance. The membrane current crossing the prejunctional membrane during the action potential upstroke tends to flow out of the cleft, but it is partly prevented from doing so by the shunt resistance constituted by the cleft volume conductor. The prejunctional upstroke gives rise to a pulse of positive potential within the cleft which induces a small capacitive current across the post-junctional membrane to yield a small positive change in the intracellular potential in the post-junctional cell. The net result is an hyperpolarization of the post-junctional cleft membrane and a slight depolarization of the rest of the cell membrane since the extracellular potential outside of the cell is zero. The magnitude of this depolarization is quite small for a flat junctional membrane and it can be increased by membrane folding and interdigitation, so as to increase the junctional membrane area by a factor of 10 or more. Even then the post-junctional depolarization does not reach threshold when the extracellular potential around the post-junctional cell is effectively zero. Threshold depolarization occurs in the presence of a large decrease of post-junctional load, by increasing the junctional membrane capacitance and/or decreasing the volume of the post-junctional cell. Assuming that the normal resistive coupling between two cardiac myocytes is 1-4 M omega, our model study indicates that electric field coupling would then be about two orders of magnitude smaller. However, substantial enhancement of the efficacy of electric field transmission was observed in the case of cells with substantial junctional membrane folding.     

缺陷富勒烯C_20分子器件的电子输运研究

运用基于第一性原理密度泛函理论(DFT)的非平衡格林函数(NEGF)方法,对正十二面体富勒烯C20分子及其有缺陷的C20分子进行了电子输运性质的研究。通过计算得出了模拟的电子透射谱线。通过比较不同缺陷的分子的传导特性,获得了C20的电子输运特点。通过比较发现,几乎所有情况下缺陷器件的传输概率在电子能量大于0.52eV时都大约是10-10,几乎没有电子透过,所以这种器件可以作为一种响应很好的电子开关;而器件中原子的缺失并没有造成电子传输路径的中断,在大多数情况下,在原子缺失处反而产生更多的传输路径,通路的增多使得器件传输能力得到提高。     

Characterization of Ca(2+)-inhibited potassium channels in the LNCaP human prostate cancer cell line.

Potassium plasma membrane channels have been studied in the LNCaP androgen-sensitive human prostate cancer cell line, derived from a lymph node of a subject with metastatic carcinoma of the prostate. Membrane currents were recorded by the patchclamp technique, using the cell-attached, cell-free and whole-cell mode. A voltage-dependent, non-inactivating potassium channel (delayed rectifier) was the most commonly observed ion channel in LNCaP cells. The slope conductance of K+ channels in a symmetrical 140 mM K+ gradient was 78 pS. In excised inside-out patches, the channel was inhibited by increasing the cytoplasmic Ca2+ concentration (with half-block at 0.5 microM Ca2+) over a wide range of membrane potentials. The K+ channel had a high sensitivity to tetraethylammonium (TEA), that reduced the single channel conductance with Kd of 280 +/- 27 microM. The K+ channel open probability was inhibited by alpha-dendrotoxin (DTX) (with a half-blocking concentration of approximately 5 nM) and mast cell degranulating peptide (MCDP) (with half-blocking concentration of approximately 70 nM) at all membrane potentials and with very slow reversibility. In view of the biophysical and pharmacological properties of K+ channels in LNCaP cells, it is not possible to classify these channels as one of the previously characterized types of voltage- or ligand-gated K+ channels in other cell lines.