Safety Implications of Plasma-Induced Effects in Living Cells – a Review of in Vitro and in Vivo Findings

Cold atmospheric plasma is a versatile new tool in the biomedical field with applications ranging from disinfection, wound healing and tissue regeneration to blood coagulation, and cancer treatment. Along with improved insights into the underlying physical, chemical and biological principles, plasma medicine has also made important advances in the introduction into the clinic. However, in the absence of a standard plasma ‘dose’ definition, the diversity of the field poses certain difficulties in terms of comparability of plasma devices, treatment parameters and resulting biological effects, particularly with regards to the question of what constitutes a safe plasma application. Data from various in vitro cytotoxic and genotoxic studies along with in vivo findings from animal and human trials are reviewed to provide an overview of the current state of knowledge on the safety of plasma for biological applications. Treatment parameters employed in clinical studies were well-tolerated but intense treatment conditions can also induce tissue damage or genotoxicity. There is a need identified to establish both guidelines and safety limits that ensure an absence of (long-term) side effects and to define treatments as safe for applications, where cell stimulation is desired, e.g. in wound healing, or those aimed at inducing cell death in the treatment of cancer

Application of Plasma Technology in Bioscience and Biomedicine

Plasma technology has been an integral part of research in life sciences for decades through its role in the manufacture and modification of material surface characteristics of many common laboratory consumables, and it is still of interest in many fields, including the treatment of biomaterials and implants. In recent years, the use of plasma at room temperature and atmospheric pressure (cold atmospheric plasma) has led to a shift in the field by putting the focus on biomedicine due to its applicability to heat-sensitive materials, including biomaterials, and expanding the traditional applicability of plasmas to cells and tissues. Consequently, biomedical applications of cold plasmas have experienced a surge in recent years due to both the evolution of plasma sources to be used under atmospheric pressure and in handy devices, and the increasing need of our society to find novel solutions to unresolved health problems. The versatility of plasmas and the vibrant advances in the field are reflected in the high number of papers collected in this Special Issue and their wide scope of topics and applicability. Cold plasmas, both at low pressure and at atmospheric pressure, are reflected in the contributions, and the latter being employed both directly on materials and cells, as well as being used to produce plasma-treated liquids which find equivalent applications to plasmas in the different areas mentioned. In the following sections we briefly discuss the most relevant findings gathered in the papers included in this Special Issue in each of the different areas concerned

Understanding The Differences Between Antimicrobial and Cytotoxic Properties of Plasma Activated Liquids

The aqueous environment plays an important role in the transmission of cold plasma effects to both prokaryotic and eukaryotic cells. The exposure of liquids to cold atmospheric plasma discharges results in the generation of secondary reactive species; specifically, hydrogen peroxide (H2O2) seems to be one of the most important amongst the reactive species contained in plasma activated liquids (PALs) in causing cytotoxicity. Detailed understanding of the effects of PALs on cells is essential to harness this new technology. Liquids acting as models for non-complex solutions were generated using a dielectric barrier discharge atmospheric cold plasma (DBD-ACP) system. The chemical characterization of the PAL included its pH and concentrations of hydrogen peroxide, nitrite, and nitrate. The antimicrobial effects of PALs on Gram-positive and Gram-negative bacteria were examined, and cytotoxicity assays were used to elucidate the cytotoxic properties of PALs. The research outcomes showed acidification of plasma activated nonbuffered solutions and differences in concentrations of hydrogen peroxide, nitrite, and nitrate. PALs with different compositions varied in their antibacterial activity and cytotoxic effects, indicating that different reactive species may be responsible for these inactivation processes. Our results suggest that antimicrobial and cytotoxic effects are distinct from each other, which may offer promising approaches for future targeted applications in medicine

Efficacy of Plasma Activated Saline in a Co‑Culture Infection Control Model

Plasma activated liquids have demonstrated antimicrobial effects and receive increasing attention due to the potential to strengthen the armoury of novel approaches against antibiotic resistant bacteria. However, the antibacterial activity and cytotoxic effects of these solutions need to be understood and balanced before exposure to humans. In this study, the antibacterial effects of plasma activated saline (PAS) were tested against Gram negative and positive bacteria, and HaCaT keratinocytes were used for cytotoxicity studies. For the first time, a co-culture model between these bacteria and eukaryotic cells under the influence of PAS has been described. Exposure of saline to plasma resulted in high concentrations of nitrate, hydrogen peroxide and a reduction of pH. PAS caused high antibacterial effects in the co-culture model, accompanied by high cytotoxic effects to the monolayer of mammalian cells. We present evidence and provide a deeper understanding for the hypothesis that upon treatment with PAS, chemical species generated in the liquid mediate high antimicrobial effects in the co-culture setup as well as mitochondrial depolarization and glutathione depletion in HaCaT cells and cell lysis due to acidic pH. In conclusion, PAS retains strong antibacterial effects in a co-culture model, which may have unintended negative biological effects on mammalian cells

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

Hydra as a Model for Screening Ecotoxicological Effects of Plasma-Treated Water

Atmospheric cold plasma (ACP) has been widely researched to generate functionalized solutions for decontamination of liquids and wastewater treatment. The present investigation demonstrates that Hydra can be used as an additional in vivo tool to monitor the impact of plasma-processed solutions on the aquatic environment

Distinct Chemistries Define the Diverse Biological Effects of Plasma Activated Water Generated with Spark and Glow Plasma Discharges

The spread of multidrug-resistant bacteria poses a significant threat to human health. Plasma activated liquids (PAL) could be a promising alternative for microbial decontamination, where different PAL can possess diverse antimicrobial efficacies and cytotoxic profiles, depending on the range and concentration of their reactive chemical species. In this research, the biological activity of plasma activated water (PAW) on different biological targets including both microbiological and mammalian cells was investigated in vitro. The aim was to further an understanding of the specific role of distinct plasma reactive species, which is required to tailor plasma activated liquids for use in applications where high antimicrobial activity is required without adversely affecting the biology of eukaryotic cells. PAW was generated by glow and spark discharges, which provide selective generation of hydrogen peroxide, nitrite and nitrate in the liquid. The PAW made by either spark or glow discharges showed similar antimicrobial efficacy and stability of activity, despite the very different reactive oxygen species (ROS) and reactive nitrogen species profiles (RNS). However, different trends were observed for cytotoxic activities and effects on enzyme function, which were translated through the selective chemical species generation. These findings indicate very distinct mechanisms of action which may be exploited when tailoring plasma activated liquids to various applications. A remarkable stability to heat and pressure was noted for PAW generated with this set up, which broadens the application potential. These features also suggest that post plasma modifications and post generation stability can be harnessed as a further means of modulating the chemistry, activity and mode of delivery of plasma functionalised liquids. Overall, these results further understanding on how PAL generation may be tuned to provide candidate disinfectant agents for biomedical application or for bio-decontamination in diverse areas

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