Nitrosylation vs. oxidation - How to modulate cold physical plasmas for biological applications.

Thiol moieties are major targets for cold plasma-derived nitrogen and oxygen species, making CAPs convenient tools to modulate redox-signaling pathways in cells and tissues. The underlying biochemical pathways are currently under investigation but especially the role of CAP derived RNS is barely understood. Their potential role in protein thiol nitrosylation would be relevant in inflammatory processes such as wound healing and improving their specific production by CAP would allow for enhanced treatment options beyond the current application. The impact of a modified kINPen 09 argon plasma jet with nitrogen shielding on cysteine as a thiol-carrying model substance was investigated by FTIR spectroscopy and high-resolution mass spectrometry. The deposition of short-lived radical species was measured by electron paramagnetic resonance spectroscopy, long-lived species were quantified by ion chromatography (NO2-, NO3-) and xylenol orange assay (H2O2). Product profiles were compared to samples treated with the so-called COST jet, being introduced by a European COST initiative as a reference device, using both reference conditions as well as conditions adjusted to kINPen gas mixtures. While thiol oxidation was dominant under all tested conditions, an Ar + N2/O2 gas compositions combined with a nitrogen curtain fostered nitric oxide deposition and the desired generation of S-nitrosocysteine. Interestingly, the COST-jet revealed significant differences in its chemical properties in comparison to the kINPen by showing a more stable production of RNS with different gas admixtures, indicating a different •NO production pathway. Taken together, results indicate various chemical properties of kINPen and COST-jet as well as highlight the potential of plasma tuning not only by gas admixtures alone but by adjusting the surrounding atmosphere as well.

Fluorescence measurements of peroxynitrite/peroxynitrous acid in cold air plasma treated aqueous solutions.

Qualitative detection of peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) as one of the key bactericidal agents produced in cold air plasma activated aqueous solutions is presented. We examined the use of the 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent dye to detect ONOO-/ONOOH in plasma activated non-buffered water (PAW) or buffered solution (PAPB) generated by DC-driven self-pulsed transient spark discharge at atmospheric pressure in ambient air. The diagnostic selectivity of H2DCFDA to reactive oxygen and nitrogen species (RONS) typical of plasma activated aqueous solutions was examined by using various scavengers of RONS. This cross-reactivity study showed the highest sensitivity of the H2DCFDA dye to ONOO-/ONOOH. However, besides ONOO-/ONOOH, H2DCFDA also exhibited sensitivity to hypochlorite anions/hypochlorous acid (OCl-/HOCl), showing that for a selective study it is important to have an idea about the possible constituents in the studied solutions. The sensitivity of H2DCFDA to other RONS even in much higher concentrations was negligible. The presence of nitrites (NO2-) and hydrogen peroxide (H2O2) in PAW led predominantly to the production of peroxynitrous acid with a strong fluorescence response of H2DCFDA in PAW. Plasma treatment of buffered solutions led to the weak response of H2DCFDA. The fluorescence induced in PAW decreased after scavenging individual reactants, namely NO2- and H2O2, as well as by scavenging the product of the peroxynitrite forming reaction, proving that the fluorescence response of H2DCFDA is primarily due to the formation of ONOO-/ONOOH. A chemical kinetics analysis of post-discharge processes and the pseudo-second order reaction between H2O2 and NO2- confirms formation of peroxynitrous acid in PAW with a rate in the order of tens of nM per second. The post-discharge evolution of the ONOOH formation rate was clearly correlated with the parallel detection of ONOO-/ONOOH by fluorescence spectroscopy using the H2DCFDA dye.

Non-touching plasma-liquid interaction - where is aqueous nitric oxide generated?

Nitric oxide is a relatively stable free radical and an important signal molecule in plants, animals, and humans with high relevance for biological processes involving inflammatory processes, e.g. wound healing or cancer. The molecule can be detected in the gas phase of non-thermal plasma jets making it a valuable tool for clinical intervention, but transport efficiency from the gas phase into the liquid phase or tissue remains to be clarified. To elucidate this fact, the nitric oxide concentration in buffered solutions is determined using electron paramagnetic resonance spectroscopy. The origin of the nitric oxide in the liquid could be excluded, therefore, potential precursors such as hydroxyl radicals, superoxide anions, atomic hydrogen and stable species (nitrite, nitrate and hydrogen peroxide) were detected and the potential formation pathway as well as ways of enhancing the production of nitric oxide by alteration of the feed gas and the surrounding gas composition during plasma treatment of the liquid have been pointed out.

Quantification of the ozone and singlet delta oxygen produced in gas and liquid phases by a non-thermal atmospheric plasma with relevance for medical treatment.

In the field of plasma medicine, the identification of relevant reactive species in the liquid phase is highly important. To design the plasma generated species composition for a targeted therapeutic application, the point of origin of those species needs to be known. The dominant reactive oxygen species generated by the plasma used in this study are atomic oxygen, ozone, and singlet delta oxygen. The species density changes with the distance to the active plasma zone, and, hence, the oxidizing potential of this species cocktail can be tuned by altering the treatment distance. In both phases (gas and liquid), independent techniques have been used to determine the species concentration as a function of the distance. The surrounding gas composition and ambient conditions were controlled between pure nitrogen and air-like by using a curtain gas device. In the gas phase, in contrast to the ozone density, the singlet delta oxygen density showed to be more sensitive to the distance. Additionally, by changing the surrounding gas, admixing or not molecular oxygen, the dynamics of ozone and singlet delta oxygen behave differently. Through an analysis of the reactive species development for the varied experimental parameters, the importance of several reaction pathways for the proceeding reactions was evaluated and some were eventually excluded.

Long-lived and short-lived reactive species produced by a cold atmospheric pressure plasma jet for the inactivation of Pseudomonas aeruginosa and Staphylococcus aureus.

Different chemical pathways leading to the inactivation of Pseudomonas aeruginosa and Staphylococcus aureus by a cold atmospheric pressure plasma jet (APPJ) in buffered and non-buffered solutions are reported. As APPJs produce a complex mixture of reactive species in solution, a comprehensive set of diagnostics were used to assess the liquid phase chemistry. This includes absorption and electron paramagnetic resonance spectroscopy in addition to a scavenger study to assess the relative importance of the various plasma produced species involved in the inactivation of bacteria. Different modes of inactivation of bacteria were found for the same plasma source depending on the solution and the plasma feed gas. The inactivation of bacteria in saline is due to the production of short-lived species in the case of argon plasma when the plasma touches the liquid. Long-lived species (ClO-) formed by the abundant amount of O. radicals produced by the plasmas played a dominant role in the case of Ar + 1% O2 and Ar + 1% air plasmas when the plasma is not in direct contact with the liquid. Inactivation of bacteria in distilled water was found to be due to the generation of short-lived species: O. &O2.- for Ar + 1% O2 plasma and O2.- (and .OH in absence of saline) for Ar plasma.

Degradation and intermediates of diclofenac as instructive example for decomposition of recalcitrant pharmaceuticals by hydroxyl radicals generated with pulsed corona plasma in water.

Seven recalcitrant pharmaceutical residues (diclofenac, 17α-ethinylestradiol, carbamazepine, ibuprofen, trimethoprim, diazepam, diatrizoate) were decomposed by pulsed corona plasma generated directly in water. The detailed degradation pathway was investigated for diclofenac and 21 intermediates could be identified in the degradation cascade. Hydroxyl radicals have been found primarily responsible for decomposition steps. By spin trap enhanced electron paramagnetic resonance spectroscopy (EPR), OH-adducts and superoxide anion radical adducts were detected and could be distinguished applying BMPO as a spin trap. The increase of concentrations of adducts follows qualitatively the increase of hydrogen peroxide concentrations. Hydrogen peroxide is eventually consumed in Fenton-like processes but the concentration is continuously increasing to about 2mM for a plasma treatment of 70min. Degradation of diclofenac is inversely following hydrogen peroxide concentrations. No qualitative differences between byproducts formed during plasma treatment or due to degradation via Fenton-induced processes were observed. Findings on degradation kinetics of diclofenac provide an instructive understanding of decomposition rates for recalcitrant pharmaceuticals with respect to their chemical structure. Accordingly, conclusions can be drawn for further development and a first risk assessment of the method which can also be applied towards other AOPs that rely on the generation of hydroxyl radicals.

Oxygen atoms are critical in rendering THP-1 leukaemia cells susceptible to cold physical plasma-induced apoptosis.

Cold physical plasma has been suggested as a powerful new tool in oncology. However, some cancer cells such as THP-1 leukaemia cells have been shown to be resistant towards plasma-induced cell death, thereby serving as a good model for optimizing plasmas in order to foster pro-apoptotic anticancer effects. A helium/oxygen radio frequency driven atmospheric plasma profoundly induced apoptosis in THP-1 cells whereas helium, humidified helium, and humidified helium/oxygen plasmas were inefficient. Hydrogen peroxide - previously shown as central plasma-derived agent - did not participate in the killing reaction but our results suggest hypochlorous acid to be responsible for the effect observed. Proteomic analysis of THP-1 cells exposed to He/O2 plasma emphasized a prominent growth retardation, cell stress, apoptosis, and a pro-immunogenic profile. Altogether, a plasma setting that inactivates previously unresponsive leukaemia cells is presented. Crucial reactive species in the plasma and liquid environment were identified and discussed, deciphering the complexity of plasma from the gas phase into the liquid down to the cellular response mechanism. These results may help tailoring plasmas for clinical applications such as oxidation-insensitive types of cancer.

Role of Ambient Gas Composition on Cold Physical Plasma-Elicited Cell Signaling in Keratinocytes.

A particularly promising medical application of cold physical plasma is the support of wound healing. This is presumably achieved by modulating inflammation as well as skin cell signaling and migration. Plasma-derived reactive oxygen and nitrogen species (ROS/RNS) are assumed the central biologically active plasma components. We hypothesized that modulating the environmental plasma conditions from pure nitrogen (N2) to pure oxygen (O2) in an atmospheric pressure argon plasma jet (kINPen) will change type and concentration of ROS/RNS and effectively tune the behavior of human skin cells. To investigate this, HaCaT keratinocytes were studied in vitro with regard to cell metabolism, viability, growth, gene expression signature, and cytokine secretion. Flow cytometry demonstrated only slight effects on cytotoxicity. O2 shielding provided stronger apoptotic effects trough caspase-3 activation compared to N2 shielding. Gene array technology revealed induction of signaling and communication proteins such as immunomodulatory interleukin 6 as well as antioxidative and proproliferative molecules (HMOX1, VEGFA, HBEGF, CSF2, and MAPK) in response to different plasma shielding gas compositions. Cell response was correlated to reactive species: oxygen-shielding plasma induces a cell response more efficiently despite an apparent decrease of hydrogen peroxide (H2O2), which was previously shown to be a major player in plasma-cell regulation, emphasizing the role of non-H2O2 ROS like singlet oxygen. Our results suggest differential effects of ROS- and RNS-rich plasma, and may have a role in optimizing clinical plasma applications in chronic wounds.

Potential of pulsed corona discharges generated in water for the degradation of persistent pharmaceutical residues.

Anthropogenic pollutants and in particular pharmaceutical residues are a potential risk for potable water where they are found in increasing concentrations. Different environmental effects could already be linked to the presence of pharmaceuticals in surface waters even for low concentrations. Many pharmaceuticals withstand conventional water treatment technologies. Consequently, there is a need for new water purification techniques. Advanced oxidation processes (AOP), and especially plasmas with their ability to create reactive species directly in water, may offer a promising solution. We developed a plasma reactor with a coaxial geometry to generate large volume corona discharges directly in water and investigated the degradation of seven recalcitrant pharmaceuticals (carbamazepine, diatrizoate, diazepam, diclofenac, ibuprofen, 17α-ethinylestradiol, trimethoprim). For most substances we observed decomposition rates from 45% to 99% for treatment times of 15-66 min. Especially ethinylestradiol and diclofenac were readily decomposed. As an inherent advantage of the method, we found no acidification and only an insignificant increase in nitrate/nitrite concentrations below legal limits for the treatment. Studies on the basic plasma chemical processes for the model system of phenol showed that the degradation is primarily caused by hydroxyl radicals.

Plasma jet's shielding gas impact on bacterial inactivation.

One of the most desired aims in plasma medicine is to inactivate prokaryotic cells and leave eukaryotic cells unharmed or even stimulate proliferation to promote wound healing. The method of choice is to precisely control the plasma component composition. Here the authors investigate the inactivation of bacteria (Escherichia coli) by a plasma jet treatment. The reactive species composition created by the plasma in liquids is tuned by the use of a shielding gas device to achieve a reactive nitrogen species dominated condition or a reactive oxygen species dominated condition. A strong correlation between composition of the reactive components and the inactivation of the bacteria is observed. The authors compare the results to earlier investigations on eukaryotic cells and show that it is possible to find a plasma composition where bacterial inactivation is strongest and adverse effects on eukaryotic cells are minimized.