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1.
Igor Marjanovič Saša Haberl Damijan Miklavčič Maša Kandušer Mojca Pavlin 《The Journal of membrane biology》2010,236(1):97-105
Knowledge of the parameters which influence the efficiency of gene electrotransfer has importance for practical implementation
of electrotransfection for gene therapy as well as for better understanding of the underlying mechanism. The focus of this
study was to analyze the differences in gene electrotransfer and membrane electropermeabilization between plated cells and
cells in a suspension in two different cell lines (CHO and B16F1). Furthermore, we determined the viability and critical induced
transmembrane voltage (ITVc) for both cell lines. In plated cells we obtained relatively little difference in electropermeabilization and gene electrotransfection
between CHO and B16F1 cells. However, significant differences between the two cell lines were observed in a suspension. CHO
cells exhibited a much higher gene electrotransfection rate compared to B16F1 cells, whereas B16F1 cells reached maximum electropermeabilization
at lower electric fields than CHO cells. Both in a suspension and on plated cells, CHO cells had a slightly better survival
rate at higher electric fields than B16F1 cells. Calculation of ITVc in a suspension showed that, for both electropermeabilization and gene electrotransfection, CHO cells have lower ITVc than B16F1 cells. In all cases, ITVc for electropermeabilization was lower than ITVc for gene electrotransfer, which is in agreement with other studies. Our results show that there is a marked difference in
the efficiency of gene electrotransfer between suspended and plated cells. 相似文献
2.
This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed
on dense cell suspensions (up to 400 × 106 cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with
a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and
the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 × 106 to 400 × 106 cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local
electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction
of cell permeabilization in suspensions with 10 × 106 and 400 × 106 cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with
numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger
than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was
mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane
and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions,
which can be attributed to cell swelling resulting from electropermeabilization. 相似文献
3.
Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent 总被引:6,自引:0,他引:6
For the evaluation of cell membrane electropermeabilization, cells are usually exposed to electric pulses in the presence of propidium iodide, a fluorescent dye activated by binding to cellular DNA. The fraction of permeabilized cells is then determined using a flow cytometer. This widely established method has several drawbacks: (i) an arbitrary choice of minimum fluorescence intensity for characterization of permeabilized cells; (ii) the inability to detect cells disintegrated because of intense electropermeabilization; and (iii) false detection of cellular ghosts devoid of fluorescence because of leakage of DNA caused by electropermeabilization. Here, we present a simple and inexpensive method that eliminates these drawbacks. The method is based on the use of a cytotoxic agent that cannot permeate through an intact plasma membrane and thus leads to selective death of the electropermeabilized cells. The amount of nonpermeabilized cells is then determined by a suitable viability test. Bleomycin at a 5-nM concentration causes no statistically significant effect on cell survival in the absence of electric pulses, yet this concentration is sufficient for lethal toxicity in electropermeabilized cells. The amount of cells surviving the exposure relative to the control gives a reliable value of the fraction of nonpermeabilized cells. 相似文献
4.
Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon. 总被引:8,自引:4,他引:4
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Transient membrane permeabilization by application of high electric field intensity pulses on cells (electropermeabilization) depends on several physical parameters associated with the technique (pulse intensity, number, and duration). In the present study, electropermeabilization is studied in terms of flow of diffusing molecules between cells and external medium. Direct quantification of the phenomenon shows that electric field intensity is a critical parameter in the induction of permeabilization. Electric field intensity must be higher than a critical threshold to make the membrane permeable. This critical threshold depends on the cell size. Extent of permeabilization (i.e., the flow rate across the membrane) is then controlled by both pulse number and duration. Increasing electric field intensity above the critical threshold needed for permeabilization results in an increase membrane area able to be permeabilized but not due to an increase in the specific permeability of the field alterated area. The electroinduced permeabilization is transient and disappears progressively after the application of the electric field pulses. Its life time is under the control of the electric field parameters. The rate constant of the annealing phase is shown to be dependent on both pulse duration and number, but is independent of electric field intensity which creates the permeabilization. The phenomenon is described in terms of membrane organization transition between the natural impermeable state and the electro-induced permeable state, phenomenon only locally induced for electric field intensities above a critical threshold and expanding in relation to both pulse number and duration. 相似文献
5.
Cholesterol has a concentration-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane potential difference modulation induced in vesicles when an external electric field is applied. The magnitude of this modulation is under the control of the membrane electrical permeability. When brought to a critical value by the external field, the membrane potential difference induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content. 相似文献
6.
The C-terminal fragment of colicin, a protein that is highly soluble in aqueous solution, is spontaneously and irreversibly inserted into the membranes of mammalian cells, which are locally permeabilized by a transmembrane voltage increase. Insertion is detected by immunodetection. This is obtained by mixing the protein with electropermeabilized cells. The same result is observed by pulsing the colicin/cell mixture. Electroinsertion is therefore obtained for the first time with a multi-fragment spanning protein. The cell viability is not affected beyond the effect of electropermeabilization. A train of low voltage repetitive transmembrane modulation, which cannot trigger membrane permeabilization, is applied a day after the electroinsertion. This induces no effect on unmodified cells but triggers the lysis of cells in which colicin has been inserted by the first electropulsation. The low-level electrical treatment is high enough to trigger the voltage gated opening of colicin and to induce the associated toxicity. A transmembrane configuration of colicin is therefore obtained by electroinsertion. The toxic effect of their voltage gating is only obtained when a critical number of voltage gated channels are activated. 相似文献
7.
H. Mussauer V.L. Sukhorukov A. Haase U. Zimmermann 《The Journal of membrane biology》1999,170(2):121-133
The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist,
a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated
phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity
DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity
solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability
of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the
results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed
effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in
molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of
the ionic equilibrium (e.g., Ca2+ sequestration) in the vicinity of the cell membrane.
Received: 19 January 1999/Revised: 1 April 1999 相似文献
8.
Poly(ADP-ribose) metabolism appears normal in EM9, a mutagen-sensitive mutant of CHO cells 总被引:5,自引:1,他引:4
EM9 is a mutagen-sensitive CHO cell whose phenotype resembles that of normal CHO cells exposed to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis. This phenotype suggested that EM9 might be defective in poly(ADP-ribose) metabolism, but we now cannot find any abnormality in the synthesis or in the degradation of poly(ADP-ribose) in permeabilized EM9 cells. Thus the effects of 3-aminobenzamide on wild-type cells may be due to the inhibition of processes other than poly(ADP-ribose) synthesis. 3-Aminobenzamide enhances the cytotoxicity of EMS toward EM9 and control cells to the same degree. 相似文献
9.
The activity of beta-adrenergic receptors at the plasma membrane level was investigated in viable, electropermeabilized C6 glioma cells. Electric field pulses were applied directly to the plated cells without any previous proteinase treatment. The affinity for isoproterenol and the density of the beta-adrenergic receptors, as judged from the number of [3H]CGP-12177 binding sites, were not affected by the electropermeabilization whereas the isoproterenol-stimulated cAMP accumulation was transiently impaired. This decrease in activity is due to an electropermeabilization-induced GTP leak. Normal activity could be obtained either by treating the cells by the electric field in a GTP-containing buffer, or by spontaneous recovery of the cells after the resealing of the plasma membrane, with a delay depending on the temperature. The activity of the receptors was not affected by the structural organization of the membrane associated to its electropermeabilization. 相似文献
10.
Parzy D Fusai T Pouvelle B Torrentino M Eustacchio F Lépolard C Scherf A Gysin J 《Microbes and infection / Institut Pasteur》2000,2(7):779-788
The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies. 相似文献
11.
Valic B Golzio M Pavlin M Schatz A Faurie C Gabriel B Teissié J Rols MP Miklavcic D 《European biophysics journal : EBJ》2003,32(6):519-528
The transmembrane potential on a cell exposed to an electric field is a critical parameter for successful cell permeabilization. In this study, the effect of cell shape and orientation on the induced transmembrane potential was analyzed. The transmembrane potential was calculated on prolate and oblate spheroidal cells for various orientations with respect to the electric field direction, both numerically and analytically. Changing the orientation of the cells decreases the induced transmembrane potential from its maximum value when the longest axis of the cell is parallel to the electric field, to its minimum value when the longest axis of the cell is perpendicular to the electric field. The dependency on orientation is more pronounced for elongated cells while it is negligible for spherical cells. The part of the cell membrane where a threshold transmembrane potential is exceeded represents the area of electropermeabilization, i.e. the membrane area through which the transport of molecules is established. Therefore the surface exposed to the transmembrane potential above the threshold value was calculated. The biological relevance of these theoretical results was confirmed with experimental results of the electropermeabilization of plated Chinese hamster ovary cells, which are elongated. Theoretical and experimental results show that permeabilization is not only a function of electric field intensity and cell size but also of cell shape and orientation. 相似文献
12.
Shirakashi R Köstner CM Müller KJ Kürschner M Zimmermann U Sukhorukov VL 《The Journal of membrane biology》2002,189(1):45-54
The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues. 相似文献
13.
Jean-Michel Escoffre Elisabeth Bellard Cécile Faurie Sarra C. Sébaï Muriel Golzio Justin Teissié Marie-Pierre Rols 《生物化学与生物物理学报:生物膜》2014
Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1 s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field. 相似文献
14.
The importance of electric field distribution for effective in vivo electroporation of tissues. 总被引:7,自引:0,他引:7
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Cells exposed to short and intense electric pulses become permeable to a number of various ionic molecules. This phenomenon was termed electroporation or electropermeabilization and is widely used for in vitro drug delivery into the cells and gene transfection. Tissues can also be permeabilized. These new approaches based on electroporation are used for cancer treatment, i.e., electrochemotherapy, and in vivo gene transfection. In vivo electroporation is thus gaining even wider interest. However, electrode geometry and distribution were not yet adequately addressed. Most of the electrodes used so far were determined empirically. In our study we 1) designed two electrode sets that produce notably different distribution of electric field in tumor, 2) qualitatively evaluated current density distribution for both electrode sets by means of magnetic resonance current density imaging, 3) used three-dimensional finite element model to calculate values of electric field for both electrode sets, and 4) demonstrated the difference in electrochemotherapy effectiveness in mouse tumor model between the two electrode sets. The results of our study clearly demonstrate that numerical model is reliable and can be very useful in the additional search for electrodes that would make electrochemotherapy and in vivo electroporation in general more efficient. Our study also shows that better coverage of tumors with sufficiently high electric field is necessary for improved effectiveness of electrochemotherapy. 相似文献
15.
Phosphatidylethanolamine synthesis through the phosphatidylserine (PtdSer) decarboxylation pathway requires PtdSer transport from the endoplasmic reticulum or mitochondrial-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PtdSer decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence was identical to part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His(6)-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PtdSer decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PtdSer decarboxylase, the recombinant S100B protein did not stimulate PtdSer decarboxylase activity and exhibited no PtdSer decarboxylase activity. These results implicated the S100B protein in the transport of PtdSer to the mitochondrial inner membrane. 相似文献
16.
Due to their spontaneous accumulation in inflamed or infected areas, blood phagocytes are potent drug vectors with specific targeting. Drug like molecule loading was obtained by use of cell electropermeabilization in which the impermeability of their plasma membrane is transiently impaired. Electrical conditions were used which allow electroloading of a drug like molecule (propidium iodide) in 70% of leukocytes in a whole blood sample while preserving in vitro functional properties. Slow release of entrapped hydrophilic molecules was observed with a half lifetime longer than 4 hours at 4 degrees C and at 37 degrees C. With an in vivo assay, using a rat model of inflammation, we showed that, as for non-pulsed cells, pulsed neutrophils accumulate 10 times more in an inflamed area than they do in control areas. Phagocyte electropermeabilization is therefore a very efficient way of drug targeting. Accumulation of electropulsed neutrophils in an area of inflammation gives targeted release of the electroloaded drug. 相似文献
17.
An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization. 总被引:6,自引:3,他引:3
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When applied on intact cell suspension, electric field pulses are known to induce membrane permeabilization (electropermeabilization) and fusion (electrofusion). These effects are triggered through a modulation of the membrane potential difference. Due to the vectorial character of the electric field effects, this modulation, which is superimposed on the resting membrane potential difference, is position-dependent on the cell surface. This explains the difference between the experimentally observed critical field strengths requested to trigger the processes of permeabilization and fusion. The critical membrane potential difference which induces membrane permeabilization can be calculated from these experimental observations. It is observed that its value is always about 200 mV for many different cell systems as we previously reported in the case of pure lipid vesicles. This is much less than assumed in most previous studies. 相似文献
18.
The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U 相似文献
19.
Jean-Michel Escoffre Thomas Portet Luc Wasungu Justin Teissié David Dean Marie-Pierre Rols 《Molecular biotechnology》2009,41(3):286-295
Cell membranes can be transiently permeabilized under application of electric pulses. This treatment allows hydrophilic therapeutic
molecules, such as anticancer drugs and DNA, to enter into cells and tissues. This process, called electropermeabilization or electroporation, has been rapidly developed over the last decade to deliver genes to tissues and organs, but there is a general agreement
that very little is known about what is really occurring during membrane electropermeabilization. It is well accepted that
the entry of small molecules, such as anticancer drugs, occurs mostly through simple diffusion after the pulse while the entry
of macromolecules, such as DNA, occurs through a multistep mechanism involving the electrophoretically driven interaction
of the DNA molecule with the destabilized membrane during the pulse and then its passage across the membrane. Therefore, successful
DNA electrotransfer into cells depends not only on cell permeabilization but also on the way plasmid DNA interacts with the
plasma membrane and, once into the cytoplasm, migrates towards the nucleus. The focus of this review is to describe the different
aspects of what is known of the mechanism of membrane permeabilization and associated gene transfer and, by doing so, what
are the actual limits of the DNA delivery into cells.
Jean-Michel Escoffre and Thomas Portet have contributed equally to this work. 相似文献
20.
Ionic-strength modulation of electrically induced permeabilization and associated fusion of mammalian cells 总被引:5,自引:0,他引:5
Application of a high electric field to cells in culture has been shown to make them both permeable and fusogenic. The molecular events involved in the phenomenon are still poorly understood. In this study we investigated the effects of the ionic strength of the pulsing buffer on the electropermeabilization and electrofusion of Chinese hamster ovary cells. Increasing the ionic strength of the pulsing medium results in an increase in sieving of transient permeant structures, but decreases the fusion index. Treatment of cells with trypsin or pronase before application of the pulses abolishes the ionic modulation of both electropermeabilization and electrofusion. A similar rate of expansion of permeabilization is obtained whatever the ionic content of the pulsing buffer, and cells fuse even at high ionic strength. This observation lends support to our hypothesis that membrane proteins play a role in electrofusion. 相似文献