Analysis of factors that contribute to and interfere with bactericidal properties of low-temperature atmospheric pressure plasmas

Bacterial resistance to antibiotics continues to be a significant concern globally. In the search for more effective antibacterial treatments, low temperature plasmas (LTPs) have arisen as an attractive alternative to traditional therapies. LTPs generate a cocktail of reactive nitrogen and oxygen species (RNOS), ultraviolet (UV) photons, electrons and electromagnetic fields. They therefore offer the potential to facilitate the localised delivery of bactericidal agents without contact to the treatment site. However, whether the distribution of RNOS in the gas phase, the presence of organic matter during treatment and the heterogeneity of bacterial populations affect the treatment outcome was unknown. Single-cell analysis was undertaken throughout to investigate the heterogeneous response of the model system Salmonella enterica to treatment with atmospheric-pressure plasma jets. In conjunction with the electrical, optical, and chemical studies, this enabled key mechanisms that drive plasma-bacteria interactions to be explored. My research has demonstrated three main points: (a) The level of DNA damage induced in single cells is determined by the spatial distribution of RNOS in the plasma effluent. This was found to be a characteristic of LTPs generated in open air. (b) The contribution of UV radiation solely to bacterial elimination and induction of DNA damage is minimal. Therefore, the bactericidal action of LTPs can be ascribed to the RNOS generated in the plasma, although the role of charged species and electric fields cannot be ruled out. (c) Preferential redox reactions between plasma-generated RNOS and external biomolecules in the environment decrease the efficacy of the treatment. This study evidences the importance of the aforementioned conditions for the development of successful antimicrobial plasma therapies. It highlights the usefulness of single cell analysis to assess heterogeneous responses in a bacterial population in response to LTPs treatments. These results were made possible only due to interdisciplinary quantitative approaches used in this project

ROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy

Physical plasmas generate unique mixes of reactive oxygen and nitrogen species (RONS or ROS). Only a bit more than a decade ago, these plasmas, operating at body temperature, started to be considered for medical therapy with considerably little mechanistic redox chemistry or biomedical research existing on that topic at that time. Today, a vast body of evidence is available on physical plasma-derived ROS, from their spatiotemporal resolution in the plasma gas phase to sophisticated chemical and biochemical analysis of these species once dissolved in liquids. Data from in silico analysis dissected potential reaction pathways of plasma-derived reactive species with biological membranes, and in vitro and in vivo experiments in cell and animal disease models identified molecular mechanisms and potential therapeutic benefits of physical plasmas. In 2013, the first medical plasma systems entered the European market as class IIa devices and have proven to be a valuable resource in dermatology, especially for supporting the healing of chronic wounds. The first results in cancer patients treated with plasma are promising, too. Due to the many potentials of this blooming new field ahead, there is a need to highlight the main concepts distilled from plasma research in chemistry and biology that serve as a mechanistic link between plasma physics (how and which plasma-derived ROS are produced) and therapy (what is the medical benefit). This inevitably puts cellular membranes in focus, as these are the natural interphase between ROS produced by plasmas and translation of their chemical reactivity into distinct biological responses. © 2019 Angela Privat-Maldonado et al

Spatial Dependence of DNA Damage in Bacteria due to Low-Temperature Plasma Application as Assessed at the Single Cell Level

Low temperature plasmas (LTPs) generate a cocktail of reactive nitrogen and oxygen species (RNOS) with bactericidal activity. The RNOS however are spatially unevenly distributed in the plasma. Here we test the hypothesis that this distribution will affect the mechanisms underpinning plasma bactericidal activity focussing on the level of DNA damage in situ. For the first time, a quantitative, single cell approach was applied to assess the level of DNA damage in bacteria as a function of the radial distance from the centre of the plasma jet. Salmonella enterica on a solid, dry surface was treated with two types of LTP: an atmospheric-pressure dielectric barrier discharge plasma jet (charged and neutral species) and a radio-frequency atmospheric-pressure plasma jet (neutral species). In both cases, there was an inverse correlation between the degree of DNA damage and the radial distance from the centre of the plasma, with the highest DNA damage occurring directly under the plasma. This trend was also observed with Staphylococcus aureus. LTP-generated UV radiation was eliminated as a contributing factor. Thus valuable mechanistic information can be obtained from assays on biological material, which can inform the development of LTP as a complementary or alternative therapy for (topical) bacterial infections

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

Modifying the tumour microenvironment : challenges and future perspectives for anticancer plasma treatments

Tumours are complex systems formed by cellular (malignant, immune, and endothelial cells, fibroblasts) and acellular components (extracellular matrix (ECM) constituents and secreted factors). A close interplay between these factors, collectively called the tumour microenvironment, is required to respond appropriately to external cues and to determine the treatment outcome. Cold plasma (here referred as ‘plasma’) is an emerging anticancer technology that generates a unique cocktail of reactive oxygen and nitrogen species to eliminate cancerous cells via multiple mechanisms of action. While plasma is currently regarded as a local therapy, it can also modulate the mechanisms of cell-to-cell and cell-to-ECM communication, which could facilitate the propagation of its effect in tissue and distant sites. However, it is still largely unknown how the physical interactions occurring between cells and/or the ECM in the tumour microenvironment affect the plasma therapy outcome. In this review, we discuss the effect of plasma on cell-to-cell and cell-to-ECM communication in the context of the tumour microenvironment and suggest new avenues of research to advance our knowledge in the field. Furthermore, we revise the relevant state-of-the-art in three-dimensional in vitro models that could be used to analyse cell-to-cell and cell-to-ECM communication and further strengthen our understanding of the effect of plasma in solid tumours