Determination of the Impact of High-Intensity Pulsed Electromagnetic Fields on the Release of Damage-Associated Molecular Pattern Molecules.

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.

High-Intensity Pulsed Electromagnetic Field-Mediated Gene Electrotransfection In Vitro.

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.

Contactless delivery of plasmid encoding EGFP in vivo by high-intensity pulsed electromagnetic field.

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 external pulsed magnetic field. Recently, HI-PEMF was applied for delivering siRNA molecules to silence enhanced green fluorescent protein (EGFP) in tumors in vivo. Still, delivered siRNA molecules were 21 base pairs long, which is 200-times smaller compared to nucleic acids such as plasmid DNA (pDNA) that are delivered in gene therapies to various targets to generate therapeutic effect. In our study, we demonstrate the use HI-PEMF treatment as a feasible noninvasive approach to achieve in vivo transfection by enabling the transport of larger molecules such as pDNA encoding EGFP into muscle and skin. We obtained a long-term expression of EGFP in the muscle and skin after HI-PEMF, in some mice even up to 230 days and up to 190 days, respectively. Histological analysis showed significantly less infiltration of inflammatory mononuclear cells in muscle tissue after the delivery of pEGFP using HI-PEMF compared to conventional gene electrotransfer. Furthermore, the antitumor effectiveness using HI-PEMF for electrotransfer of therapeutic plasmid, i.e., silencing MCAM was demonstrated. In conclusion, feasibility of HI-PEMF was demonstrated for transfection of different tissues (muscle, skin, tumor) and could have great potential in gene therapy and in DNA vaccination.

Electrotransfer of siRNA to Silence Enhanced Green Fluorescent Protein in Tumor Mediated by a High Intensity Pulsed Electromagnetic Field.

The contactless high intensity pulsed electromagnetic field (HI-PEMF)-induced increase of cell membrane permeability is similar to conventional electroporation, with the important difference of inducing an electric field non-invasively by exposing a treated tissue to a time-varying magnetic field. Due to the limited number of studies in the field of electroporation induced by HI-PEMF, we designed experiments to explore the feasibility of such a contactless delivery technique for the gene electrotransfer of nucleic acids in tissues in vivo. By using HI-PEMF for gene electrotransfer, we silenced enhanced green fluorescent protein (EGFP) with siRNA molecules against EGFP in B16F10-EGFP tumors. Six days after the transfer, the fluorescent tumor area decreased by up to 39% as determined by fluorescence imaging in vivo. In addition, the silencing of EGFP to the same extent was confirmed at the mRNA and protein level. The results obtained in the in vivo mouse model demonstrate the potential use of HI-PEMF-induced cell permeabilization for gene therapy and DNA vaccination. Further studies are thus warranted to improve the equipment, optimize the protocols for gene transfer and the HI-PEMF parameters, and demonstrate the effects of HI-PEMF on a broader range of different normal and tumor tissues.

Contactless electroporation induced by high intensity pulsed electromagnetic fields via distributed nanoelectrodes.

Pulsed electric fields (PEFs) can be used to transiently increase cell membrane permeability in procedures ranging from gene therapy to tumor eradication. Although very efficient, PEF-based therapies generally require the use of invasive electrodes, which cause pain and tissue damage. An emerging noninvasive, contactless alternative to PEFs are High Intensity Pulsed Electromagnetic Fields (HI-PEMF), whereby the electric field inside the tissue is induced remotely by external pulsed magnetic field. However, one of the current major drawbacks of HI-PEMFs is their inferior efficiency compared to PEFs. In this study we present the proof-of-concept that by adding highly conductive 5 and 20 nm gold nanoparticles (Au NPs), we can significantly potentiate the permeabilizing effect of HI-PEMFs, making it possible to permeabilize up to 80% of the cells with minimal or no effect on cell survival, compared to negligible percentage of permeabilized cells using HI-PEMF alone. Experiments, conducted on Chinese Hamster Ovary cells and Escherichia coli, suggest that Au NPs act as distributed nanoelectrodes, locally enhancing the electric field induced at the plasma membrane. Our findings open up an avenue of possibilities for combining naked as well as functionalized Au NPs with HI-PEMFs for noninvasive, remotely controlled smart drug delivery applications.

Time-Dependent Finite Element Analysis of In Vivo Electrochemotherapy Treatment.

Electrochemotherapy and irreversible electroporation are gaining importance in clinical practice for the treatment of solid tumors. For successful treatment, it is extremely important that the coverage and exposure time of the treated tumor to the electric field are within the specified range. In order to ensure successful coverage of the entire target volume with sufficiently strong electric fields, numerical treatment planning has been proposed and its use has also been demonstrated in practice. Most of numerical models in treatment planning are based on charge conservation equation and are not able to provide time course of electric current, electrical conductivity, or electric field distribution changes established in the tissue during pulse delivery. Recently, a model based on inverse analysis of experimental data that delivers time course of tissue electroporation has been introduced. The aim of this study was to apply the previously reported time-dependent numerical model to a complex in vivo example of electroporation with different tissue types and with a long-term follow-up. The model, consisting of a tumor placed in the liver with 2 needle electrodes inserted in the center of the tumor and 4 around the tumor, was validated by comparison of measured and calculated time course of applied electric current. Results of simulations clearly indicated that proposed numerical model can successfully capture transient effects, such as evolution of electric current during each pulse, and effects of pulse frequency due to electroporation effects in the tissue. Additionally, the model can provide evolution of electric field amplitude and electrical conductivity in the tumor with consecutive pulse sequences.

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.

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

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.

BACKGROUND: Electroporation 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. METHODS: Using 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. RESULTS: Acquired 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. CONCLUSIONS: The 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.

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.

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.

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.