Kristian Wende

Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells

In this study, the effect of feed gas humidity on the reactive component generation of an atmospheric-pressure argon plasma jet and its effect on human skin cells are investigated. Feed gas humidity is identified as one key parameter that strongly influences stability and reproducibility of plasma medical studies. The plasma jet is investigated by absorption spectroscopy in the ultraviolet and infrared spectral region for its ozone production depending on the humidity concentration in the feed gas. By optical emission spectroscopy the dependence of present excited plasma species such as hydroxyl radicals, molecular nitrogen, argon and atomic oxygen on the feed gas humidity is investigated. As an interface layer between the plasma jet effluent and the biological cell, a buffer solution is treated and the hydrogen peroxide (H2O2) production is studied with two independent colorimetric assays as a function of humidity admixture to the feed gas. Ultimately, the effect of varying feed gas humidity on the cell viability of indirect plasma treated adherent HaCAT cells is investigated. The highest viability is found for the driest feed gas condition. Furthermore, this work shows answers for the relevance of unwanted—or intended—feed gas humidity in plasma medical experiments and their comparatively large relevance with respect to ambient humidity. The findings will lead to more reproducible experiments in the field of plasma medicine.

Tracking plasma generated H2O2 from gas into liquid phase and revealing its dominant impact on human skin cells

The pathway of the biologically active molecule hydrogen peroxide (H2O2) from the plasma generation in the gas phase by an atmospheric pressure argon plasma jet, to its transition into the liquid phase and finally to its inhibiting effect on human skin cells is investigated for different feed gas humidity settings. Gas phase diagnostics like Fourier transformed infrared spectroscopy and laser induced fluorescence spectroscopy on hydroxyl radicals (OH) are combined with liquid analytics such as chemical assays and electron paramagnetic resonance spectroscopy. Furthermore, the viability of human skin cells is measured by Alamar Blue® assay. By comparing the gas phase results with chemical simulations in the far field, H2O2 generation and destruction processes are clearly identified. The net production rate of H2O2 in the gas phase is almost identical to the H2O2 net production rate in the liquid phase. Moreover, by mimicking the H2O2 generation of the plasma jet with the help of an H2O2 bubbler it is concluded that the solubility of gas phase H2O2 plays a major role in generating hydrogen peroxide in the liquid. Furthermore, it is shown that H2O2 concentration correlates remarkably well with the cell viability. Other species in the liquid like OH or superoxide anion radical do not vary significantly with feed gas humidity.

Identification of the biologically active liquid chemistry induced by a nonthermal atmospheric pressure plasma jet

The mechanism of interaction of cold nonequilibrium plasma jets with mammalian cells in physiologic liquid is reported. The major biological active species produced by an argon RF plasma jet responsible for cell viability reduction are analyzed by experimental results obtained through physical, biological, and chemical diagnostics. This is complemented with chemical kinetics modeling of the plasma source to assess the dominant reactive gas phase species. Different plasma chemistries are obtained by changing the feed gas composition of the cold argon based RF plasma jet from argon, humidified argon (0.27%), to argon/oxygen (1%) and argon/air (1%) at constant power. A minimal consensus physiologic liquid was used, providing isotonic and isohydric conditions and nutrients but is devoid of scavengers or serum constituents. While argon and humidified argon plasma led to the creation of hydrogen peroxide dominated action on the mammalian cells, argon-oxygen and argon-air plasma created a very different biological action and was characterized by trace amounts of hydrogen peroxide only. In particular, for the argon-oxygen (1%), the authors observed a strong negative effect on mammalian cell proliferation and metabolism. This effect was distance dependent and showed a half life time of 30 min in a scavenger free physiologic buffer. Neither catalase and mannitol nor superoxide dismutase could rescue the cell proliferation rate. The strong distance dependency of the effect as well as the low water solubility rules out a major role for ozone and singlet oxygen but suggests a dominant role of atomic oxygen. Experimental results suggest that O reacts with chloride, yielding Cl2(-) or ClO(-). These chlorine species have a limited lifetime under physiologic conditions and therefore show a strong time dependent biological activity. The outcomes are compared with an argon MHz plasma jet (kinpen) to assess the differences between these (at least seemingly) similar plasma sources.

From RONS to ROS: Tailoring Plasma Jet Treatment of Skin Cells

Finding a solution for air species contamination of atmospheric pressure plasmas in plasma medical treatment is a major task for the new field of plasma medicine. Several approaches use complex climate chambers to control the surrounding atmosphere. In this paper, ambient species are excluded in plasma-human-skin-cell treatment by ensheathing the plasma jet effluent with a shielding gas. Not only does this gas curtain protect the plasma jet effluent from inflow of air species but it also, more importantly, allows controlling the effluent reactive species composition by adjusting the mixture of the shielding gas. In the present investigations, the mixture of nitrogen to oxygen within the gas curtain around an argon atmospheric pressure plasma jet (kinpen) is varied. The resulting reactive plasma components produced in the jet effluent are thus either oxygen or nitrogen dominated. With this gas curtain, the effect of reactive oxygen species (ROS) and reactive nitrogen species (RNS) on the cell viability of indirectly plasma-treated HaCaT skin cells is studied. This human keratinocyte cell line is an established standard for a skin model system. The cell viability is determined by a fluorometric assay, where metabolically active cells transform nonfluorescent resazurin to the highly fluorescent resorufin. Plasma jet and gas curtain are characterized by numerical flow simulation as well as by optical emission spectroscopy. The generation of nitrite within the used standard cell culture medium serves as a measure for generated RNS. Measurements with the leukodye dichlorodihydrofluorescein diacetate show that, despite a variation of the shielding gas mixture, the total amount of generated reactive oxygen plus nitrogen species is constant. It is shown that a plasma dominated by RNS disrupts cellular growth less than a ROS-dominated plasma.

Impact of non-thermal plasma treatment on MAPK signaling pathways of human immune cell lines.

In the field of wound healing research non-thermal plasma (NTP) increasingly draws attention. Next to its intensely studied antibacterial effects, some studies already showed stimulating effects on eukaryotic cells. This promises a unique potential in healing of chronic wounds, where effective therapies are urgently needed. Immune cells do play an important part in the process of wound healing and their reaction to NTP treatment has yet been rarely examined. Here, we studied the impact of NTP treatment using the kinpen on apoptotic and proliferative cell signaling pathways of two human immune cell lines, the CD4(+)T helper cell line Jurkat and the monocyte cell line THP-1. Depending on NTP treatment time the number of apoptotic cells increased in both investigated cell types according to a caspase 3 assay. Western blot analysis pointed out that plasma treatment activated pro-apoptotic signaling proteins like p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase 1 and 2 (JNK 1/2) in both cell types. Stronger signals were detected in Jurkat cells at comparable plasma treatment times. Intriguingly, exposure of Jurkat and THP-1 cells to plasma also activated the pro-proliferative signaling molecules extracellular signal-regulated kinase 1/2 (ERK 1/2) and MAPK/ERK kinase 1 and 2 (MEK 1/2). In contrast to Jurkat cells, the anti-apoptotic heat shock protein 27 (HSP27) was activated in THP-1 cells after plasma treatment, indicating a possible mechanism how THP-1 cells may reduce programmed cell death. In conclusion, several signaling cascades were activated in the examined immune cell lines after NTP treatment and in THP-1 monocytes a possible defense mechanism against plasma impacts could be revealed. Therefore, plasma might be a treatment option for wound healing.

Non-thermal plasma treatment is associated with changes in transcriptome of human epithelial skin cells

Abstract Non-thermal atmospheric pressure plasma has recently gained attention in the field of biomedical and clinical applications. In the area of plasma medicine research, one promising approach is to promote wound healing by stimulation of cells involved. To understand basic molecular and cellular mechanisms triggered by plasma treatment, we investigated biological effects of an argon plasma jet kinpen on human epithelial skin cells. For assessment of transcriptome changes cell culture medium was plasma treated and applied to the HaCaT keratinocyte cell culture (indirect treatment). Consequently, whole-genome microarrays were used to analyze this interaction in detail and identified a statistically significant modification of 3,274 genes including 1,828 up- and 1,446 downregulated genes. Particularly, cells after indirect plasma treatment are characterized by differential expression of a considerable number of genes involved in the response to stress. In this regard, we found a plasma-dependent regulation of oxidative stress answer and increased expression of enzymes of the antioxidative defense system (e.g. 91 oxidoreductases). Our results demonstrate that plasma not only induces cell reactions of stress-sensing but also of proliferative nature. Consistent with gene expression changes as well as Ingenuity Pathway Analysis prediction, we propose that stimulating doses of plasma may protect epithelial skin cells in wound healing by promoting proliferation and differentiation. In conclusion, gene expression profiling may become an important tool in identifying plasma-related changes of gene expression. Our results underline the enormous clinical potential of plasma as a biomedical tool for stimulation of epithelial skin cells.

Non-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways.

Background: Non-thermal plasma provides an interesting therapeutic opportunity to control redox-based processes, e.g. wound healing. Results: The transcription factor NRF2 and downstream signaling molecules were found to act as key controllers orchestrating the cellular response. Conclusions: Plasma triggers hormesis-like processes in keratinocytes. Significance: These findings facilitate the understanding of plasma-tissue interaction and its deduced clinical application. Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.

Risk assessment of a cold argon plasma jet in respect to its mutagenicity

Cold atmospheric pressure plasmas represent a favorable option for the treatment of heat sensitive materials and human or animal tissue. Beneficial effects have been documented in a variety of medical conditions, e.g., in the treatment of chronic wounds. It is assumed that the main mechanism of the plasmas efficacy is mediated by a stimulating dissipation of energy via radiation and/or chemical energy. Although no evidence on undesired side effects of a plasma treatment has yet been presented, skepticism toward the safety of the exposure to plasma is present. However, only little data regarding the mutagenic potential of this new treatment option is available. Accordingly, we investigated the mutagenic potential of an argon plasma jet (kinpen) using different testing systems in accordance with ISO norms and multiple cell lines: a HPRT1 mutation assay, a micronucleus formation assay, and a colony formation assay. Moderate plasma treatment up to 180 s did not increase genotoxicity in any assay or cell type investigated. We conclude that treatment with the argon plasma jet kinpen did not display a mutagenic potential under the test conditions applied and may from this perspective be regarded as safe for the use in biomedical applications.

Atmospheric pressure plasma jet treatment evokes transient oxidative stress in HaCaT keratinocytes and influences cell physiology

Modern non‐thermal atmospheric pressure plasma sources enable controllable interaction with biological systems. Their future applications – e.g. wound management – are based on their unique mixture of reactive components sparking both stimulatory as well as inhibitory processes. To gain detailed understanding of plasma–cell interaction and with respect to risk awareness, key mechanisms need to be identified. This study focuses on the impact of an argon non‐thermal atmospheric pressure plasma jet (kINPen 09) on human HaCaT keratinocytes. With increasing duration, cell viability decreased. In accordance, cells accumulated in G2/M phase within the following 24 h. DNA single‐strand breaks were detected immediately after treatment and receded in the aftermath, returning to control levels after 24 h. No directly plasma‐related DNA double‐strand breaks were detected over the same time. Concurrently, DNA synthesis decreased. Coincident with treatment time, an increase in intracellular 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCFDA) conversion increased reactive oxygen species (ROS) levels. The radical scavenging activity of culture medium crucially influenced these effects. Thus, ROS changed DNA integrity, and the effectiveness of cellular defence mechanisms characterises the interaction of non‐thermal plasma and eukaryotic cells. Effects were time‐dependent, indicating an active response of the eukaryotic cells. Hence, a stimulation of eukaryotic cells using short‐term non‐thermal plasma treatment seems possible, eg in the context of chronic wound care. Long‐term plasma treatments stopped in cell proliferation and apoptosis, which might be relevant in controlling neoplastic conditions.

Surface molecules on HaCaT keratinocytes after interaction with non-thermal atmospheric pressure plasma.

Non‐thermal atmospheric‐pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma‐cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma‐cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down‐regulation of E‐cadherin and the EGFR expression occurred. The influence on α2‐ and β1‐integrins was less pronounced, and expression of ICAM‐1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H2DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two‐dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma‐cell interactions.