Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

Leibniz-Institute for Plasma Science and Technology

Physical plasma is a promising new technology regarding its antimicrobial effects. This is especially accounting for treatment of bacterial infection of chronic wounds. Plasma can be generated with different carrier gases causing various biological effects. Screening of different carrier gases and plasma generation setups is therefore needed to find suitable compositions for highly effective antimicrobial plasma treatments and other applications.

Pilot-study on the influence of carrier gas and plasma application (open resp. delimited) modifications on physical plasma and its antimicrobial effect against Pseudomonas aeruginosa and Staphylococcus aureus.

In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.

Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma.

Oxidative stress illustrates an imbalance between radical formation and removal. Frequent redox stress is critically involved in many human pathologies including cancer, psoriasis, and chronic wounds. However, reactive species pursue a dual role being involved in signaling on the one hand and oxidative damage on the other. Using a HaCaT keratinocyte cell culture model, we investigated redox regulation and inflammation to periodic, low-dose oxidative stress after two, six, eight, ten, and twelve weeks. Chronic redox stress was generated by recurrent incubation with cold physical plasma-treated cell culture medium. Using transcriptome microarray technology, we identified both acute ROS-stress responses as well as numerous adaptions after several weeks of redox challenge. We determined a differential expression (2-fold, FDR < 0.01, p < 0.05) of 260 genes that function in inflammation and redox homeostasis, such as cytokines (e.g., IL-6, IL-8, and IL-10), growth factors (e.g., CSF2, FGF, and IGF-2), and antioxidant enzymes (e.g., HMOX, NQO1, GPX, and PRDX). Apoptotic signaling was affected rather modestly, especially in p53 downstream targets (e.g., BCL2, BBC3, and GADD45). Strikingly, the cell-protective heat shock protein HSP27 was strongly upregulated (p < 0.001). These results suggested cellular adaptions to frequent redox stress and may help to better understand the inflammatory responses in redox-related diseases.

Periodic Exposure of Keratinocytes to Cold Physical Plasma: An In Vitro Model for Redox-Related Diseases of the Skin.

Cold physical plasma is an ionized gas with a multitude of components, including hydrogen peroxide and other reactive oxygen and nitrogen species. Recent studies suggest that exposure of wounds to cold plasma may accelerate healing. Upon wounding, neutrophils are the first line of defense against invading microorganisms but have also been identified to play a role in delayed healing. In this study, we examined how plasma treatment affects the functions of peripheral blood neutrophils. Plasma treatment induced oxidative stress, as assessed by the oxidation of intracellular fluorescent redox probes; reduced metabolic activity; but did not induce early apoptosis. Neutrophil oxidative burst was only modestly affected after plasma treatment, and the killing of Pseudomonas aeruginosa and Staphylococcus aureus was not significantly affected. Intriguingly, we found that plasma induced profound extracellular trap formation. This was inhibited by the presence of catalase during plasma treatment but was not replicated by adding an equivalent concentration of hydrogen peroxide. Plasma-induced neutrophil extracellular trap formation was not dependent on the activity of myeloperoxidase or NADPH oxidase 2 but seemed to involve short-lived molecules. The amount of DNA release and the time course after plasma treatment were similar to that with the common neutrophil extracellular trap inducer PMA. After neutrophil extracellular traps had formed, concentrations of IL-8 were also significantly increased in supernatants of plasma-treated neutrophils. Both neutrophil extracellular traps and IL-8 release may aid antimicrobial activity and spur inflammation at the wound site. Whether this aids or exacerbates wound healing needs to be tested.

Neutrophil extracellular trap formation is elicited in response to cold physical plasma.

Cold plasma has been successfully applied in several fields of medicine that require, for example, pathogen inactivation, implant functionalization or alteration of cellular activity. Previous studies have provided evidence that plasma supports the healing of wounds owing to its beneficial mixtures of reactive species and modulation of inflammation in cells and tissues. To investigate the wound healing activity of an atmospheric pressure plasma jet in vivo, we examined the cold plasma's efficacy on dermal regeneration in a murine model of dermal full-thickness ear wound. Over 14 days, female mice received daily plasma treatment. Quantitative analysis by transmitted light microscopy demonstrated a significantly accelerated wound re-epithelialization at days 3-9 in comparison with untreated controls. In vitro, cold plasma altered keratinocyte and fibroblast migration, while both cell types showed significant stimulation resulting in accelerated closure of gaps in scratch assays. This plasma effect correlated with the downregulation of the gap junctional protein connexin 43 which is thought to be important in the regulation of wound healing. In addition, plasma induced profound changes in adherence junctions and cytoskeletal dynamics as shown by downregulation of E-cadherin and several integrins as well as actin reorganization. Our results theorize cold plasma to be a beneficial treatment option supplementing existing wound therapies.

A cold plasma jet accelerates wound healing in a murine model of full-thickness skin wounds.

So-called cold physical plasmas for biomedical applications generate reactive oxygen and nitrogen species and the latter can trigger DNA damage at high concentrations. Therefore, the mutagenic risks of a certified atmospheric pressure argon plasma jet (kINPen MED) and its predecessor model (kINPen 09) were assessed.

Investigating the Mutagenicity of a Cold Argon-Plasma Jet in an HET-MN Model.

Distinct cytokine and chemokine patterns in chronic diabetic ulcers and acute wounds.

Multiple evidence in animal models and in humans suggest a beneficial role of cold physical plasma in wound treatment. Yet, risk assessment studies are important to further foster therapeutic advancement and acceptance of cold plasma in clinics. Accordingly, we investigated the longterm side effects of repetitive plasma treatment over 14 consecutive days in a rodent full-thickness ear wound model. Subsequently, animals were housed for 350 days and sacrificed thereafter. In blood, systemic changes of the proinflammatory cytokines interleukin 1β and tumor necrosis factor α were absent. Similarly, tumor marker levels of α-fetoprotein and calcitonin remained unchanged. Using quantitative PCR, the expression levels of several cytokines and tumor markers in liver, lung, and skin were found to be similar in the control and treatment group as well. Likewise, histological and immunohistochemical analysis failed to detect abnormal morphological changes and the presence of tumor markers such as carcinoembryonic antigen, α-fetoprotein, or the neighbor of Punc11. Absence of neoplastic lesions was confirmed by non-invasive imaging methods such as anatomical magnetic resonance imaging and positron emission tomography-computed tomography. Our results suggest that the beneficial effects of cold plasma in wound healing come without apparent side effects including tumor formation or chronic inflammation.

One Year Follow-Up Risk Assessment in SKH-1 Mice and Wounds Treated with an Argon Plasma Jet.

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.

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

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.

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

Metastatic melanoma is an aggressive and deadly disease. Therapeutic advance has been achieved by antitumor chemo- and radiotherapy. These modalities involve the generation of reactive oxygen and nitrogen species, affecting cellular viability, migration, and immunogenicity. Such species are also created by cold physical plasma, an ionized gas capable of redox modulating cells and tissues without thermal damage. Cold plasma has been suggested for anticancer therapy. Here, melanoma cell toxicity, motility, and immunogenicity of murine metastatic melanoma cells were investigated following plasma exposure in vitro. Cells were oxidized by plasma, leading to decreased metabolic activity and cell death. Moreover, plasma decelerated melanoma cell growth, viability, and cell cycling. This was accompanied by increased cellular stiffness and upregulation of zonula occludens 1 protein in the cell membrane. Importantly, expression levels of immunogenic cell surface molecules such as major histocompatibility complex I, calreticulin, and melanocortin receptor 1 were significantly increased in response to plasma. Finally, plasma treatment significantly decreased the release of vascular endothelial growth factor, a molecule with importance in angiogenesis. Altogether, these results suggest beneficial toxicity of cold plasma in murine melanomas with a concomitant immunogenicity of potential interest in oncology.

Toxicity and Immunogenicity in Murine Melanoma following Exposure to Physical Plasma-Derived Oxidants.

Pancreatic cancer is associated with a high mortality rate. In advanced stage, patients often experience peritoneal carcinomatosis. Using a syngeneic murine pancreatic cancer cell tumor model, the effect of non-thermal plasma (NTP) on peritoneal metastatic lesions was studied. NTP generates reactive species of several kinds which have been proven to be of relevance in cancer. In vitro, exposure to both plasma and plasma-treated solution significantly decreased cell viability and proliferation of 6606PDA cancer cells, whereas mouse fibroblasts were less affected. Repeated intraperitoneal treatment of NTP-conditioned medium decreased tumor growth in vivo as determined by magnetic resonance imaging, leading to reduced tumor mass and improved median survival (61 vs 52 days; p < 0.024). Tumor nodes treated by NTP-conditioned medium demonstrated large areas of apoptosis with strongly inhibited cell proliferation. Contemporaneously, no systemic effects were found. Apoptosis was neither present in the liver nor in the gut. Also, the concentration of different cytokines in splenocytes or blood plasma as well as the distribution of various hematological parameters remained unchanged following treatment with NTP-conditioned medium. These results suggest an anticancer role of NTP-treated solutions with little to no systemic side effects being present, making NTP-treated solutions a potential complementary therapeutic option for advanced tumors.

Non-thermal plasma-treated solution demonstrates antitumor activity against pancreatic cancer cells in vitro and in vivo.

Human osteosarcoma (OS) is the most common primary malignant bone tumor occurring most commonly in adolescents and young adults. Major improvements in disease-free survival have been achieved by implementing a combination therapy consisting of radical surgical resection of the tumor and systemic multi-agent chemotherapy. However, long-term survival remains poor, so novel targeted therapies to improve outcomes for patients with osteosarcoma remains an area of active research. This includes immunotherapy, photodynamic therapy, or treatment with nanoparticles. Cold atmospheric plasma (CAP), a highly reactive (partially) ionized physical state, has been shown to inherit a significant anticancer capacity, leading to a new field in medicine called "plasma oncology." The current article summarizes the potential of CAP in the treatment of human OS and reviews the underlying molecular mode of action.

Sander Bekeschus

ZIK plasmatis

Leibniz-Institute for Plasma Science and Technology

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

Sander Bekeschus

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Leibniz-Institute for Plasma Science and Technology

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

ZIK plasmatis