Cold atmospheric plasma (CAP) has potential as a new cancer treatment tool due to its room temperature biocompatibility and possible cancer cell selectivity with low side effects. This review examines the current research stage of CAP therapy for Prostate Cancer (PCa) treatment with a focus on CAP device standardization and CAP therapy clinical translation. The current CAP preclinical data is explored through the lens of PCa cellular response, PCa cells selectivity, CAP treatment type and CAP combination therapies. Finally, building on the current preclinical data, this review proposes the potential clinical application methods of CAP at different PCa stages to promote CAP therapy into clinical use.

This work is a systematic review conducted in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. A predefined protocol was followed to ensure a transparent, reproducible, and methodologically rigorous selection and analysis of the available literature on the use of cold plasma for viral inactivation. Cold atmospheric plasma (CAP) technology has emerged as a promising tool for combating viruses. This study presents a systematic review of current knowledge regarding CAP applications in virology. A comprehensive search was conducted across Scopus and Web of Science databases, including studies published between 2000 and 2024 that explored the use of CAP in virology. Following the guidelines outlined by PRISMA, 160 articles were identified and categorized into six categories. The analysis highlighted a notable increase in publications within this field since 2020, coinciding with the emergence of COVID-19. Geographically, the United States emerged as the leading contributor to research in this area, accounting for 21.9

Background/Objectives: Despite the numerous advances in glioblastoma multiforme (GBM) treatment, GBM remains as the most malignant and aggressive form of brain cancer, characterized by a very poor outcome, highlighting the ongoing need for the development of new therapeutic strategies. A novel intervention using plasma-assisted local delivery of oncology drugs was developed to mediate the drug delivery, which might improve drug uptake and/or chemotherapeutic action. Topotecan (TPT), a water-soluble topoisomerase I inhibitor with major cytotoxic effects during the S-phase of the cell cycle, was selected as the candidate drug because despite its potent antitumor activity, the systemic administration to the brain is limited due to low crossing of the blood-brain barrier. For these reasons, TPT may be repurposed for local combined therapies. Methods: We aimed to explore options for the local treatment of GBM where systematic delivery is challenging, using a combination between plasma-based technologies and TPT on a human brain cancer cell line (U-251mg). Results: The evaluation of direct TPT plasma deposition using a helium plasma jet (J-Plasma, Apyx Medical) with a nebulizer onto U-251mg cells grown in 2D or 3D culture showed a reduction in the metabolic activity and cell mass and decreased long-term survival, indicating synergistic effects between the drug and the plasma treatment. The plasma-assisted approach was confirmed using temozolomide (TMZ) as a standard drug for glioblastoma treatment, as well as with two skin cancer cell lines. Conclusions: These results revealed a pathway for new combinations and approaches to local drug application for a range of cancers.

The increase in cancer diagnoses and cancer deaths, severe side effects of existing treatments and resistance to traditional treatments have generated a need for new anticancer treatments. Glioblastoma multiforme (GBM) is the most common, malignant and aggressive brain cancer. Despite many innovations regarding GBM treatment, the final outcome is still very poor, making it necessary to develop new therapeutic approaches. Cold atmospheric plasma (CAP) as well as plasma-activated liquids (PAL) are being studied as new possible approaches against cancer. The anticancer activity of PAL such as "plasma-activated water" (PAW) is dependent on the reactive chemical compounds present in the solution. Possible combinatory effects with conventional therapies, such as chemotherapeutics, may expand the potential of PAL for cancer treatment. We aim to explore the therapeutic properties of a combination of PAW and topotecan (TPT), an antineoplastic agent with major cytotoxic effects during the S phase of the cell cycle, on a GBM cancer cell line (U-251mg). Combined treatments with PAW and TPT showed a reduction in the metabolic activity and cell mass, an increase in apoptotic cell death and a reduction in the long-term survival. Single applications of PAW+TPT treatments showed a cytotoxic effect in the short term and an antiproliferative effect in the long term, warranting future exploration of combining PAW with chemotherapeutic agents as new therapeutic approaches.

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.

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.

This chapter introduces cold plasma treatment of liquids, the interaction with liquids pertinent to the food industry, the types of plasma activated liquids (PAL), the physicochemical properties of PAL and some relevant plasma diagnostics. The antimicrobial efficacy and mechanisms of micro-biocidal action of employing plasma for treatment of liquids is also introduced. This is followed by a focus on studies describing plasma treatment of fruit juices and finally a consideration of the safety aspects pertinent to developing a cold plasma process for liquids or one that is mediated through the liquid component of a food or beverage. Cold plasma treatment of liquids should be initially developed against the critical target parameter, however, it is important to consider the entire life cycle of a product or target going through a liquid mediated plasma process.

The potential applications for cold plasma in medicine are extensive, from microbial inactivation and induction of apoptosis in cancer cells to stimulating wound healing and enhancing the blood coagulation cascade. The safe bio-medical application of cold plasma and subsequent effect on complex biological pathways requires precision and a distinct understanding of how physiological redox chemistry is manipulated. Chemical modification of biomolecules such as carbohydrates, proteins, and lipids treated with cold plasma have been characterized, however, the context of how alterations of these molecules affect cell behavior or in vivo functionality has not been determined. Thus, this study examines the cytotoxic and mutagenic effects of plasma-treated molecules in vitro using CHO-K1 cells and in vivo in Galleria mellonella larvae. Specifically, albumin, glucose, cholesterol, and arachidonic acid were chosen as representative biomolecules, with established involvement in diverse bioprocesses including; cellular respiration, intracellular transport, cell signaling or membrane structure. Long- and short-term effects depended strongly on the molecule type and the treatment milieu indicating the impact of chemical and physical modifications on downstream biological pathways. Importantly, absence of short-term toxicity did not always correlate with absence of longer-term effects, indicating the need to comprehensively assess ongoing effects for diverse biological applications.

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.

It has been shown that plasma generated in contact with liquid can be tailored to tune the composition of plasma functionalized liquids. For biomedical applications, it is necessary to understand the generation of the plasma treated liquids to modulate the composition and thus the biological response. In this work, two distinct discharge compositions were realized by modifying the location of the ground electrode in a pin-to-liquid plasma system. Through this simple modification to the configurations, the spatiotemporal characteristics of the discharge were significantly affected which, in turn, affected the composition of the generated plasma activated water (PAW). Colorimetric testing of the PAW generated from each system revealed that only one configuration was able to generate PAW with a high concentration of H2O2. Using time-, space-, and wavelength-resolved imaging of excited plasma species [OH, N2 (SPS), N2+ (FNS), and atomic O], the differences in PAW composition were linked to the differences observed in the discharge dynamics between the two configurations.

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 [...]

The microbiological profiles and responses of native microflora of endive were investigated using a model process line, to establish where a defined PFW should be optimally applied to retain or improve produce microbiological quality. The PFW processes were compared with tap water and ClO2. The antimicrobial efficacy of PFW was quantified by determining the reduction in microbial load, the microbial viability and vitality. Depending on the stage of application of PFW, up to 5 log10-cycles reduction was achieved, accompanied by a reduction of metabolic activity, but not necessarily with a decrease in metabolic vitality. Multiple application (3step-PFW-application) was more effective than single application (1-step-PFW-application) and PFW showed stronger antimicrobial effect in pre-cleaned endive. High concentrations of nitrite (315 mg l- 1) and nitrate (472 mg l- 1) in PFW were the main factors for the antimicrobial efficacy of PFW against bacteria. Furthermore, H2O2 and an acidic pH supported the mechanism of action against the endive microflora. These results identify the pathway to scale up successful industrial application of PFW targeting microbiological quality and safety of fresh leafy products. Industrial relevance: The safety, quality and shelf life of freshly cut vegetables, e.g. lettuce, are strongly influenced by the microbial load. In addition, the hygienic design of production line, and a good handling/ production practice are indispensable. This study shows that the application of PFW, as a promising non-thermal sanitation technology, enables the inactivation of native microbial contamination on fresh-cut endive depending on the process stage of application. It further describes the impact of PFW on the metabolic activity and metabolic vitality of the lettuce-associated microflora. For higher acceptance, the mechanism of action of PFW was assumed based on previous chemical analyses and compared to the industrial standard of ClO2. The results contribute to the understanding and product-specificity of PFW-induced effects on safety, quality and shelf life of fresh cut lettuce and could be a basis for a possible industrial implementation and complement of common technologies.

This study evaluated the impact of a defined plasma treated water (PTW) when applied to various stages within fresh-cut endive processing. The quality characteristic responses were investigated to establish the impact of the PTW unit processes and where PTW may be optimally applied in a model process line to retain or improve produce quality. Different stages of application of PTW within the washing process were investigated and compared to tap water and chlorine dioxide. Fresh-cut endive (Cichorium endivia L.) samples were analyzed for retention of food quality characteristics. Measurements included color, texture, and nitrate quantification. Effects on tissue surface and cell organelles were observed through scanning electron and atomic force microscopy. Overall, the endive quality characteristics were retained by incorporating PTW in the washing process. Furthermore, promising results for color and texture characteristics were observed, which were supported by the microscopic assays of the vegetal tissue. While ion chromatography detected high concentrations of nitrite and nitrate in PTW, these did not affect the nitrate concentration of the lettuce tissue post-processing and were below the concentrations within EU regulations. These results provide a pathway to scale up the industrial application of PTW to improve and retain quality characteristic retention of fresh leafy products, whilst also harnessing the plasma functionalized water as a process intervention for reducing microbial load at multiple points, whether on the food surface, within the process water or on food-processing surfaces.

Casein, β-lactoglobulin and α-lactalbumin are major milk protein allergens. In the present study, the structural modifications and antigenic response of these bovine milk allergens as induced by non-thermal treatment by atmospheric cold plasma were investigated. Spark discharge (SD) and glow discharge (GD), as previously characterized cold plasma systems, were used for protein treatments. Casein, β-lactoglobulin and α-lactalbumin were analyzed before and after plasma treatment using SDS-PAGE, FTIR, UPLC-MS/MS and ELISA. SDS-PAGE results revealed a reduction in the casein and α-lactalbumin intensity bands after SD or GD treatments; however, the β-lactoglobulin intensity band remained unchanged. FTIR studies revealed alterations in protein secondary structure induced by plasma, particularly contents of β-sheet and β-turn. The UPLC-MS/MS results showed that the amino acid compositions decreased after plasma treatments. ELISA of casein and α-lactalbumin showed a decrease in antigenicity post plasma treatment, whereas ELISA of β-lactoglobulin showed an increase in antigenicity. The study indicates that atmospheric cold plasma can be tailored to mitigate the risk of bovine milk allergens in the dairy processing and ingredients sectors.

In the field of new decontaminants, there is an increasing consensus that improved disinfection of environmental surfaces is needed in patient care facilities as an important component in the overall strategy for prevention of HAI. The interaction of cold atmospheric plasma, i.e. an ionized gas, with liquids results in complex physical and chemical processes, which offer a source of short-lived and long-lived reactive chemical species that are critical for microbial inactivation. These solutions may fulfil the urgent need for new decontaminant solutions for special purposes such as disinfection of surfaces, or use as an antiseptic for body surfaces. In this study, we explored the bactericidal effects of plasma activated liquids (PALs) on E.coli and S. aureus strains and investigated factors which influence PALs stability over time. Liquids of interest were non-complex solutions such as water and saline. An atmospheric cold plasma system using air was employed for the generation of PALs. The solutions were compared with respect to their content of long-lived reactive chemical species and bactericidal effects. Our results documented that PALs may carry different concentrations of chemical species and maintain diverse antimicrobial properties. The bactericidal activity of these solutions demonstrated high thermal stability and could be preserved over a 6 month period through specific sub-ambient storage conditions. Investigation of the inactivation processes in relation to the PALs’ chemical composition will enhance our knowledge on how prokaryotic and eukaryotic cells respond to these, and demonstrate how PALs could be a promising treatment method for future applications, with chemical and antimicrobial stability.

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.

Atmospheric cold plasma (ACP) is under investigation for an extensive range of biocontrol applications in food biosystems. However, the development of a novel intervention technology requires a thorough evaluation of the potential for negative effects and the implications for the human and animal food chains’ safety. The evaluations were performed using a contained, high-voltage, dielectric barrier discharge plasma system. The cytotoxicity of two types of food models—a liquid model (wheat model medium (WMM)) vs. a solid model (wheat grain extract (WGE)) was compared in vitro using the mammalian cell line CHO-K1. The residual toxicity of ACP treatment of grains for food purposes was assessed using the invertebrate model Tribolium castaneum, by feeding the beetles with flour produced from ACP-treated wheat grains. The cytotoxic effects and changes in the chemistry of the ACP-treated samples were more pronounced in samples treated in a liquid form as opposed to actual wheat grains. The feeding trial using T. castaneum demonstrated no negative impacts on the survivability or weight profiles of insects. Investigations into the interactions of plasma-generated species with secondary metabolites in the food matrices are necessary to ensure the safety of plasma for food applications.

The safety and quality of cereal grain supplies are adversely impacted by microbiological contamination, with novel interventions required to maximise whole grains safety and stability. The microbiological contaminants of wheat grains and the efficacy of Atmospheric Cold Plasma (ACP) for potential to control these risks were investigated. The evaluations were performed using a contained reactor dielectric barrier discharge (DBD) system; samples were treated for 0-20 min using direct and indirect plasma exposure. Amplicon-based metagenomic analysis using bacterial 16S rRNA gene and fungal 18S rRNA gene with internal transcribed spacer (ITS) region was performed to characterize the change in microbial community composition in response to ACP treatment. The antimicrobial efficacy of ACP against a range of bacterial and fungal contaminants of wheat, was assessed to include individual isolates from grains as challenge pathogens. ACP influenced wheat microbiome composition, with a higher microbial diversity as well as abundance found on the untreated control grain samples. Culture and genomic approaches revealed different trends for mycoflora detection and control. A challenge study demonstrated that using direct mode of plasma exposure with 20 min of treatment significantly reduced the concentration of all pathogens. Overall, reduction levels for B. atrophaeus vegetative cells were higher than for all fungal species tested, whereas B. atrophaeus spores were the most resistant to ACP among all microorganisms tested. Of note, repeating sub-lethal plasma treatment did not induce resistance to ACP in either B. atrophaeus or A. flavus spores. ACP process control could be tailored to address diverse microbiological risks for grain stability and safety.

Cold atmospheric plasma is a promising new non-thermal technology for improving the microbiological safety and shelf-life of food products, particularly fresh produce and minimally processed fruit and vegetables. Limited research has been conducted on the safety of plasma-treated foods for human or animal consumption. This study focuses on basic safety studies by investigating lettuce broth treated with a di-electric barrier discharge plasma device as a fresh produce model in terms of in vitro cytotoxic and mutagenic effects on mammalian cells and its in vivo toxicity on Galleria mellonella larvae. Low cytotoxic effects were detected in vitro and mutagenic events were likely to be spontaneous mutations. However, a strong response of G. mellonella larvae to injection with plasma-treated lettuce broth was observed for 5-min-treated broth, with larvae survival of less than 10%. No significant effects on quality attributes such as colour were detected and only low concentrations of peroxide were generated in the broth. This study highlights the need for more detailed investigations into the impact of plasma treatment on food components and the subsequent in vitro and in vivo effects to ensure safe implementation of plasma technology for the processing of food products.