Understanding the Potential of Plasma Activated Liquids for Biomedical Applications

Atmospheric cold plasma has evolved as a new technology for applications used in biomedicine, agriculture and food industry. Recently, treatment of liquids using various atmospheric pressure plasmas has attracted much attention owing to multiple practical applications such as water purification, surface cleaning and decontamination with impact in dentistry, wound healing and sterilisation, or cancer therapy. The over-arching aim of this study is to build a better understanding of the parameters which govern the liquid chemistry generated in a liquid exposed to cold plasma and how these translate into biological effects on pro- and eu-karyotic organisms. The objective was to investigate the effects of plasma activated liquids (PAL) generated by different custom-made plasma systems in Technological University Dublin. Non-buffered and buffered liquids treated by a high voltage dielectric barrier discharge system were used to investigate the role of the liquid composition on resultant reactive species and their bactericidal and cytotoxic effects against prokaryotic and eukaryotic cells, respectively. The impact of process and storage parameters such as temperature and storage time on chemical composition and bactericidal efficacy of plasma activated water and saline was also investigated, including the influence of supra and sub-ambient temperatures and long-term storage of up to 18 months. Bactericidal efficacy showed stability to mild heating and remained at lowest temperatures over prolonged storage, whereas changes in the physicochemical properties of solutions were observed after different storage times and temperatures. A reactive species selective spark and glow discharge set-up was used to elucidate the role of ROS and RNS in bacterial inactivation and cytotoxic effects of plasma iv activated water and saline. The effects of treatment time, mode of discharge and contact time of PAL on antimicrobial efficacy and cytotoxic effects in various cancerous and healthy cell lines were investigated to provide a primary understanding of how ROS and RNS affect different biological targets. Finally, a co-culture model consisting of bacteria and keratinocytes was developed to mimic the environment of an infected wound and provide a more complex challenge for microbial inactivation. Results showed that plasma activated saline can reduce the bacterial load under these conditions but caused cytotoxic effects with earlier onset than the bactericidal efficacy. A mechanistic approach for the mammalian cell death showed that PAL with acidic pH can cause increase of intracellular ROS and mitochondria depolarisation, reduction of glutathione, cytokine alteration and finally lysis of mammalian cells. Overall, these data demonstrate that plasma activated liquids show an efficient decontamination approach against bacteria and have anticancer effects, with the chemical composition playing a crucial role in the inactivation processes, highlighting the potential to make plasma solutions attractive for applications in biomedicine but also indicating limiting factors which require further elucidation

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

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

Cell Death Induced in Glioblastoma Cells by Plasma-Activated-Liquids (PAL) is Primarily Mediated by Membrane Lipid Peroxidation and not ROS Influx

Since first identified in 1879, plasma, the fourth state of matter, has been developed and utilised in many fields. Nonthermal atmospheric plasma, also known as cold plasma, can be applied to liquids, where plasma reactive species such as reactive Oxygen and Nitrogen species and their effects can be retained and mediated through plasma-activated liquids (PAL). In the medical field, PAL is considered promising for wound treatment, sterilisation and cancer therapy due to its rich and relatively long-lived reactive species components. This study sought to identify any potential antagonistic effect between antioxidative intracellularly accumulated platinum nanoparticles (PtNPs) and PAL. We found that PAL can significantly reduce the viability of glioblastoma U-251MG cells. This did not involve measurable ROS influx but instead lead to lipid damage on the plasma membrane of cells exposed to PAL. Although the intracellular antioxidative PtNPs showed no protective effect against PAL, this study contributes to further understanding of principle cell killing routes of PAL and discovery of potential PAL-related therapy and methods to inhibit side effects

Temperature Stability and Effectiveness of Plasma-Activated Liquids over an 18 Months Period

Non-buffered plasma-activated liquids such as water and saline have shown bactericidal effects. In the present study, we investigated the anti-bacterial efficacy and stability of plasma-activated water (PAW) and plasma-activated saline (PAS), generated using a high voltage dielectric barrier discharge system. This study compares the potential of non-buffered plasma-activated liquids (PAL) for the inactivation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) after storage of the solutions at five different temperatures for a storage time up to 18 months after their generation. The temperatures used were room temperature, 4◦ C, −16◦ C, −80◦ C, −150◦ C. Both PAW and PAS achieved 6 log reduction for both bacteria on the first day of their generation after 60 min contact time and they retained these effects after 18 months when stored at the lowest temperatures. Chemical analysis of PAL showed that plasma caused a drop in pH, generation of reactive oxygen species and nitrates, whereas no nitrites are detected in the system used. The concentrations of chemical species were affected by the storage at different temperatures and a thermocouple probe was used to investigate the freezing behaviour of the PAL

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.Science Foundation Irelan

Dose responses for ROS generator and H₂O₂.

(A) U-251 MG cells were incubated with 0.032 and 4 μg/ml PtNPs for 4 h and then treated with increasing concentrations (0 ≤ 50 mM) of ROS generator. (B) U-251 MG cells were incubated with 0.032 and 4 μg/ml PtNPs for 4 h and then treated with increasing concentrations (0 ≤ 8 mM) of H2O2. Alamar blue cell viability assay was carried out 4 h after treatment. (TIF)</p

Dose responses for PtNPs treatment.

U-251 MG cells were incubated with 0.032 and 4 μg/ml PtNPs for 1h (A), 4h (B), 8h (C), 16h (D), and 24h (E) then treated with increasing concentrations (0 ≤ 30%) of PAW. Alamar blue cell viability assay was carried out 48 h after PAW treatment.</p

A schematic illustration of the PAL generation device prototype used in this study.

A schematic illustration of the PAL generation device prototype used in this study.</p

Dose responses for simultaneous PAW treatment.

U-251 MG cells were treated with 0.032 and 4 μg/ml PtNPs and increasing concentrations (0 ≤ 30%) of PAW simultaneously and incubated for 24h (A) or 48 h (B). 0.032 and 4 μg/ml PtNPs and 25, 30, 40% of PAW were mixed at incubated at 37°C for 24 h, U-251 MG cells were treated the mixture and incubated for 24 h (C) or 48 h (D).</p