Matej Kranjc

In Situ Monitoring of Electric Field Distribution in Mouse Tumor during Electroporation

PURPOSE To investigate the feasibility of magnetic resonance (MR) electric impedance tomography ( EIT electric impedance tomography ) technique for in situ monitoring of electric field distribution during in vivo electroporation of mouse tumors to predict reversibly electroporated tumor areas. MATERIALS AND METHODS All experiments received institutional animal care and use committee approval. Group 1 consisted of eight tumors that were used for determination of predicted area of reversibly electroporated tumor cells with MR EIT electric impedance tomography by using a 2.35-T MR imager. In addition, T1-weighted images of tumors were acquired to determine entrapment of contrast agent within the reversibly electroporated area. A correlation between predicted reversible electroporated tumor areas as determined with MR EIT electric impedance tomography and areas of entrapped MR contrast agent was evaluated to verify the accuracy of the prediction. Group 2 consisted of seven tumors that were used for validation of radiologic imaging with histopathologic staining. Histologic analysis results were then compared with predicted reversible electroporated tumor areas from group 1. Results were analyzed with Pearson correlation analysis and one-way analysis of variance. RESULTS Mean coverage ± standard deviation of tumors with electric field that leads to reversible electroporation of tumor cells obtained with MR EIT electric impedance tomography (38% ± 9) and mean fraction of tumors with entrapped MR contrast agent (41% ± 13) were correlated (Pearson analysis, r = 0.956, P = .005) and were not statistically different (analysis of variance, P = .11) from mean fraction of tumors from group 2 with entrapped fluorescent dye (39% ± 12). CONCLUSION MR EIT electric impedance tomography can be used for determining electric field distribution in situ during electroporation of tissue. Implementation of MR EIT electric impedance tomography in electroporation-based applications, such as electrochemotherapy and irreversible electroporation tissue ablation, would enable corrective interventions before the end of the procedure and would additionally improve the treatment outcome.

Blumlein Configuration for High-Repetition-Rate Pulse Generation of Variable Duration and Polarity Using Synchronized Switch Control

Blumlein generators are used in different applications such as radars, lasers, and also recently in various biomedical studies, where the effects of high-voltage nanosecond pulses on biological cells are evaluated. In these studies, it was demonstrated that by applying high-voltage nanosecond pulses to cells, plasma membrane and cell organelles are permeabilized. As suggested in a recent publication, the repetition rate and polarity of nanosecond high-voltage pulses could have an important effect on the electropermeabilization process, and consequently, on the observed phenomena. Therefore, we designed a new Blumlein configuration that enables a higher repetition rate of variable duration of either bipolar or unipolar high-voltage pulses. We achieved a maximal pulse repetition rate of 1.1 MHz. However, theoretically, this rate could be even higher. We labeled endocytotic vesicles with lucifer yellow and added propidium iodide to a cell suspension for testing the cell plasma membrane integrity, so we were able to observe the permeabilization of endocytotic vesicles and the cell plasma membrane at the same time. The new design of pulse generator was built, verified, and also tested in experiments. The resulting flexibility and variability allow further in vitro experiments to determine the importance of the pulse repetition rate and pulse polarity on membrane permeabilization - both of the cell plasma membrane as well as of cell organelle membranes.

Ex Vivo and In Silico Feasibility Study of Monitoring Electric Field Distribution in Tissue during Electroporation Based Treatments

Magnetic resonance electrical impedance tomography (MREIT) was recently proposed for determining electric field distribution during electroporation in which cell membrane permeability is temporary increased by application of an external high electric field. The method was already successfully applied for reconstruction of electric field distribution in agar phantoms. Before the next step towards in vivo experiments is taken, monitoring of electric field distribution during electroporation of ex vivo tissue ex vivo and feasibility for its use in electroporation based treatments needed to be evaluated. Sequences of high voltage pulses were applied to chicken liver tissue in order to expose it to electric field which was measured by means of MREIT. MREIT was also evaluated for its use in electroporation based treatments by calculating electric field distribution for two regions, the tumor and the tumor-liver region, in a numerical model based on data obtained from clinical study on electrochemotherapy treatment of deep-seated tumors. Electric field distribution inside tissue was successfully measured ex vivo using MREIT and significant changes of tissue electrical conductivity were observed in the region of the highest electric field. A good agreement was obtained between the electric field distribution obtained by MREIT and the actual electric field distribution in evaluated regions of a numerical model, suggesting that implementation of MREIT could thus enable efficient detection of areas with insufficient electric field coverage during electroporation based treatments, thus assuring the effectiveness of the treatment.

Magnetic resonance electrical impedance tomography for measuring electrical conductivity during electroporation

The electroporation effect on tissue can be assessed by measurement of electrical properties of the tissue undergoing electroporation. The most prominent techniques for measuring electrical properties of electroporated tissues have been voltage-current measurement of applied pulses and electrical impedance tomography (EIT). However, the electrical conductivity of tissue assessed by means of voltage-current measurement was lacking in information on tissue heterogeneity, while EIT requires numerous additional electrodes and produces results with low spatial resolution and high noise. Magnetic resonance EIT (MREIT) is similar to EIT, as it is also used for reconstruction of conductivity images, though voltage and current measurements are not limited to the boundaries in MREIT, hence it yields conductivity images with better spatial resolution. The aim of this study was to investigate and demonstrate the feasibility of the MREIT technique for assessment of conductivity images of tissues undergoing electroporation. Two objects were investigated: agar phantoms and ex vivo liver tissue. As expected, no significant change of electrical conductivity was detected in agar phantoms exposed to pulses of all used amplitudes, while a considerable increase of conductivity was measured in liver tissue exposed to pulses of different amplitudes.

Assessing how electroporation affects the effective conductivity tensor of biological tissues

We report calculations of the anisotropy ratio of the electrical conductivity of a simple model of a loose connective biological tissue described as a random assembly of multiscale undeformable core-shell and controlled polydisperse spherical structures. One can estimate a 10% increase in the anisotropy ratio due to the application of electric field (duration 100 μm) above the electroporation threshold (40 kV m−1) up to 120 kV m−1. These findings are consistent with the experimental data on the field-induced anisotropy dependence of the electrical conductivity due to cell membrane electroporation.

Dynamic finite-element model for efficient modelling of electric currents in electroporated tissue

In silico experiments (numerical simulations) are a valuable tool for non-invasive research of the influences of tissue properties, electrode placement and electric pulse delivery scenarios in the process of electroporation. The work described in this article was aimed at introducing time dependent effects into a finite element model developed specifically for electroporation. Reference measurements were made ex vivo on beef liver samples and experimental data were used both as an initial condition for simulation (applied pulse voltage) and as a reference value for numerical model calibration (measured pulse current). The developed numerical model is able to predict the time evolution of an electric pulse current within a 5% error over a broad range of applied pulse voltages, pulse durations and pulse repetition frequencies. Given the good agreement of the current flowing between the electrodes, we are confident that the results of our numerical model can be used both for detailed in silico research of electroporation mechanisms (giving researchers insight into time domain effects) and better treatment planning algorithms, which predict the outcome of treatment based on both spatial and temporal distributions of applied electric pulses.

Electrochemotherapy by pulsed electromagnetic field treatment (PEMF) in mouse melanoma B16F10 in vivo

Abstract Introduction Pulsed electromagnetic field (PEMF) induces pulsed electric field, which presumably 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 cisplatin (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 platinum (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 growth, as well as the injection of CDDP only. The antitumor effect in combined treatment was related to increased drug uptake into the electroporated tumor cells, demonstrated by increased amount of Pt bound to the DNA. Approximately 2-fold increase in cellular uptake of Pt was measured. Conclusions The obtained results in 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.Introduction Pulsed electromagnetic field (PEMF) induces pulsed electric field, which presumably 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 cisplatin (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 platinum (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 growth, as well as the injection of CDDP only. The antitumor effect in combined treatment was related to increased drug uptake into the electroporated tumor cells, demonstrated by increased amount of Pt bound to the DNA. Approximately 2-fold increase in cellular uptake of Pt was measured. Conclusions The obtained results in 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.

Current density imaging sequence for monitoring current distribution during delivery of electric pulses in irreversible electroporation

BackgroundElectroporation is gaining its importance in everyday clinical practice of cancer treatment. For its success it is extremely important that coverage of the target tissue, i.e. treated tumor, with electric field is within the specified range. Therefore, an efficient tool for the electric field monitoring in the tumor during delivery of electroporation pulses is needed. The electric field can be reconstructed by the magnetic resonance electric impedance tomography method from current density distribution data. In this study, the use of current density imaging with MRI for monitoring current density distribution during delivery of irreversible electroporation pulses was demonstrated.MethodsUsing a modified single-shot RARE sequence, where four 3000 V and 100 μs long pulses were included at the start, current distribution between a pair of electrodes inserted in a liver tissue sample was imaged. Two repetitions of the sequence with phases of refocusing radiofrequency pulses 90° apart were needed to acquire one current density image. For each sample in total 45 current density images were acquired to follow a standard protocol for irreversible electroporation where 90 electric pulses are delivered at 1 Hz.ResultsAcquired current density images showed that the current density in the middle of the sample increased from first to last electric pulses by 60%, i.e. from 8 kA/m2 to 13 kA/m2 and that direction of the current path did not change with repeated electric pulses significantly.ConclusionsThe presented single-shot RARE-based current density imaging sequence was used successfully to image current distribution during delivery of short high-voltage electric pulses. The method has a potential to enable monitoring of tumor coverage by electric field during irreversible electroporation tissue ablation.

Predicting irreversible electroporation-induced tissue damage by means of magnetic resonance electrical impedance tomography

Irreversible electroporation (IRE) is gaining importance in routine clinical practice for nonthermal ablation of solid tumors. For its success, it is extremely important that the coverage and exposure time of the treated tumor to the electric field is within the specified range. Measurement of electric field distribution during the electroporation treatment can be achieved using magnetic resonance electrical impedance tomography (MREIT). Here, we show improved MREIT-enabled electroporation monitoring of IRE-treated tumors by predicting IRE-ablated tumor areas during IRE of mouse tumors in vivo. The in situ prediction is enabled by coupling MREIT with a corresponding Peleg-Fermi mathematical model to obtain more informative monitoring of IRE tissue ablation by providing cell death probability in the IRE-treated tumors. This technique can potentially be used in electroporation-based clinical applications, such as IRE tissue ablation and electrochemotherapy, to improve and assure the desired treatment outcome.

Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses

Background Cell membrane permeabilization by pulsed electromagnetic fields (PEMF) is a novel contactless method which results in effects similar to conventional electroporation. The non-invasiveness of the methodology, independence from the biological object homogeneity and electrical conductance introduce high flexibility and potential applicability of the PEMF in biomedicine, food processing, and biotechnology. The inferior effectiveness of the PEMF permeabilization compared to standard electroporation and the lack of clear description of the induced transmembrane transport are currently of major concern. Methods The PEMF permeabilization experiments have been performed using a 5.5 T, 1.2 J pulse generator with a multilayer inductor as an applicator. We investigated the feasibility to increase membrane permeability of Chinese Hamster Ovary (CHO) cells using short microsecond (15 µs) pulse bursts (100 or 200 pulses) at low frequency (1 Hz) and high dB/dt (>106 T/s). The effectiveness of the treatment was evaluated by fluorescence microscopy and flow cytometry using two different fluorescent dyes: propidium iodide (PI) and YO-PRO®-1 (YP). The results were compared to conventional electroporation (single pulse, 1.2 kV/cm, 100 µs), i.e., positive control. Results The proposed PEMF protocols (both for 100 and 200 pulses) resulted in increased number of permeable cells (70 ± 11% for PI and 67 ± 9% for YP). Both cell permeabilization assays also showed a significant (8 ± 2% for PI and 35 ± 14% for YP) increase in fluorescence intensity indicating membrane permeabilization. The survival was not affected. Discussion The obtained results demonstrate the potential of PEMF as a contactless treatment for achieving reversible permeabilization of biological cells. Similar to electroporation, the PEMF permeabilization efficacy is influenced by pulse parameters in a dose-dependent manner.