Alleviation of radiation combined skin injury in rat model by topical application of ascorbate formulation.

PURPOSE: This research endeavor was undertaken to elucidate the impact of an innovative ascorbate formulation on the regeneration process of full-thickness excision wounds in a rat model exposed to whole-body gamma irradiation, replicating conditions akin to combat or radiation emergency scenarios. MATERIALS AND METHODS: We established a comprehensive rat model by optimizing whole body γ-radiation doses (5-9 Gy) and full-thickness excision wound sizes (1-3 cm2) to mimic radiation combined injury (RCI). The developed RCI model was used to explore the healing potential of ascorbate formulation. The study includes various treatment groups (i.e., sham control, radiation alone, wound alone, radiation + wound, and radiation + wound + formulation). The ascorbate formulation was applied twice daily, with a 12-hour gap between each application, starting 1 hour after the initiation of the wound. The healing potential of the formulation in the RCI context was evaluated over 14 days through hematological, molecular, and histological parameters. RESULTS: The combination of a 5 Gy radiation dose and a 1 cm2 wound was identified as the optimal setting to develop the RCI model for subsequent studies. The formulation was used topically immediately following RCI, and then twice daily until complete healing. Treatment with the ascorbate formulation yielded noteworthy outcomes and led to a substantial reduction (p < .05) in the wound area, accelerated epithelialization periods, and an increased wound contraction rate. The formulation's localized healing response improved organ weights, normalized blood parameters, and enhanced hematopoietic and immune systems. A gene expression study revealed the treatment up-regulated TGF-ß and FGF, and down-regulated PDGF-α, TNF-α, IL-1ß, IL-6, MIP-1α, and MCP-1 (p < .05). Histopathological assessments supported the formulation's effectiveness in restoring cellular architecture and promoting tissue regeneration. CONCLUSION: Topical application of the ascorbate formulation in RCI resulted in a significant improvement in delayed wound healing, leading to accelerated wound closure by mitigating the expression of inflammatory responses.

Early cutaneous inflammatory response at different degree of burn and its significance for clinical diagnosis and management.

A complete characterization of the burn wound based on cutaneous architectural changes and inflammatory response is extremely important to provide evidence for progressive changes in the burn wound. Burn wounds are highly susceptible to conversion into deeper wounds, which need special care and attention; thereby, the complete characterization of burn wound type and their subsequent inflammatory status in the cutaneous system at the earliest is of paramount importance. Inflammatory markers at different degrees will help clinicians devise better and more specific treatment strategies for each burn type. The present study is carried out to profile pro-inflammatory gene expression along with immune cell quantification, vascular perfusion, and histopathological assessment in the cutaneous system of murine models. The study revealed that burn injury caused an immediate increase in vascular perfusion in superficial and partial-thickness burns, whereas there was a decrease in vascular perfusion in full-thickness burns. An influx of lymphocytes at the edges of burn wounds in each type of burn injury was well-orchestrated with the event of vascular perfusion. Further, pro-inflammatory gene expression profiling revealed significant upregulation vis-à-vis upregulation of TNF-α and MCP-1 genes, with an increase in the number of neutrophils following 72 h of injury that evidently cemented the conversion of superficial burn into partial-thickness burn. The molecular findings were profoundly supported by the histopathological changes. Thus, our foundational studies show distinct characteristic cutaneous changes correlated with the expression of key pro-inflammatory genes in three different types of burn injuries. Characterization of these cutaneous inflammatory responses provides a promising future for medical interventions involved with different degrees of burn injury, and it will also help in the pre-clinical testing of therapies for burn injury.

Ascorbate formulation improves healing efficacy in excisional wound mice model through interplay between pro and anti-inflammatory cytokines and angiogenic markers.

BACKGROUND AND OBJECTIVE: Biomedical research in regenerative medicine prompts researchers to formulate cost-effective therapeutics for wound healing. The present study was conducted to characterize the ascorbate based formulation vis-a-vis investigating the molecular dynamics of the formulation. MATERIALS AND METHODS: To characterize the formulation, particle size, zeta potential, thermal stability, compatibility, anti-oxidant, and permeation prospective were measured using standard protocols. The in-vitro healing potential and safety formulae were evaluated using the L929 cell line. For molecular unravelling of the pharmacodynamics of formulation, an excision wound model was used, and 54 mice were randomly and equally divided into three groups, i.e., untreated, betadine-treated, and formulation-treated, to ascertain the interplay between cytokines and chemokines and their culminative role in the release of growth factors. RESULTS: The ascorbate formulae were found to be amorphous, biocompatible, safe, and long-lasting, with particle sizes and zeta potentials of 389.7 ± 0.69 nm and -38.1 ± 0.65 mV, respectively, and anti-oxidative potential. An in-vitro study revealed that the formulation has a significant (p<0.05) migration potential and is non-toxic. Expression profiling of TGF-ß, FGF-2, VEGF, and collagen III & I showed significant (p<0.05) up-regulation, whereas significant (p<0.05) down-regulation of pro-inflammatory genes like IL-1α, IL-1ß, TNF-α, IL-6, and temporal change in CCR-5 was observed in formulae-treated animals as compared to other groups. CONCLUSION: By up-regulating angiogenic and collagen-promoting growth factor gene expression while down-regulating pro-inflammatory gene expression, ascorbate formulation promotes wound healing via extracellular matrix and granulation tissue deposition with significant improvement in tensile strength.

Bioactive Nonthermal Biocompatible Plasma Enhances Migration on Human Gingival Fibroblasts.

This study hypothesizes that the application of low-dose nonthermal biocompatible dielectric barrier discharge plasma (DBD-NBP) to human gingival fibroblasts (HGFs) will inhibit colony formation but not cell death and induce matrix metalloproteinase (MMP) expression, extracellular matrix (ECM) degradation, and subsequent cell migration, which can result in enhanced wound healing. HGFs treated with plasma for 3 min migrate to each other across the gap faster than those in the control and 5-min treatment groups on days 1 and 3. The plasma-treated HGFs show significantly high expression levels of the cell cycle arrest-related p21 gene and enhanced MMP activity. Focal adhesion kinase (FAK) mediated attenuation of wound healing or actin cytoskeleton rearrangement, and plasma-mediated reversal of this attenuation support the migratory effect of DBD-NBP. Further, this work performs computer simulations to investigate the effect of oxidation on the stability and conformation of the catalytic kinase domain (KD) of FAK. It is found that the oxidation of highly reactive amino acids (AAs) Cys427, Met442, Cys559, Met571, Met617, and Met643 changes the conformation and increases the structural flexibility of the FAK protein and thus modulates its function and activity. Low-dose DBD-NBP-induces host cell cycle arrest, ECM breakdown, and subsequent migration, thus contributing to the enhanced wound healing process.

Combined radiation burn injuries: A note.

Combined radiation injury occurs when radiation is accompanied by any other form of trauma. The past experiences of Hiroshima, Nagasaki, and Chernobyl have revealed that a large number of victims of such nuclear accidents or attacks suffer from combined radiation injuries. The possibility of a nuclear attack seems very far-fetched, but the destruction that would occur in such an event would be massive, with a huge lossof lives. Therefore, preparedness for the same should be done beforehand. The severity of combined radiation depends upon various factors, such as radiation dose, type, tissues affected, and traumas. The article focuses on combined radiation burn injury (CRBI) which may arise due to the combination of ionising radiation with thermal burns. CRBI can have varied effects on different organs like the hematopoietic, digestive, lymphatic, cardiovascular, and respiratory systems. Some of the most profound lethal effects are hematopoietic dysfunction, gastrointestinal leakage, bacterial translocation to other organ sites, pulmonary fibrosis, and pneumonitis. In this article, we have attempted to accumulate the knowledge of ongoing research on the functioning of different organ systems, which are affected due to CRBI and possible countermeasures to minimize the effects, thus improving survival.

Possible Synergies of Nanomaterial-Assisted Tissue Regeneration in Plasma Medicine: Mechanisms and Safety Concerns.

Cold atmospheric plasma and nanomedicine originally emerged as individual domains, but are increasingly applied in combination with each other. Most research is performed in the context of cancer treatment, with only little focus yet on the possible synergies. Many questions remain on the potential of this promising hybrid technology, particularly regarding regenerative medicine and tissue engineering. In this perspective article, we therefore start from the fundamental mechanisms in the individual technologies, in order to envision possible synergies for wound healing and tissue recovery, as well as research strategies to discover and optimize them. Among these strategies, we demonstrate how cold plasmas and nanomaterials can enhance each other's strengths and overcome each other's limitations. The parallels with cancer research, biotechnology and plasma surface modification further serve as inspiration for the envisioned synergies in tissue regeneration. The discovery and optimization of synergies may also be realized based on a profound understanding of the underlying redox- and field-related biological processes. Finally, we emphasize the toxicity concerns in plasma and nanomedicine, which may be partly remediated by their combination, but also partly amplified. A widespread use of standardized protocols and materials is therefore strongly recommended, to ensure both a fast and safe clinical implementation.

Modulating the Antioxidant Response for Better Oxidative Stress-Inducing Therapies: How to Take Advantage of Two Sides of the Same Medal?

Oxidative stress-inducing therapies are characterized as a specific treatment that involves the production of reactive oxygen and nitrogen species (RONS) by external or internal sources. To protect cells against oxidative stress, cells have evolved a strong antioxidant defense system to either prevent RONS formation or scavenge them. The maintenance of the redox balance ensures signal transduction, development, cell proliferation, regulation of the mechanisms of cell death, among others. Oxidative stress can beneficially be used to treat several diseases such as neurodegenerative disorders, heart disease, cancer, and other diseases by regulating the antioxidant system. Understanding the mechanisms of various endogenous antioxidant systems can increase the therapeutic efficacy of oxidative stress-based therapies, leading to clinical success in medical treatment. This review deals with the recent novel findings of various cellular endogenous antioxidant responses behind oxidative stress, highlighting their implication in various human diseases, such as ulcers, skin pathologies, oncology, and viral infections such as SARS-CoV-2.

Evaluation of Decontamination Efficacy of Electrolytically Generated Hypochlorous Acid for the Vesicating Agent: A Multimodel Study.

BACKGROUND: Sulfur Mustard is a strong vesicant and chemical warfare agent that imposes toxicity to the lungs, eyes, and skin after accidental or intended exposure. OBJECTIVES: The current study was intended to explore in vitro and in vivo decontamination properties of electrolytically generated HOCl (hypochlorous acid) against CEES (2-chloroethyle ethyle sulphide), a known sulfur mustard simulant & vesicating agent. METHODS: in vitro studies were carried out using UV spectroscopy and GC-MS methods. In vivo studies were performed in Strain A and immune-compromised mice by subcutaneous as well as prophylactic topical administration of HOCl pretreated CEES. The blister formation and mortality were considered as end-point. Histopathological study was conducted on skin samples by H & E method. DNA damage studies measuring γ-H2AX and ATM have been carried out in human blood using flow cytometry. Anti-bacterial action was tested by employing broth micro dilution methods. A comparative study was also carried out with known oxidizing agents. RESULTS: The topical application of pre-treated CEES at 5, 30 min and 1 h time points showed significant (p<0.001) inhibition of blister formation. DNA damage study showed reduced mean fluorescence intensity of DSBs nearly 17-20 times, suggesting that HOCl plays a protective role against DNA damage. Histopathology showed no sign of necrosis in the epidermis upto 5 min although moderate changes were observed at 30 min. Pretreated samples were analyzed for detection of reaction products with m/z value of 75.04, 69.08, 83.93, 85.95, 123.99, 126.00, and 108.97. HOCl showed a strong bactericidal effect at 40 ppm. The absorbance spectra of HOCl treated CEES showed lowered peaks in comparison to CEES alone and other oxidizing agents. CONCLUSION: In a nutshell, our results signify the decontamination role of HOCl for biological surface application.

Evaluation of non-thermal effect of microwave radiation and its mode of action in bacterial cell inactivation.

A growing body of literature has recognized the non-thermal effect of pulsed microwave radiation (PMR) on bacterial systems. However, its mode of action in deactivating bacteria has not yet been extensively investigated. Nevertheless, it is highly important to advance the applications of PMR from simple to complex biological systems. In this study, we first optimized the conditions of the PMR device and we assessed the results by simulations, using ANSYS HFSS (High Frequency Structure Simulator) and a 3D particle-in-cell code for the electron behavior, to provide a better overview of the bacterial cell exposure to microwave radiation. To determine the sensitivity of PMR, Escherichia coli and Staphylococcus aureus cultures were exposed to PMR (pulse duration: 60 ns, peak frequency: 3.5 GHz) with power density of 17 kW/cm2 at the free space of sample position, which would induce electric field of 8.0 kV/cm inside the PBS solution of falcon tube in this experiment at 25 °C. At various discharges (D) of microwaves, the colony forming unit curves were analyzed. The highest ratios of viable count reductions were observed when the doses were increased from 20D to 80D, which resulted in an approximate 6 log reduction in E. coli and 4 log reduction in S. aureus. Moreover, scanning electron microscopy also revealed surface damage in both bacterial strains after PMR exposure. The bacterial inactivation was attributed to the deactivation of oxidation-regulating genes and DNA damage.

Covalent Cysteine Targeting of Bruton's Tyrosine Kinase (BTK) Family by Withaferin-A Reduces Survival of Glucocorticoid-Resistant Multiple Myeloma MM1 Cells.

Multiple myeloma (MM) is a hematological malignancy characterized by plasma cells' uncontrolled growth. The major barrier in treating MM is the occurrence of primary and acquired therapy resistance to anticancer drugs. Often, this therapy resistance is associated with constitutive hyperactivation of tyrosine kinase signaling. Novel covalent kinase inhibitors, such as the clinically approved BTK inhibitor ibrutinib (IBR) and the preclinical phytochemical withaferin A (WA), have, therefore, gained pharmaceutical interest. Remarkably, WA is more effective than IBR in killing BTK-overexpressing glucocorticoid (GC)-resistant MM1R cells. To further characterize the kinase inhibitor profiles of WA and IBR in GC-resistant MM cells, we applied phosphopeptidome- and transcriptome-specific tyrosine kinome profiling. In contrast to IBR, WA was found to reverse BTK overexpression in GC-resistant MM1R cells. Furthermore, WA-induced cell death involves covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including inhibition of the hyperactivated BTK. Covalent interaction between WA and BTK could further be confirmed by biotin-based affinity purification and confocal microscopy. Similarly, molecular modeling suggests WA preferably targets conserved cysteines in the Hinge-6 region of the kinase cysteinome classification, favoring inhibition of multiple B-cell receptors (BCR) family kinases. Altogether, we show that WA's promiscuous inhibition of multiple BTK family tyrosine kinases represents a highly effective strategy to overcome GC-therapy resistance in MM.

Oxidative damage to hyaluronan-CD44 interactions as an underlying mechanism of action of oxidative stress-inducing cancer therapy.

Multiple cancer therapies nowadays rely on oxidative stress to damage cancer cells. Here we investigated the biological and molecular effect of oxidative stress on the interaction between CD44 and hyaluronan (HA), as interrupting their binding can hinder cancer progression. Our experiments demonstrated that the oxidation of HA decreased its recognition by CD44, which was further enhanced when both CD44 and HA were oxidized. The reduction of CD44-HA binding negatively affected the proliferative state of cancer cells. Our multi-level atomistic simulations revealed that the binding free energy of HA to CD44 decreased upon oxidation. The effect of HA and CD44 oxidation on CD44-HA binding was similar, but when both HA and CD44 were oxidized, the effect was much larger, in agreement with our experiments. Hence, our experiments and computations support our hypothesis on the role of oxidation in the disturbance of CD44-HA interaction, which can lead to the inhibition of proliferative signaling pathways inside the tumor cell to induce cell death.

Cold Atmospheric Plasma Increases Temozolomide Sensitivity of Three-Dimensional Glioblastoma Spheroids via Oxidative Stress-Mediated DNA Damage.

Glioblastoma multiforme (GBM) is the most frequent and aggressive primary malignant brain tumor in adults. Current standard radiotherapy and adjuvant chemotherapy with the alkylating agent temozolomide (TMZ) yield poor clinical outcome. This is due to the stem-like properties of tumor cells and genetic abnormalities in GBM, which contribute to resistance to TMZ and progression. In this study, we used cold atmospheric plasma (CAP) to enhance the sensitivity to TMZ through inhibition of antioxidant signaling (linked to TMZ resistance). We demonstrate that CAP indeed enhances the cytotoxicity of TMZ by targeting the antioxidant specific glutathione (GSH)/glutathione peroxidase 4 (GPX4) signaling. We optimized the threshold concentration of TMZ on five different GBM cell lines (U251, LN18, LN229, U87-MG and T98G). We combined TMZ with CAP and tested it on both TMZ-sensitive (U251, LN18 and LN229) and TMZ-resistant (U87-MG and T98G) cell lines using two-dimensional cell cultures. Subsequently, we used a three-dimensional spheroid model for the U251 (TMZ-sensitive) and U87-MG and T98G (TMZ-resistant) cells. The sensitivity of TMZ was enhanced, i.e., higher cytotoxicity and spheroid shrinkage was obtained when TMZ and CAP were administered together. We attribute the anticancer properties to the release of intracellular reactive oxygen species, through inhibiting the GSH/GPX4 antioxidant machinery, which can lead to DNA damage. Overall, our findings suggest that the combination of CAP with TMZ is a promising combination therapy to enhance the efficacy of TMZ towards the treatment of GBM spheroids.

Physical plasma-derived oxidants sensitize pancreatic cancer cells to ferroptotic cell death.

Despite modern therapeutic advances, the survival prospects of pancreatic cancer patients remain poor, due to chemoresistance and dysregulated oncogenic kinase signaling networks. We applied a novel kinome activity-mapping approach using biological peptide targets as phospho-sensors to identify vulnerable kinase dependencies for therapy sensitization by physical plasma. Ser/Thr-kinome specific activity changes were mapped upon induction of ferroptotic cell death in pancreatic tumor cells exposed to reactive oxygen and nitrogen species of plasma-treated water (PTW). This revealed a broad kinome activity response involving the CAMK, the AGC and CMGC family of kinases. This systems-level kinome network response supports stress adaptive switches between chemoresistant anti-oxidant responses of Kelch-like ECH-associated protein 1 (KEAP1)/Heme Oxygenase 1 (HMOX1) and ferroptotic cell death sensitization upon suppression of Nuclear factor (erythroid derived 2)-like 2 (NRF2) and Glutathione peroxidase 4 (GPX4). This is further supported by ex vivo experiments in the chicken chorioallantoic membrane assay, showing decreased GPX4 and Glutathione (GSH) expression as well as increased lipid peroxidation, along with suppressed BxPC-3 tumor growth in response to PTW. Taken all together, we demonstrate that plasma treated water-derived oxidants sensitize pancreatic cancer cells to ferroptotic cell death by targeting a NRF2-HMOX1-GPX4 specific kinase signaling network.

Challenges with Wound Infection Models in Drug Development.

Wound research is an evolving science trying to unfold the complex untold mechanisms behind the wound healing cascade. In particular, interest is growing regarding the role of microorganisms in both acute and chronic wound healing. Microbial burden plays an important role in the persistence of chronic wounds, ultimately resulting in delayed wound healing. It is therefore important for clinicians to understand the evolution of infection science and its various etiologies. Therefore, to understand the role of bacterial biofilm in chronic wound pathogenesis, various in vitro and in vivo models are required to investigate biofilms in wound-like settings. Infection models should be refined comprising an important signet of biofilms. These models are eminent for translational research to obtain data for designing an improved wound care formulation. However, all the existing models possess limitations and do not fit properly in the model frame for developing wound care agents. Among various impediments, one of the major drawbacks of such models is that the wound they possess does not mimic the wound a human develops. Therefore, a novel wound infection model is required which can imitate the human wounds. This review article mainly discusses various in vitro and in vivo models showing microbial colonization, their advantages and challenges. Apart from these models, there are also present ex vivo wound infection models, but this review mainly focused on various in vitro and in vivo models available for studying wound infection in controlled conditions. This information might be useful in designing an ideal wound infection model for developing an effective wound healing formulation.

Creation of rapid and reproducible burn in animal model with a newly developed burn device.

BACKGROUND: The currently available practices for creation of burns in the animals are mostly manual which may lead to lack of uniform wound. There is a need to develop a suitable device that could reproduce and uniformly create burn wound in animal models without the procedural variations and human variability. Present study deals with development of a burn device which has been designed for creation of animal models for burn injury. METHODS: The designed burn device comprises of two main components a heating metal stylus and the thermal sensor. Metal stylus consists of two parts with top part acts as handle and bottom part contains the aluminum probe which quickly heats and cool. The temperature monitoring sensor is attached adjacent to the tip of the probe. The temperature and timer are digitally displayed and can be adjusted as per the requirement. This device is tested for creation of uniform burn in the mice model. Animals were divided into different groups and thermal burn was generated for 60 °C, 80 °C & 100 °C respectively. Burn wound was generated dorsally on shaved skin with contact time of 40 s. Skin biopsies of burn wound were collected after 24 h for histopathology analysis to determine the burn characteristics. Blood vessels damage in the skin was detected by transillumination and digital segmentation using VesSeg tool. RESULTS: The device is able to deliver the different temperature to the animal skin. After reaching the 60 °C, 80 °C & 100 °C for 40 s respectively electronic relays shut down the device. The different groups of the animals showed significant difference in burn morphology in temperature dependent manner. Non significant variation in the burn area of different experimental groups animals was observed. All three zones vis-a-vis coagulation, stasis and hyperaemia were observed at 100 °C whereas found indistinct in 80 °C and 60 °C treated groups. Histopathological studies clearly demonstrated the differences in damage induction in stratum corneum, epidermis, dermis, collagen and hair follicle at selected thermal points. Severe blood vessels damage was observed only at 100 °C. The vascular perfusion was recorded 14% and 57% higher in 60 °C and 80 °C treated animals respectively when compared to control animals. However, at 100 °C due to highest vessel damage the perfusion was reduced to 53% compared to control. CONCLUSION: Present study demonstrates that the device is able to generate precise and uniform burn wound in mice model. The device may be useful for burn related studies and validation of burn wound care products.

Can miRNAs Serve as Potential Markers in Thermal Burn Injury: An In Silico Approach.

Burn injury has been a major cause of morbidity at global levels. They can occur by multiple agents, such as thermal radiation and chemicals. Among all, thermal burn is predominant and may require specialized treatment in some patients. Although various biomarkers are reportedly used in thermal burn for understanding the pathophysiology of the injury, their limitations prompt for the search of suitable markers that can address the depth and severity of the burn. MicroRNAs (miRNAs) are conserved noncoding molecules that seem to be the promising marker due to their role in multiple pathways and participation in different physiological processes of the body. The present review highlights the role of miRNAs in the repair of the wound and their interaction with specific genes in response to burn stress. Key miR candidates include miR-21, miR-29a, miR-378a-5p, miR-100, miR-27b, miR-200c, miR-150, miR-499-5p, miR-92a, miR-194, and miR-146b, which are identified for their respective targets involved in wound repair. Furthermore, bioinformatics and computational tools were used to confirm the miRNAs and their specific targets. Gene and miRNA expression data sets were downloaded from Research Collaboratory for Structural Bioinformatics Protein Data Bank Database and RNAComposer, respectively, and docked by PatchDock. The possible implications of the identified miRNAs could be in understanding the mechanism of burn injury. These can also be studied with the available drugs being used for burn injury. Apart from that, new intended molecules may also be tested for their effect on these miRNAs.

Vascular perfusion: A predictive tool for thermal burn injury.

Damage to the blood vascular system and their altered permeability is a prominent pathological event in case of dermal injury, particularly in burn trauma situations. Prediction of vascular perfusion at the site of damage could be an attractive tool in the estimation of thermal burn injury. A very few reports are available with reference to this tool in defining the thermal injury status. We have used the vascular perfusion estimation method as a tool in assessing the severity of thermal damage to the animal skin. To validate this tool, the mice were subjected to the thermal burn at 90 °C for 10, 20 & 30 s and excised burned skin samples were analyzed for vascular perfusion 24hr post-burn treatment. The vascular perfusion was significantly altered in a time-dependent fashion. This method also provided information regarding blood vessel damage at varied time points. The results of this study clearly indicate the severity of skin damage by the thermal burn. The finding of the present study could have greater implications in predicting the degree of burn. This method is very simple and cost-effective compared to other available modalities used for the estimation of thermal burn injury. The method certainly has the benefit of the estimation of burn injury in the animal models.

Synergistic Effects of Melittin and Plasma Treatment: A Promising Approach for Cancer Therapy.

Melittin (MEL), a small peptide component of bee venom, has been reported to exhibit anti-cancer effects in vitro and in vivo. However, its clinical applicability is disputed because of its non-specific cytotoxicity and haemolytic activity in high treatment doses. Plasma-treated phosphate buffered saline solution (PT-PBS), a solution rich in reactive oxygen and nitrogen species (RONS) can disrupt the cell membrane integrity and induce cancer cell death through oxidative stress-mediated pathways. Thus, PT-PBS could be used in combination with MEL to facilitate its access into cancer cells and to reduce the required therapeutic dose. The aim of our study is to determine the reduction of the effective dose of MEL required to eliminate cancer cells by its combination with PT-PBS. For this purpose, we have optimised the MEL threshold concentration and tested the combined treatment of MEL and PT-PBS on A375 melanoma and MCF7 breast cancer cells, using in vitro, in ovo and in silico approaches. We investigated the cytotoxic effect of MEL and PT-PBS alone and in combination to reveal their synergistic cytological effects. To support the in vitro and in ovo experiments, we showed by computer simulations that plasma-induced oxidation of the phospholipid bilayer leads to a decrease of the free energy barrier for translocation of MEL in comparison with the non-oxidized bilayer, which also suggests a synergistic effect of MEL with plasma induced oxidation. Overall, our findings suggest that MEL in combination with PT-PBS can be a promising combinational therapy to circumvent the non-specific toxicity of MEL, which may help for clinical applicability in the future.

Bacterial inactivation by plasma treated water enhanced by reactive nitrogen species.

There is a growing body of literature that recognizes the importance of plasma treated water (PTW) for inactivation of microorganism. However, very little attention has been paid to the role of reactive nitrogen species (RNS) in deactivation of bacteria. The aim of this study is to explore the role of RNS in bacterial killing, and to develop a plasma system with increased sterilization efficiency. To increase the concentration of reactive oxygen and nitrogen species (RONS) in solution, we have used vapor systems (DI water/HNO3 at different wt%) combined with plasma using N2 as working gas. The results show that the addition of the vapor system yields higher RONS contents. Furthermore, PTW produced by N2 + 0.5 wt% HNO3 vapor comprises a large amount of both RNS and ROS, while PTW created by N2 + H2O vapor consists of a large amount of ROS, but much less RNS. Interestingly, we observed more deactivation of E. Coli with PTW created by N2 + 0.5 wt% HNO3 vapor plasma as compared to PTW generated by the other plasma systems. This work provides new insight into the role of RNS along with ROS for deactivation of bacteria.

Enhancement of cellular glucose uptake by reactive species: a promising approach for diabetes therapy.

It is generally known that antidiabetic activity is associated with an increased level of glucose uptake in adipocytes and skeletal muscle cells. However, the role of exogenous reactive oxygen and nitrogen species (RONS) in muscle development and more importantly in glucose uptake is largely unknown. We investigate the effect of RONS generated by cold atmospheric plasma (CAP) in glucose uptake. We show that the glucose uptake is significantly enhanced in differentiated L6 skeletal muscle cells after CAP treatment. We also observe a significant increase of the intracellular Ca++ and ROS level, without causing toxicity. One of the possible reasons for an elevated level of glucose uptake as well as intracellular ROS and Ca++ ions is probably the increased oxidative stress leading to glucose transport.