Concentration Of Reactive Oxygen And Nitrogen Species Research Articles

Effect of Plasma-Activated Water on the Cellulase-Producing Strain Aspergillus niger A32.

To investigate the effect and mechanism of plasma-activated water (PAW) on Aspergillus niger, PAW was prepared using a needle array-plate dielectric barrier discharge plasma system. The concentrations of long-lived reactive oxygen and nitrogen species (RONS), namely, H2O2, NO2-, and NO3-, in the PAW were 48.76 mg/L, 0.046 mg/L, and 172.36 mg/L, respectively. Chemically activated water (CAW) with the same concentration of long-lived RONS was also prepared for comparison. A. niger A32 was treated with PAW and CAW. After treatment, the treated strains were observed and analyzed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to screen probable mutants. The results indicated that the pH, conductivity, and ORP values of PAW were 2.42, 1935 μS/cm, and 517.07 mV, respectively. In contrast, the pH and ORP values of CAW were 6.15 and 301.73 mV, respectively, which differed significantly from those of PAW. In addition, the conductivity of CAW showed no change. SEM and TEM analyses revealed that A. niger A32 treated with CAW exhibited less damage compared with the control. In contrast, A. niger A32 treated with PAW showed significant shrinkage, deformation, and exudate attachment over time. Following PAW treatment, after four passages, a high cellulase-producing stable mutant strain A-WW5 was screened, exhibiting a filter paper enzyme activity of 29.66 U/mL, a cellulose endonuclease activity of 13.79 U/mL, and a β-glucosidase activity of 27.13 U/mL. These values were found to be 33%, 38%, and 2.1% higher than those of the original fungus sample, respectively. In total, 116 SNPs and 61 InDels were present in the genome of the mutant strain A-WW5. The above findings indicate that the impact of PAW on A. niger is not only attributed to long-lasting H2O2, NO2-, and NO3- particles but also to other short-lived active particles; PAW is expected to become a new microbial breeding mutagen.

Reduced serum concentrations of reactive oxygen and nitrogen species following strenuous exercise in the heat are not associ-ated with an upregulation in serum antioxidative capacity

Introduction: Reactive oxygen and nitrogen species (RONS) are produced as a response to exercise and play a part in guiding the adaptive response to physical training. Exercise in the heat has been shown to further increase accumulation of RONS in systemic circulation; however, a high anti-oxidative response or heat acclimatization mechanisms could attenuate this response. In a previous study, we found reduced RONS concentrations in trained athletes following strenuous cycling in the heat. Therefore, this secondary analysis assessed whether these reduced RONS serum con-centrations are induced by an increased serum antioxidant capacity. Methods: Twelve male cyclists (V̇O2peak: 60 ± 4 ml ∙ kg-1 ∙ min-1) completed a 60-minute constant workload trial (55% peak power output, ambient temperature 30.4 ± 0.6°C) with and without ice vest in a randomized order. The core body temperature (Tcore) was measured by an ingestible capsule. Blood samples were col-lected before and after each trial to determine superoxide dismutase (SOD) and catalase (CAT) activity, total antioxidant capacity (TAC) and RONS. Due to the absence of between-group dif-ferences, data of both conditions were pooled. Results: Tcore statistically increased (p < 0.001) over the experimental trials (+6.0 ± 1.6%, effect size (ES) = 5.6). Concentrations of RONS (-17.2 ± 15.5%, p < 0.001, ES = 1.0) and TAC (-8.9 ± 22.9%, p = 0.04, ES = 0.7) statistically decreased, while the activity of CAT (+15.5 ± 84.0%, p = 0.90, ES = 0.04) and SOD (+9.2 ± 58.7%, p = 0.98, ES = 0.01) remained un-changed. Conclusions: Reduced serum RONS concentrations after strenuous cycling in the heat were not associated with upregulation of serum antioxidant capacity in trained athletes. The prooxidant-antioxidant balance may rather be regulated at a myocellular level and should be further assessed in future studies.

Precise Control of Glioma Cell Apoptosis Induced by Micro-Plasma-Activated Water (μ-PAW).

To verify the existence of plasma with the potential to kill tumor cells, this paper designed a novel helium (He) micro-plasma jet array device and detected the concentration of typical long-lived reactive oxygen and nitrogen species (RONS) with oxidative activity generated by it. The paper described a new He micro-plasma jet array device consisting of nine flexible quartz capillaries with an inner diameter of 75 μm arranged in a 3 × 3 array. Sterilized ultrapure water (up water) was first treated with the He micro-plasma jet array device to activate it to form enriched RONS micro-plasma-activated water (μ-PAW), and then μ-PAW was added to the cell culture medium (with cells) to observe the proliferation of human glioma cells. The concentration of long-lived RONS, such as nitrate (NO3-), was detected according to Beer-Lambert's law in combination with UV spectrophotometry as well as a color development method. The MTT Cell Proliferation and Cytotoxicity Assay Kit combined with the Hoechst Staining Kit were used to assess the proliferation status of the cells. The results showed that the range of RONS concentration variation could be controlled in the order of micromoles (µmol), while plasma-induced tumor cell death is apoptosis that does not affect the surrounding environment.

Spectral Characteristics of Needle Array-Plate Dielectric Barrier Discharge Plasma and Its Activated Water

The plasma-activated water (PAW), as a new environmentally friendly nonthermal processing technology, has attracted wide attention for its applications in agriculture, food, and biomedical fields. This paper used the needle array-plate dielectric barrier discharge (needle array-plate DBD) device to activate deionized water and prepare PAW simply and efficiently. It was found that the concentration of reactive oxygen and nitrogen species (RONS) generated in the process of needle array-plate DBD was large and varied. Especially in the wavelength range of 600–770 nm, the spectral intensity is stronger. And, the ionic wind is more likely to interact with the deionized water. The changes in PAW parameters and UV/Vis spectra with treatment time were measured under different voltages and needle-dielectric plate distances. Results show that increasing discharge voltage or decreasing needle-dielectric plate distance increases the concentration of RONS, ionic wind speed, water evaporation, and conductivity of the PAW and decreases pH. UV/Vis spectra results show that prolonged treatment time results in increased total absorbance and concentrations of H2O2 and NO 3 − and that a new absorption peak appears at 210 nm in the UV/Vis spectra. When the wavelength is larger than 210 nm, a redshifted new peak and color enhancement are observed. The seeds of Astragalus adsurgens Pall were treated by discharge plasma, PAW, and the combination of plasma and PAW. It was found that high voltage and long-time activated PAW could significantly increase the ROS level of seeds and seedlings after germination for 3 days, resulting in oxidative stress damage. The survival rate of seeds was lower than that under the half lethal dose. This paper provides a feasible device design for treating activated water in large quantities with high efficiency, which is important for the application of PAW and mutation breeding of A. adsurgens Pall.

Enhanced Generation of Reactive Species by Cold Plasma in Gelatin Solutions for Selective Cancer Cell Death.

Atmospheric pressure plasma jets generate reactive oxygen and nitrogen species (RONS) in liquids and biological media, which find application in the new area of plasma medicine. These plasma-treated liquids were demonstrated recently to possess selective properties on killing cancer cells and attracted attention toward new plasma-based cancer therapies. These allow for local delivery by injection in the tumor but can be quickly washed away by body fluids. By confining these RONS in a suitable biocompatible delivery system, great perspectives can be opened in the design of novel biomaterials aimed for cancer therapies. Gelatin solutions are evaluated here to store RONS generated by atmospheric pressure plasma jets, and their release properties are evaluated. The concentration of RONS was studied in 2% gelatin as a function of different plasma parameters (treatment time, nozzle distance, and gas flow) with two different plasma jets. Much higher production of reactive species (H2O2 and NO2-) was revealed in the polymer solution than in water after plasma treatment. The amount of RONS generated in gelatin is greatly improved with respect to water, with concentrations of H2O2 and NO2- between 2 and 12 times higher for the longest plasma treatments. Plasma-treated gelatin exhibited the release of these RONS to a liquid media, which induced an effective killing of bone cancer cells. Indeed, in vitro studies on the sarcoma osteogenic (SaOS-2) cell line exposed to plasma-treated gelatin led to time-dependent increasing cytotoxicity with the longer plasma treatment time of gelatin. While the SaOS-2 cell viability decreased to 12%-23% after 72 h for cells exposed to 3 min of treated gelatin, the viability of healthy cells (hMSC) was preserved (∼90%), establishing the selectivity of the plasma-treated gelatin on cancer cells. This sets the basis for designing improved hydrogels with high capacity to deliver RONS locally to tumors.

Perfluorodecalin to enhance reactive species delivery in plasma-biomaterial interactions

Treatment of biological tissue by non-thermal atmospheric pressure plasmas in air can often cause beneficial biological outcomes, commonly by generation and delivery of reactive oxygen and nitrogen species (RONS). Biological plasma treatment is often mediated through plasma-liquid interactions, wherein plasma-generated RONS are dissolved into the liquid phase and subsequently initiate liquid-phase chemistry. In this paper we report observations on plasma-liquid interactions using a biologically inert perfluorocarbon solvent—perfluorodecalin (PFD)—that is known to be useful in other biomedical applications. The present paper concerns thin films of liquid PFD applied to porous and non-porous surfaces and subsequent plasma exposure of these films. With PFD application as a thin film prior to plasma exposure, we observe evidence of delayed, but ultimately enhanced RONS delivery to non-porous surfaces and through porous underlying surfaces. We demonstrate that PFD dissolves and retains relatively high concentrations of plasma-produced RONS, especially NO2. When the thin liquid film of PFD evaporates during plasma exposure, there appears to be a relatively rapid release of dissolved RONS, potentially aiding certain applications.

On the penetration of reactive oxygen and nitrogen species generated by a plasma jet into and through mice skin with/without stratum corneum

In this work, reactive oxygen and nitrogen species (RONS) generated by a plasma jet penetrating through and left in the skin after the plasma treatment are measured, and the effects of stratum corneum (SC) on the penetration of the RONS are also investigated. It is found that the RONS generated by the plasma jet can penetrate through the skin, and that the penetration of some kinds of the RONS could be enhanced significantly by tape stripping the SC layer of the skin. Further investigations find that the typical reactive oxygen species, including OH, 1O2, O3, and H2O2, cannot penetrate through the mice skin at all (under the detection limit) no matter whether the SC layer of the skin is present or not, where the thickness of the mice skin is about 200–300 μm. This result is very different from the experimental results obtained from the muscle tissue model and gelatin model. Finally, it is found that high concentrations of long-lived RONS (H2O2, NO2−, and NO3−) are left in the skin after the plasma treatment, which means that the plasma treatment could have a long-time scale therapy effect. This finding is important for the applications of plasma medicine.

Chemical and antibacterial effects of plasma activated water: correlation with gaseous and aqueous reactive oxygen and nitrogen species, plasma sources and air flow conditions

When cold atmospheric plasma comes into contact with water and biological media, antimicrobial or antitumor effects are induced, representing great potential for applications in biomedicine and agriculture. The need to control and tune the chemical composition and biomedical effects of plasma activated water/media (PAW/PAM) is emerging. By comparing two nonthermal air plasma sources, streamer corona and transient spark, interacting with water in open and closed reactors, and by enhancing the plasma–liquid interaction by water electrospray through these discharges, we demonstrate that the plasma gaseous products strongly depend on the discharge regime, its deposited power and gas flow conditions. The streamer corona strongly leads to the formation of ozone and hydrogen peroxide, while the more energetic transient spark leads to nitrogen oxides and hydrogen peroxide. The gaseous products then determine the chemical properties of the PAW and the dominant aqueous reactive oxygen and nitrogen species (RONS). The production of hydrogen peroxide depends on water evaporation and hydroxyl radical formation, which is determined by the discharge power. A transient spark produces higher concentrations of gaseous and aqueous RONS and induces stronger antibacterial effects than a streamer corona; however, the RONS production rates per joule of deposited energy are comparable for both studied discharge regimes. The net production rate per joule of gaseous nitrogen oxides strongly correlates with that of aqueous nitrites and nitrates. The antibacterial effects of the PAW tested on Escherichia coli bacteria are determined by the aqueous RONS: in the lower power streamer corona, this is ascertained mainly by the dissolved ozone and hydrogen peroxide, and in the higher power transient spark, by the combination of hydrogen peroxide, nitrite and acidic pH, while in the transient spark in the closed reactor it is determined by the acidified nitrites present.

Effect of tissue thickness and liquid composition on the penetration of long-lifetime reactive oxygen and nitrogen species (RONS) generated by a plasma jet

Two of the key questions in plasma medicine are how deep the reactive oxygen and nitrogen species (RONS) generated by a plasma can penetrate into tissue and how the liquid (extracellular and intracellular fluid) composition affects the concentration of RONS. In this paper, different thicknesses of pig muscle tissue are used as a tissue mode to investigate the effect of tissue thickness on the penetration of RONS through tissue. Six different types of liquid (inorganic group: double-distilled water (DDW), 1% phosphate-buffered saline, 0.9% NaCl; organic group: 5% glucose, 2% serum and 10% serum solution) are used in the receiving chamber under the tissue in order to try to understand the effect of liquid composition on the penetration of RONS (H2O2, and ) generated by the plasma. It is found that when a tissue thickness of 500 µm is used the H2O2 concentrations in organic liquids are about 20–30 times higher than in DDW. The and concentrations in serum liquid are much higher than in all other liquids, which might be due to the plasma reacting with amino acids and proteins. Besides, the concentration in organic solution is higher than the concentration for the same experimental conditions. Furthermore, when the serum percentage is increased from 2% to 10%, the concentration increases dramatically but the concentration decreases significantly. This is especially true for a tissue thickness of 500 µm. One novel discovery is the RONS do not only penetrate the tissue by diffusion—there are also reactions between the plasma and the liquid which affect the final RONS concentration.

Plasma Elicits Immunogenic Death In Melanoma Cells

Development of non-thermal plasma for cancer immunotherapeutic applications has received growing attention, namely for induction of immunogenic cancer cell death (ICD). Cancer cells undergoing ICD emit signals known as damage-associated molecular patterns (DAMPs), that can attract and stimulate local immune cells [1]. Of these, membrane-bound calreticulin is a key DAMP signal that facilitates engulfment of cancer cells by dendritic cells, a critical process for the development of a specific, anti-tumor immune response [2]. Several studies have shown that plasma treatment of cancer cell lines increased the exposure of CRT on the cell surface [3, 4]. However, the mechanism by which plasma elicits ICD is not fully elucidated. We therefore studied the interaction of plasma with melanoma cells, at ICD-inducing regimes. The B16-F10 murine melanoma cell line and the A375 human melanoma cell line were treated with a microsecond-pulsed dielectric barrier discharge system over a range of energies and evaluated for CRT emission. We also studied changes to liquid chemistry following plasma treatment, as cells were not treated under dry conditions. Using electron paramagnetic resonance spectroscopy and colorimetric assays, we identified the reactive oxygen and nitrogen species (RONS) present in the liquid after treatment with plasma. By comparing the trends between the exposure of CRT on the cell membrane and the concentration of RONS in the liquid following increasing plasma treatment energies, we can gain insight into which species are most crucial for plasma-induction of ICD. Knowing this will facilitate the optimization of plasma systems for immunotherapy of cancers. Ongoing work includes evaluation of ICD induction of melanoma cells in an in vivo mouse model.

On the penetration depth of reactive oxygen and nitrogen species generated by a plasma jet through real biological tissue

In this work, an actual biological tissue is used to investigate how thick the reactive oxygen and nitrogen species (RONS) produced by a plasma jet can penetrate through the tissue. This is one of the most critical questions in plasma medicine. The concentration of RONS (O3, H2O2, OH, NO2−, NO2− + NO3−) produced by the plasma jet penetrating through different thickness of the tissue is measured. It is found that the reactive oxygen species (ROS) produced by the plasma are significantly consumed by the tissue. For the tissue thickness of 500 μm, there is only about five percent or even less of the ROS (O3, H2O2, and OH) penetrating through the tissue. On the other hand, more than 80% of the reactive nitrogen species (NO2− + NO3−) are able to penetrate through the 500 μm biological tissue. Furthermore, under certain experimental conditions, some of the RONS can penetrate through 1.25 mm of the biological tissue. Finally, besides H2O2, NO2−, and NO3−, there is some other unknown RONS that penetrate through the biological tissue.

How to assess the plasma delivery of RONS into tissue fluid and tissue

The efficacy of helium (He) and argon (Ar) plasma jets are being investigated for different healthcare applications including wound and cancer therapy, sterilisation and surface disinfections. Current research points to a potential link between the generation of reactive oxygen and nitrogen species (RONS) and outcomes in a range of biological and medical applications. As new data accrue, further strengthening this link, it becomes important to understand the controlled delivery of RONS into solutions, tissue fluids and tissues. This paper investigates the use of He and Ar plasma jets to deliver three RONS (hydrogen peroxide—H2O2, nitrite— and nitrate—) and molecular oxygen (O2) directly into deionised (DI) water, or indirectly into DI water through an agarose target. The DI water is used in place of tissue fluid and the agarose target serves as a surrogate of tissue. Direct plasma jet treatments deliver more RONS and O2 than the through-agarose treatments for equivalent treatments times. The former only deliver RONS whilst the plasma jets are ignited; the latter continues to deliver RONS into the DI water long after the plasmas are extinguished. The He plasma jet is more effective at delivering H2O2 and directly into DI water, but the Ar plasma jet is more effective at nitrating the DI water in both direct and through-agarose treatments. DI water directly treated with the plasma jets is deoxygenated, with the He plasma jet purging more O2 than the Ar plasma jet. This effect is known as ‘sparging’. In contrast, for through-agarose treatments both jets oxygenated the DI water. These results indicate that in the context of direct and indirect plasma jet treatments of real tissue fluids and tissue, the choice of process gas (He or Ar) could have a profound effect on the concentrations of RONS and O2. Irrespective of operating gas, sparging of tissue fluid (in an open wound) for long prolonged periods during direct plasma jet treatment, could have implications for healthy tissue function; whilst through-tissue plasma jet treatment may provide a method to reperfuse oxygen-starved tissue. The assays described in this paper can be readily adopted (by others) and may support the future development of plasma sources to deliver specific (metered) doses of RONS.

Effect of plasma jet diameter on the efficiency of reactive oxygen and nitrogen species generation in water

The plasma jet generation of reactive oxygen and nitrogen species (RONS) in solution is important in biology, medicine, and disinfection. Studies using a wide variety of plasma jet devices have been carried out for this purpose, making it difficult to compare the performance between devices. In this study, we compared the efficiency of RONS generation in deionized (DI) water between 3.7-mm- and 800-µm-sized helium (He) plasma jets (hereafter mm-jet and µm-jet, respectively) at different treatment distances and times. The efficiency of RONS generation was determined by considering the total amount of RONS generated in DI water with respect to the input energy and gas consumption. We found that the mm-jet generated 20% more RONS in the DI water than the µm-jet at the optimized distance. However, when the input power and He gas consumption were taken into account, we discovered that the µm-jet was 5 times more efficient in generating RONS in the DI water. Under the parameters investigated in this study, the concentration of RONS continued to increase as a function of treatment time (up to 30 min). However treatment distance had a marked effect on the efficiency of RONS generation: treatment distances of 25 and 30 mm were optimal for the mm-jet and µm-jet, respectively. Our method of comparing the efficiency of RONS generation in solution between plasma jets could be used as a reference protocol for the development of efficient plasma jet sources for use in medicine, biology, and agriculture.

Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress.

We performed molecular dynamics simulations to investigate the effect of lipid peroxidation products on the structural and dynamic properties of the cell membrane. Our simulations predict that the lipid order in a phospholipid bilayer, as a model system for the cell membrane, decreases upon addition of lipid peroxidation products. Eventually, when all phospholipids are oxidized, pore formation can occur. This will allow reactive species, such as reactive oxygen and nitrogen species (RONS), to enter the cell and cause oxidative damage to intracellular macromolecules, such as DNA or proteins. On the other hand, upon increasing the cholesterol fraction of lipid bilayers, the cell membrane order increases, eventually reaching a certain threshold, from which cholesterol is able to protect the membrane against pore formation. This finding is crucial for cancer treatment by plasma technology, producing a large number of RONS, as well as for other cancer treatment methods that cause an increase in the concentration of extracellular RONS. Indeed, cancer cells contain less cholesterol than their healthy counterparts. Thus, they will be more vulnerable to the consequences of lipid peroxidation, eventually enabling the penetration of RONS into the interior of the cell, giving rise to oxidative stress, inducing pro-apoptotic factors. This provides, for the first time, molecular level insight why plasma can selectively treat cancer cells, while leaving their healthy counterparts undamaged, as is indeed experimentally demonstrated.

Combined effect of protein and oxygen on reactive oxygen and nitrogen species in the plasma treatment of tissue

The influence of protein and molecular, ground state oxygen (O2) on the plasma generation, and transport of reactive oxygen and nitrogen species (RONS) in tissue are investigated. A tissue target, comprising a 1 mm thick gelatin film (a surrogate for real tissue), is placed on top of a 96-well plate; each well is filled with phosphate buffered saline (PBS, pH 7.4) containing one fluorescent or colorimetric reporter that is specific for one of three RONS (i.e., H2O2, NO2−, or OH•) or a broad spectrum reactive oxygen species reporter (2,7-dichlorodihydrofluorescein). A helium cold atmospheric plasma (CAP) jet contacts the top of the gelatin surface, and the concentrations of RONS generated in PBS are measured on a microplate reader. The data show that H2O2, NO2−, or OH• are generated in PBS underneath the target. Independently, measurements are made of the O2 concentration in the PBS with and without the gelatin target. Adding bovine serum albumin protein to the PBS or gelatin shows that protein either raises or inhibits RONS depending upon the O2 concentration. Our results are discussed in the context of plasma-soft tissue interactions that are important in the development of CAP technology for medicine, biology, and food manufacturing.

Slow Molecular Transport of Plasma-Generated Reactive Oxygen and Nitrogen Species and O2 through Agarose as a Surrogate for Tissue

The helium (He) atmospheric-pressure plasma jet (APPJ) delivery of reactive oxygen and nitrogen species (RONS) and molecular oxygen (O2) in deionized (DI) water was monitored in real time using in situ UV absorption spectroscopy. The He APPJ was used to treat DI water directly and through an agarose target as a surrogate for tissue (e.g., a skin barrier). For direct treatment, the RONS were generated immediately in the DI water, and the concentration of RONS continued to increase during the He APPJ treatment. But there was only a very minor increase in the total RONS concentration detected after the plasma and gas flow were switched off. The agarose target delayed the generation of RONS into the DI water, but the total RONS concentration continued to increase long after (25 min) the plasma and gas flow were switched off. Direct treatment deoxygenated the DI water, whereas treatment through agarose resulted in oxygenation of the DI water. A dynamic change in the ratio of H2O2, NO2 -, NO3 -, and O2 was detected in the DI water during He APPJ treatment and 25 min after the He and gas flow were switched off for both direct and through-agarose treatment. These results have implications for the plasma treatment of real tissue where the dynamic changes in the RONS and O2 concentrations within the tissue and tissue fluid could affect cellular and physiological processes.