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.

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.

Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms.

Extracellular vesicles (EVs) effectively suppress neuroinflammation and induce neuroprotective effects in different disease models. However, the mechanisms by which EVs regulate the neuroinflammatory response of microglia remains largely unexplored. Here, we addressed this issue by testing the action of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on immortalized human microglial cells. We found that EVs induced a rapid increase in intracellular Ca2+ and promoted significant ATP release in microglial cells after 20 min of treatment. Boyden chamber assays revealed that EVs promoted microglial migration by 20%. Pharmacological inhibition of different subtypes of purinergic receptors demonstrated that EVs activated microglial migration preferentially through the P2X4 receptor (P2X4R) pathway. Proximity ligation and co-immunoprecipitation assays revealed that EVs promote association between milk fat globule-epidermal growth factor-factor VIII (MFG-E8) and P2X4R proteins. Furthermore, pharmacological inhibition of αVß3/αVß5 integrin suppressed EV-induced cell migration and formation of lipid rafts in microglia. These results demonstrate that EVs promote microglial motility through P2X4R/MFG-E8-dependent mechanisms. Our findings provide novel insights into the molecular mechanisms through which EVs target human microglia that may be exploited for the development of new therapeutic strategies targeting disease-associated neuroinflammation.

MicroRNA-124 acts as a positive regulator of IFN-ß signaling in the lipopolysaccharide-stimulated human microglial cells.

miR-124 is ubiquitously expressed in the nervous tissue and acts as a negative regulator of neuroinflammation. In the present study, we analyzed the possible role of miR-124 in response to LPS in the human microglial cell line. Our data revealed that the miR-124 anti-inflammatory effect is based not only on the suppression of MyD88 - NFκB pathway and downregulation of pro-inflammatory cytokines IL-1ß and IL-6 but also on the enhancement of TRAM-TRIF signaling and increased IFN-ß expression. Furthermore, the NFκB reporter assay demonstrated that specific miR-124 - induced NFκB activity changes could be detected only using NFκB reporter promoters lacking ATF/CREB binding site.

Detection of intracellular biomarkers in viable cells using millisecond pulsed electric fields.

Reversible electroporation is a temporary permeabilization of cell membrane through the formation of transient pores created by short high voltage electric pulses. This method has numerous applications in biology and biotechnology and has become an important technique in molecular medicine. Reversible electroporation is usually used to transfer macromolecules into the cells. However, the delivery of large molecules such as proteins into cells without loss of cell viability remains a challenge. In our study, we investigated whether electroporation can be used for this purpose. The study was performed with the primary mouse splenocytes and Jurkat cell line. The electroporation efficacy was evaluated by flow cytometry. We used the reversible electroporation for intracellular marker detection investigating antibody and fluorescein-conjugated dextran transfer efficiency, cell viability and metabolic activity. We have found that reversible electroporation parameters can be optimized for efficient transfer of large molecules such as antibodies/proteins into live cells without a significant loss of cell viability. We conclude that a well-established and relatively easy method of reversible electroporation can be adjusted to detect intracellular biomarkers in viable cells. This is a new approach on how electroporation could be utilised in medicine and biological research to detect rare subpopulations of cells that produce specific markers and to keep cells viable. This would allow the use of these rare subpopulations of isolated cells for further research and personalized medicine.

Regenerative potential of partially differentiated mesenchymal stromal cells in a mouse model of a full-thickness skin wound.

Mesenchymal stromal cells (MSCs, known as mesenchymal stem cells) are considered to be a promising therapeutic tool for many diseases. But it is still unclear which cells are more efficient and safe for wound healing and tissue regeneration for clinical applications: undifferentiated, partially differentiated stem cells or differentiated cells. In this study, we modified MSCs with keratinocyte-conditioned medium (KCM) and examined MSCs, partially differentiated MSCs (PMSCs) and differentiated cell migration, accumulation in the wounded area as well as cell regenerative efficiency in a full-thickness skin wound model. In addition to that, the impact of intradermal and intravenous cell delivery methods of wound healing was evaluated. C57BL/6J mouse compact bone MSCs were treated with a KCM for 14 days. Flow cytometry analysis showed the appearance of keratinocyte surface markers which were absent in MSCs, whereas the specific markers for MSCs were lost. Cells were injected either intravenously or intradermally in C57BL/6J mice. Wound closure, cell migration and accumulation in the wounded area were further analysed. Wound healing was assessed by the rate of wound closure and by histological evaluation. Cells were monitored using optical imaging. We demonstrated that PMSCs showed morphology similar to keratinocyte cells, had enhanced migration and increased survival at the site of injury. PMSCs had a beneficial effect on wound healing and tissue regeneration. This effect was reinforced when these cells were injected intravenously. Due to their partial differentiation status, we assume that PMSCs can differentiate more rapidly into epidermal cell lineages thus causing faster and qualitatively improved wound healing.

Bone marrow-derived lineage-negative cells accelerate skin regeneration in vivo.

Cell-based therapy is a promising strategy for promoting tissue regeneration when conventional treatments are not effective. ehT choice of the accessible source to obtain a sufficient cell amount and the use of suitable biomaterials to improve the cell delivery efficiency are the main tasks for safe, effective, and reliable application of stem cell therapy. In this study, we have compared the influence of bone marrow-derived Lin¯ cells on skin regeneration after local transplantation with or without type I collagen-based gel in a BALB/c mice full-thickness wound model. Lin¯ cells were isolated using magnetic-associated cell sorting and identified by flow cytometry. Cytokine gene expression was examined using real-time PCR. Our results show that the bone marrow-derived Lin¯ cell population demonstrates the properties to stimulate the skin tissue regeneration. Significant accelerated wound closure was revealed after cell transplantation (P < 0.05). Histological analysis indicated the earliest inhibition of inflammation, accelerated reepithelialization, and evenly distributed skin appendages in the neodermis after Lin¯ cell transplantation with type I collagen gel. eTh significant changes in mRNA levels of cytokines TNF-α, IL-10, TGF-ß, and VEGF after Lin¯ cell transplantation were confirmed by RT-PCR (P < 0.05). eTh ability to positively control the reactions taking place during the wound healing process gives the advantage to the bone marrow Lin¯ cell population to be used as a cell source for therapy.

Caspase dependent apoptosis induced in yeast cells by nanosecond pulsed electric fields.

Saccharomyces cerevisiae yeast cells were used as a model organism to investigate the effects of various pulsed electric fields on the programed death of such cells. These were exposed to electric field pulses with field strengths (E) of up to 220kV/cm. The effects of square shaped pulses having different durations (τ=10-90ns) and different pulse numbers (pn=1-5) were then analysed. The obtained results show that nanosecond pulses can induce the death of such cells, which in turn is dependent on the electric field pulse parameters and increase with the rise in E, τ and pn. The decrease of the cells' viability was accompanied by an increase in the active form of intracellular yeast metacaspases. It was thus shown that nanosecond electric field pulses induced the caspase-dependent yeast cell death.

Quantitative evaluation of the transplanted lin(-) hematopoietic cell migration kinetics.

Stem cells take part in organogenesis, cell maturation and injury repair. The migration is necessary for each of these functions to occur. The aim of this study was to investigate the kinetics of transplanted hematopoietic lin(-) cell population (which consists mainly of the stem and progenitor cells) in BALB/c mouse contact hypersensitivity model and quantify the migration to the site of inflammation in the affected foot and other healthy organs. Quantitative analysis was carried out with the real-time polymerase chain reaction method. Spleen, kidney, bone marrow, lung, liver, damaged and healthy foot tissue samples at different time points were collected for analysis. The quantitative data normalization was performed according to the comparative quantification method. The analysis of foot samples shows the significant migration of transplanted cells to the recipient mice affected foot. The quantity was more than 1000 times higher, as compared with that of the untreated foot. Due to the inflammation, the number of donor origin cells migrating to the lungs, liver, spleen and bone marrow was found to be decreased. Our data shows that transplanted cells selectively migrated into the inflammation areas of the foot edema. Also, the inflammation caused a secondary migration in ectopic spleen of hematopoietic stem cell niches and re-homing from the spleen to the bone marrow took place.

Exosomes from Human Dental Pulp Stem Cells Suppress Carrageenan-Induced Acute Inflammation in Mice.

The primary goal of this study was to examine the effects of human dental pulp stem cell-derived exosomes on the carrageenan-induced acute inflammation in mice. Exosomes were purified by differential ultracentrifugation from the supernatants of stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) cultivated in serum-free medium. At 1 h post-carrageenan injection, exosomes derived from supernatants of 2 × 10(6) SHEDs were administered by intraplantar injection to BALB/c mice; 30 mg/kg of prednisolone and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Edema was measured at 6, 24, and 48 h after carrageenan injection. For the in vivo imaging experiments, AngioSPARK750, Cat B 750 FAST, and MMPSense 750 FAST were administered into the mouse tail vein 2 h post-carrageenan injection. Fluorescence images were acquired at 6, 24, and 48 h after edema induction by IVIS Spectrum in vivo imaging system. Exosomes significantly reduced the carrageenan-induced edema at all the time points studied (by 39.5, 41.6, and 25.6% at 6, 24, and 48 h after injection, respectively), to similar levels seen with the positive control (prednisolone). In vivo imaging experiments revealed that, both exosomes and prednisolone suppress activities of cathepsin B and matrix metalloproteinases (MMPs) at the site of carrageenan-induced acute inflammation, showing more prominent effects of prednisolone at the early stages, while exosomes exerted their suppressive effects gradually and at later time points. Our study demonstrates for the first time that exosomes derived from human dental pulp stem cells suppress carrageenan-induced acute inflammation in mice.

Impact of differently modified nanocrystalline diamond on the growth of neuroblastoma cells.

The aim of this study was to assess the impact of nanocrystalline diamond (NCD) thin coatings on neural cell adhesion and proliferation. NCD was fabricated on fused silica substrates by microwave plasma chemical vapor deposition (MPCVD) method. Different surface terminations were performed through exposure to reactive hydrogen and by UV induced oxidation during ozone treatment. Boron doped NCD coatings were also prepared and investigated. NCD surface wettability was determined by contact angle measurement. To assess biocompatibility of the NCD coatings, the neuroblastoma SH-SY5Y cell line was used. Cells were plated directly onto diamond surfaces and cultured in medium with or without fetal bovine serum (FBS), in order to evaluate the ability of cells to adhere and to proliferate. The obtained results showed that these cells adhered and proliferated better on NCD surfaces than on the bare fused silica. The cell proliferation on NCD in medium with and without FBS after 48h from plating was on average, respectively, 20 and 58% higher than that on fused silica, irrespective of NCD surface modification. Our results showed that the hydrogenated, oxygenated and boron-doped NCD coatings can be used for biomedical purposes, especially where good optical transparency is required.

Impact of diamond nanoparticles on neural cells.

Diamond nanoparticles (DNPs) are very attractive for biomedical applications, particularly for bioimaging. The aim of this study was to evaluate the impact of DNPs on neural cancer cells and thus to assess the possible application of DNPs for these cells imaging. For this purpose, the neuroblastoma SH-SY5Y cell line was chosen. Cells were cultured in medium with different concentrations (15, 50, 100 and 150 µg/ml) of DNPs. After 48 h of incubation, cell metabolic activity was evaluated by the XTT assay. For assessment of cellular metabolic activity, cells were also cultured on differently terminated nanocrystalline diamond (NCD) coatings in medium with 150 µg/ml of DNPs. Cell adhesion and morphology were evaluated by brightfield microscopy. Diamond nanoparticle internalization was determined by confocal microscopy. The obtained results showed that low concentrations (15, 50 and 100 µg/ml) of nanoparticles did not significantly affect the SH-SY5Y cell metabolic activity. However, a higher concentration (150 µg/ml) of DNPs statistically significantly reduced SH-SY5Y cell metabolic activity. After 48 h incubation with 150 µg/ml DNPs, cell metabolic activity was 23% lower than in medium without DNPs on standard tissue culture polystyrene.

Evaluation of cytotoxicity of polypyrrole nanoparticles synthesized by oxidative polymerization.

Polypyrrole (Ppy) is known as biocompatible material, which is used in some diverse biomedical applications and seeming to be a very promising for advanced biotechnological applications. In order to increase our understanding about biocompatibility of Ppy, in this study pure Ppy nanoparticles (Ppy-NPs) of fixed size and morphology were prepared by one-step oxidative polymerization and their cyto-compatibility was evaluated. The impact of different concentration of Ppy nanoparticles on primary mouse embryonic fibroblasts (MEF), mouse hepatoma cell line (MH-22A), and human T lymphocyte Jurkat cell line was investigated. Cell morphology, viability/proliferation after the treatment by Ppy nanoparticles was evaluated. Obtained results showed that Ppy nanoparticles at low concentrations are biocompatible, while at high concentrations they became cytotoxic for Jurkat, MEF and MH-22A cells, and it was found that cytotoxic effect is dose-dependent.

Skin regeneration with bone marrow-derived cell populations.

Bone marrow-derived cells of distinct differentiation level could differently influence the process of skin regeneration. The results of our study revealed that hematopoietic stem cells (HSC) population influenced the repair of injured tissue slower in comparison with lineage negative (lin⁻) cell population containing not only HSC but also cell progenitors of different differentiation levels. Wound healing process was faster in lin⁻) cell suspension treated group, the stage of proliferation was more intensive and increased number of skin appendages occurred. The adaptation of purified HSC at the site of injury was longer and the stages of wound healing took place later. The results obtained show that in further experiments the complex procedure of HSC isolation and purification could be shortened and heavy skin injuries could be successfully treated with the help of lin⁻ cell population.