14.05.2014 Nitric Oxide related Interactions of Nonthermal Dielectric-barrier Discharge Plasma with Human Skin K. Heuer, M. A. Hoffmanns, E. Demir, S. Baldus, P.C. Fuchs, P. Awackowicz, C. V. Suschek and Christian Opländer 1. International Plasma Technologies Congress 28th – 30th April 2014 Abdullah Gül University Kayseri, Turkey Target: Intact Skin Hair shaft Hair follicle Skin DBD Plasma Device Nitric oxide Effects Conclusion Stratum Stratum Stratum Stratum corneum lucidum granulosum basale Stratum papillare Epidermis Dermis Stratum reticulare Hair root Sweat gland Hypodermis There are many inter- and intraindividual differences in texture and chemical constitution of human skin 1 14.05.2014 Characterization of DBD generated plasma under clinical conditions A B 1.2 Skin DBD Plasma Device Nitric oxide Effects Fibre Optics Sample Gas Tube NO / NO2 Measurement Spectroscopy Human Skin intensity (a.u.) DBD Electrode irradiance (mW/cm2) 1 0.8 UV-A 7.27±1.78 x10-3 UV-B 1.34±0.67 x10-3 0.6 0.4 Earthed Metal Plate 0.2 Conclusion 0 0 100 200 300 400 500 600 700 800 900 1000 wavelength (nm) C gas temperature (Tg) electron density (ne) trigger frequency (ftrig) peak voltage (Up) NO2 NO 395 ± 15 K 7.46 ± 2.29x1011 cm-3 120 hz 14 kV 60.3 ± 17.7 ppm 213.2 ± 7.7 ppb Skin can influence plasma parameters NO und NO2 are generated Physiological functions of nitric oxide (NO) Skin DBD Plasma Device Nitric oxide Effects Conclusion NO. v v v v v v v Vasodilatation Blood flow Platelet adhesion Platelet aggregation Wound healing Defence against bacteria Inflammation v Collagen metabolismus v Cell parameters NO has many functions in the human body e.g. impaired wound healing is correlated with low NO levels 2 14.05.2014 Recycling/storing of nitric oxide (NO) NO. Skin DBD Plasma Device Nitric oxide nitrite (NO2-) nitrate (NO3-) nitroso-compounds (RX-NO) Effects Conclusion UV/VIS pH antioxidants metals NO. NO reacts fast with oxygen/ozone and proteins but can be stored in tissue as bioactive oxidation/reaction products Is DBD plasma safe? A Skin in vitro skin corrosion B in vitro skin irritation 2.0 2.0 1.5 1.5 Toxicity Effects Conclusion Aborbance570 Nitric oxide Aborbance570 DBD Plasma Device 1.0 0.5 0.0 con. 1.0 0.5 0.0 3 min 60 min (+) con. plasma treat. conc. IL-1α = conc. IL-1α = 4.1± 3.5 pg/ml 4.18± 2.7 pg/ml con. 60 min plasma treat. DBD plasma did not cause harmful effects in experiments using a validated skin model 3 14.05.2014 Is DBD plasma safe? TUNEL DAPI Skin DBD Plasma Device contr. 25 Toxicity Effects rel. TUNEL+cells (%) Nitric oxide Plasma 360s Conclusion (+) contr. 20 15 10 5 0 K plasma 360s UVA UV-A 100 J/cm² DBD plasma did not induce apoptosis in experiments using human skin Is DBD plasma safe? 6 Skin y = 1.91 + 3.52*exp(-0.017*x) R² = 0.96 5 DBD Plasma Device 4 Toxicity pH Nitric oxide 3 Effects Conclusion 2 1 0 0 30 60 90 120 150 180 plasma treat. (s) 2 NO2 + H2O → HNO3 + HNO2 DBD plasma decreased human skin pH 4 14.05.2014 DBD plasma effects on fluids DBD Electrode 250 Skin Sampling Port Split Skin (0.3 or 0.7 mm) Toxicity Effects Conclusion Receptor Chamber (PBS pH 7.4) Heater / Circulator Water Jacket (37°C) concentration (µM) Nitric oxide nitrite nitrate DBD Plasma Device 200 Linear (nitrite) Linear (nitrate) 150 y = 18.054x - 7.0659 R2 = 0.9831 100 50 y = 7.3611x + 1.5288 R2 = 0.9983 Stirbar 0 Magnetic Stirrer 0 2.5 5 7.5 10 plasma treatment (min) DBD plasma increased nitrite and nitrate concentrations in fluids DBD plasma effects: NO-penetration A DBD Plasma Device 0.7 mm split skin control 20 control 18 2.5 min 18 2.5 min 16 5.0 min 16 5.0 min 14 10 min 14 10 min nitrite (nmol) nitrite (nmol) Skin B 0.3 mm split skin 20 12 10 8 6 4 Nitric oxide 8 6 2 0 0 0 10 20 Effects 30 40 50 0 10 20 time (min) C 100 50 control 2.5 min 45 2.5 min 5 min 40 5.0 min 10 min Split Skin 35 10 min (0.3 or 0.7 mm) 80 Receptor Chamber 60 (PBS pH 7.4) Heater / Circulator Water Jacket 40 (37°C) Stirbar 30 25 20 15 10 20 0 50 0.7 mm split skin control nitrate (nmol) Sampling Port 40 D 0.3 mm split skin 120 30 time (min) DBD Electrode 140 nitrate (nmol) Conclusion 10 4 2 Toxicity 12 5 Magnetic Stirrer 0 10 0 20 30 time (min) 40 50 0 10 20 30 40 50 time (min) DBD plasma induced penetration of NO and NO-related products through intact human epidermis 5 14.05.2014 Skin DBD Plasma Device Nitric oxide Toxicity 160 nitrate (nmol/g skin) nitrite (nmol/g skin) DBD plasma effects on dermal NODs 140 120 100 80 60 40 20 0 2500 2000 1500 1000 500 0 K Effects 3000 2.5 min 5 min 10 min K plasma treatment Conclusion RXNO (nmol/mg prot.) NO RXNO 2.5 min 5 min 10 min plasma treatment NO2NO3 - 4 3.5 3 2.5 2 1.5 1 0.5 0 K 2.5 min 5 min 10 min plasma treatment DBD plasma increased the levels of NO-related products in human skin DBD plasma effects: in vivo 300 1-2mm 6-8 mm DBD Plasma Device Nitric oxide Toxicity Effects Conclusion blood flow (a.u.) Skin 250 200 150 100 50 after treatment 4d after treatment 0 0 10 20 30 40 50 60 time (min) DBD plasma application (90s) induced an increase in dermal microcirculation, however with side effects (slight pain, erythema and itchiness) 6 14.05.2014 Summary and conclusion Skin may influence DBD plasma parameter Skin DBD Plasma Device DBD plasma has no harmful effects in vitro Nitric oxide Application of DBD plasma lowers skin pH Toxicity Effects DBD plasma enrichs skin with NO products and enhance dermal microcirculation Conclusion In vivo application of DBD plasma under our conditions has unwanted side effects such as slight pain, erythema and itchiness DBD plasma devices represents a useful tool in medicine, but further studies are necessary to find suitable and individual treatment conditions Thanks to: Kiara Heuer Martin Hoffmanns Sabrina Baldus Erhan Demir Peter Awackowizc Paul C. Fuchs Christoph V. Suschek 7
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