Biomedical Engineering and Bioengineering Commons^™

Control Of The Electroporation Efficiency Of Nanosecond Pulses By Swinging The Electric Field Vector Direction, Vitalii Kim, Iurii Semenov, Allen S. Kiester, Mark A. Keppler, Bennett L. Ibey, Joel N. Bixler, Ruben M. L. Colunga Biancatelli, Andrei G. Pakhomov

Bioelectrics Publications

Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as “bipolar cancellation,” enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing …

Biomechanical And Biophysical Properties Of Breast Cancer Cells Under Varying Glycemic Regimens, Diganta Dutta, Xavier-Lewis Palmer, Jose Ortega-Rodas, Vasundhara Balraj, Indrani Ghosh Dastider, Surabhi Chandra

Electrical & Computer Engineering Faculty Publications

Diabetes accelerates cancer cell proliferation and metastasis, particularly for cancers of the pancreas, liver, breast, colon, and skin. While pathways linking the 2 disease conditions have been explored extensively, there is a lack of information on whether there could be cytoarchitectural changes induced by glucose which predispose cancer cells to aggressive phenotypes. It was thus hypothesized that exposure to diabetes/high glucose alters the biomechanical and biophysical properties of cancer cells more than the normal cells, which aids in advancing the cancer. For this study, atomic force microscopy indentation was used through microscale probing of multiple human breast cancer cells (MCF-7, …

Electropermeabilization Does Not Correlate With Plasma Membrane Lipid Oxidation, Olga Michel, Andrei G. Pakhomov, Maura Casciola, Jolanta Saczko, Julita Kulbacka, Olga N. Pakhomova

Bioelectrics Publications

The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) …

Modulation Of Biological Responses To 2 Ns Electrical Stimuli By Field Reversal, Esin B. Sözer, P. Thomas Vernier

Bioelectrics Publications

Nanosecond bipolar pulse cancellation, a recently discovered Phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (> 100 mu s) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). …

Transport Of Charged Small Molecules After Electropermeabilization - Drift And Diffusion, Esin B. Sözer, C. Florencia Pocetti, P. Thomas Vernier

Bioelectrics Publications

Background: Applications of electric-field-induced permeabilization of cells range from cancer therapy to wastewater treatment. A unified understanding of the underlying mechanisms of membrane electropermeabilization, however, has not been achieved. Protocols are empirical, and models are descriptive rather than predictive, which hampers the optimization and expansion of electroporation-based technologies. A common feature of existing models is the assumption that the permeabilized membrane is passive, and that transport through it is entirely diffusive. To demonstrate the necessity to go beyond that assumption, we present here a quantitative analysis of the post-permeabilization transport of three small molecules commonly used in electroporation research-YO-PRO-1, propidium, …

Dielectric Properties Of Isolated Adrenal Chromaffin Cells Determined By Microfluidic Impedance Spectroscopy, A. C. Sabuncu, M. Stacey, G. L. Graviso, N. Semenova, P. T. Vernier, N. Leblanc, I. Chatterjee, J. Zaklit

Bioelectrics Publications

Knowledge of the dielectric properties of biological cells plays an important role in numerical models aimed at understanding how high intensity ultrashort nanosecond electric pulses affect the plasma membrane and the membranes of intracellular organelles. To this end, using electrical impedance spectroscopy, the dielectric properties of isolated, neuroendocrine adrenal chromaffin cells were obtained. Measured impedance data of the cell suspension, acquired between 1 kHz and 20 MHz, were fit into a combination of constant phase element and Cole-Cole models from which the effect of electrode polarization was extracted. The dielectric spectrum of each cell suspension was fit into a Maxwell-Wagner …

Computing Spatiotemporal Heat Maps Of Lipid Electropore Formation: A Statistical Approach, Willy Wriggers, Frederica Castellani, Julio A. Kovacs, P. Thomas Vernier

Mechanical & Aerospace Engineering Faculty Publications

We extend the multiscale spatiotemporal heat map strategies originally developed for interpreting molecular dynamics simulations of well-structured proteins to liquids such as lipid bilayers and solvents. Our analysis informs the experimental and theoretical investigation of electroporation, that is, the externally imposed breaching of the cell membrane under the influence of an electric field of sufficient magnitude. To understand the nanoscale architecture of electroporation, we transform time domain data of the coarse-grained interaction networks of lipids and solvents into spatial heat maps of the most relevant constituent molecules. The application takes advantage of our earlier graph-based activity functions by accounting for …

A Balanced Approach To Adaptive Probability Density Estimation, Julio Kovacs, Cailee Helmick, Willy Wriggers

Mechanical & Aerospace Engineering Faculty Publications

Our development of a Fast (Mutual) Information Matching (FIM) of molecular dynamics time series data led us to the general problem of how to accurately estimate the probability density function of a random variable, especially in cases of very uneven samples. Here, we propose a novel Balanced Adaptive Density Estimation (BADE) method that effectively optimizes the amount of smoothing at each point. To do this, BADE relies on an efficient nearest-neighbor search which results in good scaling for large data sizes. Our tests on simulated data show that BADE exhibits equal or better accuracy than existing methods, and visual tests …

Quantitative Limits On Small Molecule Transport Via The Electropermeome - Measuring And Modeling Single Nanosecond Perturbations, Esin B. Sözer, Zachary A. Levine, P. Thomas Vernier

Bioelectrics Publications

The detailed molecular mechanisms underlying the permeabilization of cell membranes by pulsed electric fields (electroporation) remain obscure despite decades of investigative effort. To advance beyond descriptive schematics to the development of robust, predictive models, empirical parameters in existing models must be replaced with physics- and biology-based terms anchored in experimental observations. We report here absolute values for the uptake of YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity, into cells after a single electric pulse lasting only 6 ns. We correlate these measured values, based on fluorescence microphotometry of hundreds of individual cells, with a diffusion-based geometric analysis of pore-mediated …

Biophysical Tools To Study Cellular Mechanotransduction, Ismeel Muhamed, Farhan Chowdhury, Venkat Maruthamuthu

Mechanical & Aerospace Engineering Faculty Publications

The cell membrane is the interface that volumetrically isolates cellular components from the cell's environment. Proteins embedded within and on the membrane have varied biological functions: reception of external biochemical signals, as membrane channels, amplification and regulation of chemical signals through secondary messenger molecules, controlled exocytosis, endocytosis, phagocytosis, organized recruitment and sequestration of cytosolic complex proteins, cell division processes, organization of the cytoskeleton and more. The membrane's bioelectrical role is enabled by the physiologically controlled release and accumulation of electrochemical potential modulating molecules across the membrane through specialized ion channels (e.g., Na^⁺, Ca²⁺, K^⁺ channels). …

A 'Tissue Model' To Study The Barrier Effects Of Living Tissues On The Reactive Species Generated By Surface Air Discharge, Tongtong He, Dingxin Liu, Han Xu, Zhichao Liu, Dehui Xu, Dong Li, Qiosong Li, Mingzhe Rong, Michael G. Kong

Bioelectrics Publications

Gelatin gels are used as surrogates of human tissues to study their barrier effects on incoming reactive oxygen and nitrogen species (RONS) generated by surface air discharge. The penetration depth of nitrite into gelatin gel is measured in real time during plasma treatment, and the permeabilities of nitrite, nitrate, O₃ and H₂O₂ through gelatin gel films are quantified by measuring their concentrations in the water underneath such films after plasma treatment. It is found that the penetration speed of nitrite increases linearly with the mass fraction of water in the gelatin gels, and the permeabilities of …

Effects Of High Voltage Nanosecond Electric Pulses On Eukaryotic Cells (In Vitro): A Systematic Review, Tina Batista Napotink, Matej Reberšek, P. Thomas Vernier, Babara Mali, Damijan Miklavčič

Bioelectrics Publications

For this systematic review, 203 published reports on effects of electroporation using nanosecond high-voltage electric pulses (nsEP) on eukaryotic cells (human, animal, plant) in vitro were analyzed. A field synopsis summarizes current published data in the field with respect to publication year, cell types, exposure configuration, and pulse duration. Published data were analyzed for effects observed in eight main target areas (plasma membrane, intracellular, apoptosis, calcium level and distribution, survival, nucleus, mitochondria, stress) and an additional 107 detailed outcomes. We statistically analyzed effects of nsEP with respect to three pulse duration groups: A: 1–10 ns, B: 11–100 ns and C: …

Diffuse, Non-Polar Electropermeabilization And Reduced Propidium Uptake Distinguish The Effect Of Nanosecond Electric Pulses, Iurii Semenov, Christian W. Zemlin, Olga N. Pakhomova, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Ca²⁺ activation and membrane electroporation by 10-ns and 4-ms electric pulses (nsEP and msEP) were compared in rat embryonic cardiomyocytes. The lowest electric field which triggered Ca²⁺ transients was expectedly higher for nsEP (36 kV/cm)than forms EP (0.09 kV/cm) but the respective doses were similar (190 and460 mJ/g). At higher intensities, both stimuli triggered prolonged firing in quiescent cells. An increase of basal Ca²⁺ level by N10 nM in cells with blocked voltage-gated Ca²⁺ channels and depleted Ca²⁺ depot occurred at 63 kV/cm (nsEP) or 0.14 kV/cm (msEP) and was regarded as electroporation threshold. These …

Dielectric Characterization Of Coastal Cartilage Chondrocytes, Michael W. Stacey, Ahmet C. Sabuncu, Ali Beskok

Bioelectrics Publications

BACKGROUND: Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.

METHODS: Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are …

Primary Pathways Of Intracellular Ca2+ Mobilization By Nanosecond Pulsed Electric Field, Iurii Semenov, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which determines multifarious downstream effects. By using fast ratiometric Ca²⁺ imaging with Fura-2, we quantified the external Ca²⁺ uptake, compared it with Ca²⁺ release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca²⁺ channels, so that the nsPEF-induced [Ca²⁺]_i changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca2+]_i increase …

Response To "Sodium Current Inhibition By Nanosecond Pulsed Electric Field (Nspef) - Fact Or Artifact?" By Verkerk Et Al, Andrei G. Pakhomov

Bioelectrics Publications

It was nice to learn that our studies of nanosecond pulsed electric field (nsPEF) effects on membrane currents [Nesin et al., 2012; Nesin and Pakhomov, 2012] gained the attention of scientists outside the immediate field of bioelectromagnetics.

Cell Permeabilization And Inhibition Of Voltage-Gated Ca²+ And Na+ Channel Currents By Nanosecond Pulsed Electric Fields, Vasyl Nesin, Angela M. Bowman, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Previous studies have found that nanosecond pulsed electric field (nsPEF) exposure causes long-term permeabilization of the cell plasma membrane. In this study, we utilized the whole-cell patch-clamp method to study the nsPEF effect on currents of voltage-gated (VG) Ca²⁺ and Na⁺ channels (I_Ca and I_Na) in cultured GH3 and NG108 cells. We found that a single 300 or 600 ns pulse at or above 1.5-2 kV/cm caused prolonged inhibition of I_Ca and I_Na. Concurrently, nsPEF increased a non-inactivating leak current (I_leak), presumably due to the formation of nanoelectropores or larger …

Regulation Of Intracellular Calcium Concentration By Nanosecond Pulsed Electric Fields, Shaka S. Scarlett, Jody A. White, Peter F. Blackmore, Karl H. Schoenbach, Juergen Kolb

Bioelectrics Publications

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma …

Plasma Membrane Voltage Changes During Nanosecond Pulsed Electric Field Exposure, W. Frey, R. O. Price, P. F. Blackmore, R. P. Joshi, R. Nuccitelli, S. J. Beebe, K. H. Schoenbach, J. F. Kolb

Bioelectrics Publications

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of similar to 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat …

Energy-Landscape-Model Analysis For Irreversibility And Its Pulse-Width Dependence In Cells Subjected To A High-Intensity Ultrashort Electric Pulse, R. P. Joshi, Q. Hu, Karl H. Schoenbach, Stephen J. Beebe

Bioelectrics Publications

We provide a simple, but physical analysis for cell irreversibility and apoptosis in response to an ultrashort (nanosecond), high-intensity electric pulse. Our approach is based on an energy landscape model for determining the temporal evolution of the configurational probability function p(q). The primary focus is on obtaining qualitative predictions of a pulse width dependence to apoptotic cell irreversibility that has been observed experimentally. The analysis couples a distributed electrical model for current flow with the Smoluchowski equation to provide self-consistent, time-dependent transmembrane voltages. The model captures the essence of the experimentally observed pulse-width dependence, and provides a possible physical picture …