The Cytotoxic Synergy of Nanosecond Electric Pulses and Low Temperature Leads to Apoptosis

Electroporation by nanosecond electric pulses (nsEP) is an emerging modality for tumor ablation. Here we show the efficient induction of apoptosis even by a non-toxic nsEP exposure when it is followed by a 30-min chilling on ice. This chilling itself had no impact on the survival of U-937 or HPAF-II cells, but caused more than 75% lethality in nsEP-treated cells (300 ns, 1.8-7 kV/cm, 50-700 pulses). The cell death was largely delayed by 5-23 hr and was accompanied by a 5-fold activation of caspase 3/7 (compared to nsEP without chilling) and more than 60% cleavage of poly-ADP ribose polymerase (compared to less than 5% in controls or after nsEP or chilling applied separately). When nsEP caused a transient permeabilization of 83% of cells to propidium iodide, cells placed at 37 ° C resealed in 10 min, whereas 60% of cells placed on ice remained propidium-permeable even in 30 min. The delayed membrane resealing caused cell swelling, which could be blocked by an isosmotic addition of a pore-impermeable solute (sucrose). However, the block of swelling did not prevent the delayed cell death by apoptosis. The potent enhancement of nsEP cytotoxicity by subsequent non-damaging chilling may find applications in tumor ablation therapies

Technobiology Paradigm in Nanomedicine: Treating Cancer with MagnetoElectric Nanoparticles

Today, cancer is the world’s deadliest disease. Despite significant progress to find a cure, especially over the last decade, with immunotherapy rapidly becoming the state of the art, major open questions remain. Each successful therapy is not only limited to a few cancers but also has relatively low specificity to target cancer cells; although cancer cells can indeed be eradicated, many normal cells are sacrificed as collateral damage. To fill this gap, we have developed a class of multiferroic nanostructures known as magnetoelectric nanoparticles (MENs) that can be used to enable externally controlled high-specificity targeted delivery and release of therapeutic drugs on demand. First, the underlying physics of MENs was studied, as it relates to different externally applied sequences of a.c and d.c. magnetic fields to facilitate (i) high-specificity targeting driven by a physical force rather than antibody matching, (ii) a delivery mechanism that enhances cellular uptake (via nanoelectroporation) of therapeutic drugs across the cellular membrane of cancer cells only, and (iii) an externally controlled mechanism that releases the therapeutic drug on-demand. Secondly, the application of MENs as a nuclear magnetic resonance (NMR) nanoprobe was explored. The intrinsically coupled ferromagnetic and ferroelectric phases allowes the nanoparticle to be used as sensitive nanoprobe detectors of biological cells; based on the knowledge that the cellular membrane is an electrically charged medium which creates an ideal environment for MENs to distinguish between cancer and normal cells. Lastly, through in-vivo and in-vitro studies, MENs were used as drug delivery vehicle capable of crossing the blood brain barrier (BBB) and delivering recently discovered MIA690 peptide drug (via nanoelectroporation) to glioblastoma multiforme (GBM) brain cancer cells. Glioblastomas are tumors that arise from astrocytes in the brain; that are highly malignant and reproduces quickly due to their large network of blood vessels. In the following study, we report the binding efficacy of MIA690 to magnetoelecric nanoparticles as well as present an unprecedented targeted and on-demand release to glioblastoma cells through special sequences of a.c. and d.c. magnetic fields. The potential therapeutic and diagnostic impact of MENs for future medicine is beyond the scope of this study, as MENs can be used to treat any type of cancer

Effects of High Voltage Nanosecond Electric Pulses on Eukaryotic Cells (in vitro): A Systematic Review

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: 101–999 ns. The analysis confirmed that the plasma membrane is more affected with longer pulses than with short pulses, seen best in uptake of dye molecules after applying single pulses. Additionally, we have reviewed measurements of nsEP and evaluations of the electric fields to which cells were exposed in these reports, and we provide recommendations for assessing nanosecond pulsed electric field effects in electroporation studies

Disassembly of Actin Structures by Nanosecond Pulsed Electric Field is a Downstream Effect of Cell Swelling

Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance

Sviluppo delle applicazioni cliniche dell'elettroporazione nel trattamento delle metastasi cutanee e dei tumori dei tessuti molli

Introduction. The principle of cell electroporation (EP) (the temporary permeabilization of the cytoplasmatic membrane by means of electric fields) has been applied to the treatment of tumors with the intent of increasing the concentration of anticancer drugs (cisplatin and bleomycin) within the tumor cell and hence their cytotoxic effect. Until 2006, the year of standardization of the electrochemotherapy (ECT) procedure, clinical experience was limited to small heterogeneous series, mainly focused on local activity data in patients with superficial metastases. Aims. The aim of this project was to investigate the efficacy of ECT in homogeneous populations of cancer patients of different histotypes (patients with skin metastases from malignant melanoma, recurrent breast cancer on the chest wall, recurrent soft tissue sarcomas and tumors of the head and neck area). Furthermore, it was investigated the feasibility and efficacy of a new device (electric pulse generator and dedicated needle electrodes), capable of applying homogeneous electric fields also to deep tumors. Finally, we investigated, both at clinical and preclinical level, the possibility of improving the effectiveness of ECT by, respectively, the evaluation of some immune effect of treatment (i.e., the induction of the Toll-like receptors, TLRs) and the evaluation of the tumor sensitizing action to ECT of buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis. Methods. Overall, four prospective, phase-II, clinical trials were designed, three of them have been concluded and a fourth is still ongoing and patientsâ enrollment is open. Moreover, a retrospective study was performed on head and neck cancer patients. In some patients, tissue samples were analyzed before and after ECT, to evaluate the expression of TLRs. Finally, some in vitro tests were conducted on tumor cell lines to assess the sensitizing effect of BSO pre-treatment to BLM + EP administration. Results. All the histotypes showed high local response rates and ECT activity was more pronounced than standard chemotherapy regimens employed in current oncology practice. Local response translated in an appreciable local control of the treated tumors, while toxicity was limited and mainly local. TLRs levels in post-ECT tumor biopsies was not significantly different, compared with pre-ECT samples. However, the immune reaction seems to play a role since a high lymphocytic infiltrate into the electroporated lesions was associated with higher response rates. In vitro tests, BSO pretreatment enhanced the cytotoxicity of BLM + EP. Of note, we also found an increased toxicity of melphalan, a drug currently used in the treatment of locoregional melanoma, in association with EPs. Thanks to the multi-disciplinary collaboration and clinical case management, we have individuated some technical aspects that deserve further improvement in order to increase the effectiveness of treatment application and the number of patients who can benefit from it. Conclusions. ECT has proved a highly active treatment in recurrent melanoma, breast cancer, soft tissue sarcomas and cancers of the head and neck region. The preliminary clinical experience with a new device and dedicated electrodes indicates the feasibility to treat percutaneously even large, deep-seated tumors. Although ECT treatment do not seem to raise the levels of TLRs in the electroporated tumors, however the levels of lymphocytic infiltration were associated with better local response. In vitro, BSO pre-treatment is able to sensitize tumor cells to the combined treatment of BLM and EP. Electroporation deserves further investigation also in combination with melphalan, which is currently used in locoregional chemotherapy of in transit metastases from melanoma. In conclusion, ECT has the potential to be implemented in the oncology field through the multidisciplinary collaboration of oncologists, surgeons, radiation oncologists, biologists and engineers

Estudo da formação de poros nas membranas plasmática e nuclear de uma célula biológica isolada durante a nanoeletroporação

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Elétrica, Florianópolis, 2017.Este trabalho tem como objetivo analisar a formação de poros nas membranas plasmática e nuclear de uma célula biológica isolada, durante a aplicação de campos elétricos com diferentes configurações de duração e amplitude. Neste sistema, a condutividade da solução na qual a célula está imersa também é variável. A principal hipótese que direcionou este trabalho foi a de que as configurações do pulso elétrico e condutividade da solução podem fazer com que a eletroporação seja predominante na região da membrana plasmática ou da membrana nuclear (eletroporação seletiva). Esta técnica pode ser utilizada na obtenção de acesso ao citoplasma e plasma nuclear, tal como num processo de transferência genética, em que há a necessidade de condução de plasmídeos desde o meio externo até o núcleo celular, sem provocar a morte da célula. Para este estudo, foram aplicados modelos matemáticos da eletroporação, a fim de verificar variações de potencial transmembrana, densidade de poros e condutividade elétrica nas membranas, durante a aplicação dos campos elétricos. Os resultados teóricos obtidos comprovaram a hipótese proposta, apontando que a condutividade externa para valores inferiores às do citoplasma e nucleoplasma (aproximadamente 0,1 S/m) torna ainda maior o tempo necessário para o carregamento da membrana plasmática em relação à membrana nuclear. Dessa forma, quando pulsos de curta duração (em torno de 10 ns) e alta intensidade (em torno de 10 kV/cm) são aplicados, a predominância de poros ocorre na membrana nuclear, processo denominado nanoeletroporação. Além disso, este estudo analisou diferentes características dimensionais das células, tais como espessuras das membranas, raios da célula e do núcleo, condutividades do citoplasma e do plasma nuclear, e ainda as permissividades e as capacitâncias das membranas, a fim de fornecer diretrizes para as configurações dos parâmetros do pulso elétrico e condutividade.Abstract : This work aims to analyze the pore formation in the plasma and nuclear membranes of a single biological cell, during the application of electric fields with different duration and amplitude configurations. In this system, the conductivity of pore solution in which the cell is immersed is also variable. The main hypothesis that guided this work was that the electric pulse settings and solution conductivity can cause electroporation to be predominant either on the plasma membrane region or the nuclear membrane region (selective electroporation). This technique can be used to obtain access to cytoplasm and nuclear plasma, such as in a genetic transfer process, this process requires plasmids conduction from the external environment to the cell nucleus without cause cell death. For this study, mathematical models of electroporation were applied, in order to verify variations of transmembrane potential, pore density and electric conductivity of the membranes during the applications of the electric fields. The obtained theoretical results suggests the proposed hypothesis, when values of external conductivity is lower than values of cytoplasm and nucleoplasm (approximately 0,1 S/m), increases the time required of plasm membrane to load in relation to the nuclear membrane. Therefore, when pulses of short duration (around 10 ns) and high intensity (around 10 kV/cm) are applied, the predominance of pores occurs in the nuclear membrane, a process known as nanoelectroporation. Furthermore, this study analyzed different cell dimensional characteristics, such as membrane thickness, cell and nucleus radius, cytoplasm and nuclear plasma conductivities, and even permittivity and capacitance of the membranes, to provide guidelines for parameter settings of the electrical pulse and conductivity

細胞に対するナノ秒パルス電場の影響の顕微分光研究

Tohoku University中林孝和課

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world

Ultra-Low Intensity Post-Pulse Affects Cellular Responses Caused by Nanosecond Pulsed Electric Fields

High-intensity nanosecond pulse electric fields (nsPEF) can preferentially induce various effects, most notably regulated cell death and tumor elimination. These effects have almost exclusively been shown to be associated with nsPEF waveforms defined by pulse duration, rise time, amplitude (electric field), and pulse number. Other factors, such as low-intensity post-pulse waveform, have been completely overlooked. In this study, we show that post-pulse waveforms can alter the cell responses produced by the primary pulse waveform and can even elicit unique cellular responses, despite the primary pulse waveform being nearly identical. We employed two commonly used pulse generator designs, namely the Blumlein line (BL) and the pulse forming line (PFL), both featuring nearly identical 100 ns pulse durations, to investigate various cellular effects. Although the primary pulse waveforms were nearly identical in electric field and frequency distribution, the post-pulses differed between the two designs. The BL’s post-pulse was relatively long-lasting (~50 µs) and had an opposite polarity to the main pulse, whereas the PFL’s post-pulse was much shorter (~2 µs) and had the same polarity as the main pulse. Both post-pulse amplitudes were less than 5% of the main pulse, but the different post-pulses caused distinctly different cellular responses. The thresholds for dissipation of the mitochondrial membrane potential, loss of viability, and increase in plasma membrane PI permeability all occurred at lower pulsing numbers for the PFL than the BL, while mitochondrial reactive oxygen species generation occurred at similar pulsing numbers for both pulser designs. The PFL decreased spare respiratory capacity (SRC), whereas the BL increased SRC. Only the PFL caused a biphasic effect on trans-plasma membrane electron transport (tPMET). These studies demonstrate, for the first time, that conditions resulting from low post-pulse intensity charging have a significant impact on cell responses and should be considered when comparing the results from similar pulse waveforms