The Evidence of the Bystander Effect after Bleomycin Electrotransfer and Irreversible Electroporation.

One of current applications of electroporation is electrochemotherapy and electroablation for local cancer treatment. Both of these electroporation modalities share some similarities with radiation therapy, one of which could be the bystander effect. In this study, we aimed to investigate the role of the bystander effect following these electroporation-based treatments. During direct CHO-K1 cell treatment, cells were electroporated using one 100 µs duration square wave electric pulse at 1400 V/cm (for bleomycin electrotransfer) or 2800 V/cm (for irreversible electroporation). To evaluate the bystander effect, the medium was taken from directly treated cells after 24 h incubation and applied on unaffected cells. Six days after the treatment, cell viability and colony sizes were evaluated using the cell colony formation assay. The results showed that the bystander effect after bleomycin electrotransfer had a strong negative impact on cell viability and cell colony size, which decreased to 2.8% and 23.1%, respectively. On the contrary, irreversible electroporation induced a strong positive bystander effect on cell viability, which increased to 149.3%. In conclusion, the results presented may serve as a platform for further analysis of the bystander effect after electroporation-based therapies and may ultimately lead to refined application of these therapies in clinics.

Electrochemotherapy Using Doxorubicin and Nanosecond Electric Field Pulses: A Pilot in Vivo Study.

Pulsed electric field (PEF) is frequently used for intertumoral drug delivery resulting in a well-known anticancer treatment-electrochemotherapy. However, electrochemotherapy is associated with microsecond range of electrical pulses, while nanosecond range electrochemotherapy is almost non-existent. In this work, we analyzed the feasibility of nanosecond range pulse bursts for successful doxorubicin-based electrochemotherapy in vivo. The conventional microsecond (1.4 kV/cm × 100 µs × 8) procedure was compared to the nanosecond (3.5 kV/cm × 800 ns × 250) non-thermal PEF-based treatment. As a model, Sp2/0 tumors were developed. Additionally, basic current and voltage measurements were performed to detect the characteristic conductivity-dependent patterns and to serve as an indicator of successful tumor permeabilization both in the nano and microsecond pulse range. It was shown that nano-electrochemotherapy can be the logical evolution of the currently established European Standard Operating Procedures for Electrochemotherapy (ESOPE) protocols, offering better energy control and equivalent treatment efficacy.

Predicting electrotransfer in ultra-high frequency sub-microsecond square wave electric fields.

Measurement of cell transmembrane potential (TMP) is a complex methodology involving patch-clamp methods or fluorescence-based potentiometric markers, which have limited to no applicability during ultrafast charging and relaxation phenomena. In such a case, analytical methods are applied for evaluation of the voltage potential changes in biological cells. In this work, the TMP-based electrotransfer mechanism during ultra-high frequency (≥1 MHz) electric fields is studied and the phenomenon of rapid membrane charge accumulation, which is non-occurrent during conventional low-frequency electroporation is simulated using finite element method (FEM). The influence of extracellular medium conductivity (0.1, 1.5 S/m) and pulse rise/fall times (10-50 ns) TMP generation are presented. It is shown that the medium conductivity has a dramatic influence on the electroporation process in the high-frequency range of applied pulsed electric fields (PEF). The applied model allowed to grasp the differences in polarization between 100 and 900 ns PEF and enabled successful prediction of the experimental outcome of propidium iodide electrotransfer into CHO-K1 cells and the conductivity-dependent patterns of MHz range PEF-triggered electroporation were determined. The results of this study form recommendations for development and pre-evaluation of future PEF protocols and generators based on ultra-high frequency electroporation for anticancer and gene therapies.

Low concentrations of acetic and formic acids enhance the inactivation of Staphylococcus aureus and Pseudomonas aeruginosa with pulsed electric fields.

BACKGROUND: Skin infections, particularly caused by drug-resistant pathogens, represent a clinical challenge due to being a frequent cause of morbidity and mortality. The objectives of this study were to examine if low concentrations of acetic and formic acids can increase sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa to pulsed electric field (PEF) and thus, promote a fast and efficient treatment methodology for wound treatment. RESULTS: We have shown that the combination of PEF (10-30 kV/cm) with organic acids (0.1% formic and acetic acids) increased the bactericidal properties of treatment. The effect was apparent for both acids. The proposed methodology allowed to reduce the energy of electrical pulses and the inhibitory concentrations of acids, while still maintain high efficiency of bacteria eradication. CONCLUSIONS: Application of weak organic acids as bactericidal agents has many advantages over antibiotics because they do not trigger development of drug-resistance in bacteria. The combination with PEF can make the treatment effective even against biofilms. The results of this study are particularly useful for the development of new methodologies for the treatment of extreme cases of wound infections when the chemical treatment is no longer effective or hinders wound healing.

Membrane Permeabilization of Pathogenic Yeast in Alternating Sub-microsecond Electromagnetic Fields in Combination with Conventional Electroporation.

Recently, a novel contactless treatment method based on high-power pulsed electromagnetic fields (PEMF) was proposed, which results in cell membrane permeabilization effects similar to electroporation. In this work, a new PEMF generator based on multi-stage Marx circuit topology, which is capable of delivering 3.3 T, 0.19 kV/cm sub-microsecond pulses was used to permeabilize pathogenic yeast Candida albicans separately and in combination with conventional square wave electroporation (8-17 kV/cm, 100 µs). Bursts of 10, 25, and 50 PEMF pulses were used. The yeast permeabilization rate was evaluated using flow cytometric analysis and propidium iodide (PI) assay. A statistically significant (P < 0.05) combinatorial effect of electroporation and PEMF treatment was detected. Also the PEMF treatment (3.3 T, 50 pulses) resulted in up to 21% loss of yeast viability, and a dose-dependent additive effect with pulsed electric field was observed. As expected, increase of the dB/dt and subsequently the induced electric field amplitude resulted in a detectable effect solely by PEMF, which was not achievable before for yeasts in vitro.

Induction of Different Sensitization Patterns of MRSA to Antibiotics Using Electroporation.

Treatment of bacteria-associated infections is complicated and antibiotic treatment alone is often inadequate to overcome biofilm infections. Physical methods allow overcoming this problem and propose solutions that are non-dependent on drug resistance. In this work, we investigated the feasibility of pulsed electric fields for sensitization of MRSA to common antibiotics. We analyzed the efficacy of inactivation of methicillin-resistant Staphylococcus aureus in 5⁻20 kV/cm electric field separately and in combination with gentamicin, doxycycline, ciprofloxacin, sulfamethoxazole, and vancomycin. Combined treatment allowed using up to 1000-fold smaller concentrations of antibiotics to induce the same inactivation of S. aureus.

Irreversible electropermeabilization of the human pathogen Candida albicans: an in-vitro experimental study.

Pathogenic fungi cause many life-threatening infections, especially among individuals with immune system dysfunction. The antifungal drugs commonly used to suppress fungal pathogens can result in long-lasting and toxic therapy. In this work, irreversible electropermeabilization was used to investigate the dynamics of the decrease in Candida albicans colony vitality after application of a pulsed electric field (PEF) and use of antifungal drugs. The fungi were subjected to single 250-µs to 2-ms (0.5-2.5 kV/cm) pulses or repeated short 5-µs pulses, and efficacy was compared. It was shown that electropermeabilization combined with antifungal agents results in rapid and more effective treatment, eliminating more than 90% of C. albicans colony-forming units in a single procedure, which is advantageous in biomedicine. It was also observed that, because of application of PEF and use of the antifungal agents, the Candida cells form cell aggregates and average live cell size is reduced by as much as 53%.

Microsecond pulsed magnetic field improves efficacy of antifungal agents on pathogenic microorganisms.

Control and treatment of the emerging filamentous and yeast fungal diseases are of high priority in the biomedical field. This study investigated the influence of the pulsed magnetic field combined with common antifungal agents on the viability of various pathogenic fungi such as Aspergillus fumigatus, Candida albicans, and Trychophyton rubrum. Repetitive microsecond pulsed magnetic fields up to 6.1 T were applied in the study. The synergistic effect of co-applying drugs and magnetic treatment to different fungi species causing various human mycoses showed the potential for more effective and less toxic therapy.

Mitochondrial depolarization and ATP loss during high frequency nanosecond and microsecond electroporation.

It is predicted that ultra-short electric field pulses (nanosecond) can selectively permeabilize intracellular structures (e.g., mitochondria) without significant effects on the outer cell plasma membrane. Such a phenomenon would have high applicability in cancer treatment and could be employed to modulate cell death type or immunogenic response. Therefore, in this study, we compare the effects of 100 µs x 8 pulses (ESOPE - European Standard Operating Procedures on Electrochemotherapy) and bursts of 100 ns pulses for modulation of the mitochondria membrane potential. We characterize the efficacies of various protocols to trigger permeabilization, depolarize mitochondria (evaluated 1 h  after treatment), the extent of ATP depletion and generation of reactive oxygen species (ROS). Finally, we employ the most prominent protocols in the context of Ca2+ electrochemotherapy in vitro. We provide experimental proof that 7.5-12.5 kV/cm x 100 ns pulses can be used to modulate mitochondrial potential, however, the permeabilization of the outer membrane is still a prerequisite for depolarization. Similar to 100 µs x 8 pulses, the higher the permeabilization rate, the higher the mitochondrial depolarization. Nevertheless, 100 ns pulses result in lesser ROS generation when compared to ESOPE, even when the energy input is several-fold higher than for the microsecond procedure. At the same time, it shows that even the short 100 ns pulses can be successfully used for Ca2+ electrochemotherapy, ensuring excellent cytotoxic efficacy.

Calcium Electrochemotherapy for Tumor Eradication and the Potential of High-Frequency Nanosecond Protocols.

Calcium electroporation (CaEP) is an innovative approach to treating cancer, involving the internalization of supraphysiological amounts of calcium through electroporation, which leads to cell death. CaEP enables the replacement of chemotherapeutics (e.g., bleomycin). Here, we present a standard microsecond (µsCaEP) and novel high-frequency nanosecond protocols for calcium electroporation (nsCaEP) for the elimination of carcinoma tumors in C57BL/6J mice. We show the efficacy of CaEP in eliminating tumors and increasing their survival rates in vivo. The antitumor immune response after the treatment was observed by investigating immune cell populations in tumors, spleens, lymph nodes, and blood, as well as assessing antitumor antibodies. CaEP treatment resulted in an increased percentage of CD4+ and CD8+ central memory T cells and decreased splenic myeloid-derived suppressor cells (MDSC). Moreover, increased levels of antitumor IgG antibodies after CaEP treatment were detected. The experimental results demonstrated that the administration of CaEP led to tumor growth delay, increased survival rates, and stimulated immune response, indicating a potential synergistic relationship between CaEP and immunotherapy.

Improving NonViral Gene Delivery Using MHz Bursts of Nanosecond Pulses and Gold Nanoparticles for Electric Field Amplification.

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy.

Transfection by Electroporation of Cancer and Primary Cells Using Nanosecond and Microsecond Electric Fields.

Gene transfer into primary immune cells as well as into cell lines is essential for scientific and therapeutical applications. One of the methods used for gene transfer is electroporation (EP). EP is a method where a pulsed electric field (PEF) causes a highly transient permeability of the targeted cell membrane. In this work, we present the electrotransfection of CHO-K1, 4T1 cell lines, and primary murine DCs with detectable protein-encoding plasmids in the sub-microsecond range. Microsecond (µs)- and nanosecond (ns)-range pulsed electric field transfection protocols were used. The efficiency of electrotransfection was evaluated using green fluorescent protein (GFP)-encoding plasmids (4.7 kbp; p-EGFP-N1) and plasmids expressing a firefly luciferase and red fluorescent protein (tdTomato) (8.5 kbp; pcDNA3.1(+)/Luc2 = tdT)). It was shown that the used nsPEFs protocol (7 kV/cm × 300 ns × 100, 1 MHz) ensured a better transfection efficiency than µsPEFs (1.2 kV/cm × 100 µs × 8, 1 Hz). Plasmid size and concentration had a strong impact on the cell transfection efficiency too. We also showed that there were no significant differences in transfection efficiency between immature and mature DCs. Finally, the nsPEF protocols were successfully applied for the stable transfection of the CHO-K1 cell line with the linearized pcDNA3.1(+)/Luc2 = tdT plasmid. The results of the study are applicable in gene therapy and DNA vaccination studies for the derivation of optimal electrotransfection conditions.

Bioluminescent calcium mediated detection of nanosecond electroporation: Grasping the differences between 100 ns and 100 µs pulses.

Electroporation is a phenomenon of transient or irreversible permeabilization of the cell membrane after pulsed electric field treatment. Fluorescent probes are frequently used to assess the extent of permeabilization, however, as an alternative, a D-luciferin oxidation-based method can be used. In this work, we have used sequences of a microsecond (1.3 kV/cm × 100 µs) and nanosecond (12.5 kV/cm × 100 ns) pulses to trigger various levels of cell permeabilization and assessed the differences in the response using a conventional fluorescent probe (YO-PRO-1 (YP)) and D-luciferin oxidation methodology. The nanosecond pulses (n = 5-100) have been delivered with 1 kHz repetition frequency, and the results were compared with 1 MHz protocols. Additionally, the effects of extracellular Ca2+ have been assessed. Various concentrations of CaCl2 (2, 5, and 10 mM) have been used, and it was shown that the bioluminescence of the cells after electroporation depends on extracellular calcium concentration. It was shown that the changes in bioluminescence signal could be used as a marker of cell membrane permeabilization on par with YP assay when calcium is added and thus, effectively employed for analysis of electroporation phenomenon in vitro both for nanosecond and microsecond pulses.

Nanosecond electrochemotherapy using bleomycin or doxorubicin: Influence of pulse amplitude, duration and burst frequency.

Electroporation is a pulsed electric field (PEF) induced phenomenon, which effectiveness varies dependent on pulse parameters. This work focuses on nano-electrochemotherapy with bleomycin and doxorubicin to derive protocols as effective as European Standard Operating Procedures on Electrochemotherapy (ESOPE), which employ conventional microsecond range pulses. As a model, murine Lewis lung carcinoma (LLC1) cell line was used. The effects of pulse duration (100-500 ns), PEF amplitude (6-10 kV/cm) and pulse repetition frequency (10 kHz, 100 kHz, 1 MHz) were studied. A total of 75 ns protocol variations have been used. For detection of cell permeabilization, Yo-Pro-1 and flow cytometry were employed. Cell viability was evaluated 24-, 48-, or 72-hours post-electroporation. Nanosecond parametric protocols resulting in comparable treatment efficiency as ESOPE (1.3 kV/cm × 100 µs × 8) have been proposed. It was shown that high-frequency nanosecond electrochemotherapy with bleomycin or doxorubicin could be an alternative for established ESOPE protocols.

Effects of Time Delay Between Unipolar Pulses in High Frequency Nano-Electrochemotherapy.

OBJECTIVE: this work focuses on bleomycin electrochemotherapy using new modality of high repetition frequency unipolar nanosecond pulses. METHODS: As a tumor model, Lewis lung carcinoma (LLC1) cell line in C57BL mice (n = 42) was used. Electrochemotherapy was performed with intertumoral injection of bleomycin (50 µL of 1500 IU solution) followed by nanosecond and microsecond range electrical pulse delivery via parallel plate electrodes. The 3.5 kV/cm pulses of 200 and 700 ns were delivered in a burst of 200 at frequencies of 1 kHz and 1 MHz. For comparison of treatment efficiency, a standard 1.3 kV/cm x 100 µs x 8 protocol was used. RESULTS: It was shown that it is possible to manipulate the efficacy of unipolar sub-microsecond electrochemotherapy solely by the time delay between the pulses. SIGNIFICANCE: the results suggest that the sub-microsecond range pulses can be as effective as the protocols in European Standard Operating Procedures on Electrochemotherapy (ESOPE) using 100 µs pulses.

High-Frequency Nanosecond Bleomycin Electrochemotherapy and its Effects on Changes in the Immune System and Survival.

In this work, a time-dependent and time-independent study on bleomycin-based high-frequency nsECT (3.5 kV/cm × 200 pulses) for the elimination of LLC1 tumours in C57BL/6J mice is performed. We show the efficiency of nsECT (200 ns and 700 ns delivered at 1 kHz and 1 MHz) for the elimination of tumours in mice and increase of their survival. The dynamics of the immunomodulatory effects were observed after electrochemotherapy by investigating immune cell populations and antitumour antibodies at different timepoints after the treatment. ECT treatment resulted in an increased percentage of CD4+ T, splenic memory B and tumour-associated dendritic cell subsets. Moreover, increased levels of antitumour IgG antibodies after ECT treatment were detected. Based on the time-dependent study results, nsECT treatment upregulated PD 1 expression on splenic CD4+ Tr1 cells, increased the expansion of splenic CD8+ T, CD4+CD8+ T, plasma cells and the proportion of tumour-associated pro inflammatory macrophages. The Lin- population of immune cells that was increased in the spleens and tumour after nsECT was identified. It was shown that nsECT prolonged survival of the treated mice and induced significant changes in the immune system, which shows a promising alliance of nanosecond electrochemotherapy and immunotherapy.

Inactivation of Bacteria Using Bioactive Nanoparticles and Alternating Magnetic Fields.

Foodborne pathogens are frequently associated with risks and outbreaks of many diseases; therefore, food safety and processing remain a priority to control and minimize these risks. In this work, nisin-loaded magnetic nanoparticles were used and activated by alternating 10 and 125 mT (peak to peak) magnetic fields (AMFs) for biocontrol of bacteria Listeria innocua, a suitable model to study the inactivation of common foodborne pathogen L. monocytogenes. It was shown that L. innocua features high resistance to nisin-based bioactive nanoparticles, however, application of AMFs (15 and 30 min exposure) significantly potentiates the treatment resulting in considerable log reduction of viable cells. The morphological changes and the resulting cellular damage, which was induced by the synergistic treatment, was confirmed using scanning electron microscopy. The thermal effects were also estimated in the study. The results are useful for the development of new methods for treatment of the drug-resistant foodborne pathogens to minimize the risks of invasive infections. The proposed methodology is a contactless alternative to the currently established pulsed-electric field-based treatment in food processing.

Sub-microsecond electrotransfection using new modality of high frequency electroporation.

Micro-millisecond range electric field pulses have been used for decades to facilitate DNA transfer into cells and tissues, while the growing number of clinical trials underline the strong potential of DNA electroporation. In this work, we present new sub-microsecond range protocols and methodology enabling successful electrotransfection in the sub-microsecond range. To facilitate DNA transfer, a 3 kV/60 A and high frequency (1 MHz) sub-microsecond range square wave generator was applied in the study. As a model, Chinese hamster ovary (CHO-K1) cells were used. Sub-microsecond range (300-700 ns) high frequency pulsed electric fields of 2-15 kV/cm were applied. The efficiency of electrotransfection was evaluated using two green fluorescent protein encoding plasmids of different size (3.5 kbp and 4.7 kbp). It was shown that transfection efficiency cannot be effectively improved with increase of the number of pulses after a certain threshold, however, independently on the plasmid size, the proposed sub-microsecond range pulsing methodology (2-5 kV/cm; n = 250) efficiency-wise was equivalent to 1.5 kV/cm × 100 µs × 4 electroporation procedure. The results of the study are useful for further development of in vitro and in vivo methods for effective electrotransfer of DNA using shorter pulses.

Bioluminescence as a sensitive electroporation indicator in sub-microsecond and microsecond range of electrical pulses.

The cell membrane permeabilization in electroporation studies is usually quantified using fluorescent markers such as propidium iodide (PI) or YO-PRO, while Chinese Hamster Ovary cell line frequently serves as a model. In this work, as an alternative, we propose a sensitive methodology for detection and analysis of electroporation phenomenon based on bioluminescence. Luminescent mice myeloma SP2/0 cells (transfected using Luciferase-pcDNA3 plasmid) were used as a cell model. Electroporation has been studied using the 0.1-5 µs × 250 and 100 µs × 1-8 pulsing protocols in 1-2.5 kV/cm PEF range. It was shown that the bioluminescence response is dependent on the cell permeabilization state and can be effectively used to detect even weak permeabilization. During saturated permeabilization the methodology accurately predicts the losses of cell viability due to irreversible electroporation. The results have been superpositioned with permeabilization and pore resealing (1 h post-treatment) data using PI. Also, the viability of the cells was evaluated. Lastly, the SP2/0 tumors have been developed in BALB/C mice and the methodology has been tested in vivo using electrochemotherapy with bleomycin.

Influence of the electrode material on ROS generation and electroporation efficiency in low and high frequency nanosecond pulse range.

Electroporation is a widely-used methodology for permeabilization of cells using pulsed electric field (PEF). In this paper, we compare the electroporation efficiency in terms of molecular transport and the generated reactive oxygen species (ROS) between low (1 Hz) and high (1 MHz) frequency nanosecond range PEF bursts. We used aluminum, copper and stainless-steel electrodes and evaluated the influence of electrode material on ROS generation and electroporation. Bursts of 25 or 50 pulses of 7-14 kV/cm amplitude and 200 ns duration were applied, and the results were compared to those obtained using electroporation with pulses of equivalent energy in conventional microsecond range. It was determined that electroporation efficiency scales with ROS generation and is highly affected by the material of electrodes and by the applied pulsing protocols. We present experimental evidence that metal ions, and not the pH fronts near the electrodes, play a major role in generation of ROS during electroporation.