Plasma Control: A Review of Developments and Applications of Plasma Medicine Control Mechanisms.

Cold atmospheric plasmas (CAPs) within recent years have shown great promise in the field of plasma medicine, encompassing a variety of treatments from wound healing to the treatment of cancerous tumors. For each subsequent treatment, a different application of CAPs has been postulated and attempted to best treat the target for the most effective results. These treatments have varied through the implementation of control parameters such as applied settings, electrode geometries, gas flow, and the duration of the treatment. However, with such an extensive number of variables to consider, scientists and engineers have sought a means to accurately control CAPs for the best-desired effects in medical applications. This paper seeks to investigate and characterize the historical precedent for the use of plasma control mechanisms within the field of plasma medicine. Current control strategies, plasma parameters, and control schemes will be extrapolated through recent developments and successes to gain better insight into the future of the field and the challenges that are still present in the overall implementation of such devices. Proposed approaches, such as data-driven machine learning, and the use of closed-loop feedback controls, will be showcased as the next steps toward application.

Cold atmospheric plasma inactivates Aspergillus flavus and Fusarium keratoplasticum biofilms and conidia in vitro.

Introduction. Aspergillus flavus and Fusarium keratoplasticum are common causative pathogens of fungal keratitis (FK), a severe corneal disease associated with significant morbidity and vision loss. Escalating incidence of antifungal resistance to available antifungal drugs poses a major challenge to FK treatment. Cold atmospheric plasma (CAP) is a pioneering nonpharmacologic antimicrobial intervention that has demonstrated potential as a broad-spectrum antifungal treatment.Gap statement. Previous research highlights biofilm-associated resistance as a critical barrier to effective FK treatment. Although CAP has shown promise against various fungal infections, its efficacy against biofilm and conidial forms of FK pathogens remains inadequately explored.Aim. This study aims to investigate the antifungal efficacy of CAP against clinical fungal keratitis isolates of A. flavus and F. keratoplasticum in vitro.Methodology. Power parameters (22-27 kVpp, 300-400 Hz and 20-80 mA) of a dielectric barrier discharge CAP device were optimized for inactivation of A. flavus biofilms. Optimal applied voltage and total current were applied to F. keratoplasticum biofilms and conidial suspensions of A. flavus and F. keratoplasticum. The antifungal effect of CAP treatment was investigated by evaluating fungal viability through means of metabolic activity, c.f.u. enumeration (c.f.u. ml-1) and biofilm formation.Results. For both fungal species, CAP exhibited strong time-dependent inactivation, achieving greater than 80 % reduction in metabolic activity and c.f.u. ml-1 within 300 s or less, and complete inhibition after 600 s of treatment.Conclusion. Our findings indicate that CAP is a promising broad-spectrum antifungal intervention. CAP treatment effectively reduces fungal viability in both biofilm and conidial suspension cultures of A. flavus and F. keratoplasticum, suggesting its potential as an alternative treatment strategy for fungal keratitis.

Plasma-liquid interactions in the presence of organic matter-A perspective.

As investigations in the biomedical applications of plasma advance, a demand for describing safe and efficacious delivery of plasma is emerging. It is quite clear that not all plasmas are "equal" for all applications. This Perspective discusses limitations of the existing parameters used to define plasma in context of the need for the "right plasma" at the "right dose" for each "disease system." The validity of results extrapolated from in vitro studies to preclinical and clinical applications is discussed. We make a case for studying the whole system as a single unit, in situ. Furthermore, we argue that while plasma-generated chemical species are the proposed key effectors in biological systems, the contribution of physical effectors (electric fields, surface charging, dielectric properties of target, changes in gap electric fields, etc.) must not be ignored.

Depyrogenation using Plasmas: A Novel Approach for Endotoxin Deactivation Using a Dielectric Barrier Discharge at Atmospheric Pressure.

Developing a low-cost depyrogenation process is vital in extending medical applicability of polymers that can be used in medicine. We present an overview of the plasma-based depyrogenation literature and address the need to develop a non-thermal plasma-based depyrogenation process for delicate materials such as chitosan. We present a low-cost plasma apparatus to treat chitosan powder in hermetically sealed bags. We decouple the experiments into two; depyrogenation experiments for dried standard endotoxin on glass slides, and chitosan modifications analysis through FTIR spectroscopy. We demonstrate depyrogenation efficacy with up to a 4-log reduction in endotoxin levels and discuss minor changes observed in plasma-treated chitosan.

Differential Effect of Non-Thermal Plasma RONS on Two Human Leukemic Cell Populations.

Non-thermal plasma application to cancer cells is known to induce oxidative stress, cytotoxicity and indirect immunostimulatory effects on antigen presenting cells (APCs). The purpose of this study was to evaluate the responses of two leukemic cell lines-Jurkat T lymphocytes and THP-1 monocytes-to NTP-generated reactive oxygen and nitrogen species (RONS). Both cell types depleted hydrogen peroxide, but THP-1 cells neutralized it almost immediately. Jurkat cells transiently blunted the frequency-dependent increase in nitrite concentrations in contrast to THP-1 cells, which exhibited no immediate effect. A direct relationship between frequency-dependent cytotoxicity and mitochondrial superoxide was observed only in Jurkat cells. Jurkat cells were very responsive to NTP in their display of calreticulin and heat shock proteins 70 and 90. In contrast, THP-1 cells were minimally responsive or unresponsive. Despite no NTP-dependent decrease in cell surface display of CD47 in either cell line, both cell types induced migration of and phagocytosis by APCs. Our results demonstrate that cells modulate the RONS-mediated changes in liquid chemistry, and, importantly, the resultant immunomodulatory effects of NTP can be independent of NTP-induced cytotoxicity.

Catalytic oxidation of small organic molecules by cold plasma in solution in the presence of molecular iron complexes†.

The plasma-mediated decomposition of volatile organic compounds has previously been investigated in the gas phase with metal oxides as heterogeneous catalysts. While the reactive species in plasma itself are well investigated, very little is known about the influence of metal catalysts in solution. Here, we present initial investigations on the time-dependent plasma-supported oxidation of benzyl alcohol, benzaldehyde and phenol in the presence of molecular iron complexes in solution. Products were identified by HPLC, ESI-MS, FT-IR, and [Formula: see text] spectroscopy. Compared to metal-free oxidation of the substrates, which is caused by reactive oxygen species and leads to a mixture of products, the metal-mediated reactions lead to one product cleanly, and faster than in the metal-free reactions. Most noteworthy, even catalytic amounts of metal complexes induce these clean transformations. The findings described here bear important implications for plasma-supported industrial waste transformations, as well as for plasma-mediated applications in biomedicine, given the fact that iron is the most abundant redox-active metal in the human body.

Advances in Plasma Oncology toward Clinical Translation.

This Special Issue on "Advances in Plasma Oncology Toward Clinical Translation" aims to bring together cutting-edge research papers within the field in the context of clinical translation and application [...].

Study on Chemical Modifications of Glutathione by Cold Atmospheric Pressure Plasma (Cap) Operated in Air in the Presence of Fe(II) and Fe(III) Complexes.

Cold atmospheric pressure plasma is an attractive new research area in clinical trials to treat skin diseases. However, the principles of plasma modification of biomolecules in aqueous solutions remain elusive. It is intriguing how reactive oxygen and nitrogen species (RONS) produced by plasma interact on a molecular level in a biological environment. Previously, we identified the chemical effects of dielectric barrier discharges (DBD) on the glutathione (GSH) and glutathione disulphide (GSSG) molecules as the most important redox pair in organisms responsible for detoxification of intracellular reactive species. However, in the human body there are also present redox-active metals such as iron, which is the most abundant transition metal in healthy humans. In the present study, the time-dependent chemical modifications on GSH and GSSG in the presence of iron(II) and iron(III) complexes caused by a dielectric barrier discharge (DBD) under ambient conditions were investigated by IR spectroscopy, mass spectrometry and High Performance Liquid Chromatography (HPLC). HPLC chromatograms revealed one clean peak after treatment of both GSH and GSSH with the dielectric barrier discharge (DBD) plasma, which corresponded to glutathione sulfonic acid GSO3H. The ESI-MS measurements confirmed the presence of glutathione sulfonic acid. In our experiments, involving either iron(II) or iron(III) complexes, glutathione sulfonic acid GSO3H appeared as the main oxidation product. This is in sharp contrast to GSH/GSSG treatment with DBD plasma in the absence of metal ions, which gave a wild mixture of products. Also interesting, no nitrosylation of GSH/GSSG was oberved in the presence of iron complexes, which seems to indicate a preferential oxygen activation chemistry by this transition metal ion.

Nitrosylation vs. oxidation - How to modulate cold physical plasmas for biological applications.

Thiol moieties are major targets for cold plasma-derived nitrogen and oxygen species, making CAPs convenient tools to modulate redox-signaling pathways in cells and tissues. The underlying biochemical pathways are currently under investigation but especially the role of CAP derived RNS is barely understood. Their potential role in protein thiol nitrosylation would be relevant in inflammatory processes such as wound healing and improving their specific production by CAP would allow for enhanced treatment options beyond the current application. The impact of a modified kINPen 09 argon plasma jet with nitrogen shielding on cysteine as a thiol-carrying model substance was investigated by FTIR spectroscopy and high-resolution mass spectrometry. The deposition of short-lived radical species was measured by electron paramagnetic resonance spectroscopy, long-lived species were quantified by ion chromatography (NO2-, NO3-) and xylenol orange assay (H2O2). Product profiles were compared to samples treated with the so-called COST jet, being introduced by a European COST initiative as a reference device, using both reference conditions as well as conditions adjusted to kINPen gas mixtures. While thiol oxidation was dominant under all tested conditions, an Ar + N2/O2 gas compositions combined with a nitrogen curtain fostered nitric oxide deposition and the desired generation of S-nitrosocysteine. Interestingly, the COST-jet revealed significant differences in its chemical properties in comparison to the kINPen by showing a more stable production of RNS with different gas admixtures, indicating a different •NO production pathway. Taken together, results indicate various chemical properties of kINPen and COST-jet as well as highlight the potential of plasma tuning not only by gas admixtures alone but by adjusting the surrounding atmosphere as well.

Comparing Spore Resistance of Bacillus Strains Isolated from Hydrothermal Vents and Spacecraft Assembly Facilities to Environmental Stressors and Decontamination Treatments.

Submarine hydrothermal vents are inhabited by a variety of microorganisms capable of tolerating environmental extremes, making them ideal candidates to further expand our knowledge of the limitations for terrestrial life, including their ability to survive the exposure of spaceflight-relevant conditions. The spore resistance of two Bacillus spp. strains, APA and SBP3, isolated from two shallow vents off Panarea Island (Aeolian Islands, Italy), to artificial and environmental stressors (i.e., UVC radiation, X-rays, heat, space vacuum, hydrogen peroxide [H2O2], and low-pressure plasma), was compared with that of two close phylogenetic relatives (Bacillus horneckiae and Bacillus oceanisediminis). Additional comparisons were made with Bacillus sp. isolated from spacecraft assembly facilities (B. horneckiae, Bacillus pumilus SAFR-032, and Bacillus nealsonii) and the biodosimetry strain and space microbiology model organism Bacillus subtilis. Overall, a high degree of spore resistance to stressors was observed for the strains isolated from spacecraft assembly facilities, with an exceptional level of resistance seen by B. pumilus SAFR-032. The environmental isolate SBP3 showed a more robust spore resistance to UVC, X-rays, H2O2, dry heat, and space vacuum than the closely related B. horneckiae. Both strains (SBP3 and APA) were more thermotolerant than their relatives, B. horneckiae and B. oceanisediminis, respectively. SBP3 may have a novel use as a bacterial model organism for future interrogations into the potential of forward contamination in extraterrestrial environments (e.g., icy moons of Jupiter or Saturn), spacecraft sterilization and, broadly, microbial responses to spaceflight-relevant environmental stressors.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Elucidation of Plasma-induced Chemical Modifications on Glutathione and Glutathione Disulphide.

Cold atmospheric pressure plasmas are gaining increased interest in the medical sector and clinical trials to treat skin diseases are underway. Plasmas are capable of producing several reactive oxygen and nitrogen species (RONS). However, there are open questions how plasma-generated RONS interact on a molecular level in a biological environment, e.g. cells or cell components. The redox pair glutathione (GSH) and glutathione disulphide (GSSG) forms the most important redox buffer in organisms responsible for detoxification of intracellular reactive species. We apply Raman spectroscopy, mass spectrometry, and molecular dynamics simulations to identify the time-dependent chemical modifications on GSH and GSSG that are caused by dielectric barrier discharge under ambient conditions. We find GSSG, S-oxidised glutathione species, and S-nitrosoglutathione as oxidation products with the latter two being the final products, while glutathione sulphenic acid, glutathione sulphinic acid, and GSSG are rather reaction intermediates. Experiments using stabilized pH conditions revealed the same main oxidation products as were found in unbuffered solution, indicating that the dominant oxidative or nitrosative reactions are not influenced by acidic pH. For more complex systems these results indicate that too long treatment times can cause difficult-to-handle modifications to the cellular redox buffer which can impair proper cellular function.

Effects of Low-Temperature Plasma-Sterilization on Mars Analog Soil Samples Mixed with Deinococcus radiodurans.

We used Ar plasma-sterilization at a temperature below 80 °C to examine its effects on the viability of microorganisms when intermixed with tested soil. Due to a relatively low temperature, this method is not thought to affect the properties of a soil, particularly its organic component, to a significant degree. The method has previously been shown to work well on spacecraft parts. The selected microorganism for this test was Deinococcus radiodurans R1, which is known for its remarkable resistance to radiation effects. Our results showed a reduction in microbial counts after applying a low temperature plasma, but not to a degree suitable for a sterilization of the soil. Even an increase of the treatment duration from 1.5 to 45 min did not achieve satisfying results, but only resulted in in a mean cell reduction rate of 75% compared to the untreated control samples.

Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies.

Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 × 10(7) spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques.

Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws.

A special focus area of planetary protection is the monitoring, control, and reduction of microbial contaminations that are detected on spacecraft components and hardware during and after assembly. In this study, wild-type spores of Bacillus pumilus SAFR-032 (a persistent spacecraft assembly facility isolate) and the laboratory model organism B. subtilis 168 were used to study the effects of low-pressure plasma, with hydrogen alone and in combination with oxygen and evaporated hydrogen peroxide as a process gas, on spore survival, which was determined by a colony formation assay. Spores of B. pumilus SAFR-032 and B. subtilis 168 were deposited with an aseptic technique onto the surface of stainless steel screws to simulate a spore-contaminated spacecraft hardware component, and were subsequently exposed to different plasmas and hydrogen peroxide conditions in a very high frequency capacitively coupled plasma reactor (VHF-CCP) to reduce the spore burden. Spores of the spacecraft isolate B. pumilus SAFR-032 were significantly more resistant to plasma treatment than spores of B. subtilis 168. The use of low-pressure plasma with an additional treatment of evaporated hydrogen peroxide also led to an enhanced spore inactivation that surpassed either single treatment when applied alone, which indicates the potential application of this method as a fast and suitable way to reduce spore-contaminated spacecraft hardware components for planetary protection purposes.