**7. Plasma effects on skin barrier and transdermal drug delivery**

## **7.1. Chemical reactions plasma particles with the skin**

If argon plasma is used for skin treatment, the plasma produces electrons, argon ions, metastable and excited states. These particles can react with the skin directly. But, if argon or nitrogen plasma is working in atmospheric air, it makes the situation more complex because molecules of air (O2, N2, CO2 and H2O) can enter the volume of the plasma jet [73, 74]. In this case, argon can react with skin also indirectly through the excitation or dissociation or ionization process with air molecules. The reaction results in the creation of a number of species. It is difficult to find the molecule causing changes in skin. Pig skins treated by argon, nitrogen and argon/water plasma were compared by measuring ATR-FTIR spectra and monitoring of the shift of asymmetric stretching CH2 band near 2920 cm−1. A comparison of argon and nitrogen plasma jets has shown that argon can play a role in reactions with molecules of stratum corneum, but a similar effect can be achieved by nitrogen plasma itself as similar shift can be observed. A significantly higher value of shift was observed when argon with water vapours was used. The asymmetric stretching band was shifted 3.5 cm−1 (**Figure 9**). This result indicates that the stratum corneum became the most permeable after treatment of Ar plasma with water vapours. On the other hand, we observed low wavenumber shifts of the maxima of amide I and amide II (**Figure 9**).

**6.3. Plasma jet**

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Plasma jets consist usually from a gas nozzle with one, two or three electrodes [70, 71]. The plasma jet can be realised by two ways—active plasma jets (expanding plasma contains free and high energetic electrons) and remote plasma jets (plasma is potential free and consists of relaxing and recombining active species from inside the nozzle) [70]. The plasma jet in **Figure 8** consists of a Pyrex tube and a central tungsten high voltage (HV) electrode. The grounded electrode is an aluminium ring located at the end of the outer surface of the Pyrex tube. The distance between the skin sample and the outlet of the plasma jet was set to 2 mm. The sample was isolated from the holder by a 30-mm thick PVC isolator or without isolator to compare the effect of the conductive layer under the skin surface as the human body is not isolator. The treatment time of the sample was set from several seconds to 1 min. Argon, nitrogen or argon-water vapour gases were introduced into the plasma jet. The waveform of

the voltage and current of argon plasma jet is depicted in **Figure 8** [72].

**Figure 8.** Waveform of plasma jet discharge (left) [72] and plasma jet electrode (right).

**7.1. Chemical reactions plasma particles with the skin**

**7. Plasma effects on skin barrier and transdermal drug delivery**

If argon plasma is used for skin treatment, the plasma produces electrons, argon ions, metastable and excited states. These particles can react with the skin directly. But, if argon or nitrogen plasma is working in atmospheric air, it makes the situation more complex because molecules of air (O2, N2, CO2 and H2O) can enter the volume of the plasma jet [73, 74]. In this case, argon can react with skin also indirectly through the excitation or dissociation or ionization process with air molecules. The reaction results in the creation of a number of species. It is difficult to find the molecule causing changes in skin. Pig skins treated by argon, nitrogen and argon/water plasma were compared by measuring ATR-FTIR spectra and monitoring of the shift of asymmetric stretching CH2 band near 2920 cm−1. A comparison of argon and nitrogen plasma jets has shown that argon can play a role in reactions with molecules

**Figure 9.** Shift of asymmetric stretching band of CH2 to higher wavenumbers for the gases used in plasma jet discharge. Flow 10 L/min = (Ar\*; N2), 3 L/min = (Ar; Ar+H2O).

Pig skin treated by argon microplasma shows shift of asymmetric stretching CH2 band near 2920 cm−1 after 1, 3 and 5 min of irradiation comparable with the plasma jet (**Figure 10**).

**Figure 10.** Shift of asymmetric stretching band of CH2 to higher wavenumbers for Ar gas after microplasma irradiation [69].

When argon flew through the water reservoir, the argon ensured a higher concentration of water vapours in the discharge. The high shift of the asymmetric stretching band of CH2 indicates that H2O and the created OH molecules can play important roles in increasing the shift of the asymmetric stretching band of CH2 in stratum corneum. OH could be created mainly by two channels [75]:

$$Ar\_m + H\_2O \to OH + H + Ar \tag{1}$$

$$H\_2O + e \to OH + H + e \tag{2}$$

Simulation of the interaction of O and OH radicals with α-linolenic acid as a representative of fatty acid [76] showed that OH radicals most typically abstract an H atom from the fatty acids, which can lead to the creation of a double bond and also to the incorporation of alcohols or aldehyde groups, increasing hydrophilic properties of fatty acids and changing the lipid composition of the skin, causing an increase of skin permeability. Creation of these groups increases the hydrophilic character of the lipid layer. Incorporation of oxygen to stratum corneum lipids was also confirmed by increasing of C–O and C=O bonds after treatment of skin layer by atmospheric plasma [77]. Also, micropores (10 nm–1 μm in size) were observed in an artificial cell membrane system consisting of supported lipid bilayers after DBD plasma irradiation [78]. Later, it was found out that these micropores are induced transient species such as OH and OOH formed by plasma and they caused lipid peroxidation leading to truncated lipid chains, which induced pore formation [79]. These temporal pores were observed in skin after DBD plasma treatment and it was allowed to penetrate large molecules through the skin in several minutes. These pores had lifetimes of less than 5 min after treatment [68].

#### **7.2. Effect of heating on skin barrier**

Heating of the skin can increase permeability of stratum corneum [80]. Reasons of improving of transdermal delivery through the skin by increasing temperature are structural changes of stratum corneum lipids. Plasma can raise skin temperature from 30°C to more than 100°C which depends on the irradiated time, power of plasma source or used gas. Structural changes of stratum corneum lipids occur between 20 and 40°C, from orthorhombic to hexagonal ordered lipids. These changes can be indicated by CH2 symmetric stretching frequency with an increase of 0.5 cm−1 in ATR-FTIR spectra or it is possible to observe by CH2 scissoring mode where transition is revealed by splitting of the scissoring modes to produce a doublet with components at 1473 and 1463 cm−1. Ordered lipid chains change to disordered chains at around 80–90°C. But some scientific groups identified more transitions in lipid structure [52]. For example, four phase transitions in temperature ranges 35–42°C, 65–75°C, 78–86°C and 90– 115°C, respectively. Transitions below 75°C are reversible [81]. Between 35°C and 42°C two transitions can occur belonging to solid fluid transition and disruption of lipids covalently linked to corneocites at 37 and at 40°C to 'orthorhombic to hexagonal' change in structure. But too high a temperature can cause damage to the skin. Thermal conditions that cause burns of the skin are functions of the time and method of how the skin is exposed to the heat. Longer exposure of skin to a temperature higher than 43°C can lead to the formation of blisters [82]. It was found out that fast heating of skin can increase skin permeability without damaging deeper tissues. Investigation of high temperatures of up to 315°C applied to skin for 100 ms, 1 s and 5 s showed small variations between drug deliveries of calcein [82]. Exposure for 1 s or 5 s should be sufficient to equalize temperature in the full thickness of skin, but the 100 ms exposure should have influence only on stratum corneum. Between 100 and 150°C, permeability of skin was increased a few fold. This increase was attributed to lipid melting in the stratum corneum. In the range of 150–250°C, transdermal flux increased by two orders, attributable to disruption of stratum corneum keratin network structure. Above 300°C, transdermal flux increased by three orders, attributable to decomposition and vaporization of the stratum corneum [83].

## **7.3. Effect of UV radiation on skin barrier**

When argon flew through the water reservoir, the argon ensured a higher concentration of water vapours in the discharge. The high shift of the asymmetric stretching band of CH2 indicates that H2O and the created OH molecules can play important roles in increasing the shift of the asymmetric stretching band of CH2 in stratum corneum. OH could be created

Simulation of the interaction of O and OH radicals with α-linolenic acid as a representative of fatty acid [76] showed that OH radicals most typically abstract an H atom from the fatty acids, which can lead to the creation of a double bond and also to the incorporation of alcohols or aldehyde groups, increasing hydrophilic properties of fatty acids and changing the lipid composition of the skin, causing an increase of skin permeability. Creation of these groups increases the hydrophilic character of the lipid layer. Incorporation of oxygen to stratum corneum lipids was also confirmed by increasing of C–O and C=O bonds after treatment of skin layer by atmospheric plasma [77]. Also, micropores (10 nm–1 μm in size) were observed in an artificial cell membrane system consisting of supported lipid bilayers after DBD plasma irradiation [78]. Later, it was found out that these micropores are induced transient species such as OH and OOH formed by plasma and they caused lipid peroxidation leading to truncated lipid chains, which induced pore formation [79]. These temporal pores were observed in skin after DBD plasma treatment and it was allowed to penetrate large molecules through the skin in several minutes. These pores had lifetimes of less than 5 min

Heating of the skin can increase permeability of stratum corneum [80]. Reasons of improving of transdermal delivery through the skin by increasing temperature are structural changes of stratum corneum lipids. Plasma can raise skin temperature from 30°C to more than 100°C which depends on the irradiated time, power of plasma source or used gas. Structural changes of stratum corneum lipids occur between 20 and 40°C, from orthorhombic to hexagonal ordered lipids. These changes can be indicated by CH2 symmetric stretching frequency with an increase of 0.5 cm−1 in ATR-FTIR spectra or it is possible to observe by CH2 scissoring mode where transition is revealed by splitting of the scissoring modes to produce a doublet with components at 1473 and 1463 cm−1. Ordered lipid chains change to disordered chains at around 80–90°C. But some scientific groups identified more transitions in lipid structure [52]. For example, four phase transitions in temperature ranges 35–42°C, 65–75°C, 78–86°C and 90– 115°C, respectively. Transitions below 75°C are reversible [81]. Between 35°C and 42°C two transitions can occur belonging to solid fluid transition and disruption of lipids covalently

*Ar H O OH H Ar <sup>m</sup>* + ® ++ <sup>2</sup> (1)

*H O e OH H e* <sup>2</sup> +® + + (2)

mainly by two channels [75]:

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after treatment [68].

**7.2. Effect of heating on skin barrier**

The wavelength range of 100–400 nm is called UV light and is usually divided into three ranges: UVA (320–400 nm), UVB (280–320 nm) and UVC (100–280 nm). UV light can cause damage to skin. It is well-known that atmospheric plasma can generate wide range of UV light dependent on the used gas [84, 85]. UVC radiation can reach only stratum corneum and it consists of emission of excited NO molecule (200–280 nm—NO-gama system) or nitrogen molecule (120– 200 nm—LBH system) in plasma discharges. The source of UVB radiation is emission of OH and nitrogen (second positive system of nitrogen). UVA is composed mostly of second positive system of nitrogen. UVA and UVB can reach deeper layers of skin like epidermis and UVA also dermis. UV radiation can cause formation of hydroxides and epoxides, hydrogenation of double bonds and breaking of carbon chains. The effect of UV on lipids depends on their structure, and only double bonds of fatty acids are sensitive to the formation of oxygenated molecules by oxygen in the air. These changes can be amplified or weakened by the surrounding atmosphere around the treated skin. When lipids of stratum corneum like cholesterol, cholesterol sulphate, ceramide III, linolenic acid and dipalmitoylphosphatidylcholine were irradiated by UV light up to 240 min, peroxidative changes occurred only in lipids with double bonds such as cholesterol and linolenic acid [86]. But the changes are not permanent and the order of stratum corneum lipids and water-loss protection can recover after 3 days and it returns to its initial state, thanks to repair processes in the skin [87]. Reactive oxygen species like superoxide anion radicals, singlet oxygen, hydrogen peroxide and hydroxyl radical can be created by UV light. For example, superoxide anion radicals are precursors to other oxygen reactive species, and it was found that there exists a correlation between the superoxide anion radical's concentration and degree of oxidation of lipids [88]. UV light is also used for treatment of some skin diseases but delivered doses have to be controlled and maintained in safety level to not penetrate to deeper layers of skin [89].
