Electroporation-Induced Stress Response and Its Effect on Gene Electrotransfer Efficacy: In Vivo Imaging and Numerical Modeling.

OBJECTIVE: Skin is an attractive target tissue for gene transfer due to its size, accessibility, and its immune competence. One of the promising delivery methods is gene delivery by means of electroporation (EP), i.e., gene electrotransfer (GET). To assess the importance of different effects of electroporation for successful GET we investigated: stress response and transfection efficacy upon different pulse protocols. Moreover, numerical modeling was used to explain experimental results and to test the agreement of experimental results with current knowledge about GET. METHODS: Double transgenic mice Hspa1b-LucF (+/+) Hspa1b-mPlum (+/+) were used to determine the level of stress sensed by the cell in the tissue in vivo that was exposed to EP. The effect of five different pulse protocols on stress levels sensed by the exposed cells and their efficacy for gene electrotransfer for two plasmids pEGFP-C1 (EGFP) and pCMV-tdTomato was tested. RESULTS: Quantification of the bioluminescence signal intensity shows that EP, regardless of the electric pulse parameters used, increased mean bioluminescence compared to the baseline bioluminescence signal of the non-exposed skin. The results of numerical modeling indicate that thermal stress alone is not sufficient to explain the measured bioluminescence signal. Of the tested pulse protocols, the highest expression of EGFP and tdTomato was achieved with HV-MV (high voltage - medium voltage) protocols, which agrees also with numerical model. SIGNIFICANCE: Although EP is widely used as a method for gene delivery, we show that the field could benefit from the use of mathematical modeling by introducing additional parameters such as EP induced stress and electrophoretic movement of plasmids.

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

BACKGROUND: The 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. METHODS: A 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. RESULTS: According 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. CONCLUSIONS: The 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.

Mathematical model of tumor volume dynamics in mice treated with electrochemotherapy.

The effectiveness of electrochemotherapy, a local treatment using electric pulses to increase the uptake of chemotherapeutic drug, includes several antitumor mechanisms. In addition to the cytotoxic action of chemotherapeutic drug, treatment outcome also depends on antitumor immune response. In order to assess the contribution of different antitumor mechanisms to the observed treatment outcome, we designed a model of tumor volume dynamics, which is able to quantify early and late treatment effects. Model integrates characteristics of both main posttreatment processes, namely removal of lethally damaged cells from tumor volume and tumor-immune interaction. Fitting to individual responses gives the insight into the dynamics of tumor cell elimination. Two more or less clearly separable peaks can be observed from these dynamics. Model was used to quantify responses obtained after chemotherapy and electrochemotherapy with bleomycin and cisplatin in immunocompetent and immunodeficient mice. As expected, electrochemotherapy resulted in higher number of lethally damaged cells as well as in stronger immune response compared to chemotherapy alone. Additionally, bleomycin-treated tumors proved to be more immunogenic than cisplatin-treated tumors in the given range of doses.

Web-based tool for visualization of electric field distribution in deep-seated body structures and planning of electroporation-based treatments.

BACKGROUND: Treatments based on electroporation are a new and promising approach to treating tumors, especially non-resectable ones. The success of the treatment is, however, heavily dependent on coverage of the entire tumor volume with a sufficiently high electric field. Ensuring complete coverage in the case of deep-seated tumors is not trivial and can in best way be ensured by patient-specific treatment planning. The basis of the treatment planning process consists of two complex tasks: medical image segmentation, and numerical modeling and optimization. METHODS: In addition to previously developed segmentation algorithms for several tissues (human liver, hepatic vessels, bone tissue and canine brain) and the algorithms for numerical modeling and optimization of treatment parameters, we developed a web-based tool to facilitate the translation of the algorithms and their application in the clinic. The developed web-based tool automatically builds a 3D model of the target tissue from the medical images uploaded by the user and then uses this 3D model to optimize treatment parameters. The tool enables the user to validate the results of the automatic segmentation and make corrections if necessary before delivering the final treatment plan. RESULTS: Evaluation of the tool was performed by five independent experts from four different institutions. During the evaluation, we gathered data concerning user experience and measured performance times for different components of the tool. Both user reports and performance times show significant reduction in treatment-planning complexity and time-consumption from 1-2 days to a few hours. CONCLUSIONS: The presented web-based tool is intended to facilitate the treatment planning process and reduce the time needed for it. It is crucial for facilitating expansion of electroporation-based treatments in the clinic and ensuring reliable treatment for the patients. The additional value of the tool is the possibility of easy upgrade and integration of modules with new functionalities as they are developed.