Nanosecond pulsed electric fields depolarize transmembrane potential via voltage-gated K+, Ca2+ and TRPM8 channels in U87 glioblastoma cells.

Nanosecond pulsed electric fields (nsPEFs) have a variety of applications in the biomedical and biotechnology industries. Cancer treatment has been at the forefront of investigations thus far as nsPEFs permeabilize cellular and intracellular membranes leading to apoptosis and necrosis. nsPEFs may also influence ion channel gating and have the potential to modulate cell physiology without poration of the membrane. This phenomenon was explored using live cell imaging and a sensitive fluorescent probe of transmembrane voltage in the human glioblastoma cell line, U87 MG, known to express a number of voltage-gated ion channels. The specific ion channels involved in the nsPEF response were screened using a membrane potential imaging approach and a combination of pharmacological antagonists and ion substitutions. It was found that a single 10ns pulsed electric field of 34kV/cm depolarizes the transmembrane potential of cells by acting on specific voltage-sensitive ion channels; namely the voltage and Ca2+ gated BK potassium channel, L- and T-type calcium channels, and the TRPM8 transient receptor potential channel.

Genetically engineered HEK cells as a valuable tool for studying electroporation in excitable cells.

Electric pulses used in electroporation-based treatments have been shown to affect the excitability of muscle and neuronal cells. However, understanding the interplay between electroporation and electrophysiological response of excitable cells is complex, since both ion channel gating and electroporation depend on dynamic changes in the transmembrane voltage (TMV). In this study, a genetically engineered human embryonic kidney cells expressing NaV1.5 and Kir2.1, a minimal complementary channels required for excitability (named S-HEK), was characterized as a simple cell model used for studying the effects of electroporation in excitable cells. S-HEK cells and their non-excitable counterparts (NS-HEK) were exposed to 100 µs pulses of increasing electric field strength. Changes in TMV, plasma membrane permeability, and intracellular Ca2+ were monitored with fluorescence microscopy. We found that a very mild electroporation, undetectable with the classical propidium assay but associated with a transient increase in intracellular Ca2+, can already have a profound effect on excitability close to the electrostimulation threshold, as corroborated by multiscale computational modelling. These results are of great relevance for understanding the effects of pulse delivery on cell excitability observed in context of the rapidly developing cardiac pulsed field ablation as well as other electroporation-based treatments in excitable tissues.

PEDOT:PSS coated electrodes reduce intracellular oxidation and cell damage with pulsed electric field application.

Glioblastoma Multiforme is a highly lethal form of brain cancer, resistant to traditional therapeutic approaches and oftentimes hardly resectable. The application of pulsed electric fields (PEF) is gaining prominence as a highly effective approach for combating malignant tumors. However, PEF application at high voltages can generate reactive oxygen species through electrochemical events at electrodes, which can greatly affect intracellular processes and damage healthy cells. Here, we present an in depth study on the cellular impact of coating metal electrodes with an organic polymer PEDOT:PSS. We compared the effect of PEF application through coated and uncoated gold electrodes on the U87 human glioblastoma cell line. The results show that PEF application using PEDOT:PSS-coated electrodes does not induce intracellular ROS generation, even at high voltages, contrary to that observed with uncoated electrodes. PEF delivery with PEDOT:PSS coated electrodes results in minimal cell electroporation and a lower intracellular calcium response than uncoated metal electrodes. The application of the antioxidant MnTBAP allowed us to establish that superoxide generation is partially responsible for the higher intracellular calcium response observed in uncoated metal electrodes. The results demonstrate that PEDOT-coated electrodes allow for PEF application without intracellular ROS generation, with the trade-off being a diminished electroporation efficiency. These electrodes could therefore be useful for PEF application in ROS-sensitive tissues, as well as for disentangling the effect of PEFs on cells from the metabolic impact of electrolytic events arising from the electrode material.

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma.

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

Ca2+ roles in electroporation-induced changes of cancer cell physiology: From membrane repair to cell death.

The combination of Ca2+ ions and electroporation has gained attention as potential alternative to electrochemotherapy. Ca2+ is an important component of the cell membrane repair system and its presence directly influences the dynamics of the pore cycle after electroporation which can be exploited for cancer therapies. Here, the influence of Ca2+ concentration is investigated on small molecule electrotransfer and release of Calcein from 4T1, MX-1, B16F10, U87 cancer cells after cell exposure to microsecond electric pulses. Moreover, we investigated simultaneous molecule electrotransfer and intracellular calcium ion influx when media was supplemented with different Ca2+ concentrations. Results show that increased concentrations of calcium ions reduce the electrotransfer of small molecules to different lines of cancer cells as well as the release of Calcein. These effects are related with an enhanced membrane repair mechanism. Overall, we show that the efficiency of molecular electrotransfer can be controlled by regulating Ca2+ concentration in the electroporation medium. For the first time, the cause of cancer cell death in vitro from 1 mM CaCl2 concentrations is related to the irreversible loss of Ca2+ homeostasis after cell electroporation. Our findings provide fundamental insight on the mechanisms of Ca2+ electroporation that might lead to improved therapeutic outcomes.

Optical recording of electrical activity in intact neuronal networks with random access second-harmonic generation microscopy.

One of the main challenges in understanding the central nervous system is to measure the network dynamics of neuronal assemblies, while preserving the computational role of individual neurons. However, this is not possible with current techniques. In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk loading of tissue with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where action potentials were recorded without averaging across trials. These results show the strength of this technique in describing the temporal dynamics of neuronal assemblies, opening promising perspectives in understanding the computations of neuronal networks.

Visualisation of an nsPEF induced calcium wave using the genetically encoded calcium indicator GCaMP in U87 human glioblastoma cells.

Cytosolic, synthetic chemical calcium indicators are typically used to visualise the rapid increase in intracellular calcium ion concentration that follows nanosecond pulsed electric field (nsPEF) application. This study looks at the application of genetically encoded calcium indicators (GECIs) to investigate the spatiotemporal nature of nsPEF-induced calcium signals using fluorescent live cell imaging. Calcium responses to 44kV/cm, 10ns pulses were observed in U87-MG cells expressing either a plasma membrane targeted GECI (GCaMP5-G), or one cytosolically expressed (GCaMP6-S), and compared to the response of cells loaded with cytosolic or plasma membrane targeted chemical calcium indicators. Application of 100 pulses, to cells containing plasma membrane targeted indicators, revealed a wave of calcium across the cell initiating at the cathode side. A similar spatial wave was not observed with cytosolic indicators with mobile calcium buffering properties. The speed of the wave was related to pulse application frequency and it was not propagated by calcium induced calcium release.

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C.

The influence of infrared laser pulses on intracellular Ca2+ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo-4 was used to measure Ca2+ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo-4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca2+ transient with similar dose-response characteristics in hippocampal neurons and glioblastoma cells (half-maximal effective energy density EC50 of around 58 J.cm-2 ). For both type of cells, the source of the infrared-induced Ca2+ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP3 -induced Ca2+ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP3 cell signaling cascade has many potential applications including the development of optoceutical therapeutics.

Delivery devices for exposure of biological cells to nanosecond pulsed electric fields.

In this paper, delivery devices for nanosecond pulsed electric field exposure of biological samples in direct contact with electrodes or isolated are presented and characterized. They are based on a modified electroporation cuvette and two transverse electromagnetic cells (TEM cells). The devices were used to apply pulses with high intensity (4.5 kV) and short durations (3 and 13 ns). The delivery devices were electromagnetically characterized in the frequency and time domains. Field intensities of around 5, 0.5, and 12 MV m-1 were obtained by numerical simulations of the biological sample positioned in the three delivery devices. Two delivery systems had a homogenous electric field spatial distribution, and one was adapted to permit a highly localized exposure in the vicinity of a needle. Experimental biological investigations were carried out at different field intensities for five cancer cell lines. The results using flow cytometry showed that cells kept polarized mitochondrial membrane but lost plasma membrane integrity following a dose-response trend after exposure to different electric field intensities. Certain cell types (U87, MCF7) showed higher sensitivities to nsPEFs than other lines tested.

A bilayered PVA/PLGA-bioresorbable shuttle to improve the implantation of flexible neural probes.

OBJECTIVE: Neural electrophysiology is often conducted with traditional, rigid depth probes. The mechanical mismatch between these probes and soft brain tissue is unfavorable for tissue interfacing. Making probes compliant can improve biocompatibility, but as a consequence, they become more difficult to insert into the brain. Therefore, new methods for inserting compliant neural probes must be developed. APPROACH: Here, we present a new bioresorbable shuttle based on the hydrolytically degradable poly(vinyl alcohol) (PVA) and poly(lactic-co-glycolic acid) (PLGA). We show how to fabricate the PVA/PLGA shuttles on flexible and thin parylene probes. The method consists of PDMS molding used to fabricate a PVA shuttle aligned with the probe and to also impart a sharp tip necessary for piercing brain tissue. The PVA shuttle is then dip-coated with PLGA to create a bi-layered shuttle. MAIN RESULTS: While single layered PVA shuttles are able to penetrate agarose brain models, only limited depths were achieved and repositioning was not possible due to the fast degradation. We demonstrate that a bilayered approach incorporating a slower dissolving PLGA layer prolongs degradation and enables facile insertion for at least several millimeters depth. Impedances of electrodes before and after shuttle preparation were characterized and showed that careful deposition of PLGA is required to maintain low impedance. Facilitated by the shuttles, compliant parylene probes were also successfully implanted into anaesthetized mice and enabled the recording of high quality local field potentials. SIGNIFICANCE: This work thereby presents a solution towards addressing a key challenge of implanting compliant neural probes using a two polymer system. PVA and PLGA are polymers with properties ideal for translation: commercially available, biocompatible with FDA-approved uses and bioresorbable. By presenting new ways to implant compliant neural probes, we can begin to fully evaluate their chronic biocompatibility and performance compared to traditional, rigid electronics.

In vivo multiphoton nanosurgery on cortical neurons.

Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research.

Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells.

High powered, nanosecond duration, pulsed electric fields (nsPEF) cause cell death by a mechanism that is not fully understood and have been proposed as a targeted cancer therapy. Numerous chemotherapeutics work by disrupting microtubules. As microtubules are affected by electrical fields, this study looks at the possibility of disrupting them electrically with nsPEF. Human glioblastoma cells (U87-MG) treated with 100, 10 ns, 44 kV/cm pulses at a frequency of 10 Hz showed a breakdown of their interphase microtubule network that was accompanied by a reduction in the number of growing microtubules. This effect is temporally linked to loss of mitochondrial membrane potential and independent of cellular swelling and calcium influx, two factors that disrupt microtubule growth dynamics. Super-resolution microscopy revealed microtubule buckling and breaking as a result of nsPEF application, suggesting that nsPEF may act directly on microtubules.

Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts.

Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10 ns, high voltage electric pulse (35-45 kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma.

Dosimetric Characteristics of an EMF Delivery System Based on a Real-Time Impedance Measurement Device.

In this paper, the dosimetric characterization of an EMF exposure setup compatible with real-time impedance measurements of adherent biological cells is proposed. The EMF are directly delivered to the 16-well format plate used by the commercial xCELLigence apparatus. Experiments and numerical simulations were carried out for the dosimetric analysis. The reflection coefficient was less than -10 dB up to 180 MHz and this exposure system can be matched at higher frequencies up to 900 and 1800 MHz. The specific absorption rate (SAR) distribution within the wells containing the biological medium was calculated by numerical finite-difference time domain simulations and results were verified by temperature measurements at 13.56 MHz. Numerical SAR values were obtained at the microelectrode level where the biological cells were exposed to EMF including 13.56, 900, and 1800 MHz. At 13.56 MHz, the SAR values, within the cell layer and the 270-µL volume of medium, are 1.9e3 and 3.5 W/kg/incident mW, respectively.

Multicolor multiphoton microscopy based on a nanosecond supercontinuum laser source.

Multicolor multiphoton microscopy is experimentally demonstrated for the first time on a spectral bandwidth of excitation of 300 nm (full width half maximum) thanks to the implementation a nanosecond supercontinuum (SC) source compact and simple with a low repetition rate. The interest of such a wide spectral bandwidth, never demonstrated until now, is highlighted in vivo: images of glioma tumor cells stably expressing eGFP grafted on the brain of a mouse and its blood vessels network labelled with Texas Red(®) are obtained. These two fluorophores have a spectral bandwidth covering the whole 300 nm available. In parallel, a similar image quality is obtained on a sample of mouse muscle in vitro when excited with this nanosecond SC source or with a classical high rate, femtosecond and quasi monochromatic laser. This opens the way for (i) a simple and very complete biological characterization never performed to date with multiphoton processes, (ii) multiple means of contrast in nonlinear imaging allowed by the use of numerous fluorophores and (iii) other multiphoton processes like three-photon ones.

Rhodamine B as an optical thermometer in cells focally exposed to infrared laser light or nanosecond pulsed electric fields.

The temperature-dependent fluorescence property of Rhodamine B was used to measure changes in temperature at the cellular level induced by either infrared laser light exposure or high intensity, ultrashort pulsed electric fields. The thermal impact of these stimuli were demonstrated at the cellular level in time and contrasted with the change in temperature observed in the extracellular bath. The method takes advantage of the temperature sensitivity of the fluorescent dye Rhodamine B which has a quantum yield linearly dependent on temperature. The thermal effects of different temporal pulse applications of infrared laser light exposure and of nanosecond pulsed electric fields were investigated. The temperature increase due to the application of nanosecond pulsed electric fields was demonstrated at the cellular level.

Exposure to GSM RF fields does not affect calcium homeostasis in human endothelial cells, rat pheocromocytoma cells or rat hippocampal neurons.

In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an "electromagnetic smog", with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca2+) homeostasis is affected by the types of radiofrequency fields emitted by mobile phones. In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca2+ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012-2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types (human endothelial cells, PC-12 neuroblastoma and primary hippocampal neurons) that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca2+, in addition to Ca2+ signals evoked by an InsP(3)-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca2+ store emptying and store-operated Ca2+ entry following application of the Ca2+ATPase inhibitor thapsigargin. Multiple parameters (e.g., peak amplitude, integrated Ca2+ signal, recovery rates) were analysed to explore potential impact of radiofrequency field exposure on Ca2+ signals. Our data indicate that 900 MHz GSM fields do not affect either basal Ca2+ homeostasis or provoked Ca2+ signals. Even at the highest field strengths applied, which exceed typical phone exposure levels, we did not observe any changes in cellular Ca2+ signals. We conclude that under the conditions employed in our experiments, and using a highly-sensitive assay, we could not detect any consequence of RF exposure.