In this study two plasma sources were used for an in vivo treatment of human stratum corneum. The sample preparation was realised with the Cyanoacrylat stripping method, whereby a few layers of corneocytes embedded in the lipid matrix were removed from the skin of healthy volunteers. For the plasma treatment, dielectric barrier discharges with pulse durations in the microsecond as well as in the nanosecond range were applied. A comparison of these sources with respect to their biologically active components including dissipated power, gas and electron temperature, irradiance in the ultraviolet range, ozone and nitric oxide concentration is presented. Furthermore, species generated during plasma treatment on the sample surface like hydrogen peroxide, nitride or nitrate were measured using reflectometry. In addition, safety aspects for both sources were evaluated. Resulting plasma induced changes in the sample composition were investigated through X-ray photoelectron spectroscopy. The main ingredients carbon, oxygen, and nitrogen in addition to minor concentrations of sulphur were considered. A significant influence of the pulse duration on plasma characteristics was shown. A more effective formation of reactive species as well as more intense UV emission for ns-plasma was observed. Based on the determined parameters, both plasma sources are suitable for therapeutic purpose. Furthermore, significant plasma induced changes in the stratum corneum composition were reported, including an increase in nitrogen and oxygen content.
Plasma medicine is a young, rapidly growing field of research, which has a high innovation potential due to the development of the so-called “cold plasma”. A low gas temperature only a few degrees above room temperature, is characteristic for this cold plasma. Therefore, it is possible to apply a plasma treatment to temperature sensitive materials like human skin, seeds and plants, or wood surfaces [
To improve wound healing especially in chronic wounds, as well as for stimulating regenerative processes, plasma devices are already used [
Additional application fields where new therapeutic/treatment methods could be validated are tooth treatment in dental medicine, sterilisation of surgical equipment, or veterinary medicine [
Also, cold plasma is used for cosmetic treatment of nails [
The effects of plasma treatment are based on a mixture of different biologically active components, including temperature, electric current, electric fields, UV-radiation, and reactive nitrogen (RNS) as well as reactive oxygen species (ROS). For many years, single components were already used for therapeutic purposes, taking into account permissible limits for each method not to be exceeded in order to avoid adverse effects.
The thermotherapy is a well-established method for the treatment of muscle tensions and pain, and is utilized to promote blood circulation. Additionally, the cell proliferation can be stimulated by a slight increase of temperature. Otherwise, temperatures above 318 K induce a denaturation and structural alteration of proteins [
Positive impacts of radiation in the ultraviolet range on skin are anti-inflammatory and anti-itching properties, applied as part of phototherapy for treatment of psoriasis and eczema [
There are diverse influences of ROS and RNS on the human organism. ROS provokes cell damage and cell death as well as defects in the respiratory system in high doses. In addition, the stimulation of cell proliferation, skin disinfection and an improved wound healing were observed [
During plasma treatment, all components act simultaneously on the substrate. Possible synergetic effects of these components could not be clarified sufficiently yet, but especially concerning the interaction of plasma with the complex structure and composition of skin, the knowledge of different mechanisms of action is the basis to validate new therapeutic approaches.
For this purpose, an in-vitro plasma treatment on Stratum Corneum (SC) was performed. The SC-samples were produced with the Cyanoacrylat Stripping Method and comprising corneocytes embedded in a lipid matrix including ceramides, cholesterol, and free fatty acids [
The influence of the duration of the exciting high voltage pulses on the formation of various species has been validated. Therefore, two plasma sources with comparable dissipated power, voltage amplitude and pulse repetition frequency, which only differ in the pulse duration (μs- as well as ns-range) and in the resulting discharge current, were applied. The plasma devices have been characterized with respect to rotational, vibrational and electron temperature by high resolution optical emission spectroscopy in a previous study. Emission spectra and the additional use of a radiometer allow the calculation of the irradiance in the ultraviolet range. A spectral weighting factor of the ICNIRP allows the evaluation of the effective irradiance, taking into account the wavelength-depending effects of radiation on the skin. Plasma induced changes in skin samples were studied via X-ray photoelectron spectroscopy. For this purpose, the sample composition was examined before and after plasma treatment. The carbon, oxygen, nitrogen and sulphur content as well as the chemical bonds were investigated. Through this, conclusions about the structure-modifying effect of plasma treatment on SC may be drawn.
The lipids of the SC in combination with keratin form a closed-packed layer with great importance for the skin barrier function. The Cyanoacrylate Stripping method was applied for collecting non-invasive human SC. In the process, a drop of cyanoacrylate adhesive LiquiBand® (MedLogic Global Limited, Plymouth, United Kingdom) was placed on a sample holder, here an aluminium surface, pressed onto the skin and then removed after 60 seconds [
The applied plasma sources are based on the concept of dielectric barrier discharges. Both sources consist of the same electrode geometry, comprising a copper electrode covered by a ceramic (Al2O3) cylinder. The wall thickness of the dielectric barrier is about 1 mm. The discharges were ignited in ambient air at atmospheric pressure; the discharge gap was kept constant at 1 mm. The lipid stripping samples were applied as grounded counter electrodes in the setup. The two plasma sources show a comparable dissipated power, voltage amplitude and pulse repetition frequency and only differ in the pulse duration (μs- as well as ns-range) and in the resulting discharge current. Additional plasma parameters such as rotational and electron temperature have already been investigated by high resolution spectroscopy, presented in
By using these plasma sources, it is possible to draw conclusions about the influence of the pulse duration on the resulting plasma parameters and consequently the impact on plasma induced changes in the treated samples. In various studies, DBDs with pulse durations in the ns-range were applied. This excitation mode results in a quasi-uniform discharge with less streamer formation, which does not require constant discharge gaps [
μs-source | ns-source | |
---|---|---|
Peak voltage Umax | 11.6 kV | 11.7 kV |
Pulse duration tpulse | 70 μs | 600 ns |
Pulse repetition rate f | 300 Hz | 300 Hz |
Dissipated power P | 710 mW | 720 mW |
Rotational temperature Trot = gas temperature TGas | 375 K | 330 K |
Mean electron energy ε | 11 eV | 8.25 eV |
Ozone concentration cO3 | 275 ppm | 1650 ppm |
Nitric oxide concentration cNO | 580 ppm | 2980 ppm |
Absolute irradiance I | 42.5 mW/m2 | 93.4 mW/m2 |
Amongst the numerous oxygen species generated into a plasma discharge, ozone was of particular interest due to its strong oxidative effect [
Nitric oxide is a well-known representative of the RNS in plasma discharges, which has an important varying impact on human organism [
The irradiance specifies the emitted power per area from a lighting source, in this case from a plasma discharge. In medical application, the emitted radiation in the UV-range has a primary importance, since its dose depending positive or negative effects on humans [
The characteristics of ITO coated fused silica are a high transmission in the UV-range as well as the electrical conductivity, allowing the grounding of the glass electrode. The transmission function of the fused silica T(λ) was measured spectroscopically and was included in the evaluation. Furthermore, emission spectra of the discharge E(λ) were taken for calculating the spectrally resolved intensity distribution I(λ):
where Iabs is the absolute measured irradiance. The ICNIRP has given a spectral weighting factor S(λ) taking into account the wavelength-dependent effect of electro-magnetic radiation on human skin [
In addition, the ICNIRP releases policies with regard to maximum dosage of effective expose per day. The limit of Dmax = 30 J/m2 was suggested for the most sensitive type of skin and enables the calculation of a safety- limited treatment duration tmax:
In this study, the nitric (
For measuring the concentrations of these species, a reflectometric measurement with the RQflex 10 (Merck KGaA, Darmstadt, Germany) and reflectometric test strips was realized. The chemical compounds to be investigated were dissolved indistilled water from the sample surface. In the case of nitric, nitrate, and hydrogen peroxide measurements a volume of 50 μl, in the case of ammonium measurement a volume of 100 μl was required. The varying amounts of liquid arise from different manufacturer specifications on experimental procedure. The respective test strips were moisturized with the solution and the resulting change of colour was reflectometrically evaluated. The results presented here based on a sample size of five test persons with two samples each.
X-ray photoelectron spectroscopy provides information about the elemental and chemical composition. In this study the carbon, oxygen and, nitrogen as well as the sulphur content of SC were investigated. Therefore, survey spectra from 0 eV up to 1400 eV as well as high resolution scans of the C1s (284.8 eV), O1s (~532 eV), N 1s (~400 eV), and the S2p (~168 eV) peaks were taken. All binding energies of photoemission peaks were referenced to the C1s peak at 284.8 eV. For further information, a peak fitting with Gauss-Lorentzian profiles was performed on the high-resolution C1s spectra. The measurements were carried out on the XPS system PHI Versa Probe II (Physical Electronics, Inc., Minneapolis, USA) in high vacuum at 10−6 Pa. The exciting line radiation was generated by means of a monochromatized Al Kα X-ray source with a line width of 0.26 eV. The resolution of the system is <0.5 eV.
Photoelectrons driven out from the sample, induced by the high energy X-rays, have characteristic kinetic energies that directly identify each element and chemical bond present in the sample surface. The investigations were accomplished with a spot diameter of 200 μm; the information depth of the measurements is less than tennanometres. For the analysis, the photoelectron peak areas were calculated after Shirley background correction. The peak fitting was performed using MultiPak Software (Ulvac-Phi, Inc., Minneapolis, USA). Three analyses were performed per test person (two volunteers in total) before and after the plasma treatment.
During the plasma treatment, several ROS and RNS were generated within the discharge volume. This paper is limited to the evaluation of two species, ozone and nitric oxide, because they are formed in high concentrations in the plasma discharge. Also, several effects of O3 and NO are already known in medical application, like regulation of wound healing, anti-inflammatory effects or the stimulation of proliferation [
The ozone concentrations of both discharge sources are measured directly in the discharge. In the ns-plasma, a maximum concentration of approximately 1650 ± 60 ppm was determined, the μs-plasma exhibits a six times lower value (~275 ± 30 ppm) despite the same dissipated power. These results are in line with the expected effective ozone generation in ns-discharges, which was already shown in [
Due to the experimental setup, the measurement of the NO concentration is only possible within the discharge. For the μs-plasma a value of 580 ± 70 ppm, for the ns-plasma a value of 2980 ± 80 ppm was detected. With regard to NO molecules the more efficient formation of reactive species in the ns-discharge was also confirmed, with a five times higher concentration. To evaluate these results with regard to safety aspects, the DFG guidelines specifying a maximum workplace concentration of 0.5 ppm NO (0.95 mg/m3), based on an 8 h exposure time, are utilized [
In summary, a safe use of the examined discharges is ensured, if the distance to the respiratory tract does not drop below 15 cm, whereas the ozone concentration is the limiting factor. Also in the ns-discharge, a more efficient formation of reactive species is shown, and therefore major changes in the treated substrates were expected and proven by XPS (see Section 3.4).
For determining the absolute irradiance Iabs in the substrate level, the UV radiation has been detected in a wavelength range of 250 - 400 nm, whereby the detector head occupies the position of the treated substrates in the experimental setup. Thus, the results correspond to the irradiance that interacts with the sample surface during treatment. For the μs-discharge a value of 42.5 ± 0.2 mW/m2 for ns-discharge a value of 93.4 ± 10 W/m2 was calculated; the given values are based on 10 individual measurements using the radiometer.
For the subsequent evaluation of the wavelength-dependent effect of the plasma radiation, the emission spectra of both sources (data not shown) were taken into account. The emitted radiation essentially depends on the working gas used; in discharges ignited in ambient air the high nitrogen content dominates the appearance of spectra resulting in a high emission in the UVA and low proportions of UVB. The harmful short wavelength UVC radiation is effectively absorbed by oxygen and nitrogen species, so that it does not interact with the biological substrates in treatment [
Based on ICNIRP guidelines with regard to the maximum effective irradiance dosage, a limit value for treatment duration of about 3.6 h in case of μs-discharge, and 2.3 h in case of ns-discharge is determined. Consequently, lower treatment times must be complied for security reasons for ns-plasma. However, since typical exposure times in medical therapies are in the range of a few minutes, these results do not contradict dermatological applications of these plasma sources [
To estimate the interaction of the ROS and RNS with the SC formed in the plasma discharge, the occurring species concentrations on the sample surface were examined. For a comparison of the two discharge modes, the significance of the results was verified using the Student distribution (T-test). Prior to the plasma treatment, none of the tested chemical compounds could be detected on the lipid stripping; the concentrations immediately after plasma exposure are shown in
The nitric, peroxides and ammonium species were measured in a comparable concentration in the range of 0 - 3 mg/l, the nitrate concentrations are more than one order of magnitude higher. This result corresponds to our expectations, because in the here considered discharges NOx was formed by numerous reactions [
The ammonium and peroxide concentration on the substrate show very low concentrations, so harmful effects to human skin are not expected. Furthermore, the SC represents an effective barrier; cytotoxic effects of the substances are only possible in the living cells in the epidermis. On the other hand, the antiseptic and antibacterial properties of low concentrations of H2O2 are well known [
Analogous to the ozone and nitric oxide formation, a more efficient generation of the species by the ns-plas- ma is shown as well. Excluding the NH4, which has no significant statistical differences due to very large fluctuations, the measured values show differences between the two plasma sources with a significance level of p ≤ 0.0001. As well the detected nitrate concentrations are confirmed with the stronger acidifying effect of ns-plasma [
For the evaluation of plasma induced changes in SC-compositions, carbon, oxygen and nitrogen contents of untreated and plasma-treated samples were examined. In
It is already known that using the Cyanoacrylate Stripping method for SC-samples preparation results in a lipid covered sample surface. This effect is based on cohesive fractures within the lipid bilayer occurring during sample extraction [
After plasma treatment, a clear increase in oxygen and nitrogen content as well as a decrease of carbon content was shown, independent of the used DBD. This observation can be caused by two plasma induced effects: 1) The ROS and RNS present in plasma lead to an oxidation and nitration or rather nitrosation in the SC whereby oxygen and nitrogen are included in the sample; 2) The plasma treatment removes surface lipids by which the underlying corneocytes were detected. A purification of surfaces by plasma induced ablation was already shown inter alia by Iwasaki et al. [
In detail, the plasma induced changes were analysed on the basis of the carbon 1s peak. In
C1s | N1s | O1s | S2p | |
---|---|---|---|---|
Reference | 89.9 ± 1.5 | 2.3 ± 0.6 | 7.8 ± 1.2 | - |
μs-plasma | 75.4 ± 2.6 | 6.2 ± 1.2 | 18.3 ± 3.0 | 0.2 ± 0.1 |
ns-plasma | 73.8 ± 2.4 | 8.5 ± 0.9 | 17.5 ± 1.5 | 0.3 ± 0.1 |
Protein | 59.9 | 23 | 17.1 | Not specified |
Skinlipid | 90.9 | 0.23 | 8.5 | Not specified |
As additional effect, small amounts of sulphur were found after the plasma treatment. In the reference, the S2p peak could not be clearly distinguished from noise in the signal. In human skin, two chemical bonds of sulphur are known, on the one hand in the form of SIV (sulphate) as component in cholesterol sulphate, on the other hand in form of SII which can be found in disulphide bridges of proteins [
When comparing the two plasma sources, only minor differences in the composition are shown, although in case of ns-plasma a stronger impact is suspected due to the more effective formation of reactive species. As mentioned before, the plasma treatment leads to a mechanical removal of surface lipids. Consequently, it is assumed that the altered composition caused by the lipid removal overlays the plasma-induced changes and therefore no significant differences between μs- and ns-discharge sources could be shown.
In this article, two DBD sources developed for medical use which only differ in the pulse duration (μs- as well as ns-range) and in the resulting discharge current were used for in-vitro plasma treatment of SC-lipids. A significant influence of the pulse duration on characteristics of the discharge was shown, including a more effective formation of reactive species in ns-plasma. The same dissipated power results in substantial higher ozone and nitric oxide concentrations when using pulse durations in the ns-range. This behaviour is also confirmed by the reactive oxygen and nitrogen species generated on the sample surface. In addition, the ns-source emits a two times higher irradiance in the UV-range.
A safety evaluation was performed based on the determined parameters ozone, nitric oxide and UV-irradiance. No harmful effects are expected as long as the minimal distance between the discharge gap and the respiratory tract is more than 15 cm, and if a maximum application time of 2 h is not exceeded. However, this limitation in treatment time is practically irrelevant since positive therapeutic effects were achieved after only a few minutes of plasma treatment in previous clinical studies [
Plasma induced changes in the SC-composition were detected by means of XPS with regard to carbon, oxygen, and nitrogen content. A marked increase in oxygen and nitrogen content in plasma treated SC-samples was detected which can be explained by oxidation, nitrosation, and nitration of the sample surface as well as a removal of surface lipids by plasma treatment.
In summary, a major impact of pulse duration on the plasma composition was shown. With this parameter, the biological active components can be dimensioned for an individual therapeutic purpose.
This work was funded by the German Federal Ministry of Education and Research (BMBF), “PlasBaWirk”, Contract No. 03FH015IX5.
JoannaHirschberg,LeanderLoewenthal,AlexanderKrupp,SteffenEmmert,WolfgangViöl, (2016) Plasma Induced Changes in Human Lipid Composition as Revealed through XPS-Analysis. Natural Science,08,125-137. doi: 10.4236/ns.2016.83016