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Electroendocytosis: Exposure of Cells to Pulsed Low Electric Fields Enhances Adsorption and Uptake of Macromolecules
Abstract
This study demonstrates alteration of cell surface, leading to enhanced adsorption of macromolecules (bovine serum albumin (BSA), dextran, and DNA), after the exposure of cells to unipolar pulsed low electric fields (LEF). Modification of the adsorptive properties of the cell membrane also stems from the observation of LEF-induced cell-cell aggregation. Analysis of the adsorption isotherms of BSA-fluorescein isothiocyanate (FITC) to the surface of COS 5-7 cells reveals that the stimulated adsorption can be attributed to LEF-induced increase in the capacity of both specific and nonspecific binding. The enhanced adsorption was consequently followed by increased uptake. At 20 V/cm the maximal binding and subsequent uptake of BSA-FITC attached to specific sites are 6.5- and 7.4-fold higher than in controls, respectively. The nonspecific LEF-induced binding and uptake of BSA are 34- and 5.2-fold higher than in controls. LEF-enhanced adsorption is a temperature-independent process, whereas LEF-induced uptake is a temperature-dependent one that is abolished at 4 degrees C. The stimulation of adsorption and uptake is reversible, revealing similar decay kinetics at room temperature. It is suggested that electrophoretic segregation of charged components in the outer leaflet of the cell membrane is responsible for both enhanced adsorption and stimulated uptake via changes of the membrane elastic properties that enhance budding and fission processes.
... (7) Electroendocytosis, unlike electroporation, which causes nanometer-sized electropores to develop on the lipid bilayer of the plasma membrane, causes cell membrane internalization and fission via endocytotic vesicles. (8,9) It incorporates either macromolecules that are bound to membrane receptors by receptor-mediated endocytosis (10,11) or any soluble drugs by fluid-phase endocytosis (pinocytosis). (12,13) Exocytosis, a reciprocal mechanism, restores the cellular membrane area. ...
... (14) Two main types of electric pulses have reported endocytosis induced by pulsed low electric fields. (7,10,11,(13)(14)(15) The first pulse type used was bipolar symmetrical square pulses with field intensity ranging from 1.2 V/cm to 8 V/cm, pulse durations ranging from 75 µs to 580µs, frequencies ranging from 50 to 400 Hz, and total exposure times ranging from 5 to 90 minutes. A bipolar pulse was adopted to limit unidirectional electrophoresis and to reduce electrochemical deposits at the surface of the electrodes. ...
... The uptake was also linearly dependent on the pulse duration and medium conductivity. (7,10,11) These two sets of data were markedly different. ...
Electric and electromagnetic pulses have been shown to enhance the endocytosis rate, with all-or-nothing responses beyond a field strength threshold and linear responses as a function of field strength and treatment duration utilizing bipolar symmetrical and monopolar pulses, respectively. Malignant glioma (MG) is resistant to chemotherapy. The present study looked for a new electrical impulse that can aid electrochemotherapy to deliver anticancer drugs while using less electrical energy. Bipolar asymmetric electric pulses were applied to U251MG cells suspended in physiologically conductive media in the presence of molecular probes, including Bleomycin. The delivered electric pulses with a pulse duration range of 180-500 µs and a frequency range of 100-400 Hz had a low field intensity ranging from 1.5 V/cm to 7.3 V/cm. Spectrophotometric and spectrofluorometric measurements were used to investigate the impact of these variables on cancer cell survival and the molecular probe uptake induced by the electric pulses. An all-or-nothing response was observed above a specified threshold of electric field intensity of 4 V/cm. This threshold was unaffected by changes in repetition frequency or pulse duration. It was not a temperature effect that caused the molecular probe uptake to increase. When bipolar asymmetric electric pulses were applied just before electroporation, the effectiveness of the cytotoxic impact of bleomycin was increased from 80%, when employing electroporation pulses alone, to 100%.
... The amplitude of this low voltage electric field is lower by several orders of magnitude than the electroporation threshold; thus, the formation of pores in the cell membrane is vague and other mechanisms should be considered. According to the results of the Antov et al. (3) study that was based on the uptake of macromolecules through the endocytosis pathway by a low voltage electric field, we assumed that endocytosis was a mechanism for increasing the efficiency of chemotherapy (small molecules) by LVHF. Endocytosis is a cellular process by which a cell takes up extracellular macromolecules into the cell cytoplasm. ...
... It has been reported that plasmid DNA (pDNA) is internalized by cells through an endocytosis-like process when used for electrotransfection (22)(23)(24). Antov et al. (3,25) reported that a low unipolar pulse electric field with a 2.5-20 V/cm amplitude, 500 Hz frequency and exposure time of 1-10 minutes increased the uptake of macromolecules of dextran-FITC and BSA-FITC into the cells by stimulation of fluid-phase endocytosis. In another study, fluorescent-labelled macromolecules (dextran-FITC and β-galactosidase) were incorporated into membrane vesicles and cells by low pulsed electric fields that had intensities of 60 and 100 V/cm, 90 µs pulse duration, 1000 Hz and exposure times of 10 minutes. ...
... Our results showed that, in addition to 10 V/cm, the 20 V/cm intensity could induce macropinocytosis; however, an increase in electric field voltage does not induce macropinocytosis. Stimulation of fluid-phase endocytosis by a low intensity electric field (2-20 V/cm) has been reported (3,25). Clathrinmediated endocytosis is initiated upon binding of ligands to receptors. ...
Objective:
The cell membrane is a major barrier for delivery of hydrophilic drugs and molecules into the cells. Although low voltage and high frequency electric fields (LVHF) are proposed to overcome the cell membrane barrier, the mechanism of membrane permeabilization is unclear. The aim of study is to investigate endocytosis pathways as a possible mechanism for enhancing uptake of bleomycin by LVHF.
Materials and methods:
In this experimental study, MCF-7 cells were exposed to bleomycin or to electric fields with various strengths (10-80 V/cm), frequency of 5 kHz, 4000 electric pulse and 100 μs duration in the presence and absence of three endocytosis inhibitors-chlorpromazine (Cpz), amiloride (Amilo) and genistein (Geni). We determined the efficiency of these chemotherapeutic agents in each group.
Results:
LVHF, depending on the intensity, induced different endocytosis pathways. Electric field strengths of 10 and 20 V/cm stimulated the macropinocytosis route. Clathrin-mediated endocytosis was observed at electric field intensities of 10, 30, 60 and 70 V/cm, whereas induction of caveolae-mediated endocytosis was observed only at the lowest electric field intensity (10 V/cm).
Conclusion:
The results of this study imply that LVHF can induce different endocytosis pathways in MCF-7 cells, which leads to an increase in bleomycin uptake.
... Although the intensity of the induced electric field is low, researchers have recently found that pulsed low electric fields (2.5-20 V/cm) can increase cell membrane permeability by up to 10-folds [1,2]. This enhancement in cell membrane permeability leads to the increased uptake of macromolecules by cytosol. ...
... Therefore, the gap between adjacent phospholipids expands. This leads to the creation of a path through which BLM can diffuse into the cytosol [2]. ...
... Conversely, by using an alternating magnetic field that leads to the formation of a pulsed low electric field, a disturbance will occur in the arrangement of phospholipids and protein molecules within the cell membrane's structure. This effect is called electroendocytosis and we suggest this phenomenon is responsible for the increased uptake of BLM in the BLM plus 3.5 T MF group [1,2]. The cell membrane disarrangement occurs long enough (about 1 h) to pass enough BLM molecules through the cell membrane into the cytosol [1,2]. ...
Background:
In the present study, we investigated the application of pulsed magnetic field (MF) (3.5 T, 1 Hz, 8 square-wave/160 µs) permeabilization on murine breast adenocarcinoma cells when administering bleomycin (BLM) in vivo.
Objective:
This cross-over study aims to find a noninvasive method to facilitate penetration of hydrophilic anti-cancer drugs through the cancerous cells membrane into the cytosoll in order to minimize the side effects of the chemotherapy treatments of tumors.
Material and methods:
In this cross-over study, a total of 50 female Balb/c mice were tumorized via homograft. After about 2 weeks, magnetic pulses (3.5 T, 1 Hz, 8 square-wave/160 µs) were applied to tumor-bearing mice 3 min after intratumoral BLM solution injection. Tumor volume was measured every 48 h during 22 days.
Results:
The results showed that the difference between the BLM plus 3.5 T MF group versus the sham control or sham MF groups was significant. Uptake of BLM molecules by tumoral cells in the BLM plus 3.5 T MF group versus the BLM control group was 7- folds higher that this result was statistically insignificant (P<0.05, SEM=266.8676, analysis of variance).
Conclusion:
Significant cell permeabilization to BLM requires greater MF strength or exposure time. Further investigation is necessary.
... Non-permeabilizing and permeabilizing pulsed electric fields can both induce endocytosis [64,[106][107][108][109][110][111]. In this section, we focus on the effects of non-permeabilizing electric fields, because they are of particular interest for the induction of endocytic pathways. ...
... The exposure of cells to unipolar low electric fields pulses (of 20 and 43 V/cm) showed an induction of bovine serum albumin, dextran and DNA adsorption on cells surfaces and an increased uptake of these macromolecules via endocytosis-like processes [106]. While the adsorption of molecules did not depend on temperature, the internalization only occurred when low electric field pulses were applied at 24°C. ...
... The resulting asymmetric charge density between the outer and inner monolayer of the cell membrane affects the Gaussian curvature and causes membrane bending towards the inside of the cell. This phenomenon is followed by membrane fissions, which result in the formation of endocytic vesicles [106]. The absorption of molecules in the presence of a train of low electric field pulses was thus increased up to10 fold and internalization of molecules increased up to 5 fold [106]. ...
Electric fields are among physical stimuli that have revolutionized therapy. Occurring endogenously or exogenously, the electric field can be used as a trigger for controlled drug release from electroresponsive drug delivery systems, can stimulate wound healing and cell proliferation, may enhance endocytosis or guide stem cell differentiation. Electric field pulses may be applied to induce cell fusion, can increase the penetration of therapeutic agents into cells, or can be applied as a standalone therapy to ablate tumors. This review describes the main therapeutic trends and overviews the main physical, chemical and biological mechanisms underlying the actions of electric fields. Overall, the electric field can be used in therapeutic approaches in several ways. The electric field can act on drug carriers, cells and tissues. Understanding the multiple effects of this powerful tool will help harnessing its full therapeutic potential in an efficient and safe way.
... Endocytosis would be stimulated by the redistribution of the charges (i.e. reorganization of charged lipids and proteins) in the cell membrane due to the electric field [226,227]. The local lateral electrophoresis of proteins and lipids, in other words, the segregation of charged membrane components, only in the outer leaflet of the membrane would induce an asymmetric charge density between the outer and inner leaflet of the membrane thus responsible for spontaneous membrane curvature towards the cytoplasm (Fig. 11b). ...
... The local lateral electrophoresis of proteins and lipids, in other words, the segregation of charged membrane components, only in the outer leaflet of the membrane would induce an asymmetric charge density between the outer and inner leaflet of the membrane thus responsible for spontaneous membrane curvature towards the cytoplasm (Fig. 11b). The difference of charge density would also be responsible for the membrane fission into vesicles [227]. ...
... However, electric field properties in LEF are substantially different from those necessary for successful DNA electrotransfer and only macromolecules such as BSA or dextran have been investigated. The absorption at the membrane and uptake across the membrane of FITC-BSA was highly increased under LEF exposure [226,227,238]. FITC-BSA observations using microscopy show vesicle patterns and colocalization with a membrane probe (labeled DHPE) confirming the presence of vesicles enclosing the macromolecules. ...
Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.
... Scientific RepoRts | 6:19957 | DOI: 10.1038/srep19957 influence of medium conductivity vary depending on the study. It can be cell death induced by the exposure to pulses 14,16 , release or uptake of molecules 13,[16][17][18] or more indirect effects like the expression of a transfected gene 15 . Two studies focused specifically on reversible membrane permeabilization and they either concluded that conductivity does not impact the efficiency of the reversible permeabilization 14 or that lower conductivities are more efficient 17 . ...
... Scientific RepoRts | 6:19957 | DOI: 10.1038/srep19957 influence of medium conductivity vary depending on the study. It can be cell death induced by the exposure to pulses 14,16 , release or uptake of molecules 13,[16][17][18] or more indirect effects like the expression of a transfected gene 15 . Two studies focused specifically on reversible membrane permeabilization and they either concluded that conductivity does not impact the efficiency of the reversible permeabilization 14 or that lower conductivities are more efficient 17 . ...
... These results are in agreement with the studies from the group of Zimmermann who performed experiments with 'exponential pulses' in the microsecond range [17][18][19] and also with square pulses ranging from 10 to 100 ns 8 . Our results however contradict some of the previously published data indicating that the permeabilisation efficiency positively correlated with external conductivity or that there was no influence of the external medium conductivity [13][14][15][16] including one of our recent studies 22 . ...
The impact of external medium conductivity on the efficiency of the reversible permeabilisation caused by pulsed electric fields was investigated. Pulses of 12 ns, 102 ns or 100 μs were investigated. Whenever permeabilisation could be detected after the delivery of one single pulse, media of lower conductivity induced more efficient reversible permeabilisation and thus independently of the medium composition. Effect of medium conductivity can however be hidden by some saturation effects, for example when pulses are cumulated (use of trains of 8 pulses) or when the detection method is not sensitive enough. This explains the contradicting results that can be found in the literature. The new data are complementary to those of one of our previous study in which an opposite effect of the conductivity was highlighted. It stresses that the conductivity of the medium influences the reversible permeabilization by several ways. Moreover, these results clearly indicate that electropermeabilisation does not linearly depend on the energy delivered to the cells.
... Endocytosis is a general biological process in which cells take up larger particles or molecules by forming a vesicle and then internalizing it [36]. Endocytosis occurs continuously but can also be enhanced, for example, by the application of long low-voltage electric pulses (far below electroporation pulse parameters) [37][38][39][40], electroporation pulses [41], different mechanochemical stimuli [42], modulated electromagnetic fields [43] and by high-level static magnetic fields in combination with magnetic nanoparticles [44,45]. Endocytosis was also reported as a mechanism of HI-PEMF-mediated uptake of molecules [6,12,46,47], and it has been successfully used to enhance the uptake of molecules using PEF with electric fields whose values are similar to those induced by HI-PEMF [37][38][39]. ...
... Endocytosis occurs continuously but can also be enhanced, for example, by the application of long low-voltage electric pulses (far below electroporation pulse parameters) [37][38][39][40], electroporation pulses [41], different mechanochemical stimuli [42], modulated electromagnetic fields [43] and by high-level static magnetic fields in combination with magnetic nanoparticles [44,45]. Endocytosis was also reported as a mechanism of HI-PEMF-mediated uptake of molecules [6,12,46,47], and it has been successfully used to enhance the uptake of molecules using PEF with electric fields whose values are similar to those induced by HI-PEMF [37][38][39]. It is therefore reasonable to assume that high-intensity pulsed electromagnetic fields could also enhance endocytosis. ...
A high-intensity pulsed electromagnetic field (HI-PEMF) is a non-invasive and non-contact delivery method and may, as such, have an advantage over gene electrotransfer mediated by conventional electroporation using contact electrodes. Due to the limited number of in vitro studies in the field of gene electrotransfection by HI-PEMF, we designed experiments to investigate and demonstrate the feasibility of such a technique for the non-viral delivery of genetic material into cells in vitro. We first showed that HI-PEMF causes DNA adsorption to the membrane, a generally accepted prerequisite step for successful gene electrotransfection. We also showed that HI-PEMF can induce gene electrotransfection as the application of HI-PEMF increased the percentage of GFP-positive cells for two different combinations of pDNA size and concentration. Furthermore, by measuring the uptake of larger molecules, i.e., fluorescently labelled dextrans of three different sizes, we showed endocytosis to be a possible mechanism for introducing large molecules into cells by HI-PEMF.
... The real nature of the translocation step is still unknown yet. Some authors have noticed that electric fields could stimulate the uptake of various macromolecules by electro-endocytosis [28][29][30] , including plasmid DNA 31 . But, while many publications seem to confirm the involvement of endocytosis in the translocation step [32][33][34][35][36][37][38][39][40] , contradictory reports evidenced DNA entry by a non-endocytic translocation through the permeabilized membrane 18,19,26,[41][42][43] . ...
... Interestingly, there are several reports where electrotransfer is used for biomolecules distinct from nucleic acids, like proteins (β-galactosidase, bovine serum albumin or full antibody), or dextran [28][29][30]44 . However, to our knowledge, there is no report on the use of electrotransfer as a tool to induce or facilitate the intracellular penetration of viral particles. ...
Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.
... Cumulative the proline amount may be also due to changes in ion concentration in magnetized solutions in the cytosol. Water clusters can break down due to reduce the bond angle by the magnetic field and may leads to increase electric potential that rise salts solubility (Smikhina, 1981;Antov et al., 2005) and ions uptake which will stimulate the production of free radicals (Parola et al., 2005;Dhawi et al., 2009). Subsequently, the stress conditions for the plant will be simulated, resulting in producing more proline by the plant. ...
... Phenolic compounds play an imperative role in plant defense and immunity against external stressors and are synthesized in response to environmental and physiological stimuli (Balasundram et al., 2006;Napal et al., 2010). It seems increasing the number of monomer water due to magnetic fields (Zhou et al., 2000) along with the rise salts solubility (Antov et al., 2005) leading to creation of stress condition. Generally, the second method (pre-magnetized water supplemented by nutrients) with 0.1 T could affect TPC, TAC, and TFC content (Table 2). ...
... Furthermore, the decreasing viability of cells has been reported as a cytotoxic effect of electrolysis, which was the expected result of an electric current passing through the samples due to direct contact of electrodes. 26 There have been recent studies of PEF using indirect contact of electrodes demonstrating the calculation of electric field effect and the induction of biological effects. [27][28][29] Nevertheless, the electric fields of conducting these experiments are at high intensities (.1,000 V/cm) and near the verge of electric breakdown, which can be a danger of causing electric current to flow through the body. ...
... 47 However, when the transmembrane potential is lower than its threshold value, extracellular molecules are incorporated into the cells via endocytosis. 26,46 The transmembrane potential is theoretically given by ΔV m =1.5×E×a×cosθ; where E is the intensity of the applied electric field, a is the cell radius, and θ is the angle between the radial vector for a given location on cell membrane and the vector of electric field. ...
Background
Pulsed electric field (PEF) has been considered as a cell permeability enhancing agent for cancer treatment. Nevertheless, application of PEF for conventional electrochemo-therapy is usually at high intensity, and contact or even invasive electrodes are typically used, which may cause unwanted side effects. In this study, a non-invasive way of applying low intensity, non-contact PEF was adopted to study its combination effect with herb, curcumin, against pancreatic cancer cells and the mechanism involved.
Methods
The pancreatic cancer PANC-1 cells were treated with curcumin and PEF alone or in combination, and MTT assay was used to determine the viability of PANC-1 cells. Apoptosis and uptake of curcumin were analyzed by microscopy and flow cytometry. Western blot was further performed to evaluate the expression of apoptotic proteins.
Results
Our results demonstrated that PEF synergized with curcumin to inhibit the proliferation of PANC-1 cells in a field strength- and dose-dependent manner and caused apoptotic death of PANC-1 cells. The apoptotic induction of combination treatment was characterized by an increase in Bax/Bcl-2 ratio, and cleavage of caspase-8, -9, and -3. Moreover, the increase of curcumin uptake via electro-endocytosis was clearly observed in the cells following the exposure of PEF.
Conclusion
We show for the first time that a non-contact approach using low intensity electric field in a pulsed waveform could enhance the anticancer effect of low-dose curcumin on PANC-1 cells through triggering both extrinsic and intrinsic pathways. The findings highlight the potential of this alternative treatment, non-invasive electric field and curcumin, to increase therapeutic efficacy with minimum cytotoxicity and side effects, which may provide a new aspect of cancer treatment in combination of PEF and other anticancer agents.
... The opening and closing of pores could be triggered by HI-PEMF induced hydrostatic pressure with membrane deformation and additional formation and accumulation of surface charges on the membrane due to magnetic force [54], lipid oxidation, electrophoresis [11], electroporation due to an induced electric field [6], altered receptor binding or activation [55] and mechanical stress induced by magnetic and electric fields [56,57]. One of the suggested mechanisms of HI-PEMF-mediated uptake of molecules is also electro-endocytosis [11,58,59], as it has been successfully used to enhance the uptake of molecules using electric fields with values similar to those induced by HI-PEMF [60][61][62]. ...
These authors contributed equally to this work. Abstract: High-Intensity Pulsed Electromagnetic Fields (HI-PEMF) treatment is an emerging noninvasive and contactless alternative to conventional electroporation, since the electric field inside the tissue is induced remotely by an externally applied pulsed magnetic field. Recently, HI-PEMF has been successfully used in the transfer of plasmid DNA and siRNA in vivo, with no or minimal infiltration of immune cells. In addition to gene electrotransfer, treatment with HI-PEMF has also shown potential for electrochemotherapy, where activation of the immune response contributes to the treatment outcome. The immune response can be triggered by immunogenic cell death that is characterized by the release of damage-associated molecular patterns (DAMPs) from damaged or/and dying cells. In this study, the release of the best-known DAMP molecules, i.e., adenosine triphosphate (ATP), calreticulin and high mobility group box 1 protein (HMBG1), after HI-PEMF treatment was investigated in vitro on three different cell lines of different tissue origin and compared with conventional electroporation treatment parameters. We have shown that HI-PEMF by itself does not cause the release of HMGB1 or calreticulin, whereas the release of ATP was detected immediately after HI-PEMF treatment. Our results indicate that HI-PEMF treatment causes no to minimal release of DAMP molecules, which results in minimal/limited activation of the immune response.
... Depending on the PEF parameters, electroporation can be reversible or irreversible [29,30]. In mammalian cells, electroporation can be triggered at a 400-800 V/cm PEF [31], while lower field (<400 V/cm) can induce a phenomenon known as electroendocytosis, which means enhanced absorption of macromolecules after cell exposure to low electric fields [32,33]. Scientific papers focusing on the stimulating effects of high-intensity PEF also have started to appear in recent years. ...
Electrical stimulation (ES) has been frequently used in different biomedical applications both in vitro and in vivo. Numerous studies have demonstrated positive effects of ES on cellular functions, including metabolism, proliferation, and differentiation. The application of ES to cartilage tissue for increasing extracellular matrix formation is of interest, as cartilage is not able to restore its lesions owing to its avascular nature and lack of cells. Various ES approaches have been used to stimulate chondrogenic differentiation in chondrocytes and stem cells; however, there is a huge gap in systematizing ES protocols used for chondrogenic differentiation of cells. This review focuses on the application of ES for chondrocyte and mesenchymal stem cell chondrogenesis for cartilage tissue regeneration. The effects of different types of ES on cellular functions and chondrogenic differentiation are reviewed, systematically providing ES protocols and their advantageous effects. Moreover, cartilage 3D modeling using cells in scaffolds/hydrogels under ES are observed, and recommendations on reporting about the use of ES in different studies are provided to ensure adequate consolidation of knowledge in the area of ES. This review brings novel insights into the further application of ES in in vitro studies, which are promising for further cartilage repair techniques.
... As many previous reports have shown, YO-PRO-1 uptake via pores and endocytotic processes can occur during and following electrical stimulation. To explore this phenomenon, Antov et al. reported that nonpermeabilizing pulsed trains of low electric fields (LEF) in the range of 2.5-20 V/cm stimulated uptake of FITC dextran, BSA-FITC, Lucifer yellow, and PI via endocytotic pathways independent of their molecular weight and charge [42,43]. In addition, Ben-Dov et al. demonstrated that electrochemical production of protons at the anode interface is responsible for inducing the uptake of macromolecules [44]. ...
Membrane permeabilization stimulated by high-voltage electric pulses has been used to deliver cell-impermeable exogenous molecules. The electric field effect on the cells depends on various experimental parameters, such as electric field strength, the number of electric pulses, and the electroporation medium. In this study, we show the influence of the electroporation medium on membrane permeabilization stimulated by electrical short-circuiting via an aqueous droplet in dielectric oil, a novel methodology developed by our previous investigations. We investigated the membrane permeabilization by three methods, influx of calcium ions, uptake of nucleic acid-binding fluorophores (YO-PRO-1), and calcein leakage. We demonstrated that the external medium conductivity had a significant impact on the cells in all described experiments. The short-circuiting using a low-conductivity electroporation medium enhanced the formation of both transient and irreversible membrane pores. We also found that clathrin-mediated endocytosis contributed to YO-PRO-1 uptake when a cell culture medium was used as an electroporation medium.
... ECT is a tumor treatment modality that combines the administration of a poorly permeant cytotoxic drug having an intracellular target with the local application of electric pulses to improve drug diffusion into the cells (Antov et al. 2005;Domanico et al. 2015;Mir et al. 1991). Figure 1-4 The main mechanism of ECT is the increase of cell membrane permeability to drug administrated into the tumor or bloodstream (Gothelf et al. 2003;Mir 2006;Pakhomova et al. 2013;Rembiałkowska et al. 2020;Wichtowski and Murawa 2018). ...
Electrochemotherapy (ECT) as a tumor treatment modality is approved for cutaneous and subcutaneous tumors. The purpose of the present study was to examine the effect of 900 MHz radiofrequency (RF) pulse-modulated by 217 Hz EMFs similar to those emitted by mobile phones on the mechanisms of ECT in vivo including: tumor hypoxia and immune system response, and on tumor volume.4 T1 cells were injected subcutaneously into the right flank of Balb/c mice. The mice were exposed to RF fields at specific absorption rate (SAR) 2 W/kg for 10 min/day and then treated with ECT. Two protocols of ECT were used: ((70 V/cm-5 kHz) and 70 V/cm-4 kHz)). Tumor hypoxia was analyzed through HIF-1α immuonohistochemistry assay. Interleukin 4 (IL-4) and IFN-γ levels were estimated by enzyme-linked immunosorbent assay (ELISA) technique to evaluate immune system response. Also, tumors volume changes were measured for 24 days following the treatment. The results showed that pulse-modulated RF fields could increase hypoxia induced by ECT, significantly (about 13% in ECT (70 V/cm-5 kHz) and 11% in ECT (70 V/cm-4 kHz)). However, these fields did not have significant effect on immune system response (the levels of IL-4 and IFN-γ) and tumor volume changes induced by ECT. Our results indicated that pulse-modulated RF fields could not affect tumor volume changes in ECT with the frequency of 5 kHz and voltage of 70 V/cm efficacy in vivo. However, investigating the role of other environmental intervening factors on this protocol of ECT is recommended in further studies.
... Alternatively, PEF could trigger ''electroendocytosis" -an electric-field induced endocytic-like process that was first observed when DNA was internalised into large unilamellar vesicles via the formation of endosome-like vesicles when exposed to PEF [199]. ''Electroendocytosis" was later reported in different cells in vitro as well [200][201][202]. However, it remains unclear whether ''electroendocytosis" is specific to PEF or it is simply a native cellular response to membrane damage [203]. ...
Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.
... The electroendocytosis as a mechanism might have an effect of the treatment outcome, as well [37,38]. It is a phenomenon of the uptake of the macromolecules into cells, following exposure to pulsed low electric fields, which does not involve an electric breakdown of the membrane (electroporation) [39]. ...
Long duration electric pulses are frequently used to facilitate DNA electrotransfer into cells and tissues, while electroporation pulses can be combined with electrophoresis to maximize the transfection efficiency. In this work, we present the dielectrophoresis (DEP)-assisted methodology for electrotransfer of plasmid DNA (3.5 kbp pmaxGFP) into mammalian cells (CHO-K1). A prototype of an electroporation cuvette with center needle electrode for DEP-assisted transfection is presented resulting in a 1.4-fold of transfection efficiency increase compared to the electroporation-only procedure (1.4 kV/cm × 100 µs × 8). The efficiency of transfection has been compared between three DEP frequencies of 1, 100, and 1 MHz. Lastly, the effects of exposure time (1, 3, and 5 min) during the DEP application step have been determined. It is concluded that the proposed methodology and exposure setup allow a significant improvement of transfection efficiency and could be used as an alternative to the currently popular electrotransfection techniques.
... The electric stimulation has a long history of medical applications, including wound healing [34], muscle relaxation [35], and increase of blood circulation [36], often involving passage of electric current through the injury parts via direct contact between the body and implanted electrodes. However, the invasive or contact manner significantly dampens the feasibility of the treatment or even causes harms to cells or body, such as electrolysis, burns and muscle spasms [37]. Therefore, it is desirable for electrotherapy to be carried out in a non-invasive or non-contact manner. ...
As the most common type of neurodegenerative diseases (NDDs), Alzheimer's disease (AD) is thought to be caused mainly by the excessive aggregation of β-amyloid protein (Aβ). However, a growing number of studies have found that reactive oxygen species (ROS) play a key role in the onset and progression of AD. The present study aimed to probe the neuroprotective effect of high-frequency low-intensity pulsed electric field (H-LIPEF) for SH-SY5Y cells against hydrogen peroxide (H2O2) and Aβ-induced cytotoxicity. By looking in a systematic way into the frequency- and amplitude-dependent neuroprotective effect of pulsed electric field (PEF), the study finds that H-LIPEF at 200 Hz produces the optimal protective effect for SH-SY5Y cells. The underlying mechanisms were confirmed to be due to the activation of extracellular signal-regulated kinase (ERK) pathway and the downstream prosurvival and antioxidant proteins. Because the electric field can be modified to focus on specific area in a non-contact manner, the study suggests that H-LIPEF holds great potential for treating NDDs, whose effect can be further augmented with the administering of drugs or natural compounds at the same time.
... Plusieurs mécanismes ont été proposés concernant l'insertion et la translocation du complexe ADN/membrane. La molécule d'ADN pourrait appliquer une pression sur la membrane induisant la formation d'une vésicule d'endocytose (Antov et al. , 2005) ou de macropinocytose (décrit précédemment). Un autre mécanisme reposerait sur l'insertion de la molécule d'ADN dans un électropore qui induirait ensuite la formation d'une « vésicule » lipidique. ...
Au cours de ces dernières décennies, l'arsenal des thérapies anticancéreuses basées sur des principes physiques tels que les radiofréquences, les ultrasons, le laser, l'électroporation, la thérapie photo dynamique et les plasmas a considérablement augmenté. Ces approches permettent d'éradiquer les cellules cancéreuses, directement ou via la stimulation du système immunitaire. Elles sont aussi proposées en tant que méthodes de vectorisation pour délivrer des molécules d'intérêt thérapeutique. Plus récemment, est apparu un intérêt grandissant pour l'utilisation en thérapie anti-cancéreuse de liquides enrichis en espèces réactives de l'oxygène et de l'azote (RONS) via leur exposition au plasma froid. Les cellules cancéreuses peuvent ainsi être éradiquées, de façon sélective, après contact avec du liquide activé par plasma (PAL). Néanmoins, les mécanismes d'action de ces PAL sont encore peu connus à ce jour. C'est dans ce contexte que nous avons évalué le potentiel thérapeutique de différentes solutions salines exposées au jet de plasmas froids sur les cellules cancéreuses. Parallèlement, l'électroperméabilisation ou électroporation (EP) réversible connaît un essor important en raison du développement de ses applications cliniques notamment en électrochimiothérapie pour favoriser le transfert d'agent anti-cancéreux. Nous avons proposé une approche innovante reposant sur la combinaison des PAL et de l'EP, afin de potentialiser les effets cytotoxiques et génotoxiques des PAL, via la déstabilisation par l'EP de la tumeur, et obtenir ainsi une éradication plus efficace. Dans un premier temps, nous avons étudié l'effet du tampon phosphate salin (PBS) et du chlorure de sodium (NaCl) exposés au jet hélium de plasma froid, in vitro, sur la croissance, la viabilité/mort cellulaire et l'organisation structurelle de modèles 3D de tumeurs non-vascularisées (les sphéroïdes). Deux lignées cellulaires cancéreuses ont été utilisées : cellules de cancer colorectal humain HCT-116 et cellules de carcinose ovarienne péritonéale humaine SKOV-3. Nous avons mis en évidence que ces PAL induisent une cascade d'évènements cellulaires : fuite de l'ATP intracellulaire, dommages à l'ADN et dysfonction mitochondriale, provoquant la mort apoptotique des cellules des couches externes du sphéroïde dans les heures suivant le traitement (~6 heures). De plus, le PBS activé par plasma a montré une cytotoxicité et génotoxicité supérieure au NaCl, qui est corrélée avec la quantité de RONS présente dans les PAL. L'efficacité d'éradication des cellules cancéreuses de ces PAL se limitent aux cellules en périphérie des sphéroïdes. Il est nécessaire de faire plusieurs traitements pour avoir une éradication totale. Ainsi dans une deuxième partie, afin de pallier les problèmes de pénétration des PAL, nous avons proposé de les associer à l'EP réversible. La combinaison de ces deux approches a montré une potentialisation de la cytotoxicité et la génotoxicité des PAL, induisant la mort de toutes les cellules cancéreuses du sphéroïde. Cette augmentation de l'efficacité est en partie causée par des réarrangements des jonctions cellulaires suite à l'EP et à l'induction d'une mort cellulaire par autophagie.[...]
... Applying electric fields as low as 0.1 V cm −1 to a sample using capacitive coupling increased human mesenchymal stem cell invasion and proliferation [31]. Applying an electric field of 20 V cm −1 to a sample using capacitive coupling induced electroendocytosis [32]. Further, the duration of electrical treatment for typical capacitive coupling treatments, usually on the order of hours or days, greatly exceeds that of conductive coupling. ...
Most biomedical applications of electric fields use conductive coupling, where the electrodes make direct contact with a biological sample; however, this potentially contaminates the sample and hinders the workflow for large scale processes. Recent studies have reported dramatic changes in stem cell function following exposure to long duration, low intensity electric fields using capacitive coupling, where at least one of the electrodes fails to contact the cells or cell suspension. Here, we calculate the plasma membrane voltage, Vm, following treatment by intense, trapezoidal electric pulses (EPs) with durations ranging from 500 ps to 1 ms and rise- and fall-times from 200 ps to 0.3 ms using either conductive or capacitive coupling. The Vm induced by capacitive coupling is several orders of magnitude lower than conductive coupling for equivalent EP conditions while also introducing a negative polarity portion during the EP fall-time. Increasing the rise-time of an EP during capacitive coupling reduces the magnitude of Vm while increasing the EP duration extends the interval between the positive and negative portions of Vm. These calculations indicate that biological effects observed by capacitive coupling occur at much lower Vm than other electromanipulation techniques using conductive coupling, motivating further studies to assess the biophysical mechanisms induced.
... Electrosorption has been considered as the most effective desalination technique of wastewater because it has simple operation system, low cost, high efficiency, no secondary management problem and becomes environmentally friendly since it requires no chemicals for regeneration (Antov et al., 2005;Tanahashi et al., 1991). The most remarkable advantage of electrosorption technique is the ease of recovery of the adsorbent materials for their cyclic uses. ...
The world has been facing environmental challenges particularly from heavy metal pollution of water. Polyaniline (PAni) is one of the most frequently explored conducting polymers used as electrode materials in many applications including enhancing the desalination of carbonaceous electrodes. In this study, PAni modified graphite (PAni/Graphite) electrode was prepared by electrochemical polymerization of aniline in acidic solution and employed for adsorption of Fe(II) from aqueous solution under applied potentials. Characterizations of PAni/Graphite electrode were carried out by cyclic voltammetry and Fourier transform infrared (FT-IR) spectroscopy. Electrosorption of Fe(II) from aqueous solution on PAni/Graphite electrode was examined by cyclic voltammetry. The concentration of Fe(II) was monitored via cyclic voltammetry. The effects of time and potential on the adsorption were investigated and maximum adsorption of Fe(II) was found to be 0.212 mg g-1 of PAni by applying a constant potential of 0.5 V for 60 min. The desorption profile and the % recovery of Fe(II) from the surface of PAni/Graphite were also investigated and about 44 % of adsorbed Fe(II) was recovered by applying an opposite potential of-0.2 V for 60 min. This is a simple, fast, smart, cost-effective and environmental-benign technique for removal of heavy metals from aqueous solution.
... In addition to the investigation of biocompatibility of the material, the response of cells exposed to external electrical stimulus cannot be omitted. It is well documented that electrical stimulation at the cellular level can contribute to cell proliferation (93), migration (electrotaxis), differentiation (94), endocytosis (95) and permeabilization (96) of membranes. Therefore, the response of the cells against different operating conditions are required to investigate considering their viability, metabolism, spreading and shape. ...
PurposeAmong various types of external stimuli-responsive DDS, electric-responsive DDS are more promising carriers as they exploit less complex, easily miniaturized electric signal generators and the possibility of fine-tuning the electric signals. This study investigates the use of intrinsically biocompatible biopolymers in electro-simulative drug delivery to enhance the release of poorly-soluble/non-ionic drug.MethodsCMC/PLA/ZnO/CUR nanocomposite films were prepared by the dispersion of CMC and ZnO NPs in solubilized PLA/curcumin medium, followed by solvent casting step. Curcumin is poorly water-soluble and used as the model drug in this study. The films with different contents of CMC, PLA and ZnO NPs were characterized using FTIR, impedance spectroscopy, tensile testing and FESEM imaging. The in vitro drug release of the films was carried out in deionized water under DC electric field of 4.5 V.ResultsThe ionic conductivity of the films increased with increasing the CMC concentration of the film. The addition of a small amount of ZnO NPs (2%) successfully restored the tensile properties of the film. In response to the application of the electric field, the composite films released drug with a near-linear profile. There was no noticeable amount of passive diffusion of the drug from the film with the absence of the electric field.Conclusion
The outcome of this study enabled the design of an electric-responsive nanocomposite platform for the delivery of poorly water-soluble/non-ionic drugs.
... Any changes of ions organization, along the membrane change the cell electric potential. The MF may increase the electric potential which increases the nutrient uptake (Antov et al., 2005). ...
The present study was conducted during 2018 and 2019 seasons in the Experimental Research and Production Station of National Research Centre, Nubaria Region, Behira Governorate, Egypt to investigate the effect magnetic water treatment (i.e., magnetic (MW1) and un-magnetic water (MW0) under three levels of field capacity (FC, 100, 80 and 60%) on the soil moisture content, growth, yield, volatile oil production and water productivity of French basil (Ocimum basilicum, L. var Grandvert). The obtained results are summarized as follows: the irrigation intervals were converging under 100% of the FC, when compared with 80 and 60% of FC, for magnetic and un-magnetic water during growth stages of both growing seasons. The soil moisture values were increased by irrigated with magnetic water compared with un-magnetic water at 100, 80 and 60 % of the FC, during the two growing seasons. In all cuts in both seasons, the plants treated with magnetic water (MW 1) + 100% FC, showed significantly stimulation in growth characters, i.e branches (number plant-1), herb fresh and dry weights (g plant-1)/ton ha-1 (hectare) as well as volatile oil production, the exception was in case of plant height (cm) which showed a significant decrease. The results of GLC analysis of French basil volatile oil indicated that, the highest linalool content the main component {62.33 was produced with (MW 1 + 100% FC)}. The increment in volatile oil production was the same trend in water productivity under magnetic and 100% of (FC) for two growing seasons. As for high water productivity, the higher yield with lower water consumption, gives higher water productivity.
... Moderate intensity (0.45 and 0.63 kV/cm) electroporation in presence of calcium has also been demonstrated to be capable of inducing cancer cell death [14]. It should be noted that even for some proposed approaches using low-voltage (<200V/cm) electroporation to increase the permeability of cell membranes, electrolysis has been reported to the cause of cytotoxic effects resulting from the direct contact with electrodes [15]. Therefore, we suggest that the electrical stimulation in low-intensity and non-invasion manner would be more proper for anticancer treatment. ...
Most existing cancer treatments involve high-cost chemotherapy and radiotherapy, with major side effects, prompting effort to develop alternative treatment modalities. It was reported that the combination of thermal-cycling hyperthermia (TC-HT) and phenolic compound exhibited a moderate cytotoxic effect against human pancreatic cancer PANC-1 cells. In this study, we investigate the efficacy of triple combination in PANC-1 cancer cells by adopting low-intensity pulsed electric field (LIPEF) to couple with TC-HT and CGA (chlorogenic acid). The study finds that this triple combination can significantly impede the proliferation of PANC-1 cells, with only about 20% viable cells left after 24h, whereas being non-toxic to normal cells. The synergistic activity against the PANC-1 cells was achieved by inducing G2/M phase arrest and apoptosis, which were associated with up-regulation of p53 and coupled with increased expression of downstream proteins p21 and Bax. Further mechanism investigations revealed that the cytotoxic activity could be related to mitochondrial apoptosis, characterized by the reduced level of Bcl-2, mitochondrial dysfunction, and sequential activation of caspase-9 and PARP. Also, we found that the triple treatment led to the increase of intracellular reactive oxygen species (ROS) production. Notably, the triple treatment-induced cytotoxic effects and the elevated expression of p53 and p21 proteins as well as the increased Bax/Bcl-2 ratio, all could be alleviated by the ROS scavenger, N-acetyl-cysteine (NAC). These findings indicate that the combination of CGA, TC-HT, and LIPEF may be a promising modality for cancer treatment, as it can induce p53-dependent cell cycle arrest and apoptosis through accumulation of ROS in PANC-1 cells.
... However, electrical stimulation also affects the cell growth and geometry, which is usually not taken into account. It is well-known that at the cellular level, electrical stimulation can contribute to cell proliferation [26], migration (electrotaxis) [27], differentiation [28], endocytosis [29] and membrane permeabilization [30]. Particularly, at the intracellular level, changes are produced in the Ca 2+ entry modulation, in the integrin conformation, induction of plasma membrane depolarization, redistribution of the transmembrane proteins and reorganization of cytoskeletal structure. ...
Electrical stimulation is an attractive approach to tune on-demand drug release in the body as it relies on simple setups and requires typically 1 V or less. Although many studies have been focused on the development of potential smart materials for electrically controlled drug release, as well as on the exploration of different delivery mechanisms, progress in the field is slow because the response of cells exposed to external electrical stimulus is frequently omitted from such investigations. In this work, we monitor the behavior of prostate and breast cancer cells (PC-3 and MCF7, respectively) exposed to electroactive platforms loaded with curcumin, a hydrophobic anticancer drug. These consist in conducting polymer nanoparticles, which release drug molecules by altering their interactions with polymer, and electrospun polyester microfibres that contain electroactive nanoparticles able to alter the porosity of the matrix through an electro-mechanical actuation mechanism. The response of the cells against different operating conditions has been examined considering their viability, metabolism, spreading and shape. Results have allowed us to differentiate the damage induced in the cell by the electrical stimulation from other effects, as for example, the anticancer activity of curcumin and/or the presence of curcumin-loaded nanoparticles or fibres, demonstrating that these kinds of platforms can be effective when the dosage of the drug occurs under restricted conditions.
... The kink in the Arrhenius plot is not fixed at a particular temperature, as it may be shifted by various heating processes and by chemotherapy (14) or other chemical conditions (15) that modify the actual reaction (16). An electric field also may affect the kink of the Arrhenius plot (17). The Arrhenius kink, which must be overheated, corresponds well to the believed cellular phase transition observed at around 42.5˚C (18). ...
The problem with the application of conventional hyperthermia in oncology is firmly connected to the dose definition, which conventionally uses the concept of the homogeneous (isothermal) temperature of the target. Its imprecise control and complex evaluation is the primary barrier to the extensive clinical applications. The aim of this study was to show the basis of the problems of the misleading dose concept. A clear clarification of the proper dose concept must begin with the description of the limitations of the present doses in conventional hyperthermia applications. The surmounting of the limits the dose of oncologic hyperthermia has to be based on the applicability of the Eyring transition state theory on thermal effects. In order to avoid the countereffects of thermal homeostasis, the use of precise heating on the nanoscale with highly efficient energy delivery is recommended. The nano‑scale heating allows for an energy‑based dose to control the process. The main aspects of the method are the following: i) It is not isothermal (no homogeneous heating); ii) malignant cells are heated selectively; and iii) it employs high heating efficacy, with less energy loss. The applied rigorous thermodynamical considerations show the proper terminology and dose concept of hyperthermia, which is based on the energy‑absorption (such as in the case of ionizing radiation) instead of the temperature‑based ideas. On the whole, according to the present study, the appropriate dose in oncological hyperthermia must use an energy‑based concept, as it is well‑known in all the ionizing radiation therapies. We propose the use of Gy (J/kg) in cases of non‑ionizing radiation (hyperthermia) as well.
... Previous studies have reported that moderate MF is sufficient to change the plasma membrane permeability of cells and alter ion movement across the membrane. These studies also reported that an increase in calcium ion uptake is occurred due to channel activation by MF. 16,17 Teodori and coworkers showed that SMF increases the cell survival againsr damaging agents via increased Ca 2+ influx in U937 cells. 18 In the present study, the better preservation of follicles in the V2 group might be attributed to the increased membrane permeability induced by SMF and as a result caused an increase in intracellular calcium ions. ...
This study was designed to investigate the effects of applying 1 mT static magnetic field (SMF) during the vitrification process, on the viability of ovarian follicles after vitrification-warming and autotransplantation. The study was conducted in two phases. In the first phase, ovaries of female NMRI mice (6 to 8 weeks old) were randomly divided into three groups: 1- Freshly isolated ovaries fixed in Bouin solution (control group), 2- Ovaries vitrified-warmed without exposure to magnetic field (V1 group) and 3- Ovaries exposed to magnetic field during equilibration step of the vitrification process (V2 group). In the second phase, the vitrified (V1 and V2 groups) and fresh ovarian tissues were autografted into the back muscles of the mice from which the ovaries were extracted. In both phases, morphological aspects and molecular characteristics of active-apoptotic caspase-3 antibody were evaluated. Results indicated the lower percentages of morphologically intact primordial, primary and antral follicles in the V1 group (67.6, 49.5 and 17.6%, respectively) than those of control (97.3, 85.4 and 42.1%, respectively) and V2 (94.1, 78.8 and 40.9%, respectively) groups. In addition, the mean percentages of morphologically intact follicles in the V1 group were statistically lower than those in other groups, after transplantation. The rate of apoptosis in preantral follicles of the V1 group was significantly higher than that in the other groups. It was concluded that exposure of mice ovaries to SMF during vitrification resulted in greater resistance to injuries.
... Indeed, several groups demonstrated this effect, in which a decrease in σ e resulted greater electropermeabilization [58][59][60][61][62][63][64][65]. On the other hand, nearly the same number of studies reported the opposite-no change or a decrease in the efficiency of the EP treatment as the extracellular conductivity decreased [55,[66][67][68][69][70]. Only four of the above-mentioned studies evaluated the influence of σ e on nsEP-induced permeabilization. ...
Cellular effects caused by nanosecond electric pulses (nsEP) can be reduced by an electric field reversal, a phenomenon known as bipolar cancellation. The reason for this cancellation effect remains unknown. We hypothesized that assisted membrane discharge is the mechanism for bipolar cancellation. CHO-K1 cells bathed in high (16.1mS/cm; HCS) or low (1.8mS/cm; LCS) conductivity solutions were exposed to either one unipolar (300-ns) or two opposite polarity (300+300-ns; bipolar) nsEP (4-40kV/cm) with increasing interpulse intervals (0.1-50μs). Time-lapse YO-PRO-1 (YP) uptake revealed enhanced membrane permeabilization in LCS compared to HCS at all tested voltages. The time-dependence of bipolar cancellation was similar in both solutions, using either identical (22kV/cm) or isoeffective nsEP treatments (12 and 32kV/cm for LCS and HCS, respectively). However, cancellation was significantly stronger in LCS when the bipolar nsEP had no, or very short (<1μs), interpulse intervals. Finally, bipolar cancellation was still present with interpulse intervals as long as 50μs, beyond the time expected for membrane discharge. Our findings do not support assisted membrane discharge as the mechanism for bipolar cancellation. Instead they exemplify the sustained action of nsEP that can be reversed long after the initial stimulus.
... Macropinocytosis was earlier reported for the delivery of proteins, when added during the minutes following pulse delivery. One has to notice that electric pulses, applied under conditions that do not lead to "classical" permeabilization, can enhance natural endocytosis pathways [103,104]. ...
Cells are the structural and functional unit of all living organisms and exhibit fundamental properties of life. Cells are surrounded by the cell membrane and subdivided into various compartments. Pulsed electric fields (PEFs) exert profound effects on cells by interacting with the cell membrane and other cellular components. This chapter describes the biological effects of PEF at cellular and subcellular levels. First, this chapter begins with the overview of cell exposure to PEF from a biophysical point of view. Second, the interaction of PEF with biological membranes, membrane pore formation, and their physiological significance is described from multifaceted standpoints. Next, this chapter explains subcellular events induced by PEF, including the effect on cytoskeleton and signal transduction. Lastly, detailed description on irreversible electroporation and cell death by PEF is provided. The topics covered in this chapter serve as the basis for the applications of PEF in medicine, environmental science, and food and biomass processing.
... The MF may increase electric potential which increase the elements uptake. The adsorption and uptake of elements affected by MF segregated electrophoretic charges [67] which stimulate elements accumulation differently. ...
... These membrane-associated electric fields feed-back on membranes and associated proteins (Jaffe, 1977;Tsong and Astumian, 1986;Westerhoff et al., 1986;Bezanilla, 2002Bezanilla, , 2006Bezanilla, , 2008. They also control endocytosis and vesicle trafficking (Antov et al., 2005;Baluška and Wan, 2012). Relevantly, even biochemical reactions are under electric control (Aragoněs et al., 2016;Xiang and Tao, 2016), as is transcription (Pai et al., 2015b) and chromatin modification (Carneiro et al., 2011;Chernet and Levin, 2014). ...
The central nervous system (CNS) underlies memory, perception, decision-making, and behavior in numerous organisms. However, neural networks have no monopoly on the signaling functions that implement these remarkable algorithms. It is often forgotten that neurons optimized cellular signaling modes that existed long before the CNS appeared during evolution, and were used by somatic cellular networks to orchestrate physiology, embryonic development, and behavior. Many of the key dynamics that enable information processing can, in fact, be implemented by different biological hardware. This is widely exploited by organisms throughout the tree of life. Here, we review data on memory, learning, and other aspects of cognition in a range of models, including single celled organisms, plants, and tissues in animal bodies. We discuss current knowledge of the molecular mechanisms at work in these systems, and suggest several hypotheses for future investigation. The study of cognitive processes implemented in aneural contexts is a fascinating, highly interdisciplinary topic that has many implications for evolution, cell biology, regenerative medicine, computer science, and synthetic bioengineering.
... Obtained Pt amount in the tumor cells after treatment with CDDP only could be ascribed to passive diffusion and active transport mechanisms of cisplatin through the membrane, which are carrier-mediated through formed pores or via endocytosis. 70,71 In addition, it has been demonstrated that exposure of cells to train of unipolar pulsed low electric fields at strength from 1.2 up to 20 V/cm can induce electro-endocytosis. 72,73 Thus, we suspect that generated bipolar electric field of just below 0.09 V/cm by PEMF might also trigger endocytosis besides membrane permeabilization which enables the internalization of cisplatin in the cell and contributes partially to the increase of platinum amount. ...
Introduction: Pulsed electromagnetic field (PEMF) induces pulsed electric field, which increases membrane permeabilization of the exposed cells, similar to the conventional electroporation. Thus, contactless PEMF could represent a promising approach for drug delivery.
Materials and methods: Noninvasive electroporation was performed by magnetic field pulse generator connected to an applicator consisting of round coil. Subcutaneous mouse B16F10 melanoma tumors were treated with intravenously injection of CDDP (4 mg/kg), PEMF (480 bipolar pulses, at frequency of 80 Hz, pulse duration of 340 µs) or with the combination of both therapies (electrochemotherapy – PEMF + CDDP). Antitumor effectiveness of treatments was evaluated by tumor growth delay assay. In addition, the Pt uptake in tumors and serum, as well as Pt bound to the DNA in the cells and Pt in the extracellular fraction were measured by inductively coupled plasma mass spectrometry.
Results: The antitumor effectiveness of electrochemotherapy with CDDP mediated by PEMF was comparable to the conventional electrochemotherapy with CDDP, with the induction of 2.3 days and 3.0 days tumor growth delay, respectively. The exposure of tumors to PEMF only, had no effect on tumor growh, as well as the injection of CDDP only. The effect of the combined treatment was due to the increased cellular uptake of Pt in the tumors after the PEMF exposure, as well as its binding to DNA, as cellular target of CDDP. Approximately 2-fold increase in cellular uptake of Pt was measured.
Conclucion: The obtained results on mouse melanoma model in vivo demonstrate the possible use of PEMF induced electroporation for biomedical applications, such as electrochemotherapy. The main advantages of electroporation mediated by PEMF are contactless and painless application, as well as effective electroporation compared to conventional electroporation.
... Electroporation-induced endocytosis has been hypothesized for the delivery of proteins. [12][13][14][15] The possibility that endocytosis participates in DNA electrotransfer has started to emerge, [16][17][18][19][20] but the generality of this mechanism for DNA translocation across membranes is not yet a standard concept in the literature. ...
DNA electrotransfer is a successful technique for gene delivery into cells and represents an attractive alternative to virus-based methods for clinical applications including gene therapy and DNA vaccination. However, little is currently known about the mechanisms governing DNA internalization and its fate inside cells. The objectives of this work were to investigate the role of endocytosis and to quantify the contribution of different routes of cellular trafficking during DNA electrotransfer. To pursue these objectives, we performed flow cytometry and single-particle fluorescence microscopy experiments using inhibitors of endocytosis and endosomal markers. Our results show that ~50% of DNA is internalized by caveolin/raft-mediated endocytosis, 25% by clathrin-mediated endocytosis, and 25% by macropinocytosis. During active transport, DNA is routed through multiple endosomal compartments with, in the hour following electrotransfer, 70% found in Rab5 structures, 50% in Rab11-containing organelles and 30% in Rab9 compartments. Later, 60% of DNA colocalizes with Lamp1 vesicles. Because these molecular markers can overlap while following organelles through several steps of trafficking, the percentages do not sum up to 100%. We conclude that electrotransferred DNA uses the classical endosomal trafficking pathways. Our results are important for a generalized understanding of gene electrotransfer, which is crucial for its safe use in clinics.
Muscle degeneration is one the main factors that lead to the high rate of retear after a successful repair of rotator cuff tears. The current surgical practices have failed to treat patients with chronic massive rotator cuff tears. Therefore, regenerative engineering approaches are being studied to address the challenges. Recent studies showed the promising outcomes of electroactive materials on the regeneration of electrically excitable tissues such as skeletal muscle. Here, we review the most important biological mechanism of rotator cuff muscle degeneration. Further, the review covers the recent studies on electroactive materials for muscle regeneration including rotator cuff muscle. Finally, we will discuss the future direction toward the application of electroactive materials for the augmentation of rotator cuff tears.
Background
The re-emergence of infectious diseases and the increasing rate of the appearance of many antibiotic-resistant strains are major public health concerns. Zinc oxide nanoparticles (ZnO-NPs) have a great antibacterial effect. Few reports stated the antibacterial effect of low electric field (LEF).
Objective
The paper aimed to study the antibacterial effect of LEF at low frequency and investigate the antibacterial effectiveness of using LEF in synergy with ZnO-NPs.
Methods
Pseudomonas aeruginosa and Staphylococcus aureus were examined as models for Gramnegative and Gram-positive bacteria, respectively. The bacterial suspension was exposed to different concentrations of Zn-NPs ranging from 100-1600 μg/ml or 2 V/cm, 500 Hz AC electric field for 5 min. ZnO-NPs were prepared and characterized by UV-Vis spectroscopy, XRD, FTIR, TEM, and SEM. The combined effect of LEF exposure with each ZnO-NPs concentration was assessed.
Results
1600 μg/ml ZnO-NPs cause 41.93% and 48.15% death, LEF produces 20.88% and 28.03% death, and the synergetic effect causes 50.41% and 70.27% death for P. aeruginosa and S. aureus, respectively. The death percentages were correlated with DNA concentration and deformation, reactive oxygen species concentration, and ultrastructure changes.
Conclusions
LEF has antibacterial properties and can be used in combination with ZnO-NPs to increase its lethal effect.
The study aimed to evaluate the possibility to perform electrochemotherapy using nanosecond pulsed electric field (nsPEF) and low electric field (LEF) monopolar electrical impulses to alleviate the problems of conventional electroporation. Two types of pulses have been used to treat MCF-7 human breast carcinoma cell line: very low voltage (electric field strength) long trains of short unipolar electric pulses, and low frequencies of extremely intense (40kV/cm), ultra-short (10ns) electric pulses. The electropermeabilization efficiency of the formed endocytotic vesicles was measured using the cloning efficacy test. The cell viability was decreased significantly at a repetition frequency begins from 0.01 Hz by ~35% and reached complete cell loss at 1 Hz of nanosecond pulses for cells treated before with monopolar pulses at 20 V/cm in the presence of BLM with 4 µM concentration. The uptake of non-permeant drugs has been done without plasma membrane permeabilization (classical electroporation), but by endocytosis. Nanosecond electric pulses can disrupt the membrane of endocytotic vesicles and release the cytotoxic drug bleomycin.
The present study aimed to select an effective Pulsed High Magnetic Field (PHMF) stimulation protocol that would induce the Blood-Brain Barrier’s (BBB) reversible permeability to enhance brain-targeted drug delivery. PHMF was applied to the skull over the right hemisphere of 60 Wistar rats. The sham group contained other 10 rats that did not receive PHMF stimulation. The investigated parameters were repetition frequencies (0.25, 1, and 4 Hz as well as the effective low frequency combined with 10 Hz) and numbers of pulses in each train. Evans Blue Dye (EBD) uptake within the brain parenchyma was measured to select an effective PHMF stimulation protocol. BBB reversibility was evaluated by measuring EBD uptake and Gadobutrol retention, through MRI signal intensity enhancement, within brain parenchyma after exposure to the effective PHMF stimulation protocol at different time points including 0.5, 1, and 24 hours. The obtained results showed that the PHMF stimulation increased the BBB’s reversible permeability; this increase was more significant for 28 pulses with 1 Hz frequency (P < .0001). Changes in EBD uptake and MRI signal intensity in the exposed side (right hemisphere) peaked within 0.5–1 hour and returned to normal levels 24 hours after exposure to the effective protocol of PHMF stimulation (28 pulses with 1 Hz frequency). The Contrast-Enhanced MRI (CE-MRI) signal intensity confirmed the changes in EBD concentration. PHMF stimulation can be used as an effective protocol for enhancing the permeability reversibly of BBB, hence considered a potential clinical approach to brain-targeted drug delivery.
Cell membrane acts as a barrier to the entry of impermeable drugs into cells. Recent studies have suggested that using magnetic fields can enable molecules to overcome the cell membrane barrier. However, the mechanism of membrane permeabilization remains unclear. Therefore, we evaluated the increases in bleomycin (CT) uptake, a non-permanent chemotherapy agent, using high-pulsed magnetic fields and investigated whether endocytosis was involved in the process. This study exposed MCF-7 cells to magnetic fields (2.2 T strength, different number of 28 and 56 pulses, and frequency of 1 and 10 Hz) in order to investigate whether this approach could promote the cell-killing efficiency of bleomycin. The involvement of endocytosis as a possible mechanism was tested by exposing cells to three endocytosis inhibitors, namely chlorpromazine, genistein, and amiloride. Our results illustrated that magnetic fields, depending on their conditions, could induce different endocytosis pathways. In such conditions as 10 Hz-28 pulses, 10 Hz-56 pulses, and 1 Hz-56 pulse, clathrin-mediated endocytosis was observed. Moreover, macropinocytosis was induced by the 10 Hz magnetic field and caveolae-mediated endocytosis occurred in all the magnetic field conditions. The findings imply that high-pulsed magnetic fields generate different endocytosis pathways in the MCF-7 cells, thus increasing the efficiency of chemotherapy agents.
Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. In this study, IMEP was used to express the DUX4 gene into mouse tibialis anterior muscle. DUX4 is normally expressed in germ cells and early embryo, and silenced in adult muscle cells where its pathological reactivation leads to Facioscapulohumeral muscular dystrophy. DUX4 encodes a potent transcription factor causing a large deregulation cascade. Its high toxicity but sporadic expression constitutes major issues for testing emerging therapeutics. The IMEP method appeared as a convenient technique to locally express DUX4 in mouse muscles. Histological analyses revealed well delineated muscle lesions 1-week after DUX4 IMEP. We have therefore developed a convenient outcome measure by quantification of the damaged muscle area using color thresholding. This method was used to characterize lesion distribution and to assess plasmid recirculation and dose–response. DUX4 expression and activity were confirmed at the mRNA and protein levels and through a quantification of target gene expression. Finally, this study gives a proof of concept of IMEP model usefulness for the rapid screening of therapeutic strategies, as demonstrated using antisense oligonucleotides against DUX4 mRNA.
Fungal infections have become a serious medical problem due to the high infection rate and the frequent emergence of drug resistance. Ergosterol is an important structural component of the fungal cell membrane, its synthetases (squalene epoxidase (SE) and 14α-demethylase (CYP51)) are considered as the key points to block the ergosterol synthesis. In this study, we designed a series of dual-target arylamides derivatives based on the analysis of active sites (SE, CYP51). Subsequently, these target compounds were synthesized, and their antifungal activity was evaluated. Most of compounds demonstrate the potent antifungal activity against multiple Candida spp. and A. fum. In particular, the antifungal activities of compounds 10b and 11c are not only superior to positive control drugs, but also have significant inhibitory effects on drug-resistant fungi (C.alb. Strain100, C.alb. Strain103). Therefore, their action mechanism was further studied. Cellular uptake and electron microscopy observation showed that target compounds were able to enter fungal cytoplasmic region through free diffusion, and destroyed cell membrane structure. At the same time, preliminary mechanisms have demonstrated that they can affect the synthesis of ergosterol by inhibiting the activity of dual targets. It is worth noting that they also can exhibit excellent antifungal activity and low toxic side effects in vivo. Their ADMET properties and binding models were established will be useful for further lead optimization.
Among various neurodegenerative diseases (NDDs), Alzheimer's disease (AD) is the most common form. Excessive aggregation of β-amyloid protein (Aβ) is thought to be a major cause in the pathogenesis of AD. A growing number of studies also have suggested that reactive oxygen species (ROS) play a critical role in the onset and progression of AD. The present study was aimed to investigate the neuroprotective effect of the high frequency and low intensity pulsed electric field (H-LIPEF) against hydrogen peroxide (H2O2) and Aβ-induced cytotoxicity in SH-SY5Y cells. The systematic study on frequency- and amplitude-dependent neuroprotective effect of pulsed electric field (PEF) was performed, and the result showed that H-LIPEF at 200 Hz conferred the best protective effect to the SH-SY5Y cells. The underlying mechanisms were confirmed to be due to the activation of extracellular signal-regulated kinase (ERK) pathway and the downstream prosurvival and antioxidant proteins. Given the fact that electric field can be modified to focus on specific area and applicable in a non-contact manner, we propose that H-LIPEF holds great potential for neuroprotection and curing the patients with NDDs. Furthermore, it is anticipated that the combination of such physical stimulation with drugs or natural compounds will be even more effective in treating NDDs.
Most existing cancer treatments involve high-cost chemotherapy and radiotherapy, with major side effects, prompting effort to develop alternative treatment modalities. In this study, we propose a novel non-invasive treatment calling for exposure of cells to CGA, coupled with thermal-cycling hyperthermia (TC-HT) and low-intensity pulsed electric field (LIPEF). The study finds that this triple combination can significantly impede the proliferation of human pancreatic cancer PANC-1 cells, with only about 20% viable cells left after 24h, whereas being non-toxic to normal cells. The synergistic activity against the PANC-1 cells was achieved by inducing G2/M phase arrest and apoptosis, which were associated with up-regulation of p53 and coupled with increased expression of downstream proteins p21 and Bax. Further mechanism investigations revealed that the cytotoxic activity could be related to mitochondrial apoptosis, characterized by the reduced level of Bcl-2, mitochondrial dysfunction, and sequential activation of caspase-9 and PARP. Also, we found that the triple treatment led to the increase of intracellular reactive oxygen species (ROS) production. Notably, the triple treatment-induced cytotoxic effects and the elevated expression of p53 and p21 proteins as well as the increased Bax/Bcl-2 ratio, all could be alleviated by the ROS scavenger, N-acetyl-cysteine (NAC). These findings indicate that the combination of CGA, TC-HT, and LIPEF may be a promising modality for cancer treatment, as it can induce p53-dependent cell cycle arrest and apoptosis through accumulation of ROS in PANC-1 cells.
Electrosorption is a novel desalination technique that has many advantages in the treatment of sewage. However, commercially available activated carbon electrodes for electrosorption commonly have low microporosity, poor moulding performance, and low adsorption and regeneration efficiency. Here, we evaluated a novel adsorbent material, activated carbon fibre felt (ACFF), for electrosorption of chromium ions (Cr6þ) in sewage treatment. The ACFF was modified with 20% nitric acid and its modified structure was characterized. The modified ACFF was used as an adsorbing electrode to investigate its desalination effect by electrosorption. Results showed that compared with those of unmodified ACFF, the modified ACFF had more carbonyl and carboxyl groups and the specific surface area, average pore size and micropore volume of the modified ACFF also improved by 32.2%, 2.5% and 23.1%, respectively. The kinetics of Cr6þ adsorption conformed to the pseudo-second-order kinetic equation, and the adsorption isotherm conformed to the Langmuir model. In addition, the regeneration rate of the modified ACFF electrode was more than 94%. In conclusion, the modified ACFF exhibits excellent electrosorption and regeneration performance for Cr6þ removal from water and thus is of great value for promotion in sewage treatment.
Electrotransfection (ET) is a non-viral method for delivery of various types of molecules into cells both in vitro and in vivo. Close to 90 clinical trials that involve the use of ET have been performed, and approximately half of them are related to cancer treatment. Particularly, ET is an attractive technique for cancer immunogene therapy because treatment of cells with electric pulses alone can induce immune responses to solid tumors, and the responses can be further enhanced by ET of plasmid DNA (pDNA) encoding therapeutic genes. Compared to other gene delivery methods, ET has several unique advantages. It is relative inexpensive, flexible, and safe in clinical applications, and introduces only naked pDNA into cells without the use of additional chemicals or viruses. However, the efficiency of ET is still low, partly because biological mechanisms of ET in cells remain elusive. In previous studies, it was believed that pDNA entered the cells through transient pores created by electric pulses. As a result, the technique is commonly referred to as electroporation. However, recent discoveries have suggested that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA. This review will discuss current progresses in the study of biological mechanisms underlying ET, and future directions of research in this area. Understanding the mechanisms of pDNA transport in cells is critical for development of new strategies for improving the efficiency of gene delivery in tumors.
With superior biocompatibility and unique magnetic properties, iron-based nanoparticles (IBNP) are commonly encapsulated in cells and extracellular vesicles (EV) to allow for magnetic force controlled drug delivery and non-invasive tracking. Based on their natural source and similar morphology, we classify both cells and EVs as being natural lipid encapsulations (NLEs), distinguishing them from synthetic liposomes. Both their imaging contrast and drug effects are dominated by the amount of iron encapsulated in each NLE, demonstrating the importance of magnetic labeling efficiency. It is known that the membranes function as barriers to ensure that substances pass in and out in an orderly manner. The most important issue in increasing the cellular uptake of IBNPs is the interaction between the NLE membrane and IBNPs, which has been found to be affected by properties of the IBNPs as well as NLE heterogeneity. Two aspects are important for effective magnetic labelling: First, how to effectively drive membrane wrapping of the nanoparticles into the NLEs, and second, how to balance biosafety and nanoparticle uptake. In this review, we will provide a systematic overview of the magnetic labeling of NLEs with IBNPs. This article provides a summary of the applications of magnetically labeled NLEs and the labeling methods used for IBNPs. The review also analyzes the role of IBNPs physicochemical properties, especially their magnetic properties, and the heterogeneity of NLEs in the internalization pathway. At the same time, the future developmentof magnetically labeled NLEs is also discussed.
The cell membrane represents a major barrier for efficient delivery of exogenous molecules, either pharmaceuticals or genetic material, under both in vitro and in vivo conditions. The number of methods employed to attempt safe, efficient, and local drug and gene delivery has increased during the recent years. One method for membrane permeabilization, electroporation, has already been translated to clinical practice for localized anticancer drug delivery and is termed “electrochemotherapy”. Clinical trials for gene delivery using electroporation as well as drug delivery using another cell permeabilization method, sonoporation, are also underway. This review focuses on these two methods, including their fundamental principles and state-of-the-art applications. Other techniques, such as microinjection, magnetoporation, photoporation, electrospray, and hydrodynamic and ballistic gene delivery, are also discussed.
The development of new drugs depends greatly on their successful, efficient, low cost, and safe delivery into target cells or tissues. In the case of highly charged macromolecules such as nucleic acids, the therapeutic effectiveness is mainly limited by their bio-distribution within the tissue and the poor permeability of the plasma membrane of cells. For this purpose, electroporation appears as a promising method for nucleic acid delivery. Electroporation is a physical method of vectorization that consists of application of electric pulses on cells or tissues. Optimization of the pulses’ parameters leads to the transient permeabilization of the plasma membrane for molecules which otherwise cannot enter the cell. Therefore, the understanding of different principles of drug and gene delivery is necessary and needs to be taken into account according to the specificity of their delivery to tumors and/or normal tissues. This approach has been routinely used in cell biology for more than 30 years for cell transfection and in medicine in a number of clinics and hospitals through Europe to treat cutaneous cancers by increasing the toxicity of anticancer drugs (electrochemotherapy); it is also now under clinical trials for nucleic acid delivery (electrogenotherapy, electro-vaccination). The present chapter focuses on electrotransfer of nucleic acids, the nature of nucleic acids (plasmid DNA, mRNA, siRNA, LNA, etc.) which can be electrotransferred, and the mechanism of their electrotransfer.
The development of new drugs depends greatly on their successful, efficient, low cost, and safe delivery into target cells or tissues. In the case of highly charged macromolecules such as nucleic acids, the therapeutic effectiveness is mainly limited by their bio-distribution within the tissue and the poor permeability of the plasma membrane of cells. For this purpose, electroporation appears as a promising method for nucleic acid delivery. Electroporation is a physical method of vectorization that consists of application of electric pulses on cells or tissues. Optimization of the pulses’ parameters leads to the transient permeabilization of the plasma membrane for molecules which otherwise cannot enter the cell. Therefore, the understanding of different principles of drug and gene delivery is necessary and needs to be taken into account according to the specificity of their delivery to tumors and/or normal tissues. This approach has been routinely used in cell biology for more than 30 years for cell transfection and in medicine in a number of clinics and hospitals through Europe to treat cutaneous cancers by increasing the toxicity of anticancer drugs (electrochemotherapy); it is also now under clinical trials for nucleic acid delivery (electrogenotherapy, electro-vaccination). The present chapter focuses on electrotransfer of nucleic acids, the nature of nucleic acids (plasmid DNA, mRNA, siRNA, LNA, etc.) which can be electrotransferred, and the mechanism of their electrotransfer.
Several electric-based cancer treatments were developed for in situ ablation of solid tumors. The electrical parameters used for treatment range from several volts per cm delivered for a long time period, to high electric fields (300–3000 V/cm) and very high electric fields (up to 300 kV/cm, 3–300 ns pulse duration).
The treatment can be delivered as a continuous treatment or pulses. These treatments are either based on electrostimulation alone or in conjunction with chemotherapeutic drugs.
In this chapter, we summarized data on the effect of various types of electric ablation of cancer in various metastatic tumors in mice and in clinical trials. We discuss the role of electric ablation in changes that occur in the tumor microenvironment, infiltration of immune cells into the tumor, and induction of antitumor immunity. Special focus is given to the role of these responses in the elimination of residual metastatic cells, and the possible enforcement of such antitumor reactions by various immunostimulators.
Gene electrotransfer is one of the promising nonviral methods for introducing genes into the cell by using high-voltage electric pulses which transiently permeabilize cell membrane. Gene delivery, which uses electric pulses, is a complex phenomenon consisting of different steps: cell membrane electropermeabilization, formation of the contact between DNA and cell membrane, translocation across the membrane and transfer to the cell nucleus, resulting in final gene expression. In this chapter, we discuss different aspects of electric pulse-mediated plasmid DNA (pDNA) delivery. We focus on the importance of electric pulse parameters for efficient gene electrotransfer, analyze the relation between cell membrane electropermeabilization and gene electrotransfer. We present state of the art of different steps involved in electric pulse-mediated pDNA delivery. Furthermore, we focus on theoretical analysis of gene electrotransfer, calculation of pDNA mobility during electric pulse application with special emphasis on electrophoresis of highly charged pDNA. We discuss the importance of DNA availability at cell membrane level and estimate the number of DNA molecules in contact with the membrane for different concentrations of pDNA used in experiments. A novel study of DNA mobility and gene electrotransfer efficiency in cells embedded in 3D collagens will be presented. Finally, from all the data obtained in studies of mechanisms of electric pulse-mediated pDNA delivery we present also optimization of parameters for gene electrotransfer in muscle tissue using 3D numerical modeling.
Plant endocytosis emerges as an active field of contemporary plant sciences. In this chapter, control of plant endocytosis via physical forces is discussed from the perspective of structural homeostasis of the plasma membrane (PM) regulated via vesicular trafficking. Plant cells are very active in endocytosis despite high turgor pressure. Similar to neurons, plant cells are also using endocytosis and endosomes for sensing and processing of sensory information and perhaps also for rapid cell-to-cell communication of this information. As the freshly internalized endosomes are surrounded by their limiting membrane derived from the PM, these endosomes effectively amplify structural and electrical boundaries between the cellular interior and exterior. This feature is critical for the primary processing of sensory information at the PM and its further transduction into signal transduction networks permeating the eukaryotic cell. The higher the number of endosomes a cell generates and recycles, the more it is informed about its environment. Endocytosis is sensitive to diverse physical factors including mechanical, thermal, and electro-magnetic aspects of the PM. Last but not the least, blue light emerges as a physical ligand-like factor for the unique light-induced plant endocytosis. © 2012 Springer-Verlag Berlin Heidelberg. All rights are reserved.
A theory of the elasticity of lipid bilayers is proposed. Three types of strain, i. e. stretching, tilt and curvature, are distinguished and the associated stresses are identified. It is argued that in the case of vesicles (= closed bilayer films) the only elasticity controlling nonspherical shapes is that of curvature. Euler-Lagrange equations are derived for the shape in magnetic fields and under excess outside pressure. It is shown that magnetic fields can deform spherical vesicles into ellipsoids of revolution. Under excess outside pressure the spherical shape becomes unstable at a certain threshold pressure. Both effects can be influenced by a spontaneous curvature of the bilayer. Some possible experiments to determine the elastic properties are also discussed.
The structural and elastic properties of strongly curved cylindrical interface are characterised by spontaneous curvature, spontaneous area per molecule and three elastic moduli corresponding respectively to area extension, bending and mixed deformation of spontaneous state [6, 7]. All characteristics are influenced by the electrical charge of interface. We have calculated in terms of a general approach [16] the change of spontaneous geometrical characteristics and of the full set of elastic moduli of a cylindrical interface due to the electrical charge. The analysis is performed for an arbitrary dividing surface. The theoretical results are applied to the analysis of recently obtained experimental data [11]. The qualitative agreement between experimental data and theoretical predictions prove the validity of the developed approach. The quantitative comparison of results obtained with experimental data allows us to estimate the effective surface charge density of cylindrical DOPE/DOPS (3/1) monolayer in the hexagonal H$_{\rm II}$ phase. This value is equal approximately to $3\times 10^{-3}$ [ Q/m$^{2}$] .
Electrical currents near regenerating newt limbs were measured with a recently developed vibrating probe. Steady currents with local surface densities of 10 to 100 muA/cm2 or more leave the end of the stump during the first 5-10 days after amputation and are balanced by currents with densities of only 1-3 muA/cm2 that enter the intact skin around the stump. They are immediately dependent upon the entry of sodium ions into this skin and are therefore inferred to be skin-driven. The outward currents are comparable in direction, density, duration, and position to artificially imposed currents previously found sufficient to induce significant regeneration of amputated adult frog limbs. This comparison suggests that the endogenous stump currents play some causal role in initiating regeneration.
We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.
This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.
Anionic microdomains within the aortic smooth muscle cell (SMC) surface glycocalyx represent a potential barrier to the endocytosis of anionic plasma proteins. Cultured SMCs exposed briefly to cationized ferritin (CF) exhibit ultrastructural aggregations of surface anionic sites resulting in intervening areas essentially devoid of anionic charge. Preincubation of cultured aortic medial SMCs with 0.2 mg/ml CF for 1 minute at 37 C resulted in a 4-fold increase in binding and a 13-fold increase in internalization of 125I-human serum albumin (125I-HSA) relative to cells pretreated with native ferritin. When both the CF preincubation and the endocytosis were performed at 4 C, the influence of CF was abolished. Studies at 4 C indicated that CF pretreatment of SMC at 37 C induced high affinity (Kd = 1.5 nM) saturable 125I-HSA binding, in addition to low-affinity nonsaturable binding. These results were further confirmed by binding competition studies using increasing concentrations of unlabeled HSA. In contrast, low-density lipoprotein, a large anionic molecule, failed to compete with 125I-HSA for binding sites on CF-pretreated SMCs at either 4 or 37 C. Pulse-chase studies at 37 C indicated that 20-30% of internalized 125I-HSA was degraded, and 40-50% exocytosed within 24 hours in CF-treated cells. CF pretreatment of the SMCs did not significantly enhance the uptake of 14C-sucrose as a measure of fluid-phase endocytosis at 30 and 60 minutes. The results of these studies emphasize the potentially important regulatory roles of cell-surface anionic charge distribution and cationic molecules in cellular endocytosis.
Using a newly developed, extracellular vibrating electrode, we can now measure the electrical currents that traverse a single developing cell. We have studied the eggs of the common seaweed, Pelvetia, during their first 2 days of development and find that the endogenous electrical current through them includes a pulse component as well as a relatively steady component. Both of these enter the egg's growing tip and leave the rest of the embryo. The current pulses first appear a few hours after growth begins and have a characteristic shape that is independent of amplitude. They have a duration of about 100 sec, an average frequency of 1-5 per hr, and enter with peak surface intensities of 3-10 (and rarely up to 30) muA/cm(2). By the two-cell stage they account for about a fourth of the total transembryonic current. Since they may overlap to any degree and (as is documented elsewhere) are generally accompanied by peak membrane depolarizations of only 2-6 mV, their course does not seem to be voltage-controlled. Thus, they seem essentially different from action potentials. We also find that the rate at which the egg grows in length is roughly proportional to the size of the steady current traversing it.
Summary Electric impulses were found to cause transient permeability changes in the membranes of vesicles storing biogenic amines. Release of catecholamines induced by electric fields (of the order of 20 kV/cm and decaying exponentially with a decay time of about 150 μsec) was studied, using the chromaffin granules of bovine adrenomedullary cells as a vesicular model system. Far-UV-absorption spectroscopy was applied to determine the amount of catecholamines released from suspended vesicles. A polarization mechanism is suggested for the induction of short-lived permeability changes caused by electric fields. Such transient changes in permeability may possibly represent a part of the sequence of events leading to stimulated neurohumoral secretion.
A method combining electrophoresis and freeze-fracture electron microscopy is described; the method was used to determine the lateral diffusion coefficient of intramembrane particles (integral proteins) in the mitochondrial inner membrane. An electric current was passed through microsuspensions of purified, spherical inner membranes at pH 7.4, which caused an electrophoretic migration of intramembrane particles in the membrane plane into a single, crowded patch facing the positive electrode. The membrane microsuspensions were quick-frozen at specified times after the packed particles were released from the electrophoretic force and while the particles were diffusing back to a random distribution. Observed concentration gradients of intramembrane particles during this time were quantitatively compared with and found to follow a mathematical model for Fickian diffusion of particles on a spherical membrane. The results determine the kinetics of free diffusion of integral proteins at the resolution of individual proteins. The diffusion coefficient of the integral proteins in the mitochondrial inner membrane was determined to be 8.3 X 10(-10) cm2/sec at 20 degrees C, from which a root-mean-square displacement of 57 nm in 10 msec is predicted.
Scavenger receptors interact with a variety of modified proteins, mediate their endocytosis and degradation, and may play an important role in protein catabolism and pathogenic processes such as atherosclerosis, aging, and diabetes. Many scavenger receptors have been detected kinetically but few such binding proteins have actually been identified. Recently, we found that two membrane-associated proteins, gp30 and gp18, interact more avidly with albumins conformationally modified by chemical means or by surface adsorption to colloidal gold particles than with native albumin. In this study, we show that gp30 and gp18 behave similarly to other known scavenger receptors. Competition studies indicate a similar ligand binding profile to other known scavenger receptors. Polyanionic molecules (dextran sulfate, fucoidan, polyglutamic acid, polyinosinic acid, heparin) and modified albumins such as formaldehyde-treated or maleylated albumin (Mal-bovine serum albumin) competed with albumin conjugated to colloidal gold particles (A-Au) for the blotting of gp30 and gp18. A-Au and Mal-bovine serum albumin bound cultured endothelial cells with high affinity. Modified and native albumins were each internalized, but only modified albumins were then released degraded. Inhibition studies revealed that only the same molecules that were effective in blocking A-Au blotting of gp30 and gp18, also inhibited A-Au degradation. Addition of the lysosomotropic agent chloroquine resulted in more than 70% inhibition of degradation. Differential processing of A-Au by cultured smooth muscle and endothelial cells along with fibroblasts was observed in a manner consistent with gp30 and gp18 expression. Cumulatively, these results suggest that gp30 and gp18 may mediate the high affinity binding, endocytosis, and degradation of conformationally modified albumins but not native albumin.
We investigated the function of gp60, an endothelial cell membrane 60-kDa albumin-binding protein localized in caveolae, and the mechanism of its activation in regulating endothelial permeability of albumin. Gp60 organization on the bovine pulmonary microvessel endothelial cell (BPMVEC) surface was punctate as shown by immunofluorescence using an anti-gp60 antibody (Ab) conjugated with bisfunctional, N-hydroxysuccinimidyl fluorophore (Cy3). Addition of a secondary Ab to anti-gp60 Ab-treated BPMVEC induced cross-linking of gp60 as evident by increased size of fluorescent particles and cell surface gp60 clustering. Gp60 cross-linking also produced 2-3-fold increases in the endothelial cell uptake and the luminal to abluminal permeability of 125I-albumin as well as the fluid-phase tracer, horseradish peroxidase. The increased transendothelial permeability of macromolecules was the result of transcytosis as it was not associated with an increase in the paracellular pathway. Incubation of anti-gp60 Ab with BPMVEC at 37 degrees C caused internalization of gp60, and thereby reduced the uptake of the macromolecules. Activation of gp60 by either albumin (the gp60 ligand) or gp60 cross-linking induced the phosphorylation of both gp60 and caveolin-1 (the major structural caveolar protein) on tyrosine residues. Gp60 activation also phosphorylated the Src family tyrosine kinases pp60(c-Src) and Fyn. The activated pp60(c-Src) and Fyn co-immunoprecipitated with caveolin-1 in BPMVEC membrane. Protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, prevented gp60-activated macromolecule uptake and transcytosis in a concentration-dependent manner, indicating the functional significance of the PTK pathway in activating albumin transcytosis. These findings indicate that activation of gp60 stimulates the Src PTK signaling pathway, and thus regulates the transcytosis of albumin across the endothelial cell monolayer.
Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory's approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi-Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.
Electric fields can induce lateral reorganization of lipids in fluid bilayer membranes. The resulting concentration profiles readily are observed in planar-supported bilayers by epifluorescence microscopy. When a fluorescently labeled lipid was used to probe the field-induced separation of cardiolipin from egg-phosphatidylcholine, an enhanced sensitivity to the electric field was observed that is attributed to a critical demixing effect. A thermodynamic model of the system was used to analyze the results. The observed concentration profiles can be understood if the lipid mixture has a critical temperature equal to 75 degrees K. The steady-state distribution of lipids under the influence of an electric field is very sensitive to demixing effects, even at temperatures well above the critical temperature for spontaneous phase separation, and this may have significant consequences for organization and structural changes in natural cell membranes.
Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.
A large number of epidemiological and experimental studies suggest that prolonged (>100 s) weak 50-60-Hz electric and magnetic field (EMF) exposures may cause biological effects(NIEHS Working Group, NIH, 1998; Bersani, 1999). We show, however, that for typical temperature sensitivities of biochemical processes, realistic temperature variations during long exposures raise the threshold exposure by two to three orders of magnitude over a fundamental value, independent of the biophysical coupling mechanism. Temperature variations have been omitted in previous theoretical analyses of possible weak field effects, particularly stochastic resonance (Bezrukov and Vodyanoy 1997a. Nature. 385:319-321; Astumian et al., 1997 Nature. 338:632-633; Bezrukov and Vodyanoy, 1997b. Nature. 338:663; Dykman and McClintock, 1998. Nature. 391:344; McClintock, 1998;. Gammaitoni et al., 1998. Rev. Mod. Phys. 70:223-287). Although sensory systems usually respond to much shorter (approximately 1 s) exposures and can approach fundamental limits (Bialek, 1987 Annu. Rev. Biophys. Biophys. Chem. 16:455-468; Adair et al, 1998. Chaos. 8:576-587), our results significantly decrease the plausibility of effects for nonsensory biological systems due to prolonged, weak-field exposures.
Monolayer mixtures of dihydrocholesterol and phospholipids at the air-water interface are used to model membranes containing cholesterol and phospholipids. Specific, stoichiometric interactions between cholesterol and some but not all phospholipids have been proposed to lead to the formation of condensed complexes. It is reported here that an externally applied electric field of the appropriate sign can destabilize these complexes, resulting in their dissociation. This is demonstrated through the application of an electric field gradient that leads to phase separations in otherwise homogeneous monolayers. This is observed only when the monolayer composition is close to the stoichiometry of the complex. The electric field effect is analyzed with the same mean field thermodynamic model as that used previously to account for pairs of upper miscibility critical points in these mixtures. The concentrations of dihydrocholesterol, phospholipid, and complex vary strongly and sometimes discontinuously in the monolayer membrane in the field gradient. The model is an approximation to a two-dimensional liquid in which molecules freely exchange between free and complexed form so that the chemical potentials are constant throughout the membrane. The calculations are illustrated for a complex of about 15 molecules, composed of 5 cholesterol molecules and 10 phospholipid molecules.
Electric fields affect the movement and the conformation of membrane proteins and channel forming polypeptides in the membrane. Diffusion potentials, created by a potassium concentration gradient across the membrane in the presence of valinomycin, affect the circular dichroism (CD) of bacteriorhodopsin reconstituted in lipid vesicles. The changes in CD indicate that the applied electric field, irrespective of its direction, decreases the helical fraction and increases the fractions of the random and β structures. Donnan potentials, created across the vesicular bilayer membrane by polyelectrolytes, are used to induce the conformational changes in alamethicin. The induced change of CD by the electric field across the membrane was not symmetrical with respect to the field direction. Tangential electric fields induce lateral movements of membrane components. The eletrophoretic movement of photosystem I (PSI) along the membrane of hypotonically swollen thylakoid vesicles induced by low electric fields (40-80 V/cm) is studied by analyzing its electrophotoluminescence (EPL) from the different poles of the vesicle. The average apparent electric mobility, determined from the time course of the increase of EPL on the enriched hemisphere and of the decrease of EPL on the depleted hemisphere, was of the order of 3.10-5 cm²/(V s). The distribution of PSI reaches a steady state when the diffusional, electrostatic, and other counteracting forces balance the electrophoretic driving force. The electrophoretic mobility obtained from the steady state conditions was only 5 x 10⁻⁷ cm²/s. The lateral diffusion coefficient was ~5 x 10⁻⁹ cm²/s, which was obtained from the diffusional relaxation after cessation of the electric field.
Na,K-ATPase uses chemical bond energy of ATP to pump K(+) into, andNa(+) out of a cell. Both are uphill transports. During the catalyticcycle the enzyme alternates between two conformational states, E(1) andE(2). This communication describes an experiment, which employs electricfield to drive oscillation or fluctuation of enzyme conformation betweenthe E(1) and the E(2) states. It is shown that the field-inducedconformational oscillation or fluctuation leads to uphill pumping of thecation by the enzyme without consumption of ATP. Biochemical specificityof the catalysis is preserved. Data indicate that Na,K-ATPase can harvestenergy from the applied electric field to perform chemical work, and aratchet mechanism is inherent in this energy transduction process. ATheory of Electroconformational Coupling (TEC) that embodies essentialfeatures of the Brownian Ratchet successfully simulates the field-frequencyand field-amplitude optima and other features of the ion pumping activity.A four-state TEC motor can achieve high efficiency of the energytransduction, asymptotically reaching 100% under the optimal condition.Pumping by ion rectification fails to reach high efficiency. The TECconcept is also mused to understand other biological motors and engines.
In this paper the Helfrich ansatz for bending an amphiphilic monolayer is re-expressed in terms of the deviations in curvature away from a preferred value. Using this expression and a simple model of the response of amphiphiles to interfacial bending, it is demonstrated that there are strict limits to the value of the ratio of the Gaussian curvature modulus to the mean curvature modulus (κG/κ). We have made an estimate of κG/κ from measurements of the swelling behavior in water of inverse bicontinuous cubic mesophases in a system composed of 1-monoolein, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. The estimate, −0.75 ± 0.08, is in agreement with the limits set by our model of −1 < κG/κ < 0. This determination is the first to be made on an inverse bicontinuous cubic phase which is sufficiently swollen to be in a regime where first-order curvature elastic energetics should be sufficient to describe the state of the mesophase and hence provide a reliable estimate of κG/κ.
Employing a generalized Young-Laplace equation for a surfactant-loaded oil/water interface of mean curvature H and Gaussian curvature K and invoking the conventional Helfrich bending free energy expression, we analyze what interfacial configurations are compatible with mechanical and physicochemical equilibrium in Winsor type microemulsions. Our treatment is based on the notion that the Laplace pressure must be zero for an equilibrium aggregate when the microemulsion phase coexists with an excess bulk phase. When the spontaneous curvature, H-0, is different from zero, spherical and rod-shaped equilibrium droplets are possible. On the other hand, for H-0 = 0, we find that extended minimal surfaces with H = 0 can exist if the saddle splay constant k(c)BAR is positive. In order to attain stability of minimal surface structures with respect to the size variations, it is necessary to include a quadratic term in the Gaussian curvature in the free energy expression. The stability of these structures with respect to shape deformations is also studied. The theoretical scheme outlined applies equally well to minimal surface structures formed by thin symmetric bilayers.
Liposomes composed of ganglioside GM3 (GM3), Gal β 1(3←2 α NANA)→4Glc β1→1Cer, and l-α-dipalmitoylphosphatidylglycerol (DPPG) were prepared by varying the amount of GM3, and the effects of GM3 on the membrane properties of liposomes in the presence of bovine serum albumin (BSA) were examined at pH 7.4 and 37 °C in terms of adsorption amount of BSA and permeability of the liposomal bilayer membranes. GM3 incorporated into DPPG liposomes inhibited the adsorption of BSA on the liposomes, and the leakage of calcein as an aqueous-space marker from liposomes through adsorption of BSA decreased. However, a large amount of GM3 incorporated into liposomes brought about a probable phase separation in the liposomal bilayer membranes, thereby increasing their permeability. The leakage of calcein from the liposomes in the presence of BSA showed a minimum value at 0.10 mole fraction of GM3.
The present study shows a very efficient incorporation of polysaccharides and β-galactosidase into membrane vesicles and cells by low pulsed electric fields of much lower amplitudes than the threshold electric field for electroporation. The electric induced uptake of dextran-FITC, in the range of 1–2000 kD, was observed in 85% of the cell population, while preserving the cell's viability. The uptake of β-galactosidase, induced by low electric fields, revealed a 30 fold increase in the enzyme's concentration in the cytosol in comparison with its concentration in the extracellular medium. The underlying mechanism of this uptake, based on the electric field induced endocytotic-like process, emerges from the following observations: (i) The uptake is not driven by metabolic energy, since we obtained the incorporation of macromolecules into membrane vesicles or cells at low temperatures; (ii) The formation of vesicle-like structures containing the macromolecules is displayed by confocal fluorescence microscopy, which reveals the existence of massive vesicle-like structures in the cytosol, following exposure to the pulsed electric field; (iii) The formation of vesicle-like structures is supported by the observed decrease in forward light scattering and the increase in side light scattering from single exposed cells.
The adsorption and competitive adsorption of collagen and bovine serum albumin (BSA) were directly visualized and quantified using atomic force microscopy (AFM) and imaging ellipsometry. Chemically modified silicon surfaces were used as hydrophilic and hydrophobic substrates. The results showed that collagen and BSA in single component solution adsorbed onto a hydrophobic surface two times more than that onto a hydrophilic surface. The competitive adsorption between collagen and BSA showed that serum albumin preferentially adsorbed onto a hydrophobic surface, while collagen on a hydrophilic surface. In the binary solution of BSA (1 mg/ml BSA) and collagen (0.1 mg/ml), nearly 100% of the protein adsorbed onto the hydrophobic surface was BSA, but on the hydrophilic surface only about 6% was BSA. Surface affinity was the main factor controlling the competitive adsorption.
Temperature dependence of fluid-phase endocytosis was determined in two renal epithelial cell lines, MDCK cells and LLC-PK1 cells, using Lucifer Yellow or horseradish peroxidase as markers. For both cell lines, grown on solid support as a confluent monolayer, biphasic curves of marker uptake vs. temperature were obtained. The changes in slope occurred around 27°C, a critical temperature at which the lipids of the plasma membrane of MDCK cells enter in the gel state. Activation energies were significantly higher above 27°C (15–22 kcal/mol) than below that critical temperature (9–12 kcal/mol). These data indicate that changes in membrane physical state have marked effects on endocytic processes. They suggest that two mechanisms, with different activation energies are involved in the fluid phase endocytosis by renal epithelial cells in culture.
Electrification in developed countries has progressively increased the mean level of extremely low-frequency electromagnetic fields (ELF-EMFs) to which populations are exposed; these humanmade fields are substantially above the naturally occurring ambient electric and magnetic fields of approximately 10(-4) Vm(-1) and approximately 10(-13) T, respectively. Several epidemiological studies have concluded that ELF-EMFs may be linked to an increased risk of cancer, particularly childhood leukemia. These observations have been reinforced by cellular studies reporting EMF-induced effects on biological systems, most notably on the activity of components of the pathways that regulate cell proliferation. However, the limited number of attempts to directly replicate these experimental findings have been almost uniformly unsuccessful, and no EMF-induced biological response has yet been replicated in independent laboratories. Many of the most well-defined effects have come from gene expression studies; several attempts have been made recently to repeat these key findings. This review analyses these studies and summarizes other reports of major cellular responses to EMFs and the published attempts at replication. The opening sections discuss quantitative aspects of exposure to EMFs and the incidence of cancers that have been correlated with such fields. The concluding section considers the problems that confront research in this area and suggests feasible strategies.
A Landau theory of the lamellar-to-cubic phase transition of lipid bilayers in water is developed. The preference for the infinite periodic minimal surfaces G, D and P is explained and preliminary criteria for the selection of one of them are given.
Electrophoretic movement of photosystem I (PS I) along the photosynthetic membrane of hypotonically swollen thylakoid vesicles was studied by analyzing the electric field-stimulated delayed luminescence (electrophotoluminescence) emitted from PS I. The electrophoretic mobility was inferred from the differences in electrophotoluminescence (EPL) of the photosynthetic vesicles in presence and absence of trains of low amplitude (<80 V/cm) prepulses of 1 ms duration at 4 ms spacing. The average apparent electric mobility, determined from the time course of EPL increase on one hemisphere or its decrease on the other one, as function of prepulse length and intensity was of the order of 3 . 10(-5) cm(2)V(-1)s(-1). The assymetric distribution of the PS I reached a steady state when the diffusional, electrostatic, and elastic forces balanced the electrophoretic driving force. A lateral diffusion coefficient of approximately 5 . 10(-9) cm(2)s(-1) was found for the PS I complex from the diffusional relaxation after cessation of the electric field pulse train. Experimental conditions such as concentration, temperature, and viscosity of the aqueous solution were not critical for the effect. Between 23 and 150 electron charges per moving particle were estimated from the measured electrophoretic mobility.
Electrophoretic and diffusional movements of concanavalin A (Con A) receptors and acetylcholine (ACh) receptors in the plane of the plasma membrane of mononucleate, spherical Xenopus myoblasts were studied by microfluorimetry and iontophoresis. We found that (a) a uniform electric field of 10 V/cm applied along the cell surface produces a partial accumulation of both types of receptors toward the cathodal pole of the cell within 30 min: (b) post-field relaxation of the culture results in the complete recovery of the uniform distribution of the Con A receptors within 10 min; and (c) in contrast to the Con A receptor in general, accumulation of ACh receptors by the electric field results in the formation of stable, localized receptor aggregates. Theoretical analyses were carried out for the distribution of charged membrane receptors at equilibrium between electrophoresis and diffusion, and for the rate of back diffusion after the removal of the field. These analyses indicated that, at 22 degrees C, the average electrophoretic mobility of the electrophoretically mobile population of the Con A receptors is about 1.9 X 10(-3) micron/s per V/cm, while their average diffusion coefficient is 5.1 X 10(-9) cm2/s.
A steady electric field of 30 mV across a single embryonic muscle cell produces accumulation of acetylcholine receptors toward one pole of the cell within 1 h. The movement is electrophoretic in nature and the accumulation results in the formation of stable, metabolically independent receptor aggregates.
Fluorescent concanavalin A (con A)-labelling showed that an electric field of 4 V cm-1 grossly redistributed con A receptors alone the plasma membranes of living muscle cells within 4 h. This field produced a voltage drop of 12 mV across these 30 micronm-wide cells. The movement of receptors was independent of cell metabolism and seemed to be electrophoretic in nature.
Bioelectric fields may segregate charged components floating in the plasma membranes of cells by a process of electrophoresis along the membrane. Molecules in cell membranes may be sorted to different portions of the cell surface by such electrical gradients. We present here a theory to support this hypothesis.
APPLICATION of an electric pulse, at field intensities of a few kV cm-1 and of duration in the µs range, to an isotonic suspension of erythrocytes is known to cause haemolysis of the red cells1-4. Studies from different laboratories suggest that the haemolysis is due to the field-induced transmembrane potential1,3,4. Our recent experiments5 indicate that once the transmembrane potential reaches a threshold of approximately 1 V, which corresponds to an applied field of 2.2 kV cm-1, the erythrocyte membrane becomes leaky to normally impermeant ions or molecules. The permeation of solutes leads to the swelling and eventual lysis of the red cells. This type of haemolysis is known as colloid osmotic haemolysis6,7. The voltage-induced permeability change is consistent with the formation of pores in the membrane. We show here that the size of these pores can be varied in a controlled manner, and that the leaky membrane can be resealed while the haemolysis is prevented. Foreign molecules have successfully been incorporated into the resealed, but otherwise intact, erythrocytes.
Access to the cell cytoplasm in viable cells may permit direct labeling or manipulation of intracellular molecules and metabolic processes. One method to gain access to the cell cytoplasm is by electroporation, a technique that transiently creates pores in cell membranes by means of applied electrical fields. We used electroporation to introduce large-molecular-mass dextrans and proteins as probes of the cytoplasmic compartment in human gingival fibroblasts. Electrical field strength and pulse decay time were optimized to obtain cellular viability greater than 80%. Analysis by confocal microscopy and by fluorescence spectrophotometry demonstrated that a large proportion of high-molecular-mass probe was membrane-bound after electroporation. Trypsinization did not affect membrane-bound FITC-dextran but eliminated protein probe incorporated into the membrane, thereby permitting measurement of only intracellular, cytoplasmic label. Proteins of up to 66 kDa were incorporated at intracellular concentrations of 10(-15) M. After electroporation under optimal conditions, incorporated anti-vimentin antibodies were capable of binding to vimentin. Cells electroporated in the presence of RNase A exhibited significant reductions of cellular RNA. Electroporation appears to be a useful approach to probe or perturb specific cellular processes by introduction of functional molecular species into the cytoplasm of viable cells.
The influence of electric field treatments on the interaction of large unilamellar vesicles (liposomes) with animal cells was monitored by the fluorescence assay based on the use of the liposomes loaded by a dye 1-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). It was shown that application of a short electric pulse (100 microseconds of 3-4 kV/cm) to the suspension of cells in presence of vesicles resulted in significant (more than 2 times) increase of the fluorescence associated with cells. The pH-sensitivity of the excitation spectrum of the dye and its interaction with the quencher were used to determine the nature of the phenomenon as the increase of the liposome binding onto the cell surface but not the consequence of a promotion of liposome uptake into the cells by endocytosis. The higher affinity for the liposome caused by the electric field has a lifetime of several minutes. The possible relation of the effect described to the electroporation of cell membranes and to macroscopic changes in membrane structure is discussed.
A biophysical model for the equilibrium curvature of a composite membrane element is derived taking into account the mechanical bilayer properties and the adjacent charged protein layers. The minimum of the total free energy density with respect to the curvature of such a membrane curved was estimated from the sum of the electrostatic free energy density of the charges of the membrane and the elastic surface energy density due to bending the lipid bilayer membrane. It was shown that the equilibrium curvature, i.e. the spontaneous curvature, of such a charged composite sandwich-like membrane depends inversely on the bending stiffness of the lipid membrane itself and directly on the charge amount inside and outside the membrane to the second power. Furthermore the geometric and electrostatic structure of the protein layers and the physico-chemical environment conditions are involved. Corresponding to the model developed a "standard RBC" membrane element has a negative spontaneous curvature, accounting for a discocyte RBC shape. The shape change from a discocyte to a more stomatocytic shape (increase in the negative spontaneous curvature) after reducing the charges in the glycocalyx is also explained within this model.
A Coulter Counter is a measuring device for counting and sizing non-conducting particles suspended in a conducting medium. This instrument is based on the principle that the electrical resistance in a small orifice, across which a voltage is applied, will change when a suspension of non-conducting particles is sucked through it. The current or voltage pulses arising are, to a first approximation, proportional to the size of the particles (size = shape factor X volume); this means that after linear electronic amplification and subsequent pulse height analysis the size distribution of each population under investigation can be obtained. Using conventional Coulter Counters, however, the measured size distribution is generally strongly distorted (skewed) due to inhomogeneities in the electric field strength in and near of the orifice; the pulse height becomes dependent on the path and orientation of each particle in the orifice (GROVER et al., 1969a, b, 1972; THOM, 1972). As described previously (ZIMMERMANN et al., 1973) the distortion arising from the inhomogeneous field can be eliminated by hydrodynamic focusing of the suspension flow. Then the particles travel on the same pathway, parallelly oriented, along the central axis through the orifice, and a true measurement of the real size distribution of the population can be obtained.
Preilluminated chloroplast membranes, and particularly hypotonically swollen vesicles (blebs), give rise to a strong characteristic luminescence (electrophotoluminescence, EPL; Ellenson and Sauer, 1976, Photochem. Photobiol., 23:113-123; Arnold and Azzi, 1971, Photochem. Photobiol., 14:233-240) during the application of a strong external electric field. A detailed kinetic study of EPL was carried out and the initial kinetics from the field onset are reported here. The fast rise time (less than 0.2 mus) of the applied external electric field together with a high instrumental time resolution allowed the observation of a characteristic delay (lag time) between the field onset and the appearance of the induced emission. The lag time decreased with increase in the applied field strength and/or the conductivity of the suspension and is interpreted to be a consequence of (a) the necessity to reach a threshold electrical potential difference in the bleb membrane, below which no emission can be triggered, and (b) the finite time required to attain such a transmembranal field during the charging process of the membrane. A quantitative analysis, connecting the lag time, the controllable experimental parameters, and the membrane electrical characteristics is presented. Its verification was carried out in both size-selected and heterogeneous bleb populations. In the latter, experiments were consistent with the assumption that the lag time reflects the charging of the largest blebs. The results indicate (a) the possibility of directly measuring the specific membrane capacitance, yielding an estimate of Cm = 1.2 +/- 0.3 microF/cm2 (the precision being particle size-homogeneity dependent); (b) A minimal transmembranal potential difference (of approximately 240 mV) is necessary to induce electrophotoluminescence; and (c) the lag duration depends on the time elapsed between the preillumination and the external field application. Correlated with the study of ionophore effects on the lag time, this suggests additivity of the light- and field-induced transmembrane potentials in attaining the threshold for emission.
Concanavalin A (con A) receptors on the surface of cultured Xenopus myoblasts redistributed in response to monopolar, pulsed electric fields. The prefield uniform distribution of the receptors became asymmetrical, and was polarized toward the cathodal pole, in the same way as in DC fields. The extent of asymmetry depended on the duration of field exposure, pulse width (or alternatively, interpulse interval), frequency, and intensity. This relationship was most conveniently expressed by using duty cycle, a quantity determined by both pulse width and frequency. Pulses of average intensity 1.5 V/cm induced detectable asymmetry within 5 min. At the lowest average field intensity used, 0.8 V/cm, significant asymmetry was detected at 150 min. For pulses of high duty cycle (greater than 25%), steady state was reached after 30 min exposure and the steady state asymmetry was dependent on average field intensity. For low duty cycle fields, the time required to reach steady state was prolonged (greater than 50 min). Before reaching a steady state, effectiveness of the pulses, as compared with DC fields of equivalent intensity, was a function of duty cycle. A low duty cycle field (fixed number of pulses at low frequency or long interpulse interval) was less effective than high duty cycle fields or DC.
The surfaces of most cells bear a net negative charge. The imposition of an electric field parallel to the surface of the cell should produce, therefore, an electro-osmotic flow of fluid towards the cathodal side of the cell. Our analysis of a simple model of the cell surface indicates that a negatively charged mobile macromolecule will be swept by this electro-osmotic flow of fluid to the cathodal side of the cell if its zeta potential, zeta 1, is less negative than the zeta potential of the cell surface, zeta 2. Conversely, if zeta 2 is less negative than zeta 1, the negatively charged macromolecule will accumulate at the anodal side of the cell. Our experimental results demonstrate that concanavalin A (Con A) receptors on embryonic muscle cells normally accumulate at the cathodal side of the cell, but that they can be induced to accumulate at the anodal side of the cell by preincubating the myotubes either with neuraminidase, a treatment that removes negatively charged sialic acid residues, or with the lipid diI, a treatment that adds positive charges to the surface of the cell. Addition of the negatively charged lipid monosialoganglioside (GM1), on the other hand, enhances the accumulation of Con A receptors at the cathodal side of the cell.
The collagen-induced response of free platelets during aggregation was measured with an electronic particle size analyzer. Aggregation was induced in platelet-rich plasma by 0.05 mg/mL collagen. The reaction was followed turbidimetrically and samples for electronic particle size analysis were obtained during aggregation. Free platelet number decreased rapidly after the addition of collagen. This decrease preceded detectable changes in transmittance and a significant reduction in free platelet number was measured during the classical lag phase. In addition, the size distribution of free platelets at various times after the addition of collagen was not significantly altered indicating that large platelets are not preferentially involved in collagen-induced aggregation.
The stationary-state kinetic properties of a simplified two-state electro-conformational coupling model (ECC) in the presence of alternating rectangular electric potential pulses are derived analytically. Analytic expressions for the transport flux, the rate of electric energy dissipation, and the efficiency of the transducing system are obtained as a function of the amplitude and frequency of the oscillation. These formulas clarify some fundamental concept of the ECC model and are directly applicable to the interpretation and design of experiments. Based on these formulas, the reversibility and the degree of coupling of the system can be studied quantitatively. It is found that the oscillation-induced free energy transduction is reversible and tight-coupled only when the amplitude of the oscillating electric field is infinitely large. In general, the coupling is not tight when the amplitude of the electric field is finite. Furthermore, depending on the kinetic parameters of the model, there may exist a "critical" electric field amplitude, below which free energy transduction is not reversible. That is, energy may be transduced from the electric to the chemical, but not from the chemical to the electric.
We characterized binding of albumin to the apical membrane of opossum kidney (OK) cells using fluorescein isothiocyanate (FITC)-albumin (i.e., bovine serum albumin, BSA) as substrate. Functional analysis of binding data showed one specific binding site characterized by half-maximal binding (Michaelis constant, (Km) at 20 mg/l (300 nmol/l) and maximal binding capacity (Bmax) of 0.61 microgram/mg cellular protein. Excess of unlabeled albumin (BSA) inhibited binding at low concentrations of FITC-albumin completely but only partially at high concentrations. FITC-albumin binding was reversible and pH dependent. Km increased about sixfold when pH decreased from 7.4 to 5.0. The inhibitory effects of conalbumin, alpha-lactalbumin, and transferrin were significantly smaller compared with BSA. We conclude that OK cells express a high-affinity binding site for albumin on the apical membrane. This binding site is pH sensitive, binds albumin in the physiological range, and could be responsible for the effective receptor-mediated reabsorption of albumin in the proximal tubule.
Directional flow of information and energies is characteristic of many types of biochemical reactions, for instance, ion transport, energy coupling during ATP synthesis, and muscle contraction. Can a fluctuating force field, or a noise, induce such a directional flux? Previous work has shown that Na,K-ATPase of human erythrocyte can absorb free energy from an externally applied random-telegraph-noise (RTN) electric field to pump Rb+ up its concentration gradient. However, the RTN field used in these experiments was constant in amplitude and would not mimic fluctuating electric fields of a cell membrane. Here we show that electric fields which fluctuate both in life time and in amplitude, and thus, better mimicking the transmembrane electric fields of a cell, can also induce Rb+ pumping by Na,K-ATPase. A Gaussian-RTN-electric field, or a field with amplitude fluctuating according to the Gaussian distribution, with varied standard deviation (sigma), induced active pumping of Rb+ in human erythrocyte, which was completely inhibited by ouabain. Increased values for sigma led to a nonmonotonic reduction in pumping efficiency. A general formula for calculating the ion transport in a biochemical cycle induced by fluctuating electric field has been derived and applied to a simple four-state electroconformational coupling (ECC) model. It was found that the calculated efficiency in the energy coupling decreased with increasing sigma value, and this effect was relatively small and monotonic, whereas experimental data were more complex: monotonic under certain sets of conditions but nonmonotonic under different sets. The agreement in general features but disagreement in some fine features suggest that there are other properties of the electric activation process for Na,K-ATPase that cannot be adequately described by the simple ECC model, and further refinement of the ECC model is required.
We can expect that biological responses to very weak ELF electromagnetic fields will be masked by thermal noise. However, the spin of electrons bound to biologically important molecules is not strongly coupled to the thermal bath, and the effects of the precession of those spins by external magnetic fields is not bounded by thermal noise. Hence, the known role of spin orientation in the recombination of radical pairs (RP) may constitute a mechanism for the biological effects of very weak fields. That recombination will generally take place only if the valence electrons in the two radicals are in a singlet state and the effect of the magnetic field is manifest through differential spin precessions that affect the occupation of that state. Because the spin relaxation times are of the order of microseconds, any effects must be largely independent of frequency up to values of a few megahertz. Using exact calculations on an appropriately general model system, we show that one can expect small, but significant, modifications of the recombination rate by a 50 microT field only under a narrow range of circumstances: the cage time during which the two elements are together must be exceptionally long--of the order of 50 ns or longer; the hyperfine field of either radical must not be so great as to generate a precession period greater than the cage containment time; and the characteristic recombination time of the radical pair in the singlet state must be about equal to the containment time. Thus, even under such singularly favorable conditions, fields as small as 5 microT (50 milligauss) cannot change the recombination rate by as much as 1%. Hence, we conclude that environmental magnetic fields much weaker than the earth's field cannot be expected to affect biology significantly by modifying radical pair recombination probabilities.