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.

Negative effects of cancellation during nanosecond range High-Frequency calcium based electrochemotherapy in vitro.

Drug delivery using nanosecond pulsed electric fields is a new branch of electroporation-based treatments, which potentially can substitute European standard operating procedures for electrochemotherapy. In this work, for the first time, we characterize the effects of ultra-fast repetition frequency (1-2.5 MHz) nanosecond pulses (5-9 kV/cm, 200 and 400 ns) in the context of nano-electrochemotherapy with calcium. Additionally, we investigate the feasibility of bipolar symmetric (↑200 ns + ↓200 ns) and asymmetric (↑200 ns + ↓400 ns) nanosecond protocols for calcium delivery. The effects of bipolar cancellation and the influence of interphase delay (200 ns) are overviewed. Human lung cancer cell lines A549 and H69AR were used as a model. It was shown that unipolar pulses delivered at high frequency are effective for electrochemotherapy with a significant improvement in efficiency when the delay between separate pulses is reduced. Bipolar symmetric pulses trigger the cancellation phenomenon limiting applications for drug delivery and can be compensated by the asymmetry of the pulse (↑200 ns + ↓400 ns or ↑400 ns + ↓200 ns). The results of this study can be successfully used to derive a new generation of nsPEF protocols for successful electrochemotherapy treatments.

Susceptibility of various human cancer cell lines to nanosecond and microsecond range electrochemotherapy: Feasibility of multi-drug cocktails.

Electrochemotherapy (ECT) involves combining anticancer drugs with electroporation, which is induced by pulsed electric fields (PEFs), while the effects vary in effectiveness based on the specific parameters of the electrical pulses and susceptibility of the cells to a specific drug. In this work, we utilized conventional microsecond electroporation protocols (0.8 - 1.5 kV/cm × 100 µs × 8, 1 Hz) and the new modality of nanosecond pulses (4 and 8 kV/cm × 500 ns × 100, 1 kHz and 1 MHz), which are compressed into a high frequency burst. Sensitive and resistant lung, breast and ovarian human cancer cell lines were used in the study. In order to overcome drug-resistance, we have investigated the feasibility to use anticancer drug cocktails i.e., bleomycin and cisplatin combinations with metformin, vinorelbine and Dp44mT. The different susceptibility of various human cancer cells lines to electric pulses was determined, the efficacy of ECT was characterized and the type of cell death depending on the combinations of drugs was investigated. The results indicate that synergistic effects of PEFs with drug cocktails may be used to overcome drug-resistance in cancer, while the application of nsPEF provides more flexibility in parametric protocols and modulation of cancer cell death.

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.

Understanding the Immunomodulatory Effects of Bovine Colostrum: Insights into IL-6/IL-10 Axis-Mediated Inflammatory Control.

Bovine colostrum (COL), the first milk secreted by lactating cows postpartum, is a rich source of bioactive compounds that exert a significant role in the survival, growth, and immune development of neonatal calves. This study investigated the immunomodulatory effects of COL on cytokine production in vitro using a Caco-2/THP-1 macrophage co-culture model stimulated with Phorbol 12-myristate 13-acetate (PMA). COL pretreatment significantly reduced IL-6 (241.3 pg/mL) production induced by PMA (p < 0.05), while increasing IL-10 production (45.3 pg/mL), in comparison to PMA control (441.1 and 12.5 pg/mL, respectively). Further investigations revealed that the IL-6 suppressive effect of colostrum was heat-sensitive and associated with components of higher molecular mass (100 kDa). Moreover, colostrum primarily influenced THP-1 macrophages rather than Caco-2 epithelial cells. The effects of colostrum on IL-6 production were associated with reduced NF-κB activation in THP-1 macrophages. In calf-FMT transplanted C57BL/6 murine model, colostrum decreased intestinal permeability, reduced immune cell infiltration and intestinal score, and suppressed IL-6 (142.0 pg/mL) production during S. typhimurium infection, in comparison to control animals (215.2 pg/mL). These results suggest the immunomodulatory activity of bovine colostrum and its potential applications in inflammatory disorders. Further studies are needed to elucidate the underlying mechanisms and validate the findings in bovine models.

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.

The Influence of Feeding with Colostrum and Colostrum Replacer on Major Blood Biomarkers and Growth Performance in Dairy Calves.

Bovine colostrum (BC) is the first milk produced by lactating cows after parturition. BC is rich in various amino acids, proteins, and fats essential for the nutrition of the neonate calves. Despite the evident beneficial effect of BC on calves, the effect of BC on blood biomarkers is poorly understood. Calves that received BC showed significantly higher body mass at days 7 and 30 (38.54 kg and 43.42 kg, respectively) compared to the colostrum replacer group (p = 0.0064). BC induced greater quantities of blood neutrophils (0.27 × 109/L) and monocytes (4.76 × 109/L) in comparison to the colostrum replacer (0.08 and 0.06 × 109/L, respectively) (p = 0.0001). Animals that received BC showed higher levels of total serum protein (59.16 g/L) and albumin (29.96 g/L) in comparison to the colostrum replacer group (44.34 g/L and 31.58 g/L, respectively). In addition, BC induced greater intestinal mucus production in the Wistar rat model. Collectively, these results demonstrate that BC is important for the growth of calves and that it provides a significant beneficial effect on morphological and biochemical blood parameters.