Efficiency Of Gene Electrotransfer Research Articles

Effects of buffer composition and plasmid toxicity on electroporation-based non-viral gene delivery in mammalian cells using bursts of nanosecond and microsecond pulses.

Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.

The Influence of Calcium Ions on the Electrotransfer Efficiency of Plasmid DNA and Cell Viability

Gene electrotransfer (GET) is recognized as a promising technique for the development of an efficient tool for gene therapy. Such a therapy would have applications in the treatment of a variety of genetic diseases, including cancer. However, despite its wide applicability, the technique is limited by the lack of understanding of the fundamental mechanism of electroporation as well as other important factors that directly or indirectly influence its success rate. In the current study, we analyzed the impact of low concentrations (0–1 mM) of Ca2+ on the process of DNA electrotransfer using flow cytometry. The results revealed that the presence of a CaCl2 concentration as low as 0.25 mM decreased the efficiency of GET by ~1.5-fold and cell viability decreased by ~2–3-fold. In addition, we determined that the observed phenomenon of the decrease in pDNA electrotransfer due to the influence of Ca2+ was not the consequence of cell death but rather should be attributed to secondary mechanisms. The data presented in this study provide an insight into the importance of Ca2+ in the process of gene electrotransfer that may be directly applicable to in vivo settings.

Synergistic effects of an atmospheric pressure plasma jet and pulsed electric field on cells and skin.

Nonthermal atmospheric pressure plasmas produce reactive plasma species including charged particles and reactive oxygen nitrogen species, which are known to induce oxidative stress in living cells in liquid or tissue. In the meantime, pulsed electric fields have been widely used in reversible or irreversible electropermeabilization for either the delivery of plasmid DNA or inactivation of cancer cells. This work discusses the synergistic effects of nanosecond pulsed plasma jets and pulsed electric field on inactivation of pancreatic cancer cells in vitro and enhancement of plasmid DNA delivery to guinea pig skin in vivo. Higher inactivation rates of the cancer cells in suspension were obtained with combined treatment of 300-ns 50 kV/cm pulsed electric field and a 1-min exposure of a nanosecond pulsed, 250-μm plasma jet. Increased efficiency of gene electrotransfer to skin was also observed after a 3-min treatment of a nanosecond pulsed, 1-mm plasma jet. Application of the plasma alone at the same dosage did not have significant effect on gene delivery. These findings signify the dosage-dependent cell-response to both the electric fields and plasma. Importantly, the use of cold plasma to increase the sensitization of the biological cells in response to pulsed electric fields could be an effective approach to enhance the desired effects in electroporation-based applications.

Investigation of Plasmid DNA Delivery and Cell Viability Dynamics for Optimal Cell Electrotransfection In Vitro

Electroporation is an effective method for delivering plasmid DNA molecules into cells. The efficiency of gene electrotransfer depends on several factors. To achieve high transfection efficiency while maintaining cell viability is a tedious task in electroporation. Here, we present a combined study in which the dynamics of both evaluation types of transfection efficiency and the cell viability were evaluated in dependence of plasmid concentration as well as at the different number of high voltage (HV) electric pulses. The results of this study reveal a quantitative sigmoidal (R2 > 0.95) dependence of the transfection efficiency and cell viability on the distance between the cell membrane and the nearest plasmid. We propose this distance value as a new, more accurate output parameter that could be used in further optimization studies as a predictor and a measure of electrotransfection efficiency.

The Efficiency of Gene Electrotransfer in Breast-Cancer Cell Lines Cultured on a Novel Collagen-Free 3D Scaffold.

Gene Electro-Transfer (GET) is a powerful method of DNA delivery with great potential for medical applications. Although GET has been extensively studied in vitro and in vivo, the optimal parameters remain controversial. 2D cell cultures have been widely used to investigate GET protocols, but have intrinsic limitations, whereas 3D cultures may represent a more reliable model thanks to the capacity of reproducing the tumor architecture. Here we applied two GET protocols, using a plate or linear electrode, on 3D-cultured HCC1954 and MDA-MB231 breast cancer cell lines grown on a novel collagen-free 3D scaffold and compared results with conventional 2D cultures. To evaluate the electrotransfer efficiency, we used the plasmid pEGFP-C3 encoding the enhanced green fluorescent protein (EGFP) reporter gene. The novel 3D scaffold promoted extracellular matrix deposition, which particularly influences cell behavior in both in vitro cell cultures and in vivo tumor tissue. While the transfection efficiency was similar in the 2D-cultures, we observed significant differences in the 3D-model. The transfection efficiency in the 3D vs 2D model was 44% versus 15% (p < 0.01) and 24% versus 17% (p < 0.01) in HCC1954 and MDA-MB231 cell cultures, respectively. These findings suggest that the novel 3D scaffold allows reproducing, at least partially, the peculiar morphology of the original tumor tissues, thus allowing us to detect meaningful differences between the two cell lines. Following GET with plate electrodes, cell viability was higher in 3D-cultured HCC1954 (66%) and MDA-MB231 (96%) cell lines compared to their 2D counterpart (53% and 63%, respectively, p < 0.001). Based on these results, we propose the novel 3D scaffold as a reliable support for the preparation of cell cultures in GET studies. It may increase the reliability of in vitro assays and allow the optimization of GET parameters of in vivo protocols.

Numerical study of gene electrotransfer efficiency based on electroporation volume and electrophoretic movement of plasmid DNA

BackgroundThe efficiency of gene electrotransfer, an electroporation-based method for delivery of pDNA into target tissues, depends on several processes. The method relies on application of electric pulses with appropriate amplitude and pulse duration. A careful choice of electric pulse parameters is required to obtain the appropriate electric field distribution, which not only controls the electroporated volume, but also affects the movement of pDNA. We used numerical modeling to assess the influence of different types of electrodes and pulse parameters on reversibly electroporated volume and on the extent of pDNA–membrane interaction, which is necessary for successful gene electrotransfer.MethodsA 3D geometry was built representing the mice skin tissue and intradermally injected plasmid volume. The geometry of three different types of electrodes (plate, finger, needle) was built according to the configuration and placement of electrodes used in previously reported in vivo experiments of gene electrotransfer. Electric field distribution, resulting from different pulse protocols was determined, which served for calculation of reversible electroporation volume and for simulation of electrophoretic movement of pDNA. The efficiency of gene electrotransfer was evaluated in terms of predicted amount of pDNA present inside the volume of reversible electroporation at the end of pulse delivery.ResultsAccording to results of our numerical study, finger and needle electrodes provide larger amount of pDNA inside the volume of reversible electroporation than plate electrodes. However, these results are not consistent with the experiments showing that plate electrodes achieve the best transfection efficiency. Some inconsistencies were observed also by comparing the efficiencies of different high and low voltage pulse combinations, delivered by plate electrodes. The reason for inconsistencies probably lies in insufficient knowledge regarding the electroporation of stratum corneum. Namely, the size of the regions with high electrical conductivity, created by electroporation, was found to strongly affect predicted transfection efficiency.ConclusionsThe presented numerical model simulates the two most important processes involved in gene electrotransfer: electroporation of cells, and electrophoretic movement of pDNA. The inconsistencies between the model and experiments indicate incomplete knowledge of skin electroporation, or the involvement of other mechanisms, whose importance has not been yet identified.

Erratum to: Inhibition of the Innate Immune Receptors for Foreign DNA Sensing Improves Transfection Efficiency of Gene Electrotransfer in Melanoma B16F10 Cells.

Gene electrotransfer upregulate DNA pattern recognition receptors or DNA sensors, which are part of the innate immune system. In this study, we tested if addition of the cocktail of innate immune system inhibitors to the cells during gene electrotransfer (GET) can increase transfection efficiency and cell survival. The results indicate that this cocktail can decrease cytosolic DNA sensors expression after GET, and consequently increase cell survival and transfection efficiency in B16 cells, but only in highly metastatic B16F10 subtype. We demonstrated that DNA sensors expression during the transfection methods needs to be downregulated if higher transfection efficiency and better cells’ survival is needed. The inhibition of the receptors of the innate immune system can improve the transfection efficiency also for GET of malignant melanoma B16 cells, but only of highly metastatic subtype.

Inhibition of the Innate Immune Receptors for Foreign DNA Sensing Improves Transfection Efficiency of Gene Electrotransfer in Melanoma B16F10 Cells.

Gene electrotransfer upregulate DNA pattern recognition receptors or DNA sensors, which are part of the innate immune system. In this study, we tested if addition of the cocktail of innate immune system inhibitors to the cells during gene electrotransfer (GET) can increase transfection efficiency and cell survival. The results indicate that this cocktail can decrease cytosolic DNA sensors expression after GET, and consequently increase cell survival and transfection efficiency in B16 cells, but only in highly metastatic B16F10 subtype. We demonstrated that DNA sensors expression during the transfection methods needs to be downregulated if higher transfection efficiency and better cells' survival is needed. The inhibition of the receptors of the innate immune system can improve the transfection efficiency also for GET of malignant melanoma B16 cells, but only of highly metastatic subtype.

The dependence of efficiency of transmembrane molecular transfer using electroporation on medium viscosity.

In the present study, we aimed to evaluate the efficiency of drug and gene electrotransfer into cells in vitro depending on medium viscosity. Experiments were performed using Chinese hamster ovary cells. Efficiency of molecular electrotransfer depending of medium viscosity was evaluated using two different electroporation conditions: a high-voltage (HV) pulse and a combination of a high-voltage pulse and a low-voltage pulse (HV + LV). To evaluate the efficiency of molecular electrotransfer, anticancer drug bleomycin and two different plasmids coding for green fluorescent protein and luciferase were used. We found that a slight increase in medium viscosity from 1.3-1.4 mPa·s significantly decreased the transfection efficiency, both in terms of transfected cells and total protein production, which was abolished completely with an increase in medium viscosity to 6.1 mPa·s. Notably, at this medium viscosity, electrotransfer of the small anticancer drug was still efficient. Using HV and HV + LV pulse combinations, we showed that a decrease of DNA electrotransfer, especially at lower medium viscosities, can be compensated for by the LV pulse to some extent. On the other hand, the addition of the LV pulse after the HV pulse did not have any positive effect on the efficiency of bleomycin electrotransfer. These findings demonstrate that transfection is very susceptible to medium viscosity and highlights the importance of the electrophoretic component in experiments when a considerable transfection level is needed.

Endoglin (CD105) Silencing Mediated by shRNA Under the Control of Endothelin-1 Promoter for Targeted Gene Therapy of Melanoma.

Endoglin (CD105), a transforming growth factor (TGF)-β coreceptor, and endothelin-1, a vasoconstrictor peptide, are both overexpressed in tumor endothelial and melanoma cells. Their targeting is therefore a promising therapeutic approach for melanoma tumors. The aim of our study was to construct a eukaryotic expression plasmid encoding the shRNA molecules against CD105 under the control of endothelin-1 promoter and to evaluate its therapeutic potential both in vitro in murine B16F10-luc melanoma and SVEC4-10 endothelial cells and in vivo in mice bearing highly metastatic B16F10-luc tumors. Plasmid encoding shRNA against CD105 under the control of the constitutive U6 promoter was used as a control. We demonstrated the antiproliferative and antiangiogenic effects of both plasmids in SVEC4-10 cells, as well as a moderate antitumor and pronounced antimetastatic effect in B16F10-luc tumors in vivo. Our results provide evidence that targeting melanoma with shRNA molecules against CD105 under the control of endothelin-1 promoter is a feasible and effective treatment, especially for the reduction of metastatic spread.

Shock waves associated with electric pulses affect cell electro-permeabilization

New features of cell electro-permeabilization are obtained by using high field (several tens of kV/cm) with short (sub-microsecond, nanosecond) pulse duration. Arcing appears as a main safety problem when air gaps are present between electrodes. A new applicator design was chosen to obtain a closed chamber where high field pulses could be delivered in a safe way with very short pulse duration. The safety issue of the system was validated under millisecond, microsecond and nanosecond pulses. The closed chamber applicator was then checked for its use under classical electro-mediated permeabilization and electro-gene transfer (EGT). A 20 times decrease in gene expression was observed compared with classical open chambers. It was experimentally observed that shock waves were present under the closed chamber configuration of the applicator. This was not the case with an open chamber design. Electropulsation chamber design plays a role on pulsing conditions and in the efficiency of gene electro transfer.

Different Incubation Times of Cells After Gene Electrotransfer in Fetal Bovine Serum Affect Cell Viability, but Not Transfection Efficiency

Electroporation as a delivery method is increasingly important in gene therapy, not only in vivo but also in in vitro experimental systems. Different applications of gene electrotransfer require high viability of cells and high transfection efficiency of gene electrotransfer. It was already demonstrated that the addition of fetal bovine serum (FBS) immediately after gene electrotransfer leads to improved cell survival and transfection efficiency. Therefore, the aim of the study was to determine whether prolonged incubation of cells in FBS, for more than standard 5 min, can lead to increased transfection efficiency and improved cell survival. Different murine melanoma and murine and human endothelial cell lines were transfected with plasmid encoding green fluorescent protein and then incubated for different periods of time in FBS (5-30 min). Transfection efficiency was determined by flow cytometry and fluorescence microscopy and cell survival by cell viability assay. Prolonged incubation of cells in FBS after gene electrotransfer had varying effect on cell survival, which was decreased in melanoma cell lines B16F1 and B16F10, minimally affected in SVEC4-10 and HUVEC cells and increased in 2H11 cell at 30 min of incubation time in FBS. On the other hand, transfection efficiency of gene electrotransfer was not affected by long incubation of cell in FBS, regardless of the cell line used. The results of our study emphasize the importance of optimization of gene electrotransfer protocol for particular cells and specific purposes of gene electrotransfer, taking into account the importance of transfection efficiency and cell survival.

Changing electrode orientation, but not pulse polarity, increases the efficacy of gene electrotransfer to tumors in vivo

Gene electrotransfer is an established method for plasmid delivery into different tissues. Contrary to extensive in vitro studies demonstrating increased gene electrotransfer by changing the electric field direction during the pulse delivery, little is known about the efficiency of both polarities pulses in vivo. Therefore the aim of our study was to evaluate the effect of pulse polarity and orientation on the efficacy of gene electrotransfer in the murine fibrosarcoma tumor model by using the luciferase and GFP reporter gene expression plasmids. Our results demonstrated no significant difference in luciferase activity, GFP transfected area or fluorescence intensity between different sets of electric pulses. Inversion of the pulse polarity did not result in the increase of gene transfer, but non-significant enhancement up to 7-fold was detected by changing the electric field orientation in perpendicular direction. Also, transfection of surrounding skin tissue was observed, meaning that intratumoral gene electrotransfer could also result in a systemic effect. Our data indicate that tested modifications of electric pulses are not significantly affecting the efficiency of gene electrotransfer to LPB tumors. Biological factors, like tissue composition, might be of greater importance and should therefore also be taken into account when selecting the electric pulse parameters for specific tissues.

Hyaluronidase and Collagenase Increase the Transfection Efficiency of Gene Electrotransfer in Various Murine Tumors

One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy.

Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells

BackgroundGene electrotransfer is a non-viral gene delivery method that requires successful electroporation for DNA delivery into the cells. Changing the direction of the electric field during the pulse application improves the efficacy of gene delivery. In our study, we tested a pipette tip with integrated electrodes that enables changing the direction of the electric field for electroporation of cell suspension for gene electrotransfer.Materials and methodsA new pipette tip consists of four cylindrical rod electrodes that allow the application of electric pulses in different electric field directions. The experiments were performed on cell suspension of CHO cells in phosphate buffer. Plasmid DNA encoding for green fluorescent protein (GFP) was used and the efficiency of gene electrotransfer was determined by counting cells expressing GFP 24 h after the experiment.ResultsExperimental results showed that the percentage of cells expressing GFP increased when the electric field orientation was changed during the application. The GFP expression was almost two times higher when the pulses were applied in orthogonal directions in comparison with single direction, while cell viability was not significantly affected.ConclusionsWe can conclude that results obtained with the described pipette tip are comparable to previously published results on gene electrotransfer using similar electrode geometry and electric pulse parameters. The tested pipette tip, however, allows work with small volumes/samples and requires less cell manipulation.

Analysis and Comparison of Electrical Pulse Parameters for Gene Electrotransfer of Two Different Cell Lines

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 (ITV(c)) 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 ITV(c) in a suspension showed that, for both electropermeabilization and gene electrotransfection, CHO cells have lower ITV(c) than B16F1 cells. In all cases, ITV(c) for electropermeabilization was lower than ITV(c) 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.

Influence of Plasmid Concentration on DNA Electrotransfer In Vitro Using High-Voltage and Low-Voltage Pulses

DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 microl of CHO cell suspension containing 100, 10 or 1 microg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 microg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 microg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 micros) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 microg/ml and fivefold at 1 microg/ml. At 10 microg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.

Cationic and anionic lipoplexes inhibit gene transfection by electroporation in vivo

Nonviral gene therapy still suffers from low efficiency. Methods that would lead to higher gene expression level of longer duration would be a major advance in this field. Lipidic vectors and physical methods have been investigated separately, and both induced gene expression improvement. We sought to combine both chemical and physical methods. Cationic or anionic lipids can potentially destabilize the cell membrane and could consequently enhance gene delivery by a physical method such as electrotransfer. A plasmid model encoding luciferase was used, either free or associated with differently-charged lipoplexes before electrotransfer. Electrotransfer alone strongly enhanced gene expression after intramuscular and intradermal injection of naked DNA. On the other hand, cationic and anionic lipoplex formulations decreased gene expression after electrotransfer, whereas poorly-charged thiourea-based complexes, brought no benefit. Pre-injection of the lipids, followed by administration of naked DNA, did not modified gene expression induced by electroporation in the skin. The results obtained in the present study suggest that packing of DNA plasmid in lipoplexes strongly decreases the efficiency of gene electrotransfer, independently of the lipoplex charge. Non-aggregating complexes, such as poorly-charged thiourea-based complexes, should be preferred to increase DNA release.

Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization

Gene electrotransfer is a promising nonviral method that enables DNA to be transferred into living cells with electric pulses. However, there are many parameters that determine gene electrotransfer efficiency. One of the steps involved in gene electrotransfer is interaction of DNA with the cell membrane. Divalent cations in the electroporative media can influence the anchoring of DNA to the cell membrane and by that gene electrotransfer efficiency. Here we report the effect of different concentrations of Mg 2+ on electropermeabilization for small molecule (propidium iodide), gene electrotransfer and viability of the cells. We also used TOTO-1 dye to visualize DNA-cell membrane interaction for different [Mg]. For this purpose, we used different electroporative media with increasing [Mg]. Our study shows that higher [Mg] lead to higher electropermeabilization for propidium iodide and higher viability, while causing lower gene electrotransfer efficiency. Because we observed higher TOTO-1 labeled DNA at cell surface when using higher [Mg], we suggest that Mg 2+ ions can bind DNA at cell surface at such strength that cannot pass into the cell during application of electric pulses, which can lead to lower gene transfection. There may also be other mechanisms involved, since there are many steps of gene electrotransfer on which Mg 2+ ions can have an effect on. Our results also imply that membrane permeability changes are not sufficient for an efficient gene electrotransfer.

In vivo NMR imaging evaluation of efficiency and toxicity of gene electrotransfer in rat muscle

In vivo gene electrotransfer (ET) is a simple method of gene delivery in various tissues relying on the injection of plasmid DNA followed by application of electric pulses. Noninvasive tools are needed to evaluate the ET efficiency and the resulting tissue damages. In this study, we performed ET of rat tibialis muscle after injection of either a plasmid coding for luciferase or a contrast agent (CA) detected by using magnetic resonance imaging (MRI). Plasmid expression and CA intracellular trapped quantity were compared throughout the electric field intensity range 0-300 V/cm. Although the CA trapped quantity reflects only the electropermeabilization step, both measurements were correlated. MRI measurements gave easy access to tridimensional visualization of the labelled zones where the CA has been injected and the applied electric field had a value allowing permeabilization. We also performed MRI measurements of the water transverse relaxation time T2 as an indicator of tissue modification, and tested whether another CA specific for necrosis could be used to detect muscle necrosis at high electric field intensity. In conclusion, MRI measurements may bring multiparametric information upon the efficiency and tissue toxicity of an ET protocol by using a simple and safe CA.