Nano-Pulse Stimulation Induces Changes in the Intracellular Organelles in Rat Liver Tumors Treated In Situ.

BACKGROUND AND OBJECTIVES: Nano-pulse stimulation (NPS) therapy is the application of ultrafast pulses of high amplitude electrical energy to tissues to influence cell function. Unique characteristics of these pulses enable electric field penetration into the interior of cells and organelles to generate transient nanopores in both organelle and plasma membranes. The purpose of this study is to document the temporal and physical changes in intracellular organelles following NPS therapy using electron microscopy. STUDY DESIGN/MATERIALS AND METHODS: Liver tumors were induced in five buffalo rats by implanting syngeneic McA-RH7777 hepatocellular carcinoma cells into the surgically exposed livers. Tumors were allowed to grow for 1 week and then the surgically exposed livers were treated in situ with NPS energy delivered at a sufficient level to trigger regulated cell death in the tumor. Samples of NPS-treated and control tissue were removed and fixed for electron microscopy at 1 minute, 5 minutes, 30 minutes, 2 hours and 4 hours after exposure. RESULTS: Measurements of cellular organelles indicate strong swelling following NPS therapy exposure compared with untreated controls. The mean diameter of the mitochondria increased by 55% within 1 minute and then by 2.5-fold by 2 hours post-NPS therapy. The rough endoplasmic reticulum (RER) cisternae swelled immediately after NPS therapy with reduced swelling by 30 minutes and loss of structural integrity by 2 hours. The Golgi apparatus appears swollen in images collected 1 and 5 minutes after NPS therapy and was no longer detected at 30 minutes and 2 hours post-NPS therapy. By 4 hours after NPS therapy, a nascent Golgi apparatus was detected in many of the images. The plasma membrane lost its well-defined morphology and became less linear, exhibiting discontinuities as early as 1 minute post-NPS energy exposure and the nuclear envelope became subjectively less distinct over time. CONCLUSIONS: NPS therapy at sufficient energy levels causes the rapid swelling of organelles, disintegration of the RER, breaks in the plasma membrane and blurs the borders of the nuclear envelope. These changes in the mitochondria and RER are indicative of a regulated cell death process. These immediate physical changes to vital cell organelles are likely to trigger subsequent regulated cell death mechanisms observed in other studies of NPS therapy. Lasers Surg. Med. © 2020 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals, Inc.

Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy.

Intratumoral electroporation-mediated IL-12 gene therapy (IT-pIL12/EP) has been shown to be safe and effective in clinical trials, demonstrating systemic antitumor effects with local delivery of this potent cytokine. We recently optimized our IL-12 gene delivery platform to increase transgene expression and efficacy in preclinical models. Here we analyze the immunological changes induced with the new IT-pIL12/EP platform in both electroporated and distant, non-electroporated lesions. IT-pIL12/EP-treated tumors demonstrated rapid induction of IL-12-regulated pathways, as well as other cytokines and chemokines pathways, and upregulation of antigen presentation machinery. The distant tumors showed an increase in infiltrating lymphocytes and gene expression changes indicative of a de novo immune response in these untreated lesions. Flow cytometric analyses revealed a KLRG1hi CD8+ effector T-cell population uniquely present in mice treated with IT-pIL12/EP. Despite being highly activated, this population expressed diminished levels of PD-1 when re-exposed to antigen in the PD-L1-rich tumor. Other T-cell exhaustion markers appeared to be downregulated in concert, suggesting an orchestrated "armoring" of these effector T cells against T-cell checkpoints when primed in the presence of IL-12 in situ. These cells may represent an important mechanism by which local IL-12 gene therapy can induce a systemic antitumor immune response without the associated toxicity of systemic IL-12 exposure.

Effects of Pulsed Electric Fields on the Coronary Arteries in Swine.

BACKGROUND: Previous animal studies have shown no significant vascular injury from pulsed electrical field (PEF) ablation. We sought to assess the effect of PEF on swine coronary arteries. METHODS: We performed intracoronary and epicardial (near the coronary artery) PEF ablations in swine pretreated with dual antiplatelet and antiarrhythmic therapy. Intracoronary PEF was delivered using MapiT catheters (Biotronik, Berlin), whereas epicardial PEF was delivered using EPT catheters (Boston Scientific, MA). PEF pulse duration was microseconds (Nanoknife 3.0, Angio Dynamics, NY) or nanoseconds (CellFX, Pulse Biosciences, CA). RESULTS: We performed 39 intracoronary ablations in 10 swine and 20 epicardial-pericoronary ablations in 4 separate swine. Intracoronary PEF was delivered at higher energy compared with epicardial PEF (46 [interquartile range, IQR 20-85] J versus 10 [IQR 10-11] J, P < 0.01). Reversible coronary spasm occurred in 49% intracoronary ablations and 45% epicardial ablations (P=0.80). At the end study, fixed coronary stenosis was demonstrated in 44% intracoronary ablations (80% for microsecond PEF and 18% for nanosecond PEF) and 0% epicardial ablations. Visible hemorrhagic and/or fibrotic myocardial lesions were observed at necropsy with similar frequency between intracoronary and epicardial PEF (45% versus 50%, P=0.70). Nanosecond PEF (49 ablations in 11 swine), when compared with microsecond PEF (10 intracoronary ablations in 3 swine), resulted in lower energy delivery (21 [IQR 10-46] J versus 129 [IQR 24-143] J, P=0.03) and less incidence of fixed coronary stenosis (18% versus 80%, P=0.04). CONCLUSIONS: In the swine model, intracoronary PEF resulted both in significant coronary spasm and fixed coronary stenosis. Epicardial PEF, delivered at lower energy, resulted in reversible spasm but no fixed coronary stenosis.

Real-time impedance feedback to enhance cutaneous gene electrotransfer in a murine skin model.

Electric field mediated gene delivery methods have the ability to efficiently transfect cells in vivo with an excellent safety profile. The method has historically used a fixed number of electric pulses with identical characteristics in induce delivery. Electrical treatment does not typically compensate for subject-to-subject variation and other differences. This study was designed to investigate if delivery/expression could be increased using a novel electropulsation method that compensated for variation using real-time electrical impedance measurements. The method involved delivering plasmid DNA encoding luciferase to murine skin. Tissue impedance in a 1-3 KHz range was measured before electric pulses were applied. Impedance was also measured after each successive pulse. Pulsation was stopped when impedance values were reduced by either 80% or 95% relative to prepulse values. Standard/fixed pulsing parameters were also used for comparison. The results indicated that up to 15-fold increases in luciferase expression could be obtained when electrical treatment was ceased based upon impedance reductions. Furthermore, peak expression levels of all treatment groups pulsed using the novel pulsing method were statistically higher than those that employed standard pulsing. These results strongly suggest that applying pulses until a defined impedance-based endpoint results in higher expression.

Plasma facilitated delivery of DNA to skin.

Non-viral delivery of cell-impermeant drugs and DNA in vivo has traditionally relied upon either chemical or physical stress applied directly to target tissues. Physical methods typically use contact between an applicator, or electrode, and the target tissue and may involve patient discomfort. To overcome contact-dependent limitations of such delivery methodologies, an atmospheric helium plasma source was developed to deposit plasma products onto localized treatment sites. Experiments performed in murine skin showed that samples injected with plasmid DNA encoding luciferase and treated with plasma demonstrated increased levels of expression relative to skin samples that received injections of DNA alone. Increased response relative to injection alone was observed when either positive or negative voltage was used to generate the helium plasma. Quantitative results over a 26-day follow-up period showed that luciferase levels as high as 19-fold greater than the levels obtained by DNA injection alone could be achieved. These findings indicate that plasmas may compete with other physical delivery methodologies when skin is the target tissue.

Development of an adaptive electroporation system for intratumoral plasmid DNA delivery.

Intratumoral electroporation of plasmid DNA encoding the proinflammatory cytokine interleukin 12 promotes innate and adaptive immune responses correlating with anti-tumor effects. Clinical electroporation conditions are fixed parameters optimized in preclinical tumors, which consist of cells implanted into skin. These conditions have little translatability to clinically relevant tumors, as implanted models cannot capture the heterogeneity encountered in genetically engineered mouse models or clinical tumors. Variables affecting treatment outcome include tumor size, degree of vascularization, fibrosis, and necrosis, which can result in suboptimal gene transfer and variable therapeutic outcomes. To address this, a feedback controlled electroporation generator was developed, which is capable of assessing the electrochemical properties of tissue in real time. Determination of these properties is accomplished by impedance spectroscopy and equivalent circuit model parameter estimation. Model parameters that estimate electrical properties of cell membranes are used to adjust electroporation parameters for each applied pulse. Studies performed in syngeneic colon carcinoma tumors (MC38) and spontaneous mammary tumors (MMTV-PyVT) demonstrated feedback-based electroporation is capable of achieving maximum expression of reporter genes with significantly less variability and applied energy. These findings represent an advancement to the practice of gene electro-transfer, as reducing variability and retaining transfected cell viability is paramount to treatment success.

Direct Current Helium Plasma for In vivo Delivery of Plasmid DNA Encoding Erythropoietin to Murine Skin.

The use of electric fields in vivo to deliver DNA, called electroporation, has the potential to broadly impact vaccination and disease treatment. The evidence for this has emerged from a large number of recently completed and ongoing clinical trials. The methods for applying electric fields to tissues traditionally involve contact between metal electrodes and the tissue. In this study, we investigated the use of helium plasma as a noncontact method for electrically treating tissue in a manner that results in the uptake and expression of foreign DNA in murine skin. More specifically, our goal was to demonstrate that DNA encoding a model-secreted protein could be delivered, detected in the blood, and remain functional to produce its known biological effect. Murine erythropoietin (EPO) was the model-secreted protein. Results clearly demonstrated that an intradermal DNA injection followed by plasma treatment for 2 min resulted in elevated levels of EPO in the blood and corresponding hemoglobin increases that were statistically significant relative to DNA injection alone.

Impedance spectroscopy as an indicator for successful in vivo electric field mediated gene delivery in a murine model.

In vivo gene electro transfer technology has been very successful both in animal models and in clinical trials over the past 20years. However, variable transfection efficiencies can produce inconsistent outcomes. This can be due to differences in tissue architecture and/or chemical composition which may effectively create unique biological environments from subject to subject that may respond differently to the identical electric pulses. This study investigates the integration of impedance spectroscopy into the gene electro transfer process to measure murine skin impedance spectra before, during (after pulse delivery), and after gene electro transfer pulse application to determine if changes in impedance correlate with reporter gene expression. Both post-treatment impedance spectra and gene expression were dependent upon the applied electric field strength. These results indicate that alterations in tissue impedance produced by the applied electric field represent an excellent parameter to predict degrees of transfection and gene expression. These results could ultimately be used to alter pulsing parameters in order to optimize delivery/expression.

Melanoma treatment with intratumoral electroporation of tavokinogene telseplasmid (pIL-12, tavokinogene telseplasmid).

Tumors evade detection and/or clearance by the immune system via multiple mechanisms. IL-12 is a potent immunomodulatory cytokine that plays a central role in immune priming. However, systemic delivery of IL-12 can result in life-threatening toxicity and therefore has shown limited efficacy at doses that can be safely administered. We developed an electroporation technique to produce highly localized IL-12 expression within tumors leading to regression of both treated and untreated lesions in animal models and in patients with a favorable safety profile. Furthermore, intratumoral tavokinogene telseplasmid electroporation can drive cellular immune responses, converting 'cold' tumors into 'hot' tumors. Clinical trials are ongoing to determine whether intratumoral tavokinogene telseplasmid electroporation synergizes with checkpoint blockade therapy in immunologically cold tumors predicted not to respond to PD-1 antibody monotherapy.

Optimization of a plasma facilitated DNA delivery method.

Plasma-based methods have recently emerged as a technique for augmenting plasmid DNA delivery to skin. This delivery modality relies on the deposition of ionized gas molecules on to targeted cells or tissue to establish an electric field. It is hypothesized that this electric field results in the dielectric breakdown of cell membranes, making cells permeable to exogenous molecules. This in vivo investigation sought to optimize the intradermal delivery of a luciferase expressing plasmid DNA by modulating the total exposure to the plasma source and the plasmid DNA dose. Varying the plasma exposure time from 2, 5, 10, and 20 min allowed the conditions resulting in the highest expression of luciferase to be found. These conditions correlated to the 10 minute exposure time for a plasma derived from either +8 kV or -8 kV, when the generator was operated 3 cm from the epidermal tissue surface with a helium flow rate of 15 L/min. Exposing the injected flank skin for 10 min resulted in a rise of 37.3-fold for a plasma created with +8 kV and 27.1-fold for a plasma created with -8 kV. When using this treatment time with 50, 100, or 200 µg of a luciferase expressing plasmid, it was found that 100 µg resulted in the highest peak luminescence.

Electrogenetherapy of B16.F10 murine melanoma tumors with an interleukin-28 expressing DNA plasmid.

Augmented delivery of cytokine-expressing DNA plasmids to subcutaneous tumors has been demonstrated to result in a level of enhanced anti-tumor activity. One delivery enhancement method which has been evaluated is in vivo electroporation (EP), a contact-dependent delivery technique where electric pulses are hypothesized to augment the transfer of DNA into cells and tissues through the induction of temporary cell membrane pores. Previous work by members of our group, as well as others, has demonstrated the anti-tumor effects of DNA plasmids expressing the cytokines IL-12 and IL-15. In this report the potential anti-tumor activity of a relatively newly-described cytokine, IL-28, was measured when administered intratumorally as a DNA expression plasmid (designated pIL28) to established murine (B16.F10) melanoma tumors. The administration of the IL-28 expressing plasmid was performed through enhanced delivery methods. One method was EP and the other a non-contact dependent technique using a helium plasma stream. IL-28 is a member of the type III interferon family of cytokines that has been characterized as possessing potent anti-viral activity. This cytokine has been demonstrated to function as an adjuvant in small animal model vaccination protocols and stimulates CD8+ CTL responses. In addition, stimulation of anti-tumor activity has been demonstrated in several studies using IL-28. Based on these activities, it was hypothesized that this cytokine could, when delivered through a DNA expression plasmid, mediate anti-tumor activity. The results of this study indicated that enhanced delivery of pIL-28 resulted in attenuation of tumor growth, compared with non-enhanced delivery. Of note, this is the first proof-of-concept experiment, of our knowledge, documenting the ability of a non-contact dependent helium plasma-based delivery method to mediate the enhancement of an anti-tumor effect by a cytokine-expressing DNA plasmid. This suggests the use of the helium plasma delivery method as an alternative or adjunctive method to EP for the effective delivery of agents that possess potential anti-tumor activity.

Non-contact helium-based plasma for delivery of DNA vaccines. Enhancement of humoral and cellular immune responses.

Non-viral in vivo administration of plasmid DNA for vaccines and immunotherapeutics has been hampered by inefficient delivery. Methods to enhance delivery such as in vivo electroporation (EP) have demonstrated effectiveness in circumventing this difficulty. However, the contact-dependent nature of EP has resulting side effects in animals and humans. Noncontact delivery methods should, in principle, overcome some of these obstacles. This report describes a helium plasma-based delivery system that enhanced humoral and cellular antigen-specific immune responses in mice against an intradermally administered HIV gp120-expressing plasmid vaccine (pJRFLgp120). The most efficient plasma delivery parameters investigated resulted in the generation of geometric mean antibody-binding titers that were 19-fold higher than plasmid delivery alone. Plasma mediated delivery of pJRFLgp120 also resulted in a 17-fold increase in the number of interferon-gamma spot-forming cells, a measure of CD8+ cytotoxic T cells, compared with non-facilitated plasmid delivery. This is the first report demonstrating the ability of this contact-independent delivery method to enhance antigen-specific immune responses against a protein generated by a DNA vaccine.

Enhancement of antigen specific humoral immune responses after delivery of a DNA plasmid based vaccine through a contact-independent helium plasma.

Non-viral in vivo delivery of DNA, encoding for specific proteins, has traditionally relied on chemical or physical forces applied directly to tissues. Physical methods typically involve contact between an applicator/electrode and tissue and often results in transient subject discomfort. To overcome these limitations of contact-dependent delivery, a helium plasma source was utilized to deposit ionized gasses to treatment/vaccination sites without direct contact between the applicator and the tissues. The study reported here evaluated the efficacy of this strategy as an effective method to administer DNA vaccines. Balb/C mice were vaccinated with a DNA plasmid expressing an HIVgp120 envelope glycoprotein either with or without co-administration of helium plasma or electroporation. The results indicated, for the first time, the potential efficacy of helium plasma delivery for the induction and enhancement of antigen specific immune responses following DNA vaccination.

Characterization of plasma mediated molecular delivery to cells in vitro.

Ion-based strategies have recently emerged as a method to facilitate molecular delivery. These methods are attractive as they separate the applicator from the treatment site avoiding some issues encountered with other electrically driven methods. Current literature on plasma delivery has shown utility in vitro and in vivo for both drugs and genes. To advance this technology more information must become available on the mechanism responsible for delivery and the effects of ion exposure on eukaryotic cells. This in vitro investigation found that molecular delivery facilitated by a DC-based plasma follows a dose-response behavior, with optimum uptake of Sytox Green occurring in two cell lines after 600 s of exposure. In both cell lines exposure to the discharge caused no adverse effects in viability for exposure times up to 600 s. It was also found that membranes treated with ions remained permeabilized for several minutes following plasma treatment and that membrane resealing exhibited first order kinetics.