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Quantitative Study of Electroporation-Mediated Molecular Uptake and Cell Viability
Abstract
Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.
... Pore distribution models S2011 model [31] Electric field: 0-3.5 kV/cm [45]; 0-2 kV/cm [10] No. pulses: 1 Pulse shape: exponentially decaying [45]; rectangular pulses [10] Pulse duration: 50 µs to 21 ms [45]; 100 µs and 1 ms [10] calcein [45] lucifer yellow [10] electrophoresis and diffusion ...
... Pore distribution models S2011 model [31] Electric field: 0-3.5 kV/cm [45]; 0-2 kV/cm [10] No. pulses: 1 Pulse shape: exponentially decaying [45]; rectangular pulses [10] Pulse duration: 50 µs to 21 ms [45]; 100 µs and 1 ms [10] calcein [45] lucifer yellow [10] electrophoresis and diffusion ...
... Pore distribution models S2011 model [31] Electric field: 0-3.5 kV/cm [45]; 0-2 kV/cm [10] No. pulses: 1 Pulse shape: exponentially decaying [45]; rectangular pulses [10] Pulse duration: 50 µs to 21 ms [45]; 100 µs and 1 ms [10] calcein [45] lucifer yellow [10] electrophoresis and diffusion ...
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 across 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.
... Scientists have developed different multiphysics and multiscale models to describe the phenomenon 82 of electroporation and molecular transport through the electroporated membrane. Many of these 83 models have been compared qualitatively and/or quantitatively with experimental measurements of 84 small molecule uptake into cells, as well as other measurements, such as an increase in membrane 85 Electric field: 0-3.5 kV/cm [45]; 0-2 kV/cm [10] No. pulses: 1 Pulse shape: exponentially decaying [45]; rectangular pulses [10] Pulse duration: 50 µs to 21 ms [45]; 100 µs and 1 ms [10] calcein [45] lucifer yellow [10] electrophoresis and diffusion Quantitative comparison with experimental measurements of calcein uptake into DU 145 prostate cancer cells exposed to exponentially decaying pulses [45] and lucifer yellow uptake into DC3F fibroblasts exposed to rectangular pulses [10]. ...
... Scientists have developed different multiphysics and multiscale models to describe the phenomenon 82 of electroporation and molecular transport through the electroporated membrane. Many of these 83 models have been compared qualitatively and/or quantitatively with experimental measurements of 84 small molecule uptake into cells, as well as other measurements, such as an increase in membrane 85 Electric field: 0-3.5 kV/cm [45]; 0-2 kV/cm [10] No. pulses: 1 Pulse shape: exponentially decaying [45]; rectangular pulses [10] Pulse duration: 50 µs to 21 ms [45]; 100 µs and 1 ms [10] calcein [45] lucifer yellow [10] electrophoresis and diffusion Quantitative comparison with experimental measurements of calcein uptake into DU 145 prostate cancer cells exposed to exponentially decaying pulses [45] and lucifer yellow uptake into DC3F fibroblasts exposed to rectangular pulses [10]. ...
... Scientists have developed different multiphysics and multiscale models to describe the phenomenon 82 of electroporation and molecular transport through the electroporated membrane. Many of these 83 models have been compared qualitatively and/or quantitatively with experimental measurements of 84 small molecule uptake into cells, as well as other measurements, such as an increase in membrane 85 Electric field: 0-3.5 kV/cm [45]; 0-2 kV/cm [10] No. pulses: 1 Pulse shape: exponentially decaying [45]; rectangular pulses [10] Pulse duration: 50 µs to 21 ms [45]; 100 µs and 1 ms [10] calcein [45] lucifer yellow [10] electrophoresis and diffusion Quantitative comparison with experimental measurements of calcein uptake into DU 145 prostate cancer cells exposed to exponentially decaying pulses [45] and lucifer yellow uptake into DC3F fibroblasts exposed to rectangular pulses [10]. ...
... electropores in droplet interface bilayers with a time resolution of 16 ms [3]. Indirect experimental methods probing membrane conductivity [4], molecular uptake [5,6], and gene expression [7] have often been used to gain a better understanding of electroporation. However, using these methods alone tells us little about the biophysical processes involved in electroporation. ...
... In order to further validate results from models and simulations, comparisons with experimental data must also be made. In the past, good quantitative agreement has been shown between the continuum Smoluchowski electroporation model and experimental data of fluorescent dye uptake during electroporation [5,6,14]. However, almost all experimental and computational studies have focused on the case of high applied electric field strength, in which case a large number of pores are created [2,5,6,[12][13][14]17,18]. Some experimental studies have shown that when artificial lipid bilayers are clamped at a low voltage, a single pore can be observed [3,4]. ...
... In the past, good quantitative agreement has been shown between the continuum Smoluchowski electroporation model and experimental data of fluorescent dye uptake during electroporation [5,6,14]. However, almost all experimental and computational studies have focused on the case of high applied electric field strength, in which case a large number of pores are created [2,5,6,[12][13][14]17,18]. Some experimental studies have shown that when artificial lipid bilayers are clamped at a low voltage, a single pore can be observed [3,4]. ...
Current-Clamp electroporation refers to the application of a constant current across a membrane which results in voltage fluctuations due to the creation of electropores. This method allows for the measurement of electroporation across a long timescale (minutes) and facilitates the comparison between experimental and theoretical studies. Of particular interest is the claim in the literature that current-clamp electroporation results in the creation of a single pore. We simulated current-clamp electroporation using the Smoluchowski and Langevin equations and identified two possible mechanisms to explain the observed voltage fluctuations. The voltage fluctuations may be due to a single pore or a few pores growing and shrinking via a negative feedback mechanism or the opening and closing of pores in a larger population of pores. Our results suggest that current-clamp conditions do not necessarily result in the creation of a single pore. Additionally, we showed that the Langevin model is more accurate than the Smoluchowski model under conditions where there are only a few pores.
... This requirement limits protein overexpression, particularly in mammalian cells, which do not grow in perdeuterated medium. Exogenous delivery of target proteins using techniques such as microinjection 8,15 , cell-penetrating peptides 16 , creation of pores 10 and electroporation 17,18 ; limit cell viability 19 and may perturb the physiological state of the cells 20,21 by impeding homeostasis and cell growth 20 . ...
... The viability of electroporated cells after 12 h was 85-90%, comparable to the 75-85% observed by Theillet et al. 18 , i.e., 15-25% dead cells after >13 h. Overall, the higher attrition rates of electroporated versus VECT-transfected cells were consistent with the idea that the electroporated cells were more extensively damaged 19 . VECT-delivery of DARPP32 to HeLa cells. ...
... Unlike the case of VECT protein delivery, control experiments examining the supernatant of electroporated samples revealed sharp 1 H-15 N cross-peaks consistent with leakage of labeled target protein from the cells ( Supplementary Fig. 3). This likely reflects the loss of integrity of plasma and nuclear membranes and other organelles due to the electroporation process 19,27,28 . The combination of prolonged cell viability and the absence of cell leakage suggests that VECT is a simple and reliable method to deliver exogenous target proteins for longduration in-cell NMR studies. ...
High-resolution structural studies of proteins and protein complexes in a native eukaryotic environment present a challenge to structural biology. In-cell NMR can characterize atomic resolution structures but requires high concentrations of labeled proteins in intact cells. Most exogenous delivery techniques are limited to specific cell types or are too destructive to preserve cellular physiology. The feasibility of microfluidics transfection or volume exchange for convective transfer, VECT, as a means to deliver labeled target proteins to HeLa cells for in-cell NMR experiments is demonstrated. VECT delivery does not require optimization or impede cell viability; cells are immediately available for long-term eukaryotic in-cell NMR experiments. In-cell NMR-based drug screening using VECT was demonstrated by collecting spectra of the sensor molecule DARPP32, in response to exogenous administration of Forskolin. The microfluidic technique of cell volume exchange for convective transfer, VECT can be used to deliver the DARPP-32 protein into cells for in-cell NMR experiments.
... It is well known that the electric field effect to the cells depends on electric pulse parameters such as electric field strength, duration and the number of electric pulses [10,11]. If the parameters of the electric pulses are kept sufficiently low, it is possible to achieve efficient transmembrane transport without compromising cell viability [12]. However, when the electric field parameters are high, the loss in cell viability is substantial [12,13]. ...
... If the parameters of the electric pulses are kept sufficiently low, it is possible to achieve efficient transmembrane transport without compromising cell viability [12]. However, when the electric field parameters are high, the loss in cell viability is substantial [12,13]. The death of cells due to the overexposure of electric fields is the basis for non-thermal tumour ablation termed irreversible electroporation [14,15]. ...
... It is well known that the effect of the electric pulses on the cell depends on the electric pulse parameters [12,44]. Increase in either of these parameters above certain thresholds will result in the decrease of cell viability [45,46]. ...
Control of membrane permeability to exogenous compounds by membrane electroporation can lead to cell death, which is related to permanent membrane damage, oxidation stress, leakage of intracellular molecules. In this study, we show that the predominant cell death modality after the application of high voltage electric pulses is related with inability to reseal of initial pores (first stage irreversible electroporation, FirEP). After moderately strong electric pulses, initial pores reseal, however, some cell still die later on due to electric field induced cell stress which leads to delayed cell death (late-stage irreversible electroporation, LirEP). According to our data, the period in which the majority of cells commit to either pore resealing or complete loss of barrier function depends on the intensity of electric field treatment but did not exceed 35 min. Additionally, we show that after electroporation using electric pulse parameters that induce LirEP, some cells can be rescued by supplementing medium with compounds obtained from irreversibly electroporated cells. We determined that the intracellular molecules that contribute to the increase of cell viability are larger than 30 kDa. This serves to prove that the loss of intracellular compounds plays a significant role in the decrease of cell viability after electroporation.
... The main characteristics of the pulse protocol are the applied amplitude of pulse (peak voltage), duration of pulse, t i , and time interval between pulses, Δt, (or pulse (Canatella et al. 2001). Typically, the pulse duration is short in comparison with the time between pulses. ...
... The total time of PEF treatment is regulated by variation of the number of series N and it can be calculated as t PEF ¼ nNt i . The main characteristics of the pulse protocol are the applied amplitude of pulse (peak voltage), duration of pulse, t i , and time interval between pulses, Δt, (or pulse repetition rate f ¼ 1/Δt) (Canatella et al. 2001). Typically, the pulse duration is short in comparison with the time between pulses. ...
... In general, the higher electric field strength leads to better damage efficiency (Canatella et al. 2001). However, electrical power consumption can be also essential at high fields. ...
This chapter shortly presents the concepts of pulsed electric energy (PEE) generators, treatment protocols and design of batch and continuous treatment chambers (nano- and micro-scale fluidic chips, small-scale cuvettes, laboratory, pilot and industrial scale chambers). The manufacturers of small scale electroporation devices and large scale PEE equipments are also presented with detailing on the types of equipment produced.
... When a cell becomes electroporated, the process of electroporation begins with the application of an electric field on the order of 0.1 to 1.0 MV/m [58,59]. The electric field enhances the resting transmembrane potential (TMP) of the cell (-65 to -70 mV [60]) in a manner that depends on the cell radius [61,62,63,64,65]. ...
... For example, it was noticed that different molecular weight sugars would pass through the cell membrane differently following electroporation [91]. Additionally, estimates of the cellular uptake rates of fluorescent tracer molecules have been made using molecules such as Pro [65,90,92,93,94], YO-PRO-1 and its derivatives [90,93,95], ethidium homodimer [96,97], 7-AAD [98], calcein [58,97], and others [90,99,100]. ...
... Following PEF application, a cell membrane can gradually reseal [87]. The greater the duration of the permeability increase, the less viable the local cell population will become [58,85]. This loss of viability is attributed to the formation of pores within the cell membrane driven by large transmembrane potentials (TMPs) [14,67]. ...
Electroporation-based treatments are motivated by the response of biological membranes to high- intensity pulsed electric fields. These fields rearrange the membrane structure to enhance the membrane's diffusive permeability, or the degree to which a membrane allows molecules to diffuse through it, is impacted by the structure, composition, and environment in which the cell resides. Tracer molecules have been developed that are unable to pass through intact cell membranes yet enter permeabilized cells. This dissertation investigates the hypothesis that the flow of such molecules may be used to quantify the effects of the electrical stimulus and environmental conditions leading to membrane electroporation. Specifically, a series of electrical pulses that alternates between positive and negative pulses permeabilizes cells more symmetrically than a longer pulse with the same total on-time. However, the magnitude of this symmetric entry decreases for the shorter alternating pulses. Furthermore, a method for quantitatively measuring the permeability of the cell membrane was proposed and validated. From data near the electroporation threshold, the response of cells varies widely in the manner in which cells become permeabilized. This method is applied to study the transient cell membrane permeability induced by electroporation and is used to demonstrate that the cell membrane remains permeable beyond 30 min following treatment. To analyze these experimental findings in the context of physical mechanisms, computational models of molecular uptake were developed to simulate electroporation. The results of these simulations indicate that the cell's local environment during electroporation facilitates the degree of molecular uptake. We use these models to predict how manipulating both the environment of cells during electroporation affects the induced membrane permeability. These experimental and computational results provide evidence that supports the hypothesis of this dissertation and provide a foundation for future investigation and simulation of membrane electroporation.
... Electroporation is a technique that uses high-voltage electric pulses to temporarily increase membrane permeability enabling the exchange of the molecules between the cell interior and its surroundings (Canatella et al., 2001;Kotnik et al., 2019). Additionally, the permeable cell membrane has been proven to be fusogenic enabling the fusion of neighboring cells in the close contact (Zimmermann, 1982). ...
... For effective application, one should choose the electric pulses to enable cell membrane permeabilization and at the same time to preserve cell viability. The effects of electric pulse parameters involved in the process have been studied extensively (Canatella et al., 2001;Rols and Teissié, 1998;Zimmermann, 1982) while biophysical characteristics of the treated cells, did not receive enough attention. Molecular dynamics studies reveal that electroporation affects the fluid-disordered membrane phases in local membrane regions of the lipid bilayer (Reigada, 2014) while in more complex biological membranes the role of cell membrane fluidity in gene electrotransfer or cell electrofusion was not established unambiguously (Golzio et al., 2001;Kanduser et al., 2008Kanduser et al., , 2006Rols et al., 1990). ...
In the search for safe induction of cellular and humoral cancer immunity dendritic cell-based therapies hold great potential. This approach is based on manipulation of dendritic cells to activate immune system against specific cancer antigens. For the development of an effective cell vaccine platform, gene transfer, and cell fusion have been used for modification of dendritic or tumor cells to express immune (co)stimulatory signals and to load dendritic cells with tumor antigens. Both, gene transfer and cell fusion can be achieved by single technique, a cell membrane electroporation. The cell membrane exposed to external electric field becomes temporarily permeable, enabling introduction of genetic material, and also fusogenic, enabling the fusion of cells in the close contact. We tested the feasibility of combining gene electrotransfer and electrofusion into a single-step technique. We evaluated the effects of electroporation buffer, pulse parameters, and cell membrane fluidity on the efficacy of the combined method. We determined the percentage of fused cells expressing green fluorescence protein GFP in a murine cell model of melanoma B16F1, an often-used cell line in pre-clinical studies. Our results suggest that gene electrotransfer and cell electrofusion can be applied in a single procedure. The percentage of viable hybrid cells expressing GFP depends on electric pulse parameters and the composition of the electroporation buffer. Cell membrane fluidity cannot be related to the efficiency of this technique. Further optimization of electric pulse parameters and buffers has to be accomplished before this technique can be used for preparation of effective dendritic cell-based vaccines.
... Current methods of overcoming these challenges utilize either vector-based approaches, such as cationic polymers and lipid-based nanoparticle systems, or physical methods, such as electroporation and sonoporation [6][7][8][9][10][11][12]. Physical methods theoretically allow for direct delivery of molecules into the cytoplasm through disruption of the cellular membrane, circumventing the endocytotic process and need for endosomal escape. ...
... While efficient intracellular delivery can be achieved, these methods are often destructive in nature and require more complex setup which limits their usability to ex vivo and in vitro applications [10,13]. Nanoparticles have attracted attention for intracellular delivery due to their small size, high surface-area-to-volume ratio, and chemical tunability [9,10]. While efficient C 2024, 10, 37 2 of 15 cellular uptake of nanoparticles can be achieved through chemical modification, they often suffer from increased cellular toxicity, minimizing their potential impact outside of in vitro applications [14,15]. ...
In recent years, carbon nanotubes have emerged as a potentially revolutionary material with numerous uses in biomedical applications. Compared to other nanoparticles, discrete multiwalled carbon nanotubes (dMWCNTs) have been shown to exhibit advantageous characteristics such as a high surface area-to-volume ratio, biocompatibility, and unique chemical and physical properties. dMWCNTs can be modified to load various molecules such as proteins and nucleic acids and are capable of crossing the cell membrane, making them attractive delivery vehicles for biomolecules. To investigate this, we measured the impact of dMWCNTs on the number of live and dead cells present during different stages of cell proliferation. Furthermore, we used transmission electron microscopy to produce evidence suggesting that dMWCNTs enter the cytoplasm of mammalian cells via an endocytosis-like process and ultimately escape into the cytoplasm. And lastly, we used live-cell staining, qPCR, and a T-cell activation detection assay to quantify the use of dMWCNTs as a delivery vehicle for a toxic, membrane-impermeable peptide, mRNA, siRNA, and a T-cell activating synthetic dsRNA. We demonstrate successful delivery of each payload into a range of cell types, providing further evidence of dMWCNTs as a versatile delivery platform for biomolecular cargo.
... where E 0 , A 0 , k 1 , and k 2 are the regression coefficients that are related to the pulse duration and cell type, having values of 3996 V/cm, 1441 V/cm, 0.03, and 0.06, respectively, as described in the literature [35]. The probability of cell death due to EI can be calculated as follows: ...
... In addition, it should be noted that the influence of temperature on conductivity was small enough to be negligible compared with the change in electrical conductivity for electric field strength [33]. where , , , and are the regression coefficients that are related to the pulse duration and cell type, having values of 3996 V/cm, 1441 V/cm, 0.03, and 0.06, respectively, as described in the literature [35]. The probability of cell death due to EI can be calculated as follows: ...
Irreversible electroporation (IRE) is a promising nonthermal ablation technique that uses high-voltage electrical pulses to create permanent pores in the cell membrane of target tissue. Recently, endoscopic IRE with catheter-based electrodes has attracted significant attention as a potential alternative tool for gastrointestinal tumors, but it has been challenged owing to the limited electric field distribution in an in-plane electrode configuration, in which rectangular interdigitated electrodes (IDEs) are commonly used. Herein, we report an enhanced electrical injury in tissue using triangular IDEs that cause strong electric fields to be induced at the tip of the electrode fingers. A set of 10 pulses with a duration of 100 μs and a frequency of 1 Hz were delivered to the tissue, and a finite element method was used to calculate the electrical injury in the gastrointestinal model. The probability of cell death by electrical injury at the triangular IDEs increases by approximately 10 times compared to that of conventional rectangular IDEs at the same electrode distance. These results could potentially pave the way toward designing electrodes in catheter-based IRE devices.
... For reversible electroporation, several parameters such as field strength, pulse duration and number of pulses need to be carefully selected to preserve cell viability. If the pulse duration and applied field strength prevent the cell from restoring its integrity, disturbance of the electrolyte balance may trigger cell death [33,34]. Each cell type demands an optimized electroporation protocol [34,35], but once determined, up to millions of cells can be treated simultaneously. ...
... If the pulse duration and applied field strength prevent the cell from restoring its integrity, disturbance of the electrolyte balance may trigger cell death [33,34]. Each cell type demands an optimized electroporation protocol [34,35], but once determined, up to millions of cells can be treated simultaneously. However, the strong electric fields used in current electroporation techniques may still lead to significant damage or death to cells [36,37]. ...
Delivery of nanomaterials into cells is of interest for fundamental cell biological research as well as for therapeutic and diagnostic purposes. One way of doing so is by physically disrupting the plasma membrane (PM). Several methods that exploit electrical, mechanical or optical cues have been conceived to temporarily disrupt the PM for intracellular delivery, with variable effects on cell viability. However, apart from acute cytotoxicity, subtler effects on cell physiology may occur as well. Their nature and timing vary with the severity of the insult and the efficiency of repair, but some may provoke permanent phenotypic alterations. With the growing palette of nanoscale delivery methods and applications, comes a need for an in-depth understanding of this cellular response. In this review, we summarize current knowledge about the chronology of cellular events that take place upon PM injury inflicted by different delivery methods. We also elaborate on their significance for cell homeostasis and cell fate. Based on the crucial nodes that govern cell fitness and functionality, we give directions for fine-tuning nano-delivery conditions.
... 18 Increased viability loss is common with physical delivery methods that apply external stress to cell membranes to create transient pores. 20,21 Irreversible cell damage can have applications in targeted ablation of undesirable tissues such as cancerous tumors. 22 However, cell viability loss becomes disadvantageous for intracellular delivery applications meant for research, diagnostic, or therapeutic purposes. ...
... Electroporation can be optimized by controlling electric pulses parameters, as well as modifying the cell medium during exposure. 20,25,60 Sonoporation can similarly be optimized, but the sometimes violent nature of delivery by acoustic cavitation has resulted in difficulty keeping cells viable at strong conditions leading to efficient uptake. 21 Microinjection can have very high delivery efficiency and cell viability, but suffers from extremely low throughput when injecting one cell at atime. ...
Introduction
Intracellular delivery of molecules is central to applications in biotechnology, medicine, and basic research. Nanoparticle-mediated photoporation using carbon black nanoparticles exposed to pulsed, near-infrared laser irradiation offers a physical route to create transient cell membrane pores, enabling intracellular delivery. However, nanoparticle-mediated photoporation, like other physical intracellular delivery technologies, necessitates a trade-off between achieving efficient uptake of exogenous molecules and maintaining high cell viability.
Methods
In this study, we sought to shift this balance by adding serum to cells during nanoparticle-mediated photoporation as a viability protectant. DU-145 prostate cancer cells and human dermal fibroblasts were exposed to laser irradiation in the presence of carbon black (CB) nanoparticles and other formulation additives, including fetal bovine serum (FBS) and polymers.
Results
Our studies showed that FBS can protect cells from viability loss, even at high-fluence laser irradiation conditions that lead to high levels of intracellular delivery in two different mammalian cell types. Further studies revealed that full FBS was not needed: viability protection was achieved with denatured FBS, with just the high molecular weight fraction of FBS (>30 kDa), or even with individual proteins like albumin or hemoglobin. Finally, we found that viability protection was also obtained using certain neutral water-soluble polymers, including Pluronic F127, polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), and polyethylene glycol, which were more effective at increased concentration, molecular weight, or hydrophobicity.
Conclusion
Altogether, these findings suggest an interaction between amphiphilic domains of polymers with the cell membrane to help cells maintain viability, possibly by facilitating transmembrane pore closure. In this way, serum components or synthetic polymers can be used to increase intracellular delivery by nanoparticle-mediated photoporation while maintaining high cell viability.
... For example, viral-mediated transfection has high transfection efficiency, insertion induced sustainable transgene expression, but a higher chance of causing inflammatory responses and insertional mutations; 7,8 the calcium phosphate precipitation method supports large molecular weight plasmid DNA transfection but is more harmful; 9,10 while liposome transfection has no limit to the plasmid size, but is expensive and toxic to some cell types. 11,12 Electroporation, the method used in this study is a technique of introducing macromolecules, such as DNA into cells after temporary micropores are formed in the cell membrane in a high-voltage current pulse environment 13,14 . At present, electroporation technology is constantly advancing, in addition to the traditional cuvette electroporation, there are micro electroporation and nano electroporation. ...
Satellite cells are an important cellular model for studying muscle growth and development and mammalian locomotion-related molecular mechanisms. In this study, we investigated the effects of voltage, pulse duration, and DNA dosage on horse skeletal muscle satellite cells’ electroporation transfection efficiency using the eukaryotic expression plasmid Td Tomato-C1 (5.5 kb) encoding the red fluorescent protein gene mainly based on fluorescence-positive cell rate and cell survival rate. By comparison of different voltages, pulse durations, and DNA doses, horse skeletal muscle satellite cells have nearly 80% transfection efficiency under the condition of voltage 120 V, DNA dosage 7 µg/ml, and pulse duration 30 ms. This optimized electroporation condition would facilitate the application of horse skeletal muscle satellite cells in genetic studies of muscle function and related diseases.
... Current methods of overcoming these challenges utilize either vector-based approaches, such as cationic polymers and lipid-based nanoparticle systems, or physical methods, such as electroporation and sonoporation [4][5][6][7][8][9][10]. Physical methods theoretically allow for direct delivery of molecules into the cytoplasm through disruption of the cellular membrane, circumventing the endocytotic process and need for endosomal escape. ...
In recent years, carbon nanotubes have emerged as a potentially revolutionary material with numerous uses in biomedical applications. Compared to other nanoparticles, discrete multi-walled carbon nanotubes (dMWCNTs) have been shown to exhibit advantageous characteristics such as high surface area to volume ratio, biocompatibility, and unique chemical and physical properties. dMWCNTs can be modified to load various molecules such as proteins and nucleic acids and are capable of crossing the cell membrane, making them attractive delivery vehicles for biomolecules. To investigate this, we measured the impact of dMWCNTs on cell proliferation. Furthermore, we used electron microscopy to demonstrate that dMWCNTs enter the cytoplasm of mammalian cells via an endocytosis-like process. And lastly, we employed various in vitro reporter and gene assays to demonstrate dMWCNT-mediated delivery of peptides, mRNA, siRNA, and dsRNA. Our work here has helped further characterize dMWCNTs as a versatile delivery platform for biomolecular cargo.
... This increase in temperature causes the surrounding medium to produce vapor nanobubbles [44][45][46], which, when collapsed, cause stress on the membrane and generate transitory pores. Cell death results from an imbalance in the concentration of electrolytes after the formation of holes in the cell membrane [47,48], as depicted in Figure 7. The other possible mechanism for cell death is heat transfer between GNS [49][50][51] and the cell membrane. ...
A new generation of nanoscale photosensitizer agents has improved photothermal capabilities, which has increased the impact of photothermal treatments (PTTs) in cancer therapy. Gold nanostars (GNS) are promising for more efficient and less invasive PTTs than gold nanoparticles. However, the combination of GNS and visible pulsed lasers remains unexplored. This article reports the use of a 532 nm nanosecond pulse laser and polyvinylpyrrolidone (PVP)-capped GNS to kill cancer cells with location-specific exposure. Biocompatible GNS were synthesized via a simple method and were characterized under FESEM, UV–visible spectroscopy, XRD analysis, and particle size analysis. GNS were incubated over a layer of cancer cells that were grown in a glass Petri dish. A nanosecond pulsed laser was irradiated on the cell layer, and cell death was verified via propidium iodide (PI) staining. We assessed the effectiveness of single-pulse spot irradiation and multiple-pulse laser scanning irradiation in inducing cell death. Since the site of cell killing can be accurately chosen with a nanosecond pulse laser, this technique will help minimize damage to the cells around the target cells.
... For 10 6 cells/mL concentration, cell packing density is 3.5, and thus the cells should be sufficiently far apart from one another, so that they do not alter the electric field experienced by neighboring cells. [31] ECT enhances the cellular transport of drug molecules to enhance cytotoxicity up to 1000 folds. [32] ECT is a physical technique and it can target multiple phenotypes of cancer, as it utilizes the synergy of chemo drug and electrical pulses. ...
Aim:
The purpose of this research is to study the effect of electrical pulse mediated tomato lipophilic extract (TLE) on human breast cancer MCF-7 and non-tumorigenic MCF-10A cells.
Materials and methods:
MCF-7 and MCF-10A cells were treated with 50 μg/mL TLE and eight 100 μs electric pulses of different electric field intensities (800, 1000, and 1200 V/cm), and the viability was studied using real time MT assay at 24 h of treatment. In addition, we studied cell viability of both the cells at 0 h using trypan blue assay and the ability to form colonies of both cells using colony forming unit (CFU) assay for all the treatments. We also imaged the cells at 24 h using microscope.
Results:
With 50 μg/mL TLE, the cell viability of MCF-7 and MCF-10A was same (84%). When the same concentration of TLE is combined with eight electrical pulses of 1200 V/cm, the cell viability of MCF-7 and MCF-10A was 2% and 87%, respectively. These results indicate that the effect of electrical pulses mediated TLE was higher on cancerous MCF-7 cells when compared to non-cancerous MCF-10A cells.
Conclusion:
The combination of electrical pulses with TLE is an effective strategy to selectively target cancer cells in the body.
... The process results in multitude of cell damage from the strong electrical field, leading to large amount of cell death [7]. Similar to carrier mediated delivery, current electroporation techniques are only applicable to a population of cells, where uniformity and precision in dosage are beyond control [8,9]. Further, this process is only applicable to cells in suspension. ...
Electroporation is a common method of transfection due to its relatively low risk and high transfection efficiency. The most common method of electroporation is bulk electroporation which is easily performed on large quantities of cells yet results in variable levels of viability and transfection efficiency across the population. Localized electroporation is an alternative that can be administered on a similar scale but results in much more consistent with higher quality transfection and higher cell viability. This paper discusses the creation and use of a simple and cost-effective device using porous membrane for performing localized electroporation.
... Numerous efforts have been made to minimize voltage-induced cytotoxicity, including postpulse centrifugation 18 and an enhanced electroporation cell density. 19 Although clinically irrelevant, the inclusion of serum in the electroporation buffer improves cell viability with no negative effect on gene transfer and expression in mammalian cells. 20 Optimization of postelectroporation recovery also decreases the cytotoxicity for primary stem cells. ...
With the advent of new genome editing technologies and the emphasis placed on their optimization, the genetic and phenotypic correction of a plethora of diseases sit on the horizon. Ideally, genome editing approaches would provide long-term solutions through permanent disease correction instead of simply treating patients symptomatically. Although various editing machinery options exist, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated protein) editing technique has emerged as the most popular due to its high editing efficiency, simplicity, and affordability. However, while CRISPR technology is gradually being perfected, optimization is futile without accessible, effective, and safe delivery to the desired cell or tissue. Therefore, it is important that scientists simultaneously focus on inventing and improving delivery modalities for editing machinery as well. In this review, we will discuss the critical details of viral and nonviral delivery systems, including payload, immunogenicity, efficacy in delivery, clinical application, and future directions.
... Transient permeabilization of cell membranes using pulsed electric fields (electroporation) is a simple non-viral method to deliver exogenous biomolecules to a variety of cell types [1][2][3][4][5][6]. However, conventional electroporation often suffers from poor efficiency as it relies on bulk stochastic phenomena and poor control over the cell viability due to the strong electric fields used [7,8]. ...
We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidics device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real-time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecule electrotransfer using propidium iodide and large molecule electrotransfer using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).
... In conventional electroporation designs, bulk electroporation has been used for effective DNA transfection into suspension cells [15]. However, in order to obtain practical efficiency and viability for samples with large heterogeneity in cell diameter, a strong electric field was used, which might lead to the destruction of the cell membrane [16]. When evaluating the performance of electroporation, the following points should be considered: (1) electroporation efficiency and cell viability with an easy operation; (2) controllability at the single-cell level; and (3) selectivity between targeted cells and background cells (e.g., rare circulating tumor cells (CTCs) and blood cells). ...
Labeling-assisted visualization is a powerful strategy to track circulating tumor cells (CTCs) for mechanism study (e.g., tumor metastasis). Due to the rarity of CTCs in the whole blood, efficient simultaneous enrichment and labeling of CTCs are needed. Hereby, novel in situ electroporation on a previously-developed micropore-arrayed filter (PERFECT filter) is proposed. Benefiting from the ultra-small-thickness and high-porosity of the filter plus high precision pore diameter, target rare tumor cells were enriched with less damage and uniform size distribution, contributing to enhanced molecular delivery efficiency and cell viability in the downstream electroporation. Various biomolecules (e.g., small molecule dyes, plasmids, and functional proteins) were used to verify this in situ electroporation system. High labeling efficiency (74.08 ± 2.94%) and high viability (81.15 ± 3.04%, verified via live/dead staining) were achieved by optimizing the parameters of electric field strength and pulse number, ensuring the labeled tumor cells can be used for further culture and down-stream analysis. In addition, high specificity (99.03 ± 1.67%) probing of tumor cells was further achieved by introducing fluorescent dye-conjugated antibodies into target cells. The whole procedure, including cell separation and electroporation, can be finished quickly (<10 min). The proposed in situ electroporation on the PERFECT filter system has great potential to track CTCs for tumor metastasis studies.
... Strength-duration curves for unipolar pulses from 150 ns to 1 ms. Pulse duration is the major parameter that determines the electric field thresholds for diverse biological effects, including nerve stimulation, electroporation, and cell death 17,37,43,52,[57][58][59][60][61] . Most of the previous studies focused on the severity or probability of the effects rather than on the electric field thresholds and also were limited to relatively narrow PEF duration ranges. ...
Ablation therapies aim at eradication of tumors with minimal impact on surrounding healthy tissues. Conventional pulsed electric field (PEF) treatments cause pain and muscle contractions far beyond the ablation area. The ongoing quest is to identify PEF parameters efficient at ablation but not at stimulation. We measured electroporation and cell killing thresholds for 150 ns–1 ms PEF, uni- and bipolar, delivered in 10- to 300-pulse trains at up to 1 MHz rates. Monolayers of murine colon carcinoma cells exposed to PEF were stained with YO-PRO-1 dye to detect electroporation. In 2–4 h, dead cells were labeled with propidium. Electroporation and cell death thresholds determined by matching the stained areas to the electric field intensity were compared to nerve excitation thresholds (Kim et al. in Int J Mol Sci 22(13):7051, 2021). The minimum fourfold ratio of cell killing and stimulation thresholds was achieved with bipolar nanosecond PEF (nsPEF), a sheer benefit over a 500-fold ratio for conventional 100-µs PEF. Increasing the bipolar nsPEF frequency up to 100 kHz within 10-pulse bursts increased ablation thresholds by < 20%. Restricting such bursts to the refractory period after nerve excitation will minimize the number of neuromuscular reactions while maintaining the ablation efficiency and avoiding heating.
... The pulse protocol is defined by the applied amplitude, U (peak voltage); pulse duration, t p ; time interval between pulses, Δt (or pulse repetition rate f = 1/Δt); and number of pulses, n (Canatella et al. 2001). The total time of PEF treatment is defined as t PEF = nt p . ...
The chapter reviews applications of pulsed electric fields (PEF) for the efficient extraction of intercellular components from food plants. Mechanisms of cell membrane permeabilization by PEF including electroporation of plane membranes, spherical cells, cells with different shapes and sizes, and ensembles of cells and plant tissues are discussed. Different techniques to detect electroporation, PEF protocols, treatment chambers, and methods for optimization of PEF treatment are presented. Solid/liquid expression and solvent extraction assisted by PEF are described in detail. Numerous practical examples of PEF-enhanced extraction of intracellular compounds from foods (potatoes, apples, sugar crops, citruses, grapes, etc.) are presented.
... 3 Moreover, understanding the mechanics behind membrane permeability may also improve current treatment strategies, minimize drug and reagent wastage, and increase the efficacy of these treatments. 4 Chemical-based methods, such as molecular/fluorescent probes, 5−7 flow cytometry, 4,5 microfluidics devices, and other methods, 8−13 have been harnessed to understand membrane permeabilization. These techniques harness the size of cells and binding affinity of the cell membrane to various molecules for probing the cell behavior and offer excellent advantages such as high specificity, high cell-targeting accuracy, and highresolution imaging at the single-cell level. ...
Long-term nondestructive monitoring of cells is of significant importance for understanding cell proliferation, cell signaling, cell death, and other processes. However, traditional monitoring methods are limited to a certain range of testing conditions and may reduce cell viability. Here, we present a microgap, multishot electroporation (M2E) system for monitoring cell recovery for up to ∼2 h using ∼5 V pulses and with excellent cell viability using a medium cell population. Electric field simulations reveal the bias-voltage- and gap-size-dependent electric field intensities in the M2E system. In addition to excellent transparency with low cell toxicity, the M2E system does not require specialized components, expensive materials, complicated fabrication processes, or cell manipulations; it just consists of a micrometer-sized pattern and a low-voltage square-wave generator. Ultimately, the M2E system can offer a long-term and nontoxic method of cell monitoring.
... Other physical systems, such as bulk electroporation, causes inhomogeneities in the electric field around the cells, producing an uneven distribution in the pore size created in the cell membrane 32 . Cells with large holes uptake large amounts of cargo or die, while cells with very small holes may have very little or no uptake. ...
MicroRNAs (miRNAs) are small non‐coding RNAs that play key roles in post‐transcriptional gene regulation. Being involved in regulating virtually all cellular processes, from proliferation and differentiation to migration and apoptosis, they have emerged as important epigenetic players. While most interest has gone into which miRNAs are involved in specific cellular processes or pathologies, the dosage‐dependent effects of miRNAs remain vastly unexplored. Different doses of miRNAs can cause selective downregulation of target genes, in turn, determining what signalling pathways and cellular responses are triggered. To explore this behaviour, the effects of incremental miRNA dosage need to be studied, however current delivery methods for miRNAs are unable to control how much miRNA enters a cell. Here, we present an approach based on a nanostraw‐electroporation delivery platform that decouples the delivery from biological mechanisms (e.g. endocytosis) to enable precise control over the amount of miRNA delivered, along with demonstrating ratiometric intracellular delivery into primary dermal fibroblasts for miR‐181a and miR‐27a. In addition, we show that the nanostraw delivery platform allows efficient delivery of miRNAs into primary keratinocytes, opening new opportunities for successful miRNA delivery into this hard‐to‐transfect cell type.
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... where N(t) is the pore density on the membrane. The cell model simulated in this Letter is based on DU 145 prostate cancer cells, 19 whose dielectric properties are listed in the supplementary material. ...
Exposed to the nanosecond pulsed electric field (nsPEF), biological cells can be stretched in the direction parallel to the electric field
direction. A multiphysics model to investigate electrodeformation of a spherical cell with double-layered plasma membrane accounting for
both electroporation and dielectric relaxation of the membrane is proposed. Transmembrane potential, Maxwell stress tensor, total elastic
strain energy, and deformation degree, the typical influential factors and indicators for electroporation and electrodeformation, are probed
via the above multiphysics model under the action of unipolar and bipolar nsPEFs. The results suggest that the double-layered model can
reflect the experimental cellular deformation more accurately than the single-layered model in that the long axis of the ellipsoid is stretched
several micrometers in the double-layered model, while it is stretched several nanometers in the single-layered model. And merging the effect
of dielectric relaxation into the model leads to a relatively lighter but faster deformation extent, and applying bipolar nsPEF alleviates the
stretch for electrodeformation quantified with the lower aspect ratio of two principal radii of the ellipsoidal cell and the lower elastic strain
energy. Our model can reflect the temporal evolution of electroporation and electrodeformation procedure more accurately, which is instructive
to exert the nsPEF in biochemical experiments and clinical applications
... Bulk electroporation is another popular non-viral method for intracellular delivery. Despite its success in delivery of wide range of cargos into most types of cell, including hard-to-transfect cells, high cell mortality is still a major challenge (9). In addition, due to their bulk nature, cationic lipids/polymers and electroporation do not offer uniform and dosage-controlled delivery across cell population (10). ...
Intracellular delivery of cargos for cell engineering plays a pivotal role in transforming medicine and biomedical discoveries. Recent advances in microfluidics and nanotechnology have opened up new avenues for efficient, safe, and controllable intracellular delivery, as they improve precision down to the single-cell level. Based on this capability, several promising micro- and nanotechnology approaches outperform viral and conventional non-viral techniques in offering dosage-controlled delivery and/or intracellular delivery of large cargos. However, to achieve this level of precision and effectiveness, they are either low in throughput, limited to specific cell types (e.g., adherent vs. suspension cells), or complicated to operate with. To address these challenges, here we introduce a versatile and simple-to-use intracellular delivery microfluidic platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP). Hundreds of acoustic microstreaming vortices form the production line of the AESOP platform, wherein hundreds of thousands of cells are trapped, permeabilized, and mixed with exogenous cargos. Using AESOP, we show intracellular delivery of a wide range of molecules (from <1 kDa to 2 MDa) with high efficiency, cell viability, and dosage-controlled capability into both suspension and adherent cells and demonstrate throughput at 1 million cells/min per single chip. In addition, we demonstrate AESOP for two gene editing applications that require delivery of large plasmids: i) eGFP plasmid (6.1 kbp) transfection, and ii) CRISPR-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and sgRNA. Compared to alternative platforms, AESOP not only offers dosage-controlled intracellular delivery of large plasmids (>6kbp) with viabilities over 80% and comparable delivery efficiencies, but also is an order of magnitude higher in throughput, compatible with both adherent and suspension cell lines, and simple to operate.
... Conversely, bulk physical membrane disruption methods (i.e., bulk-electroporation and mechanoporation systems) subject the entire cell surface to high electric fields or mechanical stresses that may lead to an undesirable intracellular stress response or reduced viability. [9][10][11][12] Other commonly used delivery methods, like lipid-vesicle carriers and viral vectors, are limited by low delivery efficiency, 13 lack of dosage uniformity, 2 and cytotoxicity. 14 The nondestructive mechanism and precise dosage control enabled by the NFP-E system are advantageous for applications that require uniform delivery, temporal precision, high cell viability, and single-cell control (i.e., single-cell gene editing and monoclonal cell-line generation). ...
Single-cell delivery platforms like microinjection and nanoprobe electroporation enable unparalleled control over cell manipulation tasks but are generally limited in throughput. Here, we present an automated single-cell electroporation system capable of automatically detecting cells with artificial intelligence (AI) software and delivering exogenous cargoes of different sizes with uniform dosage. We implemented a fully convolutional network (FCN) architecture to precisely locate the nuclei and cytosol of six cell types with various shapes and sizes, using phase contrast microscopy. Nuclear staining or reporter fluorescence was used along with phase contrast images of cells within the same field of view to facilitate the manual annotation process. Furthermore, we leveraged the near-human inference capabilities of the FCN network in detecting stained nuclei to automatically generate ground-truth labels of thousands of cells within seconds, and observed no statistically significant difference in performance compared to training with manual annotations. The average detection sensitivity and precision of the FCN network were 95±1.7% and 90±1.8%, respectively, outperforming a traditional image-processing algorithm (72±7.2% and 72±5.5%) used for comparison. To test the platform, we delivered fluorescent-labeled proteins into adhered cells and measured a delivery efficiency of 90%. As a demonstration, we used the automated single-cell electroporation platform to deliver Cas9–guide RNA (gRNA) complexes into an induced pluripotent stem cell (iPSC) line to knock out a green fluorescent protein–encoding gene in a population of ~200 cells. The results demonstrate that automated single-cell delivery is a useful cell manipulation tool for applications that demand throughput, control, and precision.
... Electroporation offers a transient and stable transfection of RNP for different types of cells, such as human CD34 + hemopoietic stem/progenitor cells (HSPCs) [60,[93][94][95][96][97][98][99][100], human embryonic stem cells (hESCs) [101], human primary neonatal fibroblast cells [101], human induced pluripotent stem cells (iPSCs) [102], human B cells [103], human CD4 + T cells [104], CAR-T cells [105], human embryonic kidney (HEK) 293T cells [106,107], mouse CD8 + T cell [108], mouse neural stem cells [109], mouse skin stem cells [110], mouse pronuclear-stage embryos [111], mature primary mouse innate lymphocyte cells [112], rabbit fibroblast cells [102], green alga Chlamydomonas reinhardtii [113,114], Trypanosoma cruzi [115]. However, the high voltage pulses during electroporation usually cause substantial cell death [116,117]. An alternate approach is to introduce a single-cell electroporation using a nanofountain probe system, which allows efficient transfection of precise amount of RNP with high cell ability [118]. ...
CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
... Typically, higher electric field strengths lead to better damage efficiency (Bazhal 2001;Bouzrara 2001;Praporscic 2005;Toepfl 2006;Shynkaryk 2007); however, electrical power consumption and ohmic heating also become essential at high electric fields. More detailed experiments have also shown that electroporation efficiency may depend on the parameters of the pulse, such amplitude (or electric field strength E), shape, duration t i , number of repetitions n, and intervals between pulses Δt (Canatella et al. 2001(Canatella et al. , 2004. A typical PEF protocol for bipolar pulses of a near-rectangular shape is presented in Figure 2.7. ...
Extraction is an important operation in food engineering, enabling the recovery of valuable soluble components from raw materials. With increasing energy costs and environmental concerns, industry specialists are looking for improved techniques requiring less solvents and energy consumption. Enhancing Extraction Processes in the Food Industry is a comprehensive resource providing clear descriptions of the latest extraction methods and instruments used in food laboratories. The book begins with an overview of solvent extraction technology. It examines pulsed electric fields and their effect on food engineering, and the potential and limitations of microwave-assisted extraction. It explores diffusion processes and reviews what is known about electrical discharge processes in the extraction of biocompounds. Next, the book summarizes current knowledge on conventional and innovative techniques for the intensification of extractions from food and natural products, focusing on environmental impacts. It reviews recent developments in supercritical CO2 extraction of food and food products, describes the pressurized hot water extraction (PHWE) process, and examines future trends for PHWE. The book also examines essential oil extraction, and the tools and techniques of high pressure-assisted extraction. The authors demonstrate its application using litchi and longan fruits as examples. The final chapters focus on extrusion-assisted extraction, gas-assisted mechanical expression, mechanochemically assisted extraction, reverse micellar extraction, and aqueous two-phase extraction. The book concludes with a chapter on the treatment of soybeans through enzyme-assisted aqueous processing, examining the economics involved as well as the development of the process. A solid review of modern approaches that enhance extraction processes, this volume is destined to pave the way for future research and development in the field.
... The use of electroporation to transform both bacterial and higher cells became very popular throughout the 1980s. The mechanism by which electroporation occurs is not well understood and hence, like chemical transformation, the development of protocols for particular applications has usually been achieved empirically by adjusting electric pulse parameters (amplitude, duration, number and inter-pulse interval) (Ho and Mittal, 1996;Canatella et al., 2001). Two main factors seem to influence the formation of electropores -the types of cell that are used, and the amplitude and duration of the electric pulse that is applied to them. ...
... However, cell viability decreased with increasing acoustic pressure. This phenomenon was predictable given the cell damage attributable to the abundant acoustic energy (Canatella et al. 2001;Hallow et al. 2006). To optimize the delivery application, this experiment selected 0.6 MPa as the optimum PNP. ...
Studies have determined that ultrasound-activated microbubbles can increase the membrane permeability of tumor cells by triggering membrane perforation (sonoporation) to improve drug loading. However, because of the distinct cavitation events adjacent to each cell, the degree of drug loading appeared to be heterogeneous. The relationship between the long-term fate trend and the degree of drug loading remains unclear. To investigate the time-lapse viability of diversity loading cells, fluorescein isothiocyanate-dextran (FITC-dextrans) was used as a molecular model mixed with 2% v/v SonoVue microbubbles (Bracco, Milan, Italy) and exposed to various peak negative pressures (0.25 MPa, 0.6 MPa, 1.2 MPa), 1 MHz frequency and 300 μs pulse duration. To select a suitable parameter, the cavitation activity was measured, and the cell analysis was performed by flow cytometry under these acoustic pressures. The sonoporated cells were then categorized into 3 sub-groups by flow cytometry according to the various fluorescence intensity distributions to analyze their long-term fate. We observed that the stable cavitation occurred at 0.25 MPa and microbubbles underwent ultra-harmonic emission, and obvious broadband signals were observed at 0.6 MPa and 1.2 MPa, suggesting the occurs of inertial cavitation. The cell analysis further showed the maximum delivery efficiency and cell viability at 0.6 MPa, and it was selected for the following experiment. The categorization displayed that the fluorescence intensity of FITC-dextrans in sub-groups 2 and 3 were approximate 5.62-fold and 19.53-fold higher than that in sub-group 1, respectively. After separation of these sub-groups, the apoptosis and necrosis ratios in all 3 sub-groups of sonoporated cells gradually increased with increasing culture time and displayed no significant difference in either the apoptosis (p > 0.05) or necrosis (p > 0.05) ratio after 6 h and 24 h of culture, respectively. Further analysis using Western blot verified that the long-term fate of sonoporated cells involves the mitochondrial signaling proteins. These results provide better insight into the role of cavitation-enhanced permeability and a critical guide for acoustic cavitation designs.
... The process results in multitude of cell damage from the strong electrical field, leading to large amount of cell death [7]. Similar to carrier mediated delivery, current electroporation techniques are only applicable to a population of cells, where uniformity and precision in dosage are beyond control [8,9]. Further, this process is only applicable to cells in suspension. ...
Electroporation is a common method of transfection due to its relatively low risk and high transfection efficiency. The most common method of electroporation is bulk electroporation which is easily performed on large quantities of cells yet results in variable levels of viability and transfection efficiency across the population. Localized electroporation is an alternative that can be administered on a similar scale but results in much more consistent with higher quality transfection and higher cell viability. This chapter discusses the creation and use of a simple and cost-effective device using porous membrane for performing localized electroporation.
... where E 0 (399 600 V/m), A 0 (144 100 V/m), k 1 (0.03) and k 2 (0.06) are the regression coefficients and are related to the pulse duration and cell type. Because there was no available data for digestive tumor, the regression coefficients were obtained from an in vitro study of prostate cancer cells [44]. The probability of cell death due to EI can be calculated as follows: ...
Introduction: Irreversible electroporation (IRE) combined with a catheter-based electrode during endoscopy is a potential alternative treatment method for digestive tract tumors. The aim of this study was to investigate the electrical injury (EI) and thermal injury (TI) to the digestive tract via numerical analyses and to evaluate the role and impact of electrode configurations and pulse settings on the efficacy and outcomes of IRE.
Materials and methods: A finite element method was used to solve the numerical model. A digestive tract model having 4-mm-thick walls and two catheter-based electrode configuration models were constructed. The distributions of electric fields, temperature, electrical conductivity, tissue injury and limitation on the pulse number required for IRE were calculated and compared.
Results: Electrode length is an important geometric parameter for electrodes in the monopolar model (MPM), while electrode spacing affects the outcomes in the bipolar model (BPM). Increasing the pulse voltage reduces the pulse number required for tissue ablation, while increasing the risk of TI. In total, there were 6 NT-IRE protocols, 12 thermal-IRE protocols and 30 TI protocols. All of the NT-IRE protocols were set in BPMs with a voltage of 0.50 kV. With increasing electrode spacing, the minimum pulse number decreased. However, thermal effects were inevitable in the MPM.
Conclusions: The electrode configuration and pulse settings are adjusted to achieve NT-IRE synergistically. The BPM is more reliable for achieving NT-IRE in 4-mm-thick digestive wall. Future in vitro and in vivo studies are needed to support and validate this conclusion.
1] 和食物偏好 [2-4] 。动 物的学习记忆过程与其控制能量平衡的神经以及 生理基础之间存在着非常密切的关系, 各种内分 泌信号可作用于脑内的神经元, 以此来调节食物 的摄入量和体重 [5] 。尽管学习记忆主要是在哺乳 动物和鸟类中进行研究 [6] , 但最近的研究报道, 鱼类的各种行为活动都受到学习记忆的影响 [7] 。由 于觅食环境的复杂性和食物的变化性, 学习和记忆 在鱼类觅食中起着非常重要的作用 [8] 。最近的几项 研究表明, 在几种鱼类中的研究都显示, 学习记忆 加强了其觅食行为, 例如大麻哈鱼(Oncorhynchus keta) [9] 、孔雀鱼(Poecilia reticulata) [10-11] 和翘嘴鳜 (Siniperca chuatsi) [12] , 训练有素的大西洋鲑(Salmo salar)幼鱼接受猎物的概率大大提高 [2] 。而学习记 忆能否在鱼类食性转变如驯食等过程中发挥作用, 目前尚不明确。 作为学习记忆相关基因之一的原癌基因 c-Fos (proto-oncogene c-Fos), 其表达是小鼠海马区依 赖性非空间记忆的形成和反复记忆所必需的 [13] 。 研究指出早期生长反应因子 1 (early growth response 1, Egr1, 也被称为 Zif268)对记忆巩固和持 久记忆起到重要作用。学习记忆基因 Zif268 敲除 小鼠出现长期记忆受损, 但短期记忆仍保留正常 功能 [14] 。研究报道食欲调控相关基因包括厌食神 经肽(pro-opiomelanocortin, Pomc)、促食神经肽 Y (neuropeptide y, Npy)、 刺鼠色蛋白相关蛋白(agouti related peptide, Agrp)以及黑色素浓集激素(melanin-concentrating hormone, Mch)等 [15-16] 。 摄食相关基因
Cells exposed to a pulsed electric field undergo electrodeformation (ED) and electroporation (EP) under the action of electric field stress, and this paper proposes a coupled model of EP and ED that considers the change in Young's modulus. The model considers the cytoplasmic membrane as a porous viscoelastic material and decreases in Young's modulus due to many pores generated on the plasma membrane after EP onset is further included. The results show that the degree of ED in this model is significantly larger than in previous models. This is mainly due to the generation of a large number of pores in the cell membrane, which increases the membrane porosity and causes significant decreases in Young's modulus, leading to the weakened ability of the cell to resist ED. The degree of cell EP and ED can be mitigated by increasing the pulse delay between H-FIRE pulses based on this model, which is consistent with previous studies. Our model can more accurately reflect the cell ED process by considering the decreases in Young's modulus of the cell membrane during EP. It can also provide theoretical guidance for biochemical experiments using H-FIRE pulses.
Microalgae as the photosynthetic organisms offer enormous promise in a variety of industries, such as the generation of high-value byproducts, biofuels, pharmaceuticals, environmental remediation, and others. With the rapid advancement of gene editing technology, CRISPR/Cas system has evolved into an effective tool that revolutionised the genetic engineering of microalgae due to its robustness, high target specificity, and programmability. However, due to the lack of robust delivery system, the efficacy of gene editing is significantly impaired, limiting its application in microalgae. Nanomaterials have become a potential delivery platform for CRISPR/Cas systems due to their advantages of precise targeting, high stability, safety, and improved immune system. Notably, algal-mediated nanoparticles (AMNPs), especially the microalgae-derived nanoparticles, are appealing as a sustainable delivery platform because of their biocompatibility and low toxicity in a homologous relationship. In addition, living microalgae demonstrated effective and regulated distribution into specified areas as the biohybrid microrobots. This review extensively summarised the uses of CRISPR/Cas systems in microalgae and the recent developments of nanoparticle-based CRISPR/Cas delivery systems. A systematic description of the properties and uses of AMNPs, microalgae-derived nanoparticles, and microalgae microrobots has also been discussed. Finally, this review highlights the challenges and future research directions for the development of gene-edited microalgae.
Graphical Abstract
The impact of cell shape on cell membrane permeabilization by pulsed electric fields is not fully understood. For certain applications, cell survival and recovery post-treatment is either desirable, as in gene transfection, electrofusion, and electrochemotherapy, or is undesirable, as in tumor and cardiac ablations. Understanding of how morphology affects cell viability post-electroporation may lead to improved electroporation methods. In this study, we use precisely aligned nanofiber networks within a microfluidic device to reproducibly generate elongated cells with controlled orientations to an applied electric field. We show that cell viability is significantly dependent on cell orientation, elongation, and spread. Further, these trends are dependent on the external buffer conductivity. Additionally, we see that cell survival for elongated cells is still supported by the standard pore model of electroporation. Lastly, we see that manipulating the cell orientation and shape can be leveraged for increased transfection efficiencies when compared to spherical cells. An improved understanding of cell shape and pulsation buffer conductivity may lead to improved methods for enhancing cell viability post-electroporation by engineering the cell morphology, cytoskeleton, and electroporation buffer conditions.
Cell poration technologies offer opportunities not only to understand the activities of biological molecules but also to investigate genetic manipulation possibilities. Unfortunately, transferring large molecules that can carry huge genomic information is challenging. Here, we demonstrate electromechanical poration using a core-shell-structured microbubble generator, consisting of a fine microelectrode covered with a dielectric material. By introducing a microcavity at its tip, we could concentrate the electrical field with the application of electric pulses and generate microbubbles for electromechanical stimulation of cells. Specifically, the technology enables transfection with molecules that are thousands of kDa even into osteoblasts and Chlamydomonas, which are generally considered to be difficult to inject. Notably, we found that the transfection efficiency can be enhanced by adjusting the viscosity of the cell suspension, which was presumably achieved by remodeling of the membrane cytoskeleton. The applicability of the approach to a variety of cell types opens up numerous emerging gene engineering applications.
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.
Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5–6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes. Sen and colleagues describe a protocol for the fabrication of silicon hollow-needle arrays to achieve tissue nanotransfection of mouse skin.
A high‐throughput non‐viral intracellular delivery platform is introduced for the transfection of large cargos with dosage‐control. This platform, termed Acoustic‐Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min⁻¹ per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9‐mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage‐controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.
The application of recombinant DNA technology to the production of protein therapeutics has undergone tremendous progress over the past decades. Considerable scientific effort has been devoted to developing robust processes that produce large amounts of complex proteins with the desired product quality attributes. An overview of the contributions from the four major disciplines working in concert to deliver these processes and methods will be covered. This article will discuss some of the tools, methods, and approaches used to produce, purify, formulate, and analyze the drugs of modern biotechnology. Future trends in each of the disciplines will also be presented.
In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow‐through microfluidics, engineered substrates, and automated probe‐based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate‐based electroporation platforms and high throughput, high control methods in general.
One of the promising technologies that can inactivate microorganisms without heat is pulsed electric field (PEF) treatment. The aim of this study was to examine the influence of PEF treatment (2.9 kV cm−1, 100 Hz, 5000 pulses in trains mode of 500 pulses with a pulse duration of 10 µs) on Saccharomyces cerevisiae eradication and resealing in different conditions, such as current density (which is influenced by the medium conductivity), the sort of medium (phosphate buffered saline (PBS) vs. yeast malt broth (YMB) and a combined treatment of PEF with the addition of preservatives. When the S. cerevisiae were suspended in PBS, increasing the current density from 0.02 to 3.3 A cm−2 (corresponding to a total specific energy of 22.04 to 614.59 kJ kg−1) led to an increase of S. cerevisiae eradication. At 3.3 A cm−2, a total S. cerevisiae eradication was observed. However, when the S. cerevisiae in PBS was treated with the highest current density of 3.3 A cm−2, followed by dilution in a rich YMB medium, a phenomenon of cell membrane resealing was observed by flow cytometry (FCM) and CFU analysis. The viability of S. cerevisiae was also examined when the culture was exposed to repeating PEF treatments (up to four cycles) with and without the addition of preservatives. This experiment was performed when the S. cerevisiae were suspended in YMB containing tartaric acid (pH 3.4) and ethanol to a final concentration of 10% (v/v), which mimics wine. It was shown that one PEF treatment cycle led to a reduction of 1.35 log10, compared to 2.24 log10 when four cycles were applied. However, no synergic effect was observed when the preservatives, free SO2, and sorbic acid were added. This study shows the important and necessary knowledge about yeast eradication and membrane recovery processes after PEF treatment, in particular for application in the liquid food industry.
To achieve high efficiency of electroporation and to minimize unwanted side effects, the electric field parameters must be optimized. Recently, it was suggested that biphasic high-frequency irreversible electroporation (H-FIRE) pulses reduce muscle contractions. However, it was also shown for sub-microsecond biphasic pulses that the opposite polarity phase of the pulse cancels the effect of the first phase if the interphase delay is short enough. We investigated the effect of interphase and interpulse delay (ranging from 0.5 to 10,000 µs) of 1 µs biphasic H-FIRE pulses on cell membrane permeabilization, on survival of four mammalian cell lines and determined metal release from aluminum, platinum and stainless steel electrodes. Biphasic H-FIRE pulses were compared to 8 × 100 µs monophasic pulses. We show that a longer interphase and interpulse delay results in lower cell survival, while the effects on cell membrane permeabilization are ambiguous. The cancellation effect was observed only for the survival of one cell line. Application of biphasic H-FIRE pulses results in lower metal release from electrodes but the interphase and interpulse delay does not have a large effect. The electrode material, however, importantly influences metal release – the lowest release was measured from platinum and the highest from aluminum electrodes.
Trehalose synthase converts maltose into trehalose in a single conversion step via intramolecular transformation and is thus useful for industrial production. In this study, we synthesized a thermophilic trehalose synthase from Thermus antranikianii (TaTS), which was recombinantly expressed in Escherichia coli BL21(DE3). The recombinant TaTS showed the highest activity at pH 7.0 and 60°C, with the maximum trehalose yield (76.8%) obtained at pH 7.0 and 30°C. TaTS activity was stable over a wide pH and temperature range of 6-10 and 4-70°C, respectively, over 6 h of incubation. The enzyme activity was strongly inhibited by Co2+, Cu2+, Zn2+, sodium dodecyl sulfate, and Tris. TaTS showed a 1.48-fold higher catalytic efficiency (kcat/Km) for maltose than for trehalose. Overall, these results demonstrate the good application potential of the recombinant enzyme TaTS in the efficient conversion of trehalose from maltose, with superior environmental tolerance to other trehalose synthases reported.
Electrical Manipulation of Cells provides an authoritative and up-to-date review of the field, covering all the major techniques in a single source. The book features broad coverage that ranges from the mechanisms of action of external electrical fields on biological material to the ways in which electrical stimuli are employed to manipulate cells. Bringing together the work of leading international authorities, the book covers membrane breakdown, gene delivery, electroporation, electrostimulation, cell movement, hybridoma production, plant protoplasts, electrorotation and stimulation, and electromagnetic stimulation.
For each topic, the authors discuss the relevance of the approach to the current state of the art of biotechnology. Electrical Manipulation of Cells is an unmatched source of information for anyone involved in the manipulation of cells, particularly biotechnologists, cell biology, microbiologists, biophysicists and plant scientists. For researchers, the book provides technical material that ccan be employed in their own work. Students will gain thorough appreciation of the applications of this important technique.
Electrochemotherapy is a novel technique to enhance the delivery of chemotherapeutic drugs into tumor cells. In this procedure, electric pulses are delivered to cancerous cells, which induce membrane permeabilization, to facilitate the passage of cytotoxic drugs through the cell membrane. This study examines how electric fields interact with and polarize a system of cells. Specifically, we consider how cell density and organization impact on induced cell transmembrane potential due to an external electric field. First, in an infinite volume of spherical cells, we examined how cell packing density impacts on induced transmembrane potential. With high cell density, we found that maximum induced transmembrane potential is suppressed and that the transmembrane potential distribution is altered. Second, we considered how orientation of cell sheets and strands, relative to the applied field, affects induced transmembrane potential. Cells that are parallel to the field direction suppress induced transmembrane potential, and those that lie perpendicular to the applied field potentiate its effect. Generally, we found that both cell density and cell organization are very important in determining the induced transmembrane potential resulting from an applied electric field.
1 Dielectrophoresis and Rotation of Cells.- 2 Cellular Spin Resonance.- 3 Dielectrophoresis: Behavior of Microorganisms and Effect of Electric Fields on Orientation Phenomena.- 4 The Relaxation Hysteresis of Membrane Electroporation.- 5 Electrical Breakdown of Lipid Bilayer Membranes: Phenomenology and Mechanism.- 6 Stochastic Model of Electric Field-Induced Membrane Pores.- 7 Theory of Electroporation.- 8 Leaks Induced by Electrical Breakdown in the Erythrocyte Membrane.- 9 Electroporation of Cell Membranes: Mechanisms and Applications.- 10 Electrofusion Kinetics: Studies Using Electron Microscopy and Fluorescence Contents Mixing.- 11 Electrofusion of Lipid Bilayers.- 12 Role of Proteases in Electrofusion of Mammalian Cells.- 13 Electrofusion of Mammalian Cells and Giant Unilamellar Vesicles.- 14 Cell Fusion and Cell Poration by Pulsed Radio-Frequency Electric Fields.- 15 The Mechanism of Electroporation and Electrofusion in Erythrocyte Membranes.- 16 Producing Monoclonal Antibodies by Electrofusion.- 17 Generation of Human Hybridomas by Electrofusion.- 18 Gaining Access to the Cytosol: Clues to the Control and Mechanisms of Exocytosis and Signal Transduction Coupling.- 19 Gene Transfer by Electroporation: A Practical Guide.- 20 Electropermeabilization and Electrosensitivity of Different Types of Mammalian Cells.- 21 Molecular Genetic Applications of Electroporation.- 22 Plant Gene Transfer Using Electrofusion and Electroporation.- 23 Electric Field-Induced Fusion and Cell Reconstitution with Preselected Single Protoplasts and Subprotoplasts of Higher Plants.- 24 Critical Evaluation of Electromediated Gene Transfer and Transient Expression in Plant Cells.- 25 Transformation Studies in Maize and Other Cereals.- 26 Cells in Electric Fields: Physical and Practical Electronic Aspects of Electro Cell Fusion and Electroporation.- 27 External Electric Field-Induced Transmembrane Potentials in Biological Systems: Features, Effects, and Optical Monitoring.
Electrochemotherapy is a new therapeutic approach providing delivery into cell interiors of nonpermeant drugs with intracellular targets. It is based on the local application of short and intense electric pulses that transiently permeabilize cells in tissues. To date, its main application has been the treatment of tumor nodules when the electric pulses are associated with nonpermeant drugs having high intrinsic cytotoxicity. The most convenient drug is bleomycin, a currently used anticancer drug, but cytotoxicity of cisplatin is also increased in vivo by means of this original drug delivery approach. The efficacy of this new method is based on the following mechanisms: (i) electropermeabilization of cells in tissues; (ii) use of nonpermeant drugs having a high intrinsic cytotoxicity; (iii) existence of vascular effects due to the permeabilizing electric pulses; (iv) complementary role of the host's immune system. Preclinical trials have shown the efficacy of this new therapeutic modality in various tumor models. Clinical trials are in progress, demonstrating its feasibility in humans as well as the interest of the method.
Electrochemotherapy is a novel treatment which consists of a combination of a chemotherapeutic agent and pulsed electric fields. This relatively new treatment modality relies on the physical effects of locally applied electric fields to temporarily destabilize cell membranes in the presence of a drug to allow increased uptake of the agent into the cytosol. Electrochemotherapy has been used effectively in preclinical and clinical studies. The therapy was shown to be effective regardless of histologic type of tumor including head and neck squamous cell carcinoma, melanoma, basal cell carcinoma, adenocarcinoma and Kaposi's sarcoma. Objective response rates ranging from 72 percent to 100 percent have been reported from these trials. These responses were obtained with minimal adverse side effects. A review of the clinical data for this novel drug delivery method is presented.
Electroporation is well known to depend non-linearly on the magnitude and duration of the change ΔU(t) in transmembrane voltage. In the case of cell suspension experiments, an electric field Ee(t) within the electrolyte causes ΔU(t), which is governed by both the size and shape of a cell, and also by Ee(t). It is therefore important to determine the magnitude and time dependence of the electric field to which cells are actually exposed in electroporation experiments. This can be significantly different from the nominal field En, which is calculated by using electrode voltages and geometries alone. Throughout we used single, nominally exponential pulses with time constants τpulse ranging from about 0.6 to 5 ms and found that Ee was always less than En. In order to determine the actual electric field pulse, we measured the voltage across the electrodes, the current through the cuvette, the temperature rise of the pulsing medium, and the voltage across two special electrodes placed within the cuvette. From these measurements we calculated the field strength inside the cuvette using two different methods. In addition, we compared the measured temperature rise with that expected from the electrical power dissipation. In some cases there was much larger (“anomalous”) heating, due to interfacial electrochemical heat production; for one pulsing solution Te(t) was about 30 K larger than expected. These effects are important for experiments aimed at elucidating the electroporation mechanism, comparing results obtained under different conditions, and guiding applications.
The relative importance of pulse field strength E and pulse length tau 1/2 (half decay time of an exponential decay pulse) on the stable transfection frequency for HeLa or HUT-78 cells was investigated. Cells were transfected with plasmids containing the promoter and drug resistant genes pRSVgpt or pRSVneo by electroporation. The stable transfection frequency was assayed using the marker rescue technique. The transfection frequency increases with increasing values of E tau 1/2. For a given pulse length, the transfection frequency is proportional to the power of the pulse (E2 tau 1/2). Pulses with half decay times of 2.2 to 4.6 ms appear to be more efficient than 0.275 to 0.31 ms for stable transfection of HeLa cells.
Transient membrane permeabilization by application of high electric field intensity pulses on cells (electropermeabilization) depends on several physical parameters associated with the technique (pulse intensity, number, and duration). In the present study, electropermeabilization is studied in terms of flow of diffusing molecules between cells and external medium. Direct quantification of the phenomenon shows that electric field intensity is a critical parameter in the induction of permeabilization. Electric field intensity must be higher than a critical threshold to make the membrane permeable. This critical threshold depends on the cell size. Extent of permeabilization (i.e., the flow rate across the membrane) is then controlled by both pulse number and duration. Increasing electric field intensity above the critical threshold needed for permeabilization results in an increase membrane area able to be permeabilized but not due to an increase in the specific permeability of the field alterated area. The electroinduced permeabilization is transient and disappears progressively after the application of the electric field pulses. Its life time is under the control of the electric field parameters. The rate constant of the annealing phase is shown to be dependent on both pulse duration and number, but is independent of electric field intensity which creates the permeabilization. The phenomenon is described in terms of membrane organization transition between the natural impermeable state and the electro-induced permeable state, phenomenon only locally induced for electric field intensities above a critical threshold and expanding in relation to both pulse number and duration.
Fluorescent and fluorescent-labeled molecules were used with calibrated flow cytometric fluorescence measurements of electrically pulsed cells (intact yeast: Saccharomyces cerevisiae) to demonstrate a method for determining the net number of molecules transported into electroporated cells. For the conditions used, a single pulse of width 50 microseconds and magnitude 8.0 +/- 0.5 kV/cm resulted in an average net molecular uptake which is large, n = 1.4 x 10(5) molecules of 70 kDa FITC-dextran (supplied extracellular concentration of 500 microM), and n = 1.0 x 10(8) molecules of 660 Da propidium iodide (PI; 80 microM). Both molecules were present in pulsed cells at less than equilibrium values, consistent with a transient uptake mechanism. Intracellular FITC-dextran is present in soluble form, while PI is predominantly bound to nucleic acids. A broad, statistically significant distribution of molecular uptake was also observed. Such quantitative determinations should be important for guiding applications of electroporation, and for testing models of electroporation mechanisms. Further, the use of PI, which is well established as a membrane exclusion dye, provides additional support for the interpretation that both PI and FITC-dextran were internalized as a result of an electrical pulse.
The uptake of fluorescence-labeled dextrans by adherent 10T 1/2 murine fibroblasts following electric field pulse application was used as a criterion for the efficiency of electropermeation. The cells in monolayers were permeated by immersing a coaxial electrode in culture dishes. The percentage of cells which exhibited fluorescence uptake following electric field pulse application was measured at independently varying pulse field strength and pulse length. Dextrans with molecular weights equal to or higher than 41,000 dalton require higher field strength or longer pulse time to penetrate the cells. There is no detectable advantage of using a rectangular pulse against using an exponential decay pulse of similar power. The uptake was proportional to the product of the pulse amplitude and duration over the experimental range of 40-950 microseconds and 0.1-14.5 kV/cm. Cell survival decreases at the upper end of this range. The result provides a direct comparison of electric parameters which so far have not been standardized with regard to cell electropermeation.
DC-3F cells were submitted to electric square wave pulses in the presence of a d[alpha]octothymidylate 32P labelled in the 5' position. Radioactivity was incorporated in a voltage-dependent manner and reached a maximum for a field intensity of 1300-1400 V.cm-1. Growth curves and parallel cloning efficiency experiments indicated that cell viability was not altered by electric pulses, alone or in the presence of the oligothymidylate, below a field intensity of 1300 V.cm-1. Using affinity chromatography we extracted the incorporated oligonucleotide and showed that it was not degraded during the electropermeabilization experiment time.
Spheroids of the human prostatic adenocarcinoma cell line DU 145 were used to study experimental radioimmunotherapy. Spheroids were incubated with the 131I-labelled monoclonal E4 antibody until the radionuclide immunoconjugate had bound the 5 to 6 outermost cell layers of the spheroids. A set of 50 spheroids were exposed, either immediately or 48 hr after antibody incubation and washings, to a dilute trypsin solution with the aim of stripping off cells from the spheroid surface. Stripped cells were collected in fractions corresponding to defined spherical shells. Cells were subsequently plated for clonogenic growth. The technique of automated sequential trypsinization of spheroids followed by a clonogenic survival assay permits studies on therapeutic efficacy for radionuclide immunoconjugates on cells from different layers of spheroids. In addition, the absorbed doses throughout a spheroid were calculated. The binding and retention kinetics of the radionuclide immunoconjugate and the excess of 131I-E4 in the culture medium during incubation are factors that were all accounted for in the calculations. If the calculated absorbed doses were inserted into the linear-quadratic survival model and the low dose rate was taken into account, survival values were well in accordance with the experimentally obtained values. The results demonstrate that the 131I-labelled E4 antibody is capable of sterilizing cultured tumour cells that have bound the radionuclide immunoconjugate and, by means of radiation "cross-fire", those cells located in close proximity.
Electroporation is believed to involve a temporary structural rearrangement of lipid bilayer membranes, which results in ion and molecular transport across the membrane. The results of a quantitative study of molecular transport due to electroporation caused by a single exponential pulse are presented; transport of four molecules of different physical characteristics across erythrocyte ghost membranes is examined as a function of applied field strength. Flow cytometry is used to quantitatively measure the number of molecules transported for 10(4) to 10(5) individual ghosts for each condition. This study has four major findings: 1) Net transport first increases with field strength, but reaches a plateau at higher field strengths. Significant transport is found at or below 1 kV/cm, and transport plateaus begin at field strengths between 2 and 5 kV/cm depending on the molecule transported. 2) A single population of ghosts generally exists, but exhibits a wide distribution in the amount of molecular transport. 3) Under the conditions used, the direction of transport across the ghost membrane does not appear to affect molecular transport significantly. 4) Large numbers of ghosts may be destroyed by the electroporation procedure.
Molecular uptake of a charged fluorescent molecule (calcein; 623 Da, z = -4) was quantitatively determined at the single cell level using flow cytometry. Dilutely suspended cells were exposed to one exponential pulse (tau p approximately 300 microseconds) for different field strength values. For an asymmetric cell such as the yeast Saccharomyces cerevisiae a significant variation in the number of molecules taken up by individual cells was expected for physical reasons. By carrying out several thousand individual cell measurements for each pulse condition, we found that the number of molecules per cell varies significantly within the cell population, and that this population distribution changes markedly as the field strength is varied. Surprisingly, in spite of significant changes in this distribution with field strength, the average uptake per cell reaches a non-equilibrium plateau for which the uptake per cell is much smaller than the product of the mean cell volume and the supplied extracellular concentration. These observations of different field-dependent cell population distributions of uptake support the hypotheses that (1) electroporation is a transmembrane voltage-responsive phenomenon, so that cells of different sizes, shapes and orientation, respond differently to even a spatially uniform applied field, (2) population average measurements of electroporation behavior can be incomplete and misleading, and (3) transport of small charged molecules is due to electrophoresis through the pores of a dynamically changing pore population.
Electroporation is believed to involve the creation of aqueous pathways in lipid bilayer membranes by transient elevation of the transmembrane voltage to approximately 1 V. Here, results are presented for a quantitative study of the number of bovine serum albumin (BSA) molecules transported into erythrocyte ghosts caused by electroportion. 1) Uptake of BSA was found to plateau at high field strength. However, this was not necessarily an absolute maximum in transport. Instead, it represented the maximum effect of increasing field strength for a particular pulse protocol. 2) Maximum uptake under any conditions used in this study corresponded to approximately one-fourth of apparent equilibrium with the external solution. 3) Multiple and longer pulses each increased uptake of BSA, where the total time integral of field strength correlated with uptake, independent of inter-pulse spacing. 4) Pre-pulse adsorption of BSA to ghost membranes appears to have increased transport. 5) Most transport of BSA probably occurred by electrically driven transport during pulses; post-pulse uptake occurred, but to a much lesser extent. Finally, approaches to increasing transport are discussed.
The superoxide (O2-)-generating NADPH oxidase of human neutrophils consists of membrane-bound and cytosolic proteins that assemble in the plasma membrane of activated cells. To date, most of our understanding of the assembly of the NADPH oxidase has been obtained through the use of a cell-free assay, and a number of peptides that mimic regions of NADPH oxidase proteins have been shown to block oxidase assembly using this assay. However, the cell-free assay provides an incomplete representation of the assembly and regulation of the NADPH oxidase in vivo, and it has become necessary to develop methods for introducing biomolecules, such as peptides, into intact neutrophils where their effects can be investigated. One such method is electropermeabilization. Although this method has been used previously with human neutrophils, it has not been well characterized. We report here a detailed characterization of the electropermeabilized neutrophil assay system, including optimal conditions for membrane electropermeabilization with maximal retention of functional capacity, optimal conditions for analyzing the effects of experimental peptides, quantification of internalized peptide concentration, and molecular size limits for diffusion of molecules into these cells. Our results demonstrate that optimal neutrophil permeabilization (98-100%) can be achieved using significantly lower electrical fields than previously reported, resulting in the retention of higher levels of O2(-)-generating activity. We also found that biomolecules as large as 2.3 kDa readily diffuse into permeabilized cells. Analysis of flavocytochrome b peptides that were shown previously to inhibit NADPH oxidase activity in a cell-free assay demonstrated that these peptides also blocked O2- production in electropermeabilized human neutrophils; although at higher effective concentrations than in the cell-free system. Thus, electropermeabilized neutrophils provide a model system for evaluating the effects of peptides and other pharmacological agents in intact cells which closely mimic neutrophils in vivo.
The color change of electroporated intact immunoglobulin G receptor (Fc gammaR-) mouse B cells (line IIA1.6) after direct electroporative transfer of the dye SERVA blue G (Mr 854) into the cell interior is shown to be dominantly due to diffusion of the dye after the electric field pulse. Hence the dye transport is described by Fick's first law, where, as a novelty, time-integrated flow coefficients are introduced. The chemical-kinetic analysis uses three different pore states (P) in the reaction cascade (C <==> P1 <==> P2 <==> P3), to model the sigmoid kinetics of pore formation as well as the biphasic pore resealing. The rate coefficient for pore formation k(p) is dependent on the external electric field strength E and pulse duration tE. At E = 2.1 kV cm(-1) and tE = 200 micros, k(p) = (2.4 +/- 0.2) x 10(3) s(-1) at T = 293 K; the respective (field-dependent) flow coefficient and permeability coefficient are k(f)0 = (1.0 +/- 0.1) x 10(-2) s(-1) and P0 = 2 cm s(-1), respectively. The maximum value of the fractional surface area of the dye-conductive pores is 0.035 +/- 0.003%, and the maximum pore number is Np = (1.5 +/- 0.1) x 10(5) per average cell. The diffusion coefficient for SERVA blue G, D = 10(-6) cm2 s(-1), is slightly smaller than that of free dye diffusion, indicating transient interaction of the dye with the pore lipids during translocation. The mean radii of the three pore states are r(P1) = 0.7 +/- 0.1 nm, r(P2) = 1.0 +/- 0.1 nm, and r(P3) = 1.2 +/- 0.1 nm, respectively. The resealing rate coefficients are k(-2) = (4.0 +/- 0.5) x 10(-2) s(-1) and k(-3) = (4.5 +/- 0.5) x 10)(-3) s(-1), independent of E. At zero field, the equilibrium constant of the pore states (P) relative to closed membrane states (C) is K(p)0 = [(P)]/[C] = 0.02 +/- 0.002, indicating 2.0 +/- 0.2% water associated with the lipid membrane. Finally, the results of SERVA blue G cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative delivery of drugs that are similar in structure to SERVA blue G, for instance, bleomycin, which has been used successfully in the new discipline of electrochemotherapy.
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