Hydrogen Peroxide and Beyond - the Potential of High-Voltage Plasma Activated Liquids Against Cancerous Cells

Abstract: The use of plasma-activated liquids such as PBS, medium or simply plasma-activated water (PAW) has been receiving increasing attention for applications in cancer treatments. Amongst the reactive species contained in these solutions, hydrogen peroxide appears to play a pivotal role in causing cytotoxic effects. While H2O2 concentrations can be correlated with reduced cell viability and growth and used as an indicator of the potential efficacy of a plasma-activated water, comparisons to standard H2O2 kill curves demonstrate a potency in PAW which exceeds H2O2 associated toxicity, indicating that other plasma-generated species play an important role. Using a high-voltage dielectric barrier atmospheric cold plasma (DBD-ACP) system, we demonstrate the generation of plasma-activated water with high cytotoxic potential and good storage stability. The potency of the activated solutions can be modulated using system or process characteristics such as voltage level, treatment time and post-treatment storage time and target-related characteristics such as surface to volume ratio. All of these parameters were found to impact cell viability in a hydrogen peroxide concentration; correlated manner. The susceptibility of two cancer cell lines to PAW was similar to that observed for two non-cancer cell lines and the toxicity of plasma-activated water exceeded that of the corresponding hydrogen peroxide concentrations. This study examines the role of H2O2 in PAW-mediated cytotoxic effects on different mammalian cell lines and investigates the effects beyond H2O2 employing a set-up where short-lived reactive species can be discounted and activated liquids with long-term stability are generated. Here we investigate the cytotoxic mediators generated in water specific to high-voltage DBD-ACP

Controlled Cytotoxicity of Plasma Treated Water Formulated By Open-air Hybrid Mode Discharge

Plasma‐activated liquids (PAL) attract increasing interest with demonstrated biological effects. Plasma exposure in air produces stable aqueous reactive species which can serve as chemical diagnostics of PAL systems. Here, we tailor aqueous reactive species inside plasma‐activated water (PAW) through treating water with AC air spark and glow discharges in contact with water. Chemical probing demonstrated species specificity between two types of PAW. Spark discharge PAW contains urn:x-wiley:14381656:media:ppap201600207:ppap201600207-math-0006 and urn:x-wiley:14381656:media:ppap201600207:ppap201600207-math-0007, while urn:x-wiley:14381656:media:ppap201600207:ppap201600207-math-0008and urn:x-wiley:14381656:media:ppap201600207:ppap201600207-math-0009 are generated in glow discharge PAW. Species formation in different PAWs have been discussed in terms of discharge mechanisms and liquid phase chemistry process. Species specificity can provide richer parametric spaces for producing PALs with controlled impact and dosage achievable by combining discharge modes or mixing different PALs

The Potential of Cold Plasma for Safe and Sustainable Food Production

In-package decontamination of foods using cold plasma has advanced this technology as a unit process for fresh foods decontamination and shelf-life extension. Chemical residues of agricultural pesticides of varying structure can be degraded to safe or less-toxic structures using cold plasma. Cold-plasma-mediated control of contaminants, along with the promotion of seed germination and plant growth, offers alternatives to current pesticides and fertilizers for agriculture. Controlling plasma reactive species formulations in dry and liquid delivery formats advances the potential for understanding and successful translation to multiple points along the agriculture and food sectors. Employing predictive microbiology, process optimization tools and a systems approach with controlled reactive species formulations may achieve risk- or problem-tailored solutions for whole food systems. Cold plasma science and technology is increasingly investigated for translation to a plethora of issues in the agriculture and food sectors. The diversity of the mechanisms of action of cold plasma, and the flexibility as a standalone technology or one that can integrate with other technologies, provide a rich resource for driving innovative solutions. The emerging understanding of the longer-term role of cold plasma reactive species and follow-on effects across a range of systems will suggest how cold plasma may be optimally applied to biological systems in the agricultural and food sectors. Here we present the current status, emerging issues, regulatory context, and opportunities of cold plasma with respect to the broad stages of primary and secondary food production

Controlling Brochothrix Thermosphacta as a Spoilage Risk Using in Package Atmospheric Cold Plasma

Brochothrix thermosphacta is a predominant spoilage microorganism in meat and its control in processing environments is important to maintain meat product quality. Atmospheric cold plasma is of interest for control of pathogenic and spoilage microorganisms in foods. This study ascertained the potential of dielectric barrier discharge atmospheric cold plasma (DBD-ACP) for control of B. thermosphacta in response to key parameters such as treatment time, voltage level, interactions with media composition and post treatment storage conditions. Challenge populations were evaluated as suspensions in PBS, as biofilms in meat model medium and surface attached on raw lamb chops under MAP. ACP treatment (80kV) for 30s inactivated B. thermosphacta populations below detection in PBS, while 5 min treatment achieved a 2 Log cycle reduction using a complex meat model medium and attached cells, suggesting useful control for meat processing facilities. A ‘worst case scenario challenge’ of high population density on a nutritious medium in a biofilm matrix was evaluated using a surface inoculated lamb chop and the antimicrobial efficacy of plasma was reduced but still apparent over the 10 day storage period. However, there is scope to further enhance microbial control leading to meat storage life extension through adjusting the modality of treatmen

Investigation of mechanisms involved in germination enhancement of wheat (Triticum aestivum) by cold plasma: Effects on seed surface chemistry and characteristics

Recent reports indicate that atmospheric cold plasma (ACP) treatment of seeds can enhance their germination, however, the mechanisms of action are not yet entirely clear. In the present work, we report on the effects of plasma treatment on wheat seed germination and seedling growth. Additionally, changes in the surface chemistry and characteristics of the wheat seeds exposed to plasma were investigated. Treatments of 30–60 s significantly enhanced the germination rate and showed positive effects on seedling growth. ACP resulted in changes of seed surface and chemical characteristics including water uptake and contact angle values. Changes in seed pH and total titratable acidity, as well as nitrites, nitrates, and malondialdehyde concentrations were also recorded

The potential of atmospheric air cold plasma for control of bacterial contaminants relevant to cereal grain production

The aim of this work was to investigate the efficacy of dielectric barrier discharge atmospheric cold plasma (DBD ACP) against bacteria associated with grains quality and safety. ACP inactivation efficacy was tested against biofilms formed by different strains of E. coli, Bacillus and Lactobacillus in grain model media and against B. atrophaeus endospores either in grain media or attached on abiotic surfaces. Effects were dependent on bacterial strain, media composition and mode of ACP exposure. ACP treatment for 5min reduced E. coli spp., B. subtilis and Lactobacillus spp. biofilms by \u3e3 log10, whereas insignificant reductions were achieved for B. atrophaeus. ACP treatment of 5–20min reduced B. atrophaeus spores in liquids by \u3e5 log10. Treatment for 30min reduced spores on hydrophobic surface by \u3e6 log10, whereas maximum of 4.4 log reductions were achieved with spores attached to hydrophilic surface. Microscopy demonstrated that ACP caused significant damage to spores. In package ACP treatment has potential to inactivate grain contaminants in the form of biofilms, as well as spores and vegetative cells. Industrial relevance This study demonstrates that ACP technology is a promising tool for effective bio-decontamination which offers a wide range of possible applications including inactivation of microorganisms on cereal grains. However, due to the nature of the microbial contamination of grains and complex grain structures it may be necessary to optimise the potential for surface inactivation at several stages of grain processing and storage to enhance ACP efficacy against bacterial endospores

Assessment of the disinfection capacity and eco-toxicological impact of atmospheric cold plasma for treatment of food industry effluents

Generation of wastewater is one of the main environmental sustainability issues across food sector industries. The constituents of food process effluents are often complex and require high energy and processing for regulatory compliance. Wastewater streams are the subject of microbiological and chemical criteria, and can have a significant eco-toxicological impact on the aquatic life. Thus, innovative treatment approaches are required to mitigate environmental impact in an energy efficient manner. Here, dielectric barrier discharge atmospheric cold plasma (ACP) was evaluated for control of key microbial indicators encountered in food industry effluent. This study also investigated the eco-toxicological impact of cold plasma treatment of the effluents using a range of aquatic bioassays. Continuous ACP treatment was applied to synthetic dairy and meat effluents. Microbial inactivation showed treatment time dependence with significant reduction in microbial populations within 120 s, and to undetectable levels after 300 s. Post treatment retention time emerged as critical control parameter which promoted ACP bacterial inactivation efficiency. Moreover, ACP treatment for 20 min achieved significant reduction (≥2 Log10) in Bacillus megaterium endospores in wastewater effluent. Acute aquatic toxicity was assessed using two fish cell lines (PLHC-1 and RTG-2) and a crustacean model (Daphnia magna). Untreated effluents were toxic to the aquatic models, however, plasma treatment limited the toxic effects. Differing sensitivities were observed to ACP treated effluents across the different test bio-assays in the following order: PLHC-1 \u3e RTG-2 ≥ D. magna; with greater sensitivity retained to plasma treated meat effluent than dairy effluent. The toxic effects were dependent on concentration and treatment time of the ACP treated effluent; with 30% cytotoxicity in D. magna and fish cells observed after 24 h of exposure to ACP treated effluent for concentrations up to 5%. The findings suggest the need to employ wider variety of aquatic organisms for better understanding and complete toxicity evaluation of long-term effects. The study demonstrates the potential to tailor ACP system parameters to control pertinent microbial targets (mono/poly-microbial, vegetative or spore form) found in complex and nutritious wastewater effluents whilst maintaining a safe eco-toxicity profile for aquatic species

Mechanism of Inactivation by High Voltage Atmospheric Cold Plasma Differs between Escherichia coli and Staphylococcus aureus

Atmospheric cold plasma (ACP) is a promising non-thermal technology effective against a wide range of pathogenic microorganisms. Reactive oxygen species (ROS) play a crucial inactivation role when air or other oxygen containing gases are used. With strong oxidative stress, cells can be damaged by lipid peroxidation, enzyme inactivation and DNA cleavage. Identifying ROS and understanding their role is important to advance ACP applications to a range of complex microbiological issues. In this study, the inactivation efficacy of in-package, high voltage (80 kVRMS) ACP (HVACP) and the role of intracellular ROS were investigated. Two mechanisms of inactivation were observed where reactive species were found to either react primarily with the cell envelope or to damage intracellular components. E. coli was inactivated mainly by cell leakage and low level DNA damage. Conversely, S. aureus was mainly inactivated by intracellular damage with significantly higher levels of intracellular ROS observed and little envelope damage. However, for both bacteria studied, increasing treatment time had a positive effect on intracellular ROS levels generated

The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using \u3ci\u3e Listeria monocytogenes\u3c/i\u3e Knockout Mutants

Listeria monocytogenes is an opportunistic intracellular pathogen commonly associated with serious 16 infections and multiple food-borne outbreaks. In this study, we investigated the influence of 17 atmospheric cold plasma (80 kV, 50 Hz) on L. monocytogenes (EGD-e) and its knockout mutants of 18 sigB, rsbR, prfA, gadD and lmo0799 genes at different treatment time intervals. Further, to ascertain if 19 sub-lethal environmental stress conditions could influence L. monocytogenes survival and growth 20 responses, atmospheric cold plasma (ACP) resistance was evaluated for the cultures exposed to cold 21 (4°C) or acid (pH 4) stress for 1 h. The results demonstrate that both wild-type and knockout mutants 22 were similarly affected after 1 min exposure to ACP (p \u3e 0.05), with a difference in response noted 23 only after 3 min of treatment. While all L. monocytogenes strains exposed to acid/cold stress were 24 hypersensitive to ACP treatment and were significantly reduced or inactivated within 1 min of 25 treatment (p \u3c 0.05). The results indicate sigB and prfA are important for general stress resistance and 26 biofilm respectively, loss of these two genes significantly reduced bacterial resistance to ACP 27 treatment. In addition, exposure to sub-lethal 1min ACP increased the gene expression of stress 28 associated genes. SigB showed the highest gene expression, increasing by 15.60 fold, followed by 29 gadD2 (7.19) and lmo0799 (8.6) after 1 min exposure. Overall, an increase in gene expression was 30 seen in all stress associated genes analyzed both at 1 min treatment; while long treatment time reduced 31 the gene expression and some cases down-regulated prfA and gadD3 gene expression. By comparing 32 the response of mutants under ACP exposure to key processing parameters, the experimental results 33 presented here provide a baseline for understanding the bacterial genetic response and resistance to 34 cold plasma stress and offers promising insights for optimizing ACP applications