The application of atmospheric-pressure plasma jets (APPJs) in medicine often involves interaction with saline environments. This study examines how the salinity of the target liquid influences APPJ behaviour and plasma-induced liquid chemistry. The APPJ is used to treat two types of targets: (a) deionized water (DIW) and (b) an agarose gel made in DIW, with both targets made with and without the inclusion of physiologically-relevant 150 mM NaCl. APPJ is operated with argon gas at an applied voltage of 1.60 kV (rms), current of 1.95 mA (rms), frequency of approximate to 35kHz, and output power of approximate to 0.55W. The coupling of the APPJ with the DIW or NaCl solution establishes a stronger conductive path for the flow of current/charge towards the target. The magnitude of charge/current flow is higher for the 150 mM NaCl solution due to the presence of more conductive ions inside the solution. The higher flow of charges towards NaCl solution leads to a stronger emission from excited species at the plasma-liquid interface and this is confirmed through optical emission spectroscopy. It was found that the APPJ produced more hydrogen peroxide ( H2O2) and nitrites ( NO2-), as markers of reactive oxygen and nitrogen species, respectively, in DIW compared to 150 mM NaCl. However, the oxidation potential of agarose gel was qualitatively enhanced with the addition of NaCl. These contrasting results suggest that measurement of H2O2 and NO2- is not reliable to predict the oxidative potential of all media, particularly with saline media. We attribute the discrepancy in our result to the formation of potent oxidant hypochlorous acid (HOCl) in saline media, which inhibits the formation of H2O2 as described in the manuscript through the differing chemical reaction pathways that take place when the APPJ interacts with DIW and NaCl solution, and their respective rate coefficients.

Nitric oxide (NO)-releasing coatings have promising potential for biomedical applications notably in implant safety and wound dressing by promoting healing and reducing bacterial growth. Yet, the production of NO-films remains difficult through classic approaches. In this study, plasma polymerized NO-coatings are produced using a helium-isopentyl nitrite mixture under two power settings and deposited on aluminum samples. Analyses of the plasma phase by mass spectroscopy reveal the presence of nitrosoxy groups (O-NO) in monomer and quasi-monomer at low power, and a higher fragmentation rate at high power. Static and no-static samples are made and analyzed by X-ray photoelectron spectroscopy showing the presence of these group for both power conditions, with a better retention on the sample's center for the latest. This study investigates the production of nitric oxide-films through atmospheric-pressure plasma jet using a helium-isopentyl nitrite mixture, on both static and nonstatic samples. Experimental parameters, including input power and nozzle-sample distances are confronted to compare their influence on the deposited films to optimize the retention of nitrosoxy groups (O-NO) on the sample's surface. image

While blood-contacting materials are widely deployed in medicine in vascular stents, catheters, and cannulas, devices fail in situ because of thrombosis and restenosis. Furthermore, microbial attachment and biofilm formation is not an uncommon problem for medical devices. Even incremental improvements in hemocompatible materials can provide significant benefits for patients in terms of safety and patency as well as substantial cost savings. Herein, a novel but simple strategy is described for coating a range of medical materials, that can be applied to objects of complex geometry, involving plasma-grafting of an ultrathin hyperbranched polyglycerol coating (HPG). Plasma activation creates highly reactive surface oxygen moieties that readily react with glycidol. Irrespective of the substrate, coatings are uniform and pinhole free, comprising O─C─O repeats, with HPG chains packing in a fashion that holds reversibly binding proteins at the coating surface. In vitro assays with planar test samples show that HPG prevents platelet adhesion and activation, as well as reducing (>3 log) bacterial attachment and preventing biofilm formation. Ex vivo and preclinical studies show that HPG-coated nitinol stents do not elicit thrombosis or restenosis, nor complement or neutrophil activation. Subcutaneous implantation of HPG coated disks under the skin of mice shows no evidence of toxicity nor inflammation.

This paper presents and explains the principle behind anelectrochemical method to enhance the antimicrobial action of plasma-activated hydrogel therapy (PAHT) in the context of wound decontamination. The process involves grounding and hydratingpoly(vinyl alcohol) (PVA) hydrogel films during treatment with a helium (He) plasma jet. This electrochemically enhances production of hydrogen peroxide (H2O2), which is amajor antibacterial agent produced in the PVA hydrogel. Production of H2O2 is shownto be electrically enhanced through electron dissociation reactions, and through reactions associated with excited state species, metastables and ultra-violet (UV) photolysis. H2O2 production is chemically enhanced through the He flow of the plasma jet dehydrating the PVA hydrogel, which fuels the electrochemical dependent reactions associated with H2O2 production. The electrochemical process produces an unprecedented 3.4 mM of H2O2 in the PVA hydrogel. Production of other molecules such as reactive nitrogen species (RNS) are also enhanced by the same method. The electrochemically enhanced PAHT is highly effective ateradicating common wound pathogens Escherichia coli and Pseudomonas aeruginosa and mildly effective against Staphylococcus aureus. Overall, this study shows that the new PAHT dressing offers a promising alternative to antibiotics and silver-based dressings for controlling infection and stimulating healing in wounds. An electrochemical approach is investigated to enhance the antimicrobial action of plasma-activated hydrogel therapy (PAHT). The paper explores the electrochemical mechanisms that underpin increased hydrogen peroxide (H2O2) production in PAHT and how this enhances PAHT's antibacterial efficacy against prevalent wound pathogens. Electrochemically enhanced PAHT is shown to be a promising new wound dressing to overcome escalating challenges in antimicrobial resistance. image

Silver is an antimicrobial commonly used within wound care chiefly in advanced dressings or in a topical cream form, such as silver sulfadiazine (SSD). Although silver is effective at controlling the growth of many common wound bacteria, it can be cytotoxic and can build up in tissue, stalling the healing process. Here, we demonstrate the development of an on-demand release system for delivery of silver from a composite hydrogel comprising sodium polyacrylate particles dispersed in a cryo-crosslinked polyvinyl alcohol carrier gel. Application of cold atmospheric plasma (CAP) jet to the silver loaded hydrogel resulted in controlled release of silver. This release is thought to occur due to the formation of nitrous acids in the hydrogel by the CAP, resulting in protonation of carboxylate groups in the hydrogel and subsequent gel de-swelling due to the reduction in interchain charge repulsion. The location of silver within the sodium polyacrylate particles was probed using scanning electron microscopy and EDX imaging. The released silver inhibited the growth of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus and significantly reduced the viable cell count of the P. aeruginosa biofilm.

Chronic wound infections are a silent pandemic in danger of becoming a global healthcare crisis. Innovations to control infections and improve healing are required. In the context of this challenge, researchers are exploiting plasma-activated hydrogel therapy (PAHT) for use either alone or in combination with other antimicrobial strategies. PAHT involves the cold atmospheric pressure plasma activation of hydrogels with reactive oxygen and nitrogen species to decontaminate infections and promote healing. This opinion article describes PAHT for wound treatment and provides an overview of current research and outstanding challenges in translating the technology for medical use. A 'blueprint' of an autonomous PAHT is presented in the final section that can move the management and treatment of wounds from the clinical setting to the community.

Whilst blood‐contacting materials are widely deployed in medicine in vascular stents, catheters and cannulas, devices fail in‐situ because of thrombosis and restenosis. Furthermore, microbial attachment and biofilm formation is not an uncommon problem for medical devices. Even incremental improvements in hemocompatible materials could provide significant benefits for patients in terms of safety and patency as well as substantial cost savings.Herein, we describe a novel but simple strategy for coating a range of medical materials, that can be applied to objects of complex geometry, involving plasma‐grafting of an ultra‐thin hyperbranched polyglycerol coating (HPG). Plasma activation creates highly reactive surface oxygen moieties that readily react with glycidol. Irrespective of the substrate, coatings are uniform and pinhole free, comprising O‐C‐O repeats, with HPG chains packing in a fashion that holds reversibly binding proteins at the coating surface.In vitro assays with planar test samples show that HPG prevents platelet adhesion and activation, as well as reducing (>3log) bacterial attachment and preventing biofilm formation. Ex vivo and preclinical studies show that HPG‐coated nitinol stents do not elicit thrombosis or restenosis, nor complement or neutrophil activation. Subcutaneous implantation of HPG coated disks under the skin of mice showed no evidence of toxicity nor inflammation. This article is protected by copyright. All rights reserved

We present the concept of a versatile drug-loaded composite hydrogel that can be activated using an argon-based cold atmospheric plasma (CAP) jet to deliver both a drug and CAP-generated molecules, concomitantly, in a tissue target. To demonstrate this concept, we utilized the antibiotic gentamicin that is encapsulated in sodium polyacrylate (PAA) particles, which are dispersed within a poly(vinyl alcohol) (PVA) hydrogel matrix. The final product is a gentamicin-PAA-PVA composite hydrogel suitable for an on-demand triggered release using CAP. We show that by activating using CAP, we can effectively release gentamicin from the hydrogel and also eradicate the bacteria effectively, both in the planktonic state and within a biofilm. Besides gentamicin, we also successfully demonstrate the applicability of the CAP-activated composite hydrogel loaded with other antimicrobial agents such as cetrimide and silver. This concept of a composite hydrogel is potentially adaptable to a range of therapeutics (such as antimicrobials, anticancer agents, and nanoparticles) and activatable using any dielectric barrier discharge CAP device.

Here, we present a novel plasma treatment regime—plasma fractionation, analogous to the concept of dose fractionation in radiotherapy, which could see the application in plasma-based cancer treatment. In plasma fractionation, a single acute dose of plasma is divided into multiple small dosages (fractionated dosages) and administered to the cells in vitro at 24-h intervals. We utilized a helium plasma jet and studied the effects of plasma fractionation in an immortalized keratinocyte line (HaCaT) and a squamous cell carcinoma line (A431). The effects were assessed over three cell seeding densities—8000, 3500, and 1000 cells/well. Our results show that at all seeding densities, plasma fractionation produced lower levels of cell death in both cell types compared to the same dose administered as a single plasma treatment. This highlights the potential of plasma fractionation as a potentially safer method to conduct plasma treatments in the future. We also show that A431 cells were more sensitive to a single acute plasma treatment than HaCaT cells, at cell densities that are subconfluent (1000 cells/well). A similar difference in sensitivity between HaCaT cells and A431 cells was not observed on exogenous treatment with hydrogen peroxide, pointing to the importance of other shorter lived plasma components.

The global clinical and socioeconomic impact of chronic wounds is substantial. The main difficulty that clinicians face during the treatment of chronic wounds is the risk of infection at the wound site. Infected wounds arise from an accumulation of microbial aggregates in the wound bed, leading to the formation of polymicrobial biofilms that can be largely resistant to antibiotic therapy. Therefore, it is essential for studies to identify novel therapeutics to alleviate biofilm infections. One innovative technique is the use of cold atmospheric plasma (CAP) which has been shown to possess promising antimicrobial and immunomodulatory properties. Here, different clinically relevant biofilm models will be treated with cold atmospheric plasma to assess its efficacy and killing effects. Biofilm viability was assessed using live dead qPCR, and morphological changes associated with CAP evaluated using scanning electron microscopy (SEM). Results indicated that CAP was effective against Candida albicans and Pseudomonas aeruginosa, both as mono-species biofilms and when grown in a triadic model system. CAP also significantly reduced viability in the nosocomial pathogen, Candida auris. Staphylococcus aureus Newman exhibited a level of tolerance to CAP therapy, both when grown alone or in the triadic model when grown alongside C. albicans and P. aeruginosa. However, this degree of tolerance exhibited by S. aureus was strain dependent. At a microscopic level, biofilm treatment led to subtle changes in morphology in the susceptible biofilms, with evidence of cellular deflation and shrinkage. Taken together, these results indicate a promising application of direct CAP therapy in combatting wound and skin-related biofilm infections, although biofilm composition may affect the treatment efficacy.

Diagnosing biofilm infections has remained a constant challenge for the last 50 years. Existing diagnostic methods struggle to identify the biofilm phenotype. Moreover, most methods of biofilm analysis destroy the biofilm making the resultant data interpretation difficult. In this study we introduce Fourier Transform Infra-Red (FTIR) spectroscopy as a label-free, non-destructive approach to monitoring biofilm progression. We have utilised FTIR in a novel application to evaluate the chemical composition of bacterial biofilms without disrupting the biofilm architecture. S. epidermidis (RP62A) was grown onto calcium fluoride slides for periods of 30 min-96 h, before semi-drying samples for analysis. We report the discovery of a chemical marker to distinguish between planktonic and biofilm samples. The appearance of new proteins in biofilm samples of varying maturity is exemplified in the spectroscopic data, highlighting the potential of FTIR for identifying the presence and developmental stage of a single biofilm.

Current methods used to treat non-muscle invasive bladder cancer are inadequate due to a high recurrence rate after surgery and the occurrence of adverse events such as interstitial pneumonia following intravesical instillation therapy. Low-temperature plasma is a new form of physical therapy that provides a rich source of reactive oxygen species (ROS). Oxidative solutions, created by pre-treatment of aqueous media with plasma before application to target cells, lead to the destruction of cancer cells through oxidative stress pathways. This study focuses on the effects of plasma-activated media (PAM) in bladder cancer cells. PAM treatment increases oxidative stress that leads to cell cycle arrest and concomitantly depolarises the mitochondrial membrane leading to increased mitochondrial ROS production. Cell cycle arrest and increased mitochondrial ROS production led to an increase in caspase 3/cytochrome c activity, which might explain the induction of apoptosis in bladder cancer cells in vitro and in a bladder cancer tumour in vivo. These observations highlight the potential of plasma activated solutions as a new adjuvant therapy in the clinical treatment of bladder cancer.

This study investigates how addition of a 2nd ground electrode in an argon plasma jet influences the production of hydrogen peroxide (H 2 O 2 ) in deionised water (DIW). Briefly, plasma is ignited by purging argon gas through a quartz tube at 1 l min −1 and applying a sinusoidal voltage of 7 kV (peak–peak) at 23.5 kHz to a high voltage stainless steel needle electrode sealed inside the quartz tube surrounded by one or two copper ring(s) that served as the ground electrode(s) situated downstream of the high voltage electrode. The mechanisms of H 2 O 2 production are investigated through the electrical and optical plasma properties and chemical analysis of the treated DIW. We discover that the addition of a 2nd ground electrode results in higher accumulation of charges on the inner wall surface of the quartz tube of the plasma jet assembly resulting in an increase in the discharge current and dissipated power. This further leads to an increase in the electron temperature that more than doubles the H 2 O 2 production through dissociative recombination of water vapour molecules, whilst still maintaining a biological tissue tolerable gas temperature. The double ground electrode plasma jet is shown to be highly effective at reducing the growth of common wound pathogens ( Pseudomonas aeruginosa and Staphylococcus aureus ) in both planktonic and biofilm states whilst inducing a low level of cytotoxicity in HaCaT keratinocyte skin-like cells under certain conditions. The information provided in this study is useful in understanding the complex physicochemical processes that influence H 2 O 2 production in plasma jets, which is needed to optimise the development of plasma sources for clinical applications.

Wounds can commonly become infected with polymicrobial biofilms containing bacterial and fungal microorganisms. Microbial colonization of the wound can interfere with sufficient healing and repair, leading to high rates of chronicity in certain individuals, which can have a huge socioeconomic burden worldwide. One route for alleviating biofilm formation in chronic wounds is sufficient treatment of the infected area with topical wound washes and ointments. Thus, the primary aim here was to create a complex in vitro biofilm model containing a range of microorganisms commonly isolated from the infected wound milieu. These polymicrobial biofilms were treated with three conventional anti-biofilm wound washes, chlorhexidine (CHX), povidone-iodine (PVP-I), and hydrogen peroxide (H2O2), and efficacy against the microorganisms assessed using live/dead qPCR. All treatments reduced the viability of the biofilms, although H2O2 was found to be the most effective treatment modality. These biofilms were then co-cultured with 3D skin epidermis to assess the inflammatory profile within the tissue. A detailed transcriptional and proteomic profile of the epidermis was gathered following biofilm stimulation. At the transcriptional level, all treatments reduced the expression of inflammatory markers back to baseline (untreated tissue controls). Olink technology revealed a unique proteomic response in the tissue following stimulation with untreated and CHX-treated biofilms. This highlights treatment choice for clinicians could be dictated by how the tissue responds to such biofilm treatment, and not merely how effective the treatment is in killing the biofilm.

The study compares how acetyl donor molecules tetraacetylethylenediamine (TAED) and pentaacetate glucose (PAG) improve the antibacterial efficacy of solutions activated with a low-temperature atmospheric-pressure argon plasma jet. Plasma activation of solubilised TAED and PAG produce solutions with different chemical compositions and oxidative potentials. Both acetyl donor molecules enhance the hydrogen peroxide (H2O2) concentration in solution with TAED being more effective compared to PAG. However, PAG is more effective at forming peracetic acid (PAA) from reaction of its acetyl donor groups with plasma generated H2O2. The enhanced oxidative potential of plasma activated TAED and PAG solutions were shown to significantly improve bactericidal activity against common wound pathogens Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus compared to plasma activated water produced without acetyl donors. Furthermore, the oxidative capacity of plasma activated PAG was least affected by the bacterial oxidative defence enzyme catalase, attributed to the high concentration of PAA produced in this formulation. Overall, the above data show that acetyl donors may help improve next generation of antimicrobial formulations produced by plasma, which might help combat increasing problems of antimicrobial resistance.

A conical assembly of six plasma jets arranged in a rectangular pattern for biomedical applications is presented. The conical configuration increases the separation distance between individual tubes within the assembly that reduces interference between individual plasma jets and enables the jets to converge at the output, facilitating more uniform treatment as opposed to plasma jet arrays operated in parallel. Electrical and optical diagnostics of the plasma discharges and measurements of H2O2 and NO2− production in de-ionized water are used to characterize the potential suitability of the device for biomedical applications. Particularly, it was found that the efficiency in H2O2 (an important bacterial disinfection agent) production by the conical assembly of six plasma jets was more than nine-fold higher compared to its single plasma jet counterpart and that this could be achieved at a biocompatible temperature of below 300 K. Therefore, the device may find use in biomedical applications, particularly where larger area treatments are required such as for certain wounds and cancer tumors that can span areas of tens of cm2.

Improved retention of desirable chemical structures during plasma polymerization requires rational tailoring of plasma-phase conditions. Using ethyl trimethylacetate, we studied the effects of pressure and power on the contribution of intact molecular ions to deposition and retention of ester groups. The abundance of protonated molecular ions in plasmas varies with pressure and power, but the functionality of plasma polymers, assessed by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry, is not correlated. Together with high ion flux, the ion energy distribution was found to be a key parameter and needs to be tailored to enable the soft landing of ions on the surface after traversing the sheath. The compromise between the abundance of ions and their energy distribution is optimal near the transition between the alpha and gamma plasma phases.

The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV-radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test, respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, which did not resemble that for UV implying that CAP-produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences.

Current methods used to treat non-muscle invasive bladder cancer are inadequate due to a high recurrence rate after surgery and occurrence of adverse events such as interstitial pneumonia following intravesical instillation therapy. Low-temperature plasma is a new form of physical therapy that provides a rich source of reactive oxygen species (ROS). Oxidative solutions, created by pre-treatment of aqueous media with plasma before application to target cells, lead to the destruction of cancer cells through oxidative stress pathways. This study focuses on the effects of plasma activated media (PAM) in bladder cancer cells. PAM treatment increases up regulation of p21 and down regulation of Cyclin D and CdK4 leading to cell cycle arrest, and concomitantly depolarises the mitochondrial membrane leading to increased mitochondrial ROS production. Cell cycle arrest and increased mitochondrial ROS production induce apoptosis in bladder cancer cells in vitro and in a bladder cancer tumour in vivo via the pro-apoptotic caspase 3/cytochrome c pathway. These observations highlight the potential of plasma activated solutions as a new adjuvant therapy in the clinical treatment of bladder cancer.