We present an alternative technique of monosex-male reversal for Nile tilapia using transient square pulse-electric field inductions to enhance sex reversal rate of tilapia eggs by methyltestosterone ...(Oreochromis niloticus L., Chitralada). Male tilapia is preferred for aquaculture because of better size and growth rate. The egg membrane was permeabilised by an electric field pulse allowing a minimalized androgen hormone dose of 17alpha-methyltestosterone (MT) (μg l−1 concentration) to achieve monosex sex-male reversal. The technique achieved large-scale inductions of 2000 eggs rather than for individual eggs and was aimed at commercialization (Thailand petty patent no. 13516). The eggs of O. niloticus obtained from our parent breeding stocks were carefully selected to be in the segmentation-pharyngula stage (2–3 dpf - day post fertilization) for electrical inductions in a pulsed-electric field generated between narrow parallel-plate electrodes. The electrodes were powered with an adjustable signal pulse-generator (SPG) developed for on-site operations. The induction medium was prepared using HEPES buffer mixed with a minimized concentration of MT. We experimentally optimized inductions by adjusting the number of square wave pulses, pulse amplitude, pulse durations (period) and a mark-space ratio (pulse width: negative half of period) for the optimum results. A dose of 350 V (electric field intensities of 87.50 kV m−1) of 3 square pulses with 100 μs pulse-period (50:50 mark space ratio) gave the maximum rate of male-sex reversal rate (MSR) of 89.42 ± 2.28 (SD)%, hatching rate (HR) of 93.33 ± 0.58% and survival rate (SR) of 87.49 ± 1.71%, respectively. The off-spring rate (OSR) or sex ratio of the controls were 59.71 ± 3.00% male and 46.33 ± 1.53% female from a hatching rate (HR) of 91.33 ± 1.53% and a survival rate (SR) of 85.08 ± 4.16%, respectively.
•Tilapia eggs were permeabilised using electric field induction.•Methyltestosterone (MT) could then penetrate eggs for male sex reversal.•Very large minimalision of amounts of MT used compared to feeding fry with MT.•Male-sex reversal (MSR) 89.4%, hatching rate (HR) 93.3%, survival rate (SR) 87.5%.•A large scale device was developed to treat ≈ 2000 eggs/batch.
In our study, we used bleomycin to evaluate the permeabilization caused by nanosecond duration electric pulses (nanopulses). Bleomycin is a non permeant molecule which can be used both as a sensitive ...and quantitative marker to evaluate cell electropermeabilization. Indeed, the penetration of as few as 500 molecules is sufficient to entail a major biological effect: cell death. We show that one single nanopulse with a duration of 10ns and a field strength of 40kV/cm is sufficient to allow the uptake of at least 500 molecules of bleomycin in 20% of the cells when the external bleomycin concentration is 3μM. When the external bleomycin concentration is reduced by a 100 fold, the same levels of cytotoxicity require an increase of about 25 times in the number of pulses. These results are in favor of the fact that each nanopulse creates new pores or defects on the cell membrane even if most of these pores can reseal between two consecutive pulses. Results also suggest that the cell permeability observed with classical markers when a large number of pulses are delivered results from the large number of nanopores or defects of the cell membrane created by the train of nanopulses.
► Bleomycin (Mr 1500) is a very sensitive cell permeabilization marker. ► Pulses of field strength E=40kV/cm and duration 10ns induce permeabilization. ► One pulse is sufficient for cross-membrane transport of bleomycin. ►These pulses allow thus the penetration of molecules of 1500Da. ► Each pulse creates new pores or defects on the cell membrane.
Intense nanosecond pulsed electric field (nsPEF) is a novel modality for cell activation and nanoelectroporation. Applications of nsPEF in research and therapy are hindered by a high electric field ...requirement, typically from 1 to over 50 kV/cm to elicit any bioeffects. We show how this requirement can be overcome by engaging temporal summation when pulses are compressed into high-rate bursts (up to several MHz). This approach was tested for excitation of ventricular cardiomyocytes and peripheral nerve fibers; for membrane electroporation of cardiomyocytes, CHO, and HEK cells; and for killing EL-4 cells. MHz compression of nsPEF bursts (100–1000 pulses) enables excitation at only 0.01–0.15 kV/cm and electroporation already at 0.4–0.6 kV/cm. Clear separation of excitation and electroporation thresholds allows for multiple excitation cycles without membrane disruption. The efficiency of nsPEF bursts increases with the duty cycle (by increasing either pulse duration or repetition rate) and with increasing the total time “on” (by increasing either pulse duration or number). For some endpoints, the efficiency of nsPEF bursts matches a single “long” pulse whose amplitude and duration equal the time-average amplitude and duration of the bursts. For other endpoints this rule is not valid, presumably because of nsPEF-specific bioeffects and/or possible modification of targets already during the burst. MHz compression of nsPEF bursts is a universal and efficient way to lower excitation thresholds and facilitate electroporation.
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•Efficacy of nanosecond pulsed electric field was tested up to MHz frequencies.•Temporal summation enabled low-field bioeffects above a critical frequency.•MHz bursts of nanosecond stimuli activated cells at 10–100 lower thresholds.•MHz compression separated stimulation from electroporation at higher voltages.
•Critical assessment of models describing electroporation and molecular transport.•None of the models can be universily applied to the full range of experimental data.•None of the models can be ...considered truly validated by experimental data.•Guidelines for further development of mechanistic models of electroporation.•Guidelines for reporting experimental measurements to support modeling.
Electroporation has become a powerful tool for nonviral delivery of various biomolecules such as nucleic acids, proteins, and chemotherapeutic drugs to virtually any living cell by exposing the cell membrane to an intense pulsed electric field. Different multiphysics and multiscale models have been developed to describe the phenomenon of electroporation and predict molecular transport through the electroporated membrane. In this paper, we critically examine the existing mechanistic, single-cell models which allow spatially and temporally resolved numerical simulations of electroporation-induced transmembrane transport of small molecules by confronting them with different experimental measurements. Furthermore, we assess whether any of the proposed models is universal enough to describe the associated transmembrane transport in general for all the different pulse parameters and small molecules used in electroporation applications. We show that none of the tested models can be universally applied to the full range of experimental measurements. Even more importantly, we show that none of the models has been compared to sufficient amount of experimental data to confirm the model validity. Finally, we provide guidelines and recommendations on how to design and report experiments that can be used to validate an electroporation model and how to improve the development of mechanistic models.
When high-amplitude, short-duration pulsed electric fields are applied to cells and tissues, the permeability of the cell membranes and tissue is increased. This increase in permeability is currently ...explained by the temporary appearance of aqueous pores within the cell membrane, a phenomenon termed electroporation. During the past four decades, advances in fundamental and experimental electroporation research have allowed for the translation of electroporation-based technologies to the clinic. In this review, we describe the theory and current applications of electroporation in medicine and then discuss current challenges in electroporation research and barriers to a more extensive spread of these clinical applications.
Electric fields are among physical stimuli that have revolutionized therapy. Occurring endogenously or exogenously, the electric field can be used as a trigger for controlled drug release from ...electroresponsive drug delivery systems, can stimulate wound healing and cell proliferation, may enhance endocytosis or guide stem cell differentiation. Electric field pulses may be applied to induce cell fusion, can increase the penetration of therapeutic agents into cells, or can be applied as a standalone therapy to ablate tumors. This review describes the main therapeutic trends and overviews the main physical, chemical and biological mechanisms underlying the actions of electric fields. Overall, the electric field can be used in therapeutic approaches in several ways. The electric field can act on drug carriers, cells and tissues. Understanding the multiple effects of this powerful tool will help harnessing its full therapeutic potential in an efficient and safe way.
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Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the ...membrane integrity takes minutes and is only partially dependent on Ca2+. We explored the impact of Ca2+ on the kinetics of membrane resealing by monitoring the entry of a YO-PRO-1 dye (YP) in BPAE and HEK cells. Ca2+ was promptly removed or added after the electric pulse (EP) by a fast-step perfusion. YP entry increased sharply after the EP and gradually slowed down following either a single- or a double-exponential function. In BPAE cells permeabilized by a single 300- or 600-ns EP at 14 kV/cm in a Ca2+-free medium, perfusion with 2 mM of external Ca2+ advanced the 90% resealing and reduced the dye uptake about twofold. Membrane restoration was accomplished by a combination of fast, Ca2+-independent resealing (τ = 13–15 s) and slow, Ca2+-dependent processes (τ ~70 s with Ca2+ and ~ 110 s or more without it). These time constants did not change when the membrane damage was doubled by increasing EP duration from 300 to 600 ns. However, injury by microsecond-range EP (300 and 600 μs) took longer to recover even when the membrane initially was less damaged, presumably because of the larger size of pores made in the membrane. Full membrane recovery was not prevented by blocking both extra- and intracellular Ca2+ (by loading cells with BAPTA or after Ca2+ depletion from the reticulum), suggesting the recruitment of unknown Ca2+-independent repair mechanisms.
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•Cell membrane lesions after electric injury can be repaired in the absence of Ca2+•Ca2+ facilitates resealing after membrane injury by nano- and microsecond pulses•Injury triggers resealing processes with time constants from 10 to 15 s to over 100 s•The time constants depend on the type but not on the severity of the injury•External Ca2+ affects largely the slow resealing processes
•Implementation of electropermeabilization-assisted into liquid biphasic flotation.•Greener and non-thermal technique for the permeabilization of cell membrane.•Optimized conditions achieved yield ...extracted fucoxanthin of 16.09 ± 0.27 mg/g.•Extracted fucoxanthin exhibited satisfactory antioxidant activities based on TEAC and TPC.
Electropermeabilization-assisted liquid biphasic flotation (LBF) system is an emerging extraction system facilitated by the adsorptive bubble separation and the electroporation in an aqueous two-phase medium. This integrative extraction system is well suited for the direct recovery of intracellular biocompounds from cells without the needs of discrete steps of mid- or down-stream bioprocessing. The potential of electropermeabilization-assisted LBF system in bioseparation was demonstrated in this study by using the diatom Chaetoceros calcitrans (C. calcitrans) as the source of fucoxanthin, which is a promising antioxidant highly demanded by food and pharmaceutical industries. The extraction performances of LBF and electropermeabilization-assisted LBF system were comprehensively evaluated under the optimal operating conditions. Comparatively, the optimized LBF and electropermeabilization-assisted LBF systems achieved the satisfactory yields of fucoxanthin, i.e., 14.78 mg/g and 16.09 mg/g, respectively. The good recovery of fucoxanthin using electropermeabilization-assisted LBF system could be attributed to the higher release of fucoxanthin from the electrotreated C. calcitrans, allowing a higher partition of fucoxanthin to the top phase of LBF system (236.72 as compared to 152.15 from LBF system). In addition, the fucoxanthin extracted suing both methods exhibit satisfactory antioxidant activities. The application of electropermeabilization-assisted LBF system in the extraction of fucoxanthin is a greener and highly efficient bioprocessing route which can be prospectively extended to other biocompound extraction from microalgal sources.
Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules ...to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials.
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•Electroporative uptake of propidium iodide increases when actin networks are chemically disrupted.•Electroporative uptake of propidium iodide is a temperature dependent process.•Activation energy barrier of electroporation is reduced when the actin networks are disrupted.•The observed reduction in activation energy barrier is possibly due to altered cell membrane mechanical properties.
Objective: Electroporation-based therapies (EBTs) employ high voltage pulsed electric fields (PEFs) to permeabilize tumor tissue; this results in changes in electrical properties detectable using ...electrical impedance spectroscopy (EIS). Currently, commercial potentiostats for EIS are limited by impedance spectrum acquisition time (<inline-formula><tex-math notation="LaTeX">\sim </tex-math></inline-formula>10 s); this timeframe is much larger than pulse periods used with EBTs (<inline-formula><tex-math notation="LaTeX">\sim </tex-math></inline-formula>1 s). In this study, we utilize rapid EIS techniques to develop a methodology for characterizing electroporation (EP) and thermal effects associated with high-frequency irreversible EP (H-FIRE) in real-time by monitoring inter-burst impedance changes. Methods: A charge-balanced, bipolar rectangular chirp signal is proposed for rapid EIS. Validation of rapid EIS measurements against a commercial potentiostat was conducted in potato tissue using flat-plate electrodes and thereafter for the measurement of impedance changes throughout IRE treatment. Flat-plate electrodes were then utilized to uniformly heat potato tissue; throughout high-voltage H-FIRE treatment, low-voltage inter-burst impedance measurements were used to continually monitor impedance change and to identify a frequency at which thermal effects are delineated from EP effects. Results: Inter-burst impedance measurements (1.8 kHz - 4.93 MHz) were accomplished at 216 discrete frequencies. Impedance measurements at frequencies above <inline-formula><tex-math notation="LaTeX">\sim </tex-math></inline-formula>1 MHz served to delineate thermal and EP effects in measured impedance. Conclusion: We demonstrate rapid-capture (<inline-formula><tex-math notation="LaTeX">< < </tex-math></inline-formula>1 s) EIS which enables monitoring of inter-burst impedance in real-time. For the first time, we show impedance analysis at high frequencies can delineate thermal effects from EP effects in measured impedance. Significance: The proposed waveform demonstrates the potential to perform inter-burst EIS using PEFs compatible with existing pulse generator topologies.
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