Sabrina Baldus

The topical use of non-thermal dielectric barrier discharge (DBD): Nitric oxide related effects on human skin

Dielectric barrier discharge (DBD) devices generate air plasma above the skin containing active and reactive species including nitric oxide (NO). Since NO plays an essential role in skin physiology, a topical application of NO by plasma may be useful in the treatment of skin infections, impaired microcirculation and wound healing. Thus, after safety assessments of plasma treatment using human skin specimen and substitutes, NO-penetration through the epidermis, the loading of skin tissue with NO-derivates in vitro and the effects on human skin in vivo were determined. After the plasma treatment (0-60 min) of skin specimen or reconstructed epidermis no damaging effects were found (TUNEL/MTT). By Franz diffusion cell experiments plasma-induced NO penetration through epidermis and dermal enrichment with NO related species (nitrite 6-fold, nitrate 7-fold, nitrosothiols 30-fold) were observed. Furthermore, skin surface was acidified (~pH 2.7) by plasma treatment (90 s). Plasma application on the forearms of volunteers increased microcirculation fourfold in 1-2 mm and twofold in 6-8 mm depth in the treated skin areas. Regarding the NO-loading effects, skin acidification and increase in dermal microcirculation, plasma devices represent promising tools against chronic/infected wounds. However, efficacy of plasma treatment needs to be quantified in further studies and clinical trials.

Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide

The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal “cold” atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H2O2 (~200 μM). Fibroblast viability was reduced by single DBD treatments (60–300 s; ~77–66%) or exposure to freshly DBD-treated PBS (60–300 s; ~75–55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well-balanced in order to avoid possible unwanted side effects such as a delayed healing process.

A dielectric barrier discharge terminally inactivates RNase A by oxidizing sulfur-containing amino acids and breaking structural disulfide bonds

RNases are among the most stable proteins in nature. They even refold spontaneously after heat inactivation, regaining full activity. Due to their stability and universal presence, they often pose a problem when experimenting with RNA. We investigated the capabilities of nonthermal atmospheric-pressure plasmas to inactivate RNase A and studied the inactivation mechanism on a molecular level. While prolonged heating above 90 °C is required for heat inactivating RNase A, direct plasma treatment with a dielectric barrier discharge (DBD) source caused permanent inactivation within minutes. Circular dichroism spectroscopy showed that DBD-treated RNase A unfolds rapidly. Raman spectroscopy indicated methionine modifications and formation of sulfonic acid. A mass spectrometry-based analysis of the protein modifications that occur during plasma treatment over time revealed that methionine sulfoxide formation coincides with protein inactivation. Chemical reduction of methionine sulfoxides partially restored RNase A activity confirming that sulfoxidation is causal and sufficient for RNase A inactivation. Continued plasma exposure led to over-oxidation of structural disulfide bonds. Using antibodies, disulfide bond over-oxidation was shown to be a general protein inactivation mechanism of the DBD. The antibodys heavy and light chains linked by disulfide bonds dissociated after plasma exposure. Based on their ability to inactivate proteins by oxidation of sulfur-containing amino acids and over-oxidation of disulfide bonds, DBD devices present a viable option for inactivating undesired or hazardous proteins on heat or solvent-sensitive surfaces.

Atomic oxygen dynamics in an air dielectric barrier discharge: a combined diagnostic and modeling approach

Cold atmospheric pressure plasmas are a promising alternative therapy for treatment of chronic wounds, as they have already shown in clinical trials. In this study an air dielectric barrier discharge (DBD) developed for therapeutic use in dermatology is characterized with respect to the plasma produced reactive oxygen species, namely atomic oxygen and ozone, which are known to be of great importance to wound healing. To understand the plasma chemistry of the applied DBD, xenon-calibrated two-photon laser-induced fluorescence spectroscopy and optical absorption spectroscopy are applied. The measured spatial distributions are shown and compared to each other. A model of the afterglow chemistry based on optical emission spectroscopy is developed to cross-check the measurement results and obtain insight into the dynamics of the considered reactive oxygen species. The atomic oxygen density is found to be located mostly between the electrodes with a maximum density of cm. Time resolved measurements reveal a constant atomic oxygen density between two high voltage pulses. The ozone is measured up to 3?mm outside the active plasma volume, reaching a maximum value of cm between the electrodes.

FTIR spectroscopy of cysteine as a ready-to-use method for the investigation of plasma-induced chemical modifications of macromolecules

A rapid screening method for the investigation of plasma-induced chemical modifications was developed by analyzing cysteine using Fourier Transform Infrared (FTIR) spectroscopy. Cysteine is a key amino acid in proteins due to the presence of a thiol group which provides unique structural features by offering the possibility to form disulfide bonds. Its chemical composition makes cysteine a well-suited model for the investigation of plasma-induced modifications at three functional groups—the amino, the carboxyl and the thiol group—all highly abundant in proteins. FTIR spectroscopy is present in most physical laboratories and offers a fast way to assess changes in the chemical composition of cysteine substrates due to plasma treatment and to compare different treatment conditions or plasma sources with each other. Significant changes in the fingerprint spectra of cysteine samples treated with a dielectric barrier discharge (DBD) compared to untreated controls were observed using a FTIR spectrometer. The loss of the thiol signal and the simultaneous increase of bands originating from oxidized sulfur and nitrogen species indicate that the thiol group of cysteine is modified by reactive oxygen and nitrogen species during DBD treatment. Furthermore, other plasma-induced modifications, such as changes of the amino and carbonyl groups, could be observed. Complementary mass spectrometry measurements confirmed these results.

Unraveling the interactions between cold atmospheric plasma and skin-components with vibrational microspectroscopy

Using infrared and Raman microspectroscopy, the authors examined the interaction of cold atmospheric plasma with the skins built-in protective cushion, the outermost skin layer stratum corneum. Following a spectroscopic analysis, the authors could identify four prominent chemical alterations caused by plasma treatment: (1) oxidation of disulfide bonds in keratin leading to a generation of cysteic acid; (2) formation of organic nitrates as well as (3) of new carbonyl groups like ketones, aldehydes and acids; and (4) reduction of double bonds in the lipid matter lanolin, which resembles human sebum. The authors suggest that these generated acidic and NO-containing functional groups are the source of an antibacterial and regenerative environment at the treatment location of the stratum corneum. Based upon the authors results, the authors propose a mechanistic view of how cold atmospheric plasmas could modulate the skin chemistry to produce positive long-term effects on wound healing: briefly, cold atmospheric plasmas have the potential to transform the skin itself into a therapeutic resource.

Implications of electron heating and non-uniformities in a VHF-CCP for sterilization of medical instruments

A capacitively coupled plasma driven at a frequency of 81.36 MHz from the VHF-band is investigated by means of optical emission spectroscopy (OES) and multipole resonance probe (MRP). The discharge is operated with hydrogen, yielding an electropositive discharge, as well as oxygen, yielding an electronegative discharge, and mixtures of both. Pressure is varied from Pa to Pa. Homogeneity of the discharge is investigated by CCD camera recordings as well as spatially resolved multipole resonance probe measurements. The results indicate the presence of electromagnetic edge effects as well as standing wave effects. Furthermore, a largely homogeneous discharge can be achieved with hydrogen as process gas at a pressure of –10 Pa. With increasing pressure as well as with increasing oxygen content, the discharge appears less homogeneously. The transition from an electropositive to an electronegative discharge leads to a change in electron heating mechanisms, with pronounced local maxima of electron density at the sheath edges. A comparison of OES and MRP results reveal a significant difference in electron density, which can be explained by a non-Maxwellian distribution function of electrons.

Phase resolved analysis of the homogeneity of a diffuse dielectric barrier discharge

Cold atmospheric pressure plasmas have already proven their ability of supporting the healing process of chronic wounds. Especially simple configurations like a dielectric barrier discharge (DBD), comprising of one driven electrode which is coated with a dielectric layer, are of interest, because they are cost-effective and easy to handle. The homogeneity of such plasmas during treatment is necessary since the whole wound should be treated evenly. In this investigation phase resolved optical emission spectroscopy is used to investigate the homogeneity of a DBD. Electron densities and reduced electric field distributions are determined with temporal and spatial resolution and the differences for applied positive and negative voltage pulses are studied.

Analyzing the interactions between a DBD and skin components using Raman microspectroscopy

Summary form only given. Atmospheric pressure plasmas like dielectric barrier discharges (DBD) have shown noticeable positive effects on stimulation of wound healing, especially treatment of chronic wounds. Due to this, the therapeutic use of plasma devices is gaining importance although the interaction of plasma with skin and the processes taking place in skin are poorly understood. To get an insight into aforementioned interactions, a DBD, operating in air as process gas, is used for treatment of skin model components. DBD is characterized concerning plasma parameters and fluxes of important species like ozone or nitric oxide are determined. As a model component keratin was chosen because it is the most abundant protein in the stratum corneum, the upper layer of skin. After treatment, chemical changes of keratin are analyzed using Raman microspectroscopy. Raman spectroscopy is a non-destructive vibrational spectroscopic technique. It is capable of probing vibrational modes of functional groups of biological material. Hence, chemical changes of functional groups induced by plasma treatment will be detected. Chemical alterations in keratin are visible in the Raman spectrum already after a few seconds of treatment time. After longer treatment times a large oxidizing effect of the plasma generated reactive species is apparent which leads pH reduction after plasma treatment. Results achieved by this investigation contribute to the understanding of interaction between plasma and skin as well as wound healing mechanisms.

Risk assessment of a plasma device for therapeutic use in dermatology

Summary form only given. Therapeutic use in dermatology of plasma devices is a relevant topic in ongoing research. These devices are operated at atmospheric pressure in ambient air or other gases for the treatment of skin diseases or disinfection of skin surface. Due to the fact that the interaction of plasma with human skin is not fully understood, further research is needed to make sure that there is no damage done to the human body by plasma treatment.