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1.
J Cardiovasc Dev Dis ; 10(12)2023 Dec 07.
Article in English | MEDLINE | ID: mdl-38132658

ABSTRACT

Electroporation is used in medicine for drug and gene delivery, and as a nonthermal ablation method in tumor treatment and cardiac ablation. Electroporation involves delivering high-voltage electric pulses to target tissue; however, this can cause effects beyond the intended target tissue like nerve stimulation, muscle contractions and pain, requiring use of sedatives or anesthetics. It was previously shown that adjusting pulse parameters may mitigate some of these effects, but not how these adjustments would affect electroporation's efficacy. We investigated the effect of varying pulse parameters such as interphase and interpulse delay while keeping the duration and number of pulses constant on nerve stimulation, muscle contraction and assessing pain and electroporation efficacy, conducting experiments on human volunteers, tissue samples and cell lines in vitro. Our results show that using specific pulse parameters, particularly short high-frequency biphasic pulses with short interphase and long interpulse delays, reduces muscle contractions and pain sensations in healthy individuals. Higher stimulation thresholds were also observed in experiments on isolated swine phrenic nerves and human esophagus tissues. However, changes in the interphase and interpulse delays did not affect the cell permeability and survival, suggesting that modifying the pulse parameters could minimize adverse effects while preserving therapeutic goals in electroporation.

2.
Pharmaceutics ; 14(12)2022 Dec 02.
Article in English | MEDLINE | ID: mdl-36559197

ABSTRACT

The exact mechanisms of nucleic acid (NA) delivery with gene electrotransfer (GET) are still unknown, which represents a limitation for its broader use. Further, not knowing the effects that different experimental electrical and biological parameters have on GET additionally hinders GET optimization, resulting in the majority of research being performed using a trial-and-error approach. To explore the current state of knowledge, we conducted a systematic literature review of GET papers in in vitro conditions and performed meta-analyses of the reported GET efficiency. For now, there is no universal GET strategy that would be appropriate for all experimental aims. Apart from the availability of the required electroporation device and electrodes, the choice of an optimal GET approach depends on parameters such as the electroporation medium; type and origin of cells; and the size, concentration, promoter, and type of the NA to be transfected. Equally important are appropriate controls and the measurement or evaluation of the output pulses to allow a fair and unbiased evaluation of the experimental results. Since many experimental electrical and biological parameters can affect GET, it is important that all used parameters are adequately reported to enable the comparison of results, as well as potentially faster and more efficient experiment planning and optimization.

3.
Pharmaceutics ; 14(9)2022 Aug 30.
Article in English | MEDLINE | ID: mdl-36145573

ABSTRACT

Gene electrotransfer (GET) is considered one of the most efficient, safe, reproducible, and cost-effective methods of gene therapy, in which a gene is delivered to the cells in the form of a plasmid DNA vector by a method known as electroporation. To achieve successful electroporation, cells must be exposed to sufficiently high electric fields generated by short-duration, high-voltage electrical pulses that result in a temporary increase in plasma membrane permeability. The electrical pulses are generated by pulse generators (electroporators) and delivered to the cells via electrodes (applicators). However, there is a lack of standardized pulse delivery protocols as well as certified clinical pulse generators and applicators for gene delivery. In this paper, the development of a new pulse generator, applicator, and pulse delivery protocol for GET to skin cells is presented. A numerical model of electroporated skin developed and tested for two electrode configurations and two different pulse delivery protocols is also presented. An alternative pulse delivery protocol was proposed. The developed pulse generator, applicator, and the proposed pulse delivery protocol were then used in vivo for GET to skin cells in mice. The results showed high efficiency of the proposed pulse delivery protocol for the purpose of GET in mouse skin cells. Specifically, electroporation with the developed pulse generator, applicator, and proposed pulse delivery protocol resulted in higher gene expression in skin cells compared to the currently used pulse generator, applicator, and pulse delivery protocol.

4.
Sci Rep ; 12(1): 8019, 2022 05 16.
Article in English | MEDLINE | ID: mdl-35577873

ABSTRACT

To minimize neuromuscular electrical stimulation during electroporation-based treatments, the replacement of long monophasic pulses with bursts of biphasic high-frequency pulses in the range of microseconds was suggested in order to reduce muscle contraction and pain sensation due to pulse application. This treatment modality appeared under the term high-frequency electroporation (HF-EP), which can be potentially used for some clinical applications of electroporation such as electrochemotherapy, gene electrotransfer, and tissue ablation. In cardiac tissue ablation, which utilizes irreversible electroporation, the treatment is being established as Pulsed Field Ablation. While the reduction of muscle contractions was confirmed in multiple in vivo studies, the reduction of pain sensation in humans was not confirmed yet, nor was the relationship between muscle contraction and pain sensation investigated. This is the first study in humans examining pain sensation using biphasic high-frequency electroporation pulses. Twenty-five healthy individuals were subjected to electrical stimulation of the tibialis anterior muscle with biphasic high-frequency pulses in the range of few microseconds and both, symmetric and asymmetric interphase and interpulse delays. Our results confirm that biphasic high-frequency pulses with a pulse width of 1 or 2 µs reduce muscle contraction and pain sensation as opposed to currently used longer monophasic pulses. In addition, interphase and interpulse delays play a significant role in reducing the muscle contraction and/or pain sensation. The study shows that the range of the optimal pulse parameters may be increased depending on the prerequisites of the therapy. However, further evaluation of the biphasic pulse protocols presented herein is necessary to confirm the efficiency of the newly proposed HF-EP.


Subject(s)
Electrochemotherapy , Electroporation , Electroporation/methods , Humans , Muscle Contraction , Pain , Sensation
5.
Clin Ther ; 42(8): 1535-1548.e2, 2020 08.
Article in English | MEDLINE | ID: mdl-32768246

ABSTRACT

PURPOSE: Electrochemotherapy is increasingly entering into national and international guidelines, requiring formal evaluation of treatment costs and cost-effectiveness to ensure that its uptake provides value to budget-constrained health care systems. This study analyzed the early cost-effectiveness of electrochemotherapy in patients with Stage IIIc/IV skin melanoma in clinical practice in Slovenia. The costs of electrochemotherapy were compared to those of the standard of care, consisting of palliative treatment and therapy for symptoms. METHODS: wThe study enrolled 23 patients treated with electrochemotherapy at the Institute of Oncology (Ljubljana, Slovenia). The mean cost of electrochemotherapy was estimated using patient-specific cost data on electrochemotherapy procedures and subsequent follow-up. Quality-adjusted life-years (QALYs) were estimated by collecting EQ-5D-3L questionnaires at baseline, after complete or partial response following the treatment, and after a relapse of skin lesions. A discrete-time Markov model was built to estimate the lifetime costs and consequences of using electrochemotherapy compared to standard of care, from the perspective of the Slovenian health care system. The analysis was conducted separately in the whole patient sample and in the subset of patients with bleeding lesions. Deterministic and probabilistic sensitivity analyses were conducted to test model assumptions and to characterize the uncertainty around model parameters. FINDINGS: In the whole patient population, electrochemotherapy for skin melanoma Stage IIIc/IV was expected to increase QALYs by 0.29 (95% credible interval [CrI], 0.10-0.50), at the higher cost of 6568 EUR (95% CrI, 4593-8928) in comparison to the standard of care. At the cost-effectiveness threshold of 20,000 EUR/QALY, the estimated probabilities of electrochemotherapy being cost-effective compared to standard of care were 0.30 and 0.91 in the whole patient sample and in patients with bleeding lesions, respectively. In the whole sample population, a 50% reduction in the price of the electrodes was expected to increase the probability of electrochemotherapy being cost-effective from 0.30 to ~0.64. IMPLICATIONS: The findings from this cost-effectiveness analysis of data from clinical practice were based on a small sample size (ie, 23 patents), which made the subgroup of patients with bleeding lesions very small. Therefore, the findings in this patient population should be carefully interpreted.


Subject(s)
Electrochemotherapy/economics , Melanoma/economics , Skin Neoplasms/economics , Aged , Aged, 80 and over , Cost-Benefit Analysis , Female , Health Care Costs , Humans , Male , Melanoma/drug therapy , Middle Aged , Quality-Adjusted Life Years , Skin Neoplasms/drug therapy
6.
Sci Rep ; 10(1): 9149, 2020 06 04.
Article in English | MEDLINE | ID: mdl-32499601

ABSTRACT

In gene electrotransfer and cardiac ablation with irreversible electroporation, treated muscle cells are typically of elongated shape and their orientation may vary. Orientation of cells in electric field has been reported to affect electroporation, and hence electrodes placement and pulse parameters choice in treatments for achieving homogeneous effect in tissue is important. We investigated how cell orientation influences electroporation with respect to different pulse durations (ns to ms range), both experimentally and numerically. Experimentally detected electroporation (evaluated separately for cells parallel and perpendicular to electric field) via Ca2+ uptake in H9c2 and AC16 cardiomyocytes was numerically modeled using the asymptotic pore equation. Results showed that cell orientation affects electroporation extent: using short, nanosecond pulses, cells perpendicular to electric field are significantly more electroporated than parallel (up to 100-times more pores formed), and with long, millisecond pulses, cells parallel to electric field are more electroporated than perpendicular (up to 1000-times more pores formed). In the range of a few microseconds, cells of both orientations were electroporated to the same extent. Using pulses of a few microseconds lends itself as a new possible strategy in achieving homogeneous electroporation in tissue with elongated cells of different orientation (e.g. electroporation-based cardiac ablation).


Subject(s)
Cell Shape/physiology , Electroporation/methods , Animals , Cell Line , Cell Movement/physiology , Fura-2/chemistry , Humans , Microscopy, Fluorescence , Myocytes, Cardiac/cytology , Rats
7.
Bioelectrochemistry ; 132: 107442, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31923714

ABSTRACT

It was recently suggested that applying high-frequency short biphasic pulses (HF-IRE) reduces pain and muscle contractions in electrochemotherapy and irreversible ablation treatments; however, higher amplitudes with HF-IRE pulses are required to achieve a similar effect as with monophasic pulses. HF-IRE pulses are in the range of a microseconds, thus, the so-called cancellation effect could be responsible for the need to apply pulses of higher amplitudes. In cancellation effect, the effect of first pulse is reduced by the second pulse of opposite polarity. We evaluated cancellation effect with high-frequency biphasic pulses on CHO-K1 in different electroporation buffers. We applied eight bursts of 1-10 µs long pulses with inter-phase delays of 0.5 µs - 10 ms and evaluated membrane permeability and cell survival. In permeability experiments, cancellation effect was not observed in low-conductivity buffer. Cancellation effect was, however, observed in treatments with high-frequency biphasic pulses looking at survival in all of the tested electroporation buffers. In general, cancellation effect depended on inter-phase delay as well as on pulse duration, i.e. longer pulses and longer interphase delay cause less pronounced cancellation effect. Cancellation effect could be partially explained by the assisted discharge and not by the hyperpolarization by the chloride channels.


Subject(s)
Electroporation/methods , Animals , CHO Cells , Cell Membrane Permeability , Cricetulus
8.
Radiol Oncol ; 53(2): 194-205, 2019 06 01.
Article in English | MEDLINE | ID: mdl-31194692

ABSTRACT

Background In electrochemotherapy (ECT), chemotherapeutics are first administered, followed by short 100 µs monopolar pulses. However, these pulses cause pain and muscle contractions. It is thus necessary to administer muscle relaxants, general anesthesia and synchronize pulses with the heart rhythm of the patient, which makes the treatment more complex. It was suggested in ablation with irreversible electroporation, that bursts of short high-frequency bipolar pulses could alleviate these problems. Therefore, we designed our study to verify if it is possible to use high-frequency bipolar pulses (HF-EP pulses) in electrochemotherapy. Materials and methods We performed in vitro experiments on mouse skin melanoma (B16-F1) cells by adding 1-330 µM cisplatin and delivering either (a) eight 100 µs long monopolar pulses, 0.4-1.2 kV/cm, 1 Hz (ECT pulses) or (b) eight bursts at 1 Hz, consisting of 50 bipolar pulses. One bipolar pulse consisted of a series of 1 µs long positive and 1 µs long negative pulse (0.5-5 kV/cm) with a 1 µs delay in-between. Results With both types of pulses, the combination of electric pulses and cisplatin was more efficient in killing cells than cisplatin or electric pulses only. However, we needed to apply a higher electric field in HF-EP (3 kV/cm) than in ECT (1.2 kV/cm) to obtain comparable cytotoxicity. Conclusions It is possible to use HF-EP in electrochemotherapy; however, at the expense of applying higher electric fields than in classical ECT. The results obtained, nevertheless, offer an evidence that HF-EP could be used in electrochemotherapy with potentially alleviated muscle contractions and pain.


Subject(s)
Antineoplastic Agents/administration & dosage , Cisplatin/administration & dosage , Electrochemotherapy/methods , Melanoma, Experimental/drug therapy , Muscle Contraction , Pain, Procedural/prevention & control , Animals , Cell Line, Tumor , Cell Membrane Permeability/physiology , Cell Survival , Electrochemotherapy/adverse effects , Electroporation/methods , In Vitro Techniques , Indicators and Reagents/administration & dosage , Mice , Propidium/administration & dosage , Time Factors
9.
IEEE Trans Biomed Eng ; 66(12): 3526-3533, 2019 12.
Article in English | MEDLINE | ID: mdl-30908188

ABSTRACT

Nanosecond electroporation of cell organelles is being studied since more than a decade, but it is still not entirely understood. Unique prototype hardware equipment and challenging measuring methods may also be a contributing reason for this situation. In the scope of this paper, we improve the performance of the high-voltage nanosecond pulse generator by introducing silicon carbide (SiC) mosfets. We developed a new high-voltage diode opening switch (DOS)-nanosecond pulse generator for laboratory use for in vitro experiments in electroporation cuvettes. Analysis and comparison of the most commonly used switching technologies in pulse generators were made. The device is designed by two parallel two-stage Marx-bank circuits with SiC mosfets that generates up to 200 A in the resonant network. A driving circuit for stable simultaneous switching of SiC mosfets was developed. The developed generator can deliver from 500 V to more than 6 kV, approximately 8 ns pulses to a 50 Ω load. Even though the amplitude of the output pulse is not as high as expected, the multiplication factor [Formula: see text] is still approximately 9, which is an improvement compared to the previously published linear DOS generator. Measurement and evaluation process is described in detail. Additionally, we emphasize on the size of an error that occurs during measurements.


Subject(s)
Carbon Compounds, Inorganic/chemistry , Electroporation/instrumentation , Nanotechnology/instrumentation , Semiconductors , Silicon Compounds/chemistry , Equipment Design
10.
Bioelectrochemistry ; 122: 103-114, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29621662

ABSTRACT

In electroporation-based medical treatments, excitable tissues are treated, either intentionally (irreversible electroporation of brain cancer, gene electrotransfer or ablation of the heart muscle, gene electrotransfer of skeletal muscles), or unintentionally (excitable tissues near the target area). We investigated how excitable and non-excitable cells respond to electric pulses, and if electroporation could be an effective treatment of the tumours of the central nervous system. For three non-excitable and one excitable cell line, we determined a strength-duration curve for a single pulse of 10ns-10ms. The threshold for depolarization decreased with longer pulses and was higher for excitable cells. We modelled the response with the Lapicque curve and the Hodgkin-Huxley model. At 1µs a plateau of excitability was reached which could explain why high-frequency irreversible electroporation (H-FIRE) electroporates but does not excite cells. We exposed cells to standard electrochemotherapy parameters (8×100µs pulses, 1Hz, different voltages). Cells behaved similarly which indicates that electroporation most probably occurs at the level of lipid bilayer, independently of the voltage-gated channels. These results could be used for optimization of electric pulses to achieve maximal permeabilization and minimal excitation/pain sensation. In the future, it should be established whether the in vitro depolarization correlates to nerve/muscle stimulation and pain sensation in vivo.


Subject(s)
Cell Membrane Permeability , Cell Membrane/metabolism , Electroporation/methods , Animals , CHO Cells , Cell Line , Cricetulus , Electrochemotherapy , Electroporation/instrumentation , Equipment Design , Humans , Mice
11.
BMC Microbiol ; 16(1): 148, 2016 07 12.
Article in English | MEDLINE | ID: mdl-27405351

ABSTRACT

BACKGROUND: In the context of spore contamination involved in bio-terrorism and food preservation, the development of new techniques for spore inactivation is an important challenge. RESULTS: Here, a successful application of electric arc discharges resulting in spore death was reported. Two types of electric arcs were compared, different with respect to their durations. The discharges with 0.5 µs duration induced a small inactivation area of 0.6 % of surface treated around their point of entry into the sample, while those with 20 µs duration induced a much larger inactivation area from 7 to 55 % of surface treated roughly proportional to the number of discharges delivered. In particular, 50 discharges of 20 µs duration induced inactivation in more than 55% of surface treated at an inactivation rate above 3.6 log10. CONCLUSIONS: These results are promising and warrant developing electric arcing as a novel method for spore inactivation.


Subject(s)
Bacillus pumilus/physiology , Spores, Bacterial/physiology , Colony Count, Microbial , Electricity , Microbial Viability , Water
12.
Biochim Biophys Acta ; 1858(11): 2689-2698, 2016 11.
Article in English | MEDLINE | ID: mdl-27372268

ABSTRACT

High-frequency bipolar electric pulses have been shown to mitigate undesirable muscle contraction during irreversible electroporation (IRE) therapy. Here, we evaluate the potential applicability of such pulses for introducing exogenous molecules into cells, such as in electrochemotherapy (ECT). For this purpose we develop a method for calculating the time course of the effective permeability of an electroporated cell membrane based on real-time imaging of propidium transport into single cells that allows a quantitative comparison between different pulsing schemes. We calculate the effective permeability for several pulsed electric field treatments including trains of 100µs monopolar pulses, conventionally used in IRE and ECT, and pulse trains containing bursts or evenly-spaced 1µs bipolar pulses. We show that shorter bipolar pulses induce lower effective membrane permeability than longer monopolar pulses with equivalent treatment times. This lower efficiency can be attributed to incomplete membrane charging. Nevertheless, bipolar pulses could be used for increasing the uptake of small molecules into cells more symmetrically, but at the expense of higher applied voltages. These data indicate that high-frequency bipolar bursts of electrical pulses may be designed to electroporate cells as effectively as and more homogeneously than conventional monopolar pulses.


Subject(s)
Cell Membrane/metabolism , Electroporation/methods , Propidium/metabolism , Single-Cell Analysis/methods , Animals , Biological Transport , CHO Cells , Cell Membrane Permeability , Cricetulus , Electrodes , Membrane Potentials/physiology , Single-Cell Analysis/instrumentation
13.
Bioelectrochemistry ; 110: 1-12, 2016 Aug.
Article in English | MEDLINE | ID: mdl-26946156

ABSTRACT

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: 1-10ns, B: 11-100ns and C: 101-999ns. 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.


Subject(s)
Electricity , Eukaryotic Cells , Animals , Eukaryotic Cells/cytology , Humans , Statistics as Topic
14.
IEEE Trans Biomed Eng ; 62(10): 2535-43, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26389644

ABSTRACT

GOAL: We aimed to develop a system for controlled exposure of biological samples to conditions they experience when lightning strikes their habitats. METHODS: We based the generator on a capacitor charged via a bridge rectifier and a dc-dc converter, and discharged via a relay, delivering arcs similar to natural lightning strokes in electric current waveform and similarly accompanied by acoustic shock waves. We coupled the generator to our exposure chamber described previously, measured electrical and acoustic properties of arc discharges delivered, and assessed their ability to inactivate bacterial spores. RESULTS: Submicrosecond discharges descended vertically from the conical emitting electrode across the air gap, entering the sample centrally and dissipating radially toward the ring-shaped receiving electrode. In contrast, longer discharges tended to short-circuit the electrodes. Recording at 341 000 FPS with Vision Research Phantom v2010 camera revealed that initial arc descent was still vertical, but became accompanied by arcs leaning increasingly sideways; after 8-12 µs, as the first of these arcs formed direct contact with the receiving electrode, it evolved into a channel of plasmified air and short-circuited the electrodes. We eliminated this artefact by incorporating an insulating cylinder concentrically between the electrodes, precluding short-circuiting between them. While bacterial spores are highly resistant to electric pulses delivered through direct contact, we showed that with arc discharges accompanied by an acoustic shock wave, spore inactivation is readily obtained. CONCLUSION: The presented system allows scientific investigation of effects of arc discharges on biological samples. SIGNIFICANCE: This system will allow realistic experimental studies of lightning-triggered horizontal gene transfer and assessment of its role in evolution.


Subject(s)
Gene Transfer, Horizontal/radiation effects , Lightning , Models, Theoretical , Research/instrumentation , Spores, Bacterial/radiation effects , Bacillus/radiation effects , Electricity , Equipment Design , Sound
15.
Sci Rep ; 3: 3382, 2013 Nov 29.
Article in English | MEDLINE | ID: mdl-24287643

ABSTRACT

Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.


Subject(s)
Cell Fusion/methods , Electroporation/methods , Pulse/methods , Animals , CHO Cells , Cell Size , Cricetulus , Electricity , Hybridomas/cytology , Melanoma, Experimental , Mice
16.
Int J Pharm ; 457(1): 214-23, 2013 Nov 30.
Article in English | MEDLINE | ID: mdl-24076397

ABSTRACT

Electroporation can be used as an active enhancement method for intra- and transdermal drug delivery. Differences in response of skin to electric pulses depend on their amplitude, duration and number and have been a point of interest in the past. While protocols consisting of the same repetitive, mostly exponentially decaying pulses have been used before, this study is focused on comparing different combinations of square wave short high voltage (HV) and longer low voltage (LV) electroporation pulses. Our in vitro experimental results show that longer LV pulses significantly increase subsequent passive transport of calcein through dermatomed pig skin, while short HV pulses alone result in negligible calcein passive transdermal transport. Surprisingly, when the long LV pulses are preceded by short duration HV pulses, the total calcein transported is reduced significantly. This result is explained using a theoretical physics based model of individual local transport region (LTR) evolution during the applied LV pulse. The theoretical model shows that HV pulses alter the structure of the stratum corneum in such a way that when the LV pulses are applied, insufficient thermal energy is generated to initiate LTR expansion. Together, the experimental results and theoretical predictions show that the total pulse energy alone cannot account for total solute transport: that the order of the types of pulses administered must also be considered. Our findings open a direction for further improvement of the method using new protocols.


Subject(s)
Drug Delivery Systems , Electroporation , Skin/metabolism , Administration, Cutaneous , Animals , Swine
17.
J Membr Biol ; 246(11): 851-9, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23831956

ABSTRACT

Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.


Subject(s)
Gene Expression , Green Fluorescent Proteins/genetics , Animals , CHO Cells , Cell Membrane Permeability , Cricetinae , Cricetulus , Electric Stimulation , Electroporation , Green Fluorescent Proteins/biosynthesis , HCT116 Cells , Humans , Nuclear Envelope/metabolism , Time Factors , Transfection
18.
J Membr Biol ; 245(10): 643-50, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22864453

ABSTRACT

Inactivation of microorganisms with pulsed electric fields is one of the nonthermal methods most commonly used in biotechnological applications such as liquid food pasteurization and water treatment. In this study, the effects of microsecond and nanosecond pulses on inactivation of Escherichia coli in distilled water were investigated. Bacterial colonies were counted on agar plates, and the count was expressed as colony-forming units per milliliter of bacterial suspension. Inactivation of bacterial cells was shown as the reduction of colony-forming units per milliliter of treated samples compared to untreated control. According to our results, when using microsecond pulses the level of inactivation increases with application of more intense electric field strengths and with number of pulses delivered. Almost 2-log reductions in bacterial counts were achieved at a field strength of 30 kV/cm with eight pulses and a 4.5-log reduction was observed at the same field strength using 48 pulses. Extending the duration of microsecond pulses from 100 to 250 µs showed no improvement in inactivation. Nanosecond pulses alone did not have any detectable effect on inactivation of E. coli regardless of the treatment time, but a significant 3-log reduction was achieved in combination with microsecond pulses.


Subject(s)
Electricity , Escherichia coli/radiation effects , Food Preservation , Water Microbiology , Water Purification
19.
J Vis Exp ; (55)2011 Sep 12.
Article in English | MEDLINE | ID: mdl-21931297

ABSTRACT

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.


Subject(s)
DNA/administration & dosage , Gene Transfer Techniques , Plasmids/genetics , Animals , CHO Cells , Cricetinae , Cricetulus , DNA/chemistry , DNA/genetics , Electroporation/methods , Gene Transfer Techniques/instrumentation , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Plasmids/chemistry
20.
IEEE Trans Biomed Eng ; 58(11): 3279-88, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21900067

ABSTRACT

Electroporation-based applications require the use of specific pulse parameters for a successful outcome. When recommended values of pulse parameters cannot be set, similar outcomes can be obtained by using equivalent pulse parameters. We determined the relations between the amplitude and duration/number of pulses resulting in the same fraction of electroporated cells. Pulse duration was varied from 150 ns to 100 ms, and the number of pulses from 1 to 128. Fura 2-AM was used to determine electroporation of cells to Ca(2+). With longer pulses or higher number of pulses, lower amplitudes are needed for the same fraction of electroporated cells. The expression derived from the model of electroporation could describe the measured data on the whole interval of pulse durations. In a narrower range (0.1-100 ms), less complex, logarithmic or power functions could be used instead. The relation between amplitude and number of pulses could best be described with a power function or an exponential function. We show that relatively simple two-parameter power or logarithmic functions are useful when equivalent pulse parameters for electroporation are sought. Such mathematical relations between pulse parameters can be important in planning of electroporation-based treatments, such as electrochemotherapy and nonthermal irreversible electroporation.


Subject(s)
Electroporation/methods , Models, Biological , Animals , CHO Cells , Calcium/chemistry , Calcium/metabolism , Cricetinae , Cricetulus , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Fura-2/chemistry , Fura-2/metabolism , Temperature
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