Combination Of Electroporation Research Articles

Folate functionalized gold-coated magnetic nanoparticles effect in combined electroporation and radiation treatment of HPV-positive oropharyngeal cancer.

The rate of HPV-positive oropharyngeal cancer incidence is increasing, especially in the young population. While these patients show good responses to radiotherapy. The major complication of radiotherapy is normal tissue involvement. Thus, finding an effective treatment method is essential. Multimodal therapy with the lowest side effect could be an effective treatment method. Theranostic gold magnetic core-shell nanostructure was developed as aplatform for multimodal therapy of HPV-positive oropharyngeal cancer. The folate functionalized gold-magnetic core-shell nanostructure has been synthesized in a stepwise approach and characterized with various techniques including TEM, UV-Vis, and FTIR spectroscopy. KB was selected as a host for HPV and folate receptor-positive cancer. HGF as normal cell lines was selected. Both cell lines have been treated with nanoparticles, electric field and radiotherapy, either separately or in combination. Cell viability and apoptosis rate were determined by MTT and flow cytometry assay. In addition, cellular uptake of the nanoparticles was measured by ICP-OES analysis. The average size of folate functionalized gold-magnetic core-shell nanostructure was 13.8 ± 6.4nm. A characteristic plasmonic peak of gold nanoshells was observed in the UV-Vis spectrum. The functionalization of synthesized nanostructure was confirmed with FTIR spectroscopy. None of the treatments alone can cause a significant death in cancerous cells. Combination treatments can increase cancer cell mortality and increase the proportion of apoptotic cells in them. Furthermore, it has been observed that the electric field enhanced the cellular uptake of nanoparticles just in cancerous cells. Based on our findings, we conclude that the combination of folate functionalized nanoparticles and electroporation opens a new way to improve radiation therapy efficacy of HPV-positive cancers.

Impedance analysis of adherent cells after in situ electroporation-mediated delivery of bioactive proteins, DNA and nanoparticles in \xb5L-volumes

Specific intracellular manipulation of animal cells is a persistent goal in experimental cell biology. Such manipulations allow precise and targeted interference with signaling cascades, metabolic pathways, or bi-molecular interactions for subsequent tracking of functional consequences. However, most biomolecules capable of molecular recognition are membrane impermeable. The ability to introduce these molecules into the cytoplasm and then to apply appropriate readouts to monitor the corresponding cell response could prove to be an important research tool. This study describes such an experimental approach combining in situ electroporation (ISE) as a means to efficiently deliver biomolecules to the cytoplasm with an impedance-based, time-resolved analysis of cell status using electric cell-substrate impedance sensing (ECIS). In this approach, gold-film electrodes, deposited on the bottom of regular culture dishes, are used for both electroporation and monitoring. The design of the electrode layout and measurement chamber allows working with sample volumes as small as 10 µL. A miniaturized setup for combined electroporation and impedance sensing (µISE-ECIS) was applied to load different adherent cells with bioactive macromolecules including enzymes, antibodies, nucleic acids and quantum dot nanoparticles. The cell response after loading the cytoplasm with RNase A or cytochrome c (in the presence or absence of caspase inhibitors) was tracked by non-invasive impedance readings in real-time.

The effects of iontophoresis and electroporation on transdermal delivery of indomethacin evaluated in vitro and in vivo

The objective of this study was to evaluate the transdermal delivery of indomethacin in vitro and in vivo enhanced by iontophoresis and/or electroporation through several barriers. Excised Wistar rat skin, pig skin, human epidermal membrane and cellulose membrane were tested for the in vitro indomethacin permeation, whereas drug concentration of in the plasma was analyzed to monitor the percutaneous absorption in Wistar rat in vivo. Iontophoresis enhanced the in vitro transdermal delivery of indomethacin as compared to the passive diffusion. Pulsing of high voltages, as known as electroporation, followed by iontophoresis did result in a synergistic effect in contrast to iontophoresis or electroporation alone. The in vitro result indicated that higher drug permeation could be induced during and after the application of iontophoresis. In some case, the permeation could benefit from the electroporation in term of cumulative amount within 12 hr. Furthermore, the combination of electroporation and iontophoresis resulted in a higher permeation amount than iontophoresis or electroporation alone did. Both iontophoresis and the combination protocol could reduce interspecies difference in vitro. The in vivo results were similar to that of in vitro ones. Iontophoresis and electroporation/iontophoresis resulted in higher AUC (area under curve) of blood concentration profiles of indomethacin. In addition, the combination protocol significantly induced higher transepidermal water loss (TEWL). It is concluded that the in vivo permeation results did correspond to the enhancement efficiency of the in vitro experiments.

The combination of electroporation and electrolysis (E2) employing different electrode arrays for ablation of large tissue volumes.

BackgroundThe combination of electroporation with electrolysis (E2) has previously been introduced as a novel tissue ablation technique. E2 allows the utilization of a wide parameter range and may therefore be a suitable technology for development of tissue-specific application protocols. Previous studies have implied that it is possible to achieve big lesions in liver in a very short time. The goal of this study was to test a variety of electrode configurations for the E2 application to ablate large tissue volumes.Materials and methods27 lesions were performed in healthy porcine liver of five female pigs. Four, two and bipolar electrode-arrays were used to deliver various E2 treatment protocols. Liver was harvested approx. 20h after treatment and examined with H&E and Masson’s trichrome staining, and via TUNEL staining for selective specimen.ResultsAll animals survived the treatments without complications. With four electrodes, a lesion of up to 35x35x35mm volume can be achieved in less than 30s. The prototype bipolar electrode created lesions of 50x18x18mm volume in less than 10s. Parameters for two-electrode ablations with large exposures encompassing large veins were found to be good in terms of vessel preservation, but not optimal to reliably close the gap between the electrodes.ConclusionThis study demonstrates the ability to produce large lesions in liver within seconds at lower limits of the E2 parameter space at different electrode configurations. The applicability of E2 for single electrode ablations was demonstrated with bipolar electrodes. Parameters for large 4-electrode ablation volumes were found suitable, while parameters for two electrodes still need optimization. However, since the parameter space of E2 is large, it is possible that for all electrode geometries optimal waveforms and application protocols for specific tissues will emerge with continuing research.

A Review of Electroporation-based Antitumor Skin Therapies and Investigation of Betulinic Acid-loaded Ointment.

Electrochemotherapy is a novel treatment for cutaneous and subcutaneous tumors utilizing the combination of electroporation and chemotherapeutic agents. Since tumors have an increasing incidence nowadays as a result of environmental and genetic factors, electrochemotherapy could be a promising treatment for cancer patients. The aim of this article is to summarize the novel knowledge about the use of electroporation for antitumor treatments and to present a new application of electrochemotherapy with a well-known plant derived antitumor drug betulinic acid. For the review we have searched the databases of scientific and medical research to collect the available publications about the use of electrochemotherapy in the treatment of various types of cancer. By the utilization of the available knowledge, we investigated the effect of electroporation on the penetration of a topically applied betulinic acid formulation into the skin by ex vivo Raman spectroscopy on hairless mouse skin. Raman measurements have demonstrated that the penetration depth of betulinic acid can be remarkably ameliorated by the use of electroporation, so this protocol can be a possibility for the treatment of deeper localized cancer nodules. Furthermore, it proved the influence of various treatment times, since they caused different spatial distributions of the drug in the skin. The review demonstrates that electrochemotherapy is a promising tool to treat different kinds of tumors with high efficiency and with only a few moderate adverse effects. Moreover, it presents a non-invasive method to enhance the penetration of antitumor agents, which can offer novel prospects for antitumor therapies.

Abstract 3722: Efficacy of irreversible electroporation in combination with liposome-nvp-bez235 for head and neck cancer

Abstract Introduction: Irreversible electroporation (IRE) is an emerging, minimally invasive ablation technique with the advantage of not being affected by heat sink effect and the ability of ablation in close vicinity of major blood vessels. One limitation is local recurrence at the original ablated site. We hypothesize that combining IRE with a chemotherapeutic drug could prevent recurrence. NVP-BEZ235 (BEZ) is a dual PI3K/mTOR inhibitor that has shown promise for treating advanced solid malignancies, including sensitizing head and neck squamous cell carcinoma to radiotherapy. However, clinical trials revealed low bioavailability and high toxicity due to high dose oral administration over a long period of time. Thus, we formulated a liposomal BEZ (L-BEZ) and studied its antitumor effect in combination with IRE and compared the results with orally delivered BEZ (oral-BEZ). Methods: IRE was performed using ECM 830 (BTX Harvard Apparatus) at various field strengths. BEZ was loaded into liposome (L-BEZ) by hydration-sonication method. The in vitro and in vivo efficacy was tested against head and neck cancer cell line, HN5 cells and in nude mice bearing HN5 xenografts (n=4/group). Mice were divided into control (no treatment), IRE (2500 V/cm for 99 pulses), IRE + L-BEZ (IRE at 2500 V/cm for 99 pulses and 7 doses of L-BEZ) and IRE + oral-BEZ (IRE at 2500 V/cm for 99 pulses and 21 doses of oral-BEZ) groups. Tumor size was monitored for 60 days. Results: The hydrodynamic volume of NVP-BEZ ranged from 100-500nm. Maximum drug loading was achieved at 2.7mg/mL of BEZ. Electroporation disrupted nanoparticle's integrity even at the lowest tested field strength (250 V/cm) and released BEZ. At the highest field strength (2500 V/cm), approximately 5% of cells survived with IRE, while cells electroporated at 500 V/cm increased cell viability (114%) as compared to the untreated group (100 %). Combination of electroporation and L-BEZ significantly decreased cell viability at 500 V/cm (p<0.05). In vivo, IRE + L-BEZ suppressed tumor growth the longest, as compared with control (no treatment), IRE, and IRE + oral-BEZ. It was also the only group that resulted in no palpable tumor from day 30 to day 60. Survival studies show a significant difference between IRE + L-BEZ and the other groups. IRE + L-BEZ group had 100% animal survival on day 60. IRE + oral-BEZ had 50%, while IRE and control groups had 0% animal survival, on day 60. Also, the total administered BEZ amount in IRE + L-BEZ group was only 6.6% of that in IRE + oral-BEZ group. Conclusion: Incomplete electroporation increases the viability of surviving cells both in vitro and in vivo. Co-delivery of L-BEZ enhances the antitumor efficacy of IRE alone. L-BEZ also decreases BEZ exposure compared to oral-BEZ. Thus, L-BEZ in combination with IRE potentially ensures complete eradication of surviving cells left after IRE. Citation Format: Li Tian, Lucas Wang, Yang Qiao, Saisree Ravi, Ashley Chang, Thomas Alexander Rogers, Linfeng Lu, Adam D. Melancon, Marites Pasuelo Melancon. Efficacy of irreversible electroporation in combination with liposome-nvp-bez235 for head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3722.

Radiosensitizing Effect of Electrochemotherapy: A Systematic Review of Protocols and Efficiency.

Electrochemotherapy (ECT) is a combination of electroporation (EP) and chemotherapy and has been reported as a potential radiosensitizing agent for radiation therapy. The main objective of this study was to systematically review of the literature to evaluate the effectiveness of ECT in sensitization of tumors to ionization radiation. In addition, the clinical considerations and mechanisms of action of radiosensitizing effect are discussed. Nine studies were included in this review. Bleomycin and cisplatin showed radiosensitizing effects in combined protocols with EP. EP enhances the cytotoxicity of bleomycin and cisplatin by factor of 1000 and 100, respectively. The mechanism of action of these drugs is induction of single and double strand breaks in DNA molecule. Moreover, the two main mechanisms of EP are increasing drug uptake in the tumor cells and generating reactive oxygen species. A single session ECT before radiotherapy can significantly enhance the tumor response. ECT is effective for different cell lines and tumors with different levels of radiosensitivity. Our findings show that ECT can be further translated into the clinic and can be matched by singledose irradiation as well as in the fractionated regime.

Intracellular Delivery of Bleomycin by Combined Application of Electroporation and Sonoporation in Vitro.

In this study, we aimed to determine whether the combination of electroporation (EP) and ultrasound (US) waves (sonoporation) can result in an increased intracellular delivery of anticancer drug bleomycin. CHO cells were treated with electric pulses (1 or 8 high voltage pulses of 800 or 1200V/cm, 100μs or 1 low voltage pulse of 100 or 250V/cm, 100ms) and with 880kHz US of 320 or 500kPa peak negative pressure, 100% duty cycle, applied for 2s in the presence or absence of exogenously added contrast agent microbubbles. Various sequential or simultaneous combinations of EP and sonoporation were used. The results of the study showed that i) sequential treatment of cells by EP and sonoporation enhanced bleomycin electrosonotransfer at the reduced energy of electric field and US; ii) sequential combination of EP and sonoporation induced a summation effect which at some conditions was more prominent when the cells were treated first by EP and then by sonoporation; iii) the most efficient intracellular delivery of bleomycin was achieved by the simultaneous application of cell EP and sonoporation resulting in percentage of reversibly porated cells above the summation level; and iv) compared with sequential application of EP and sonoporation, simultaneous use of electric pulses and US increased cell viability in the absence of bleomycin.

4:33 PM, Abstract No. 153 - Cell killing efficacy of combined electroporation and drug delivery of NVP-BEZ235

4:33 PM, Abstract No. 153 - Cell killing efficacy of combined electroporation and drug delivery of NVP-BEZ235

4:42 PM, Abstract No. 154 - Efficacy and safety of hepatic arterial embolization compared to portal vein embolization for the preoperative induction of liver hypertrophy: results of animal experiments in swine

Cell killing efficacy of combined electroporation and drug delivery of NVP-BEZ235 Y. Qiao, J. Zhao, P. Lee, J. Jody Vykoukal, M.J. Wallace, C. Li, S. Gupta, M.P. Melancon; Interventional Radiology, University of Texas M.D. Anderson Cancer Center, Houston, TX; Cancer Systems Imaging, University of Texas M.D. Anderson Cancer Center, Houston, TX; SUNY Upstate Medical University, Syracuse, NY; Translational Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX

Locally enhanced chemotherapy by electroporation: clinical experiences and perspective of use of electrochemotherapy.

Electroporation is used to enhance drug diffusion and gene delivery into the cytosol. The combination of electroporation and cytotoxic drugs, electrochemotherapy (ECT), is used to treat metastatic tumor nodules located at the skin and subcutaneous tissue. The objective response rate following a single session of treatment exceeds 80%, with minimal toxicity for the patients. The efficacy of ECT in the bone and visceral metastasis is currently investigated, and Phase II studies have been completed. ECT has been used to treat skin primary tumors, except melanoma, and is under investigation for locally advanced pancreatic cancer. Early evidence suggests that treatment of tumor nodules with ECT recruits components of the immune system and eliciting a systemic immune response against cancer is a challenging clinical perspective. Considering the proven safety in several different clinical applications electroporation should be viewed as a clinical platform technology with wide perspectives for use in ECT, gene therapy and DNA vaccination.

Better damage of chicory tissue by combined electroporation and ohmic heating for solute extraction

Damage of chicory tissue by combined electroporation and ohmic heating is studied for better solute extraction. Moderate (400–1000V/cm) and high (10,000V/cm) PEF treatments were applied varying pause duration between the trains of individual pulses. Ohmic heating was induced with increase of the number of trains N. Temperature dependence of tissue damage degree Z is evaluated for the different PEF intensities. With higher ohmic heating, chicory tissue is faster and better damaged. Electric field strengths of 600–800V/cm combined with ohmic heating permit to enhance noticeably the solute extraction from chicory tissue. The solute diffusivity D for the different PEF treatments, is nearly the same for same values of Z. Chicory tissue treated to the same damage degree (Z=0.8–1.0) using different PEF conditions (800, 1000 and 10,000V/cm) has nearly the same diffusivity.Combined electroporation/ohmic heating pretreatment by moderate PEF (400–1000V/cm) presents an interesting alterative for the treatment of high product throughputs (e.g. in the case of inulin production from chicory).

Cell uptake mechanisms of PAMAM G4-FITC dendrimer in human myometrial cells

The high incidence and severity of diseases which involve smooth muscle dysfunction dictates the need of continued search for novel therapeutic strategies to treat these conditions. Dendrimers are branched macromolecules with multiple end-groups that can be functionalized for applications which include drug delivery. There is no data regarding the cellular uptake mechanisms used by dendrimers in smooth muscle human myometrial cells (HMC). Polyamidoamine G4 dendrimers were conjugated with fluorescein isothiocyanate (FITC) and the resulting conjugate (G4-FITC) was characterized using high-performance liquid chromatography, nuclear magnetic resonance, and atomic force microscopy. G4-FITC showed to have no significant effect on the primary culture HMC viability up to 48 h. HMC incubated with G4-FITC were analyzed by laser confocal microscopy. Peri-nuclear fluorescence distribution was observed at 5 h of incubation or more (24, 36, and 48 h). At 24 h, G4-FITC partially co-localized with lysotracker. Uptake of G4-FITC by HMC was slightly inhibited by filipin (8.0 ± 3.9 %) and significantly inhibited by chlorpromazine (63.5 ± 3.7 %). In non-electroporated HMC, G4-FITC was never observed inside the cell nucleus. Interestingly, we detected G4-FITC inside the nuclear domain of some electroporated cells. Thus, electroporation changed intracellular G4-FITC localization. Isolated nuclei of HMC incubated with G4-FITC showed fluorescence signal inside the nuclear domain. The results suggest that in HMC, G4-FITC is taken up by clathrin-mediated endocytosis with endosomal and lysosomal localization at 24 h. The combination of electroporation and dendrimers could be an interesting technology to electrotransfer drugs into smooth muscle cells cytosol and nuclei.

Elektrochemotherapie von Hauttumoren

Electrochemotherapy represents a new innovative technology using a combination of electroporation and application of very low dose cytotoxic drugs for the treatment of cutaneous and subcutaneous skin tumors and metastases. Based on a high number of preclinical and clinical studies this procedure has been established as a simple, safe and very effective tumor treatment whose costs are acceptable. The procedure can be performed as a single treatment of localized disease as well as a repeated palliative treatment of cutaneous metastases regardless of the tumor type. Because of the lack of significant side effects and of short duration the procedure can be performed on an out-patient basis. Several studies led to the validation of standard operating procedures for its use in daily clinical practice. This treatment has become established in many different European countries.

Electrochemotherapy for cutaneous and subcutaneous tumor lesions: a novel therapeutic approach

Electroporation uses pulsed, high-intensity electric fields to temporarily increase cell membrane permeability by creation of pores, through which small molecules, such as chemotherapeutic agents, can diffuse inside cells before they reseal. The combination of electroporation with the administration of otherwise low-permeant cytotoxic drugs is known as electrochemotherapy (ECT). The two most commonly used drugs are bleomycin and cisplatin. ECT has already been proven to be effective in diverse tumor histotypes, including melanoma and basal and squamous cell carcinoma, Kaposi sarcoma, and breast cancer, also in those cases nonresponding to classical chemotherapies or other loco-regional treatment modalities, with a good safety profile. ECT can be proposed as loco-regional therapy for disseminated cutaneous and subcutaneous tumor lesions as alternative treatment modality to conventional therapies or as palliative care, in order to improve patients' quality of life.

Constant voltage ‘Iron’tophoresis

The objective of this study was to investigate the feasibility of rapid administration of iron via transdermal route as an alternative to parenteral route of administration. In vitro drug delivery studies were carried out using porcine epidermis mounted on Franz diffusion cells. The effect of chemical permeation enhancers and physical techniques (constant voltage iontophoresis, electroporation and combination of electroporation with iontophoresis) on the transport of ferric pyrophosphate (FPP) was studied. Transepidermal water loss (TEWL) and electrical resistance were measured in order to see the effect of these techniques on the skin barrier function. The amount of FPP permeated was not enhanced significantly with the use of any of the enhancers (P > 0.05). It was found that constant voltage iontophoresis (0.5, 2 or 4 V) for about 30 min across electroporated epidermis (120 V, 100 pulses, 10 ms at 5 Hz) enhanced the delivery of FPP over control in the range of 2- to 42-fold. Hence, a therapeutically required dose of iron could be delivered by transdermal route using electrically-mediated techniques.

Synergistic electrochemotherapy on cancer cells by photodynamically active cytostatic agents

Two reliable methods have been combined: i) the electroporation of the cell membranes for facilitating the sensitizer incorporation into hystiocytic human lymphoma cells U-937 and (ii) the photodynamics applied by excitation of natural and synthetic sensitizers for cancer therapy. In the case of cytostatic sensitizers as daunomycin or actinomycin their photooxidation of guanine in DNA was added to their known dark medical efficacy for the first time. Several applications of single d.c. pulses and continuous visible light irradiations were performed, which resulted in about five times higher efficacy by 14 min of irradiation after the electroporation than the ordinary photodynamic effect itself on intact cell membranes. Yielding about 90% killing rate by a combination of electroporation and photooxidation this synergism will be the basis of an extended electrochemotherapy by light irradiation according to the photodynamic mechanism type 1 for the treatment of malignant cells and tissues. Analogous results – including the first synergistic treatment of tumor mice according to photodynamic mechanism type 2 – were discussed, too.

Pharmacokinetics of transdermal absorption of Sinomenine in patch under electroporation condition

Objective:To study the pharmacokinetics of the transdermal absorption of Sinomenine in patch under electroporation condition by comparing with the transdermal absorption of passive diffusion. Methods: Sinomenine was taken as a model drug and was given to rabbits by passive diffusion (PD group) or by electroporation (EP group). The plasma drug concentration was determined by HPLC. The statistical moment theory was used for analyzing the values of plasma drug concentration.Results: The AUC0→∞ was 43.396 in the EP group, which was 1.32 folds that of the PD group. The mean residence time (MRT) was 20.025 in the EP group, which was 86% of the PD group. The cmax was 1.825 in the EP group, which was 1.31 folds that of the PD group. The decrease of tmax in the EP group was 4 hours earlier than that in the PD group. Their elimination rate constants λ were the same. The drug-time curve of EP group was more smooth and slow when declining compared with that of the PD group.Conclusion: The combination of electroporation and the patch can improve the speed and extent of transdermal absorption of sinomenine, and thus enhance the bioavailability.

Upgrade of the Electroporation Device KEA-MOBIL

For the production of synthetic bio-fuel the use of green biomass enables additional resources of raw material supplementary to the commonly used dry material. An electroporation assisted drying process gives the opportunity of a more efficient dewatering than only pressing and subsequent drying. Experiments showed a lower relative humidity of the pressed material after electroporation compared to pressing the raw material only. Moreover, the drying process of the electroporated material was much shorter. This is because the pores formed in the cell membranes foster a better extraction of water during pressing and enhance the diffusion process during the drying process. The paper presents exemplarily the experimental results for green rye. For the electroporation-assisted drying process the electric contact to the plant material has to be established by means of the juice inside the plants only rather than adding additional water. Hence, for experiments in larger scale, the existing electroporation device KEA-MOBIL has been equipped with a new combined electroporation reactor and press. The reactor has a plate electrode system, whose upper electrode additionally serves as a piston to apply mechanical force to extract juice for a good electrical contact between plant material and the electrodes. A two-stage Marx generator serves as a pulse source. To reduce the insulation distance to the walls of the shielding cabin the Marx generator is operated in ground symmetric configuration. The device has been designed for a batch-wise throughput of 50 kg/h. An automatic feeding device enables the alternating use of two test vessels. The device has been tested on-site on a farm with green rye and grass.

Gene delivery to cells on a miniaturized multiwell plate for high-throughput gene function analysis

The transfectional microarray is a promising tool to conduct high-throughput analysis of gene function. In this study, a miniaturized multiwell plate was prepared by applying a silicone rubber sheet with holes on a gold electrode. The combination of electroporation and a miniaturized multiwell plate successfully achieved spatially controlled gene delivery with absence of cross-contamination between genes. Furthermore, we found that gene delivery efficiency was not dependent on conditions such as plasmid loading, electric field strength, and pulse duration.