**2. Plasma discharges**

These days many types of plasma discharges exist. If we want to use plasma in medical applications (plasma drug delivery in our case), mostly atmospheric discharges are required. Atmospheric discharges can be generated from DC, through low frequency AC, radiofrequency to microwave frequencies. Generally, we can divide plasma sources into three categories:

**3. Stratum corneum structure**

[13], with the permission of the American Vacuum Society).

centration of K, Na, ceramides and fillagrin (**Figure 3**).

The stratum corneum is an upper layer of the skin. This layer is the main barrier of the skin which protects the body against loss of water and ensures that molecules from outside will not enter the body. The stratum corneum is composed of corneocyte cells placed in a lipidrich matrix. Human stratum corneum (thickness of 10–20 μm) contains from 10 to 25 layers of corneocytes. Corneocytes are composed of fibrillary keratin and water inside a cornified envelope. The cornified envelope is composed of crosslinked fillagrin, loricrin and involucrin [25]. Lipids in the lipid matrix are organized in lamellar layers following a tri-layer broadnarrow-broad arrangement [26]. The width of the tri-layer structures can be 6 or 13 nm. These lamellae can be packed in dense orthorhombic, less dense hexagonal or disordered liquid phase [25]. The lipid matrix consists of ceramides (41%), cholesterol (27%), cholesterol esters (10%), fatty acids (9%) with a small fraction of cholesterol sulfate (2%) [27] and glucosylceramides. Lipid chains are usually in a solid crystalline or gel state. At higher temperatures, lipids change their state to a liquid crystalline and they are more permeable (**Figure 2**).

**Figure 1.** Microplasma discharge at work (left). Schematic of electrode and skin treatment (right) (reproduced from Ref.

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After disruption of the stratum corneum, cholesterol synthesis leads to repairing of the barrier and it starts 90 min after barrier disruption [28]. The renewal of the stratum corneum occurs every 14 days [29]. Lipid-rich matrix is used for the transdermal delivery (TDD)—intercellular pathway, and it is composed of hydrophilic domain—head of ceramides and lipophilic domain—tail of cermaides. The stratum corneum can be divided into three main layers with different compositions and barrier functions [30]. The layers can be characterized by the con-

The concentration of Na is high in the whole stratum corneum and the concentration of K is high in the upper layer but low in the rest of the stratum corneum. The concentration of ceramides decreases from the upper layer to the lower layer. The concentration of arginine, which is the product of fillagrin, is the highest in the middle layer and lowest in the rest of the stratum corneum. The upper layer of the stratum corneum with dimension of 2/5 of the whole thickness (~ 8 μm) appears as a layer which allows passive flow of ions inside and


**Figure 1.** Microplasma discharge at work (left). Schematic of electrode and skin treatment (right) (reproduced from Ref. [13], with the permission of the American Vacuum Society).

#### **3. Stratum corneum structure**

studied these days. Research is mainly oriented to wound healing [5], cancer treatment [6], dermatology [7] and drug delivery to the cells [8]. Drug delivery through the skin is also studied very intensively and for a very long time [9, 10] but using plasma is a relatively new approach and only some studies exist [11–15]. For successful penetration of the drug through the skin, it is necessary to know the composition and structure of the stratum corneum and its lipid barrier. The stratum corneum is the main barrier of the skin, so knowledge of the function(s) of the molecules which participate in this barrier is crucial. Plasma sources can produce different kinds of particles with lifetimes from several nanoseconds to several seconds [16–18]. The configuration of using a specific gas or mixture of gases can prefer certain type of particles. Each particle can have different effects on the skin such as penetration depth, reactivity, and the ability to convert to more reactive particles. The effects of the particles to carbon and hydrocarbon surfaces will be described. The success of using of plasma for transdermal delivery of certain molecules and also feasibility of using model drug, Cyclosporine A, will be presented.

These days many types of plasma discharges exist. If we want to use plasma in medical applications (plasma drug delivery in our case), mostly atmospheric discharges are required. Atmospheric discharges can be generated from DC, through low frequency AC, radiofrequency to microwave frequencies. Generally, we can divide plasma sources into three categories:

**1.** Discharges where plasma is directly in contact with the sample (skin). In the case of direct plasma treatment of the tissues or skin, the human body serves as one of the electrodes and partial current flows through the tissue. The plasma has a low temperature (up to 45°C). The sample is several millimeters from plasma source and active species directly treat sample [19].

**2.** Plasma is blown out by gas flow to the sample (skin) from the place where the plasma was created. The distance between the plasma nozzle and the sample can be set from several millimeters to centimeters [20]. The diameter of the plasma plume can reach the diameter

**3.** Plasma is not in contact with the sample (skin). The sample is treated by long living particles such as certain radicals, ions, metastable species or particles with very long lifetimes (for example, ozone). Plasma usually has a very high temperature up to several thousand degrees or very small dimensions. A typical example of high temperature plasma source for medical treatment is "PLASON," which is used for the production of NO radicals [22], or microwave plasma torches [23]. On the other hand, a high temperature is sometimes necessary for the production of certain type of species. Sources with low dimensions are surface plasma discharges. Microplasma DBD (**Figure 1**) is a plasma source used in Shimizu et al. [11], where electrodes had a thickness in micrometer dimensions that allowed a decrease of ignition voltage to hundreds of Volts. Advantages of surface DBD sources is that they can be very large; and the electrode can be very thin and placed on polymer which can copy the surface of treated sample [24]. A disadvantage is that the distance between the electrode and the

**2. Plasma discharges**

102 Plasma Medicine - Concepts and Clinical Applications

of the needle [21].

sample must be very short, approximately 1–2 mm.

The stratum corneum is an upper layer of the skin. This layer is the main barrier of the skin which protects the body against loss of water and ensures that molecules from outside will not enter the body. The stratum corneum is composed of corneocyte cells placed in a lipidrich matrix. Human stratum corneum (thickness of 10–20 μm) contains from 10 to 25 layers of corneocytes. Corneocytes are composed of fibrillary keratin and water inside a cornified envelope. The cornified envelope is composed of crosslinked fillagrin, loricrin and involucrin [25]. Lipids in the lipid matrix are organized in lamellar layers following a tri-layer broadnarrow-broad arrangement [26]. The width of the tri-layer structures can be 6 or 13 nm. These lamellae can be packed in dense orthorhombic, less dense hexagonal or disordered liquid phase [25]. The lipid matrix consists of ceramides (41%), cholesterol (27%), cholesterol esters (10%), fatty acids (9%) with a small fraction of cholesterol sulfate (2%) [27] and glucosylceramides. Lipid chains are usually in a solid crystalline or gel state. At higher temperatures, lipids change their state to a liquid crystalline and they are more permeable (**Figure 2**).

After disruption of the stratum corneum, cholesterol synthesis leads to repairing of the barrier and it starts 90 min after barrier disruption [28]. The renewal of the stratum corneum occurs every 14 days [29]. Lipid-rich matrix is used for the transdermal delivery (TDD)—intercellular pathway, and it is composed of hydrophilic domain—head of ceramides and lipophilic domain—tail of cermaides. The stratum corneum can be divided into three main layers with different compositions and barrier functions [30]. The layers can be characterized by the concentration of K, Na, ceramides and fillagrin (**Figure 3**).

The concentration of Na is high in the whole stratum corneum and the concentration of K is high in the upper layer but low in the rest of the stratum corneum. The concentration of ceramides decreases from the upper layer to the lower layer. The concentration of arginine, which is the product of fillagrin, is the highest in the middle layer and lowest in the rest of the stratum corneum. The upper layer of the stratum corneum with dimension of 2/5 of the whole thickness (~ 8 μm) appears as a layer which allows passive flow of ions inside and

**3.1. Corneocytes**

**3.2. Role of ceramides**

ceramide 1 [33] (**Figure 4**).

Corneocytes contain a lipid envelope bounded to the exterior protein envelope. Corneocytes of the lipid envelope function as a semipermeable membrane that allows water molecules to penetrate but not the larger hygroscopic molecules [31]. Intercellular lipid lamellae and lipids of the lipid envelope of corneocytes are connected through an ester bond (R─CO─O─R1) [32].

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The percentages of some ceramides in the lipid matrix are 29.9% of ceramide 6, 21.7% of ceramide 2, 14.8% of ceramide 4, 13.9% of ceramide 5, 11.9% of ceramide 3 and 7.8% of

The permeability of this membrane can be increased by decreasing the concentration of cholesterol and increasing the amount of short-tailed fatty acids and unsaturated fatty acids. A decrease of ceramides also decreases the barrier function of the skin. Ceramides form a multilayer lamellar structure with other lipids. Ceramides also act as a water modulator and their decrease also decreases the water-holding capacity [34]. The majority of ceramides of the stratum corneum has an even number of carbons and the most abundant has 44 and 46 carbons. Ceramides with number of carbons higher than 60 were also observed in a non-negligible concentration [35]. The permeability of the synthetic lipid membrane was not changed with the length of the ceramides. The changes in head groups of ceramides also did not change the permeability [36]. Other research showed that the chain length of ceramides has a huge influence on the barrier permeability. It was also observed that a synthetic barrier composed of a limited amount of ceramides (3 in this case) has some effect on barrier properties. A pig skin containing lamellae with a wider distribution of ceramides has higher permeability than synthetic lamellae with three ceramides. This can be explained by mismatches between various lengths in the lipid lamellae. The wider distribution of ceramide chains affects lamellar

**Figure 4.** Structure of some ceramides fatty acid and cholesterol in the lipid matrix [33].

**Figure 2.** Model of the stratum corneum and with lateral and lamellar organization of lipids in the lipid matrix [26].

**Figure 3.** Three layers of the stratum corneum characterized by normalized amount of Na, K, arginine and ceramides [30]. The thickness of red, green blue and violet rectangular indicates relative concentration. I. Denotes the upper layer, II. Denotes the middle layer, and III. Denotes the lower layer.

outside. The amount of Na and K can be changed by the influence of the outer environment. Liquids and ions can easily penetrate the corneocytes in this layer and intracellular Na can be flushed out when corneocytes absorb water or other liquids. The middle layer of the stratum corneum appears as a first real barrier. The thickness is approximately 2/5 of the whole stratum cornea as in the previous case. Ions such as K+ and Cr6+ cannot penetrate into this layer. Hydration of the skin is a function of the second layer because of the high concentration of arginine. It was observed that Cr3+ was able to penetrate into the middle layer of the stratum corneum, but not into the third layer, which is the reason why the third layer can be called a second barrier. The third layer has a thickness of 1/5 of the stratum corneum (~ 4 μm) and the barrier function of the stratum corneum increases toward the viable epidermis.

#### **3.1. Corneocytes**

Corneocytes contain a lipid envelope bounded to the exterior protein envelope. Corneocytes of the lipid envelope function as a semipermeable membrane that allows water molecules to penetrate but not the larger hygroscopic molecules [31]. Intercellular lipid lamellae and lipids of the lipid envelope of corneocytes are connected through an ester bond (R─CO─O─R1) [32].

#### **3.2. Role of ceramides**

outside. The amount of Na and K can be changed by the influence of the outer environment. Liquids and ions can easily penetrate the corneocytes in this layer and intracellular Na can be flushed out when corneocytes absorb water or other liquids. The middle layer of the stratum corneum appears as a first real barrier. The thickness is approximately 2/5 of

**Figure 3.** Three layers of the stratum corneum characterized by normalized amount of Na, K, arginine and ceramides [30]. The thickness of red, green blue and violet rectangular indicates relative concentration. I. Denotes the upper layer,

**Figure 2.** Model of the stratum corneum and with lateral and lamellar organization of lipids in the lipid matrix [26].

into this layer. Hydration of the skin is a function of the second layer because of the high concentration of arginine. It was observed that Cr3+ was able to penetrate into the middle layer of the stratum corneum, but not into the third layer, which is the reason why the third layer can be called a second barrier. The third layer has a thickness of 1/5 of the stratum corneum (~ 4 μm) and the barrier function of the stratum corneum increases toward the

and Cr6+ cannot penetrate

the whole stratum cornea as in the previous case. Ions such as K+

II. Denotes the middle layer, and III. Denotes the lower layer.

104 Plasma Medicine - Concepts and Clinical Applications

viable epidermis.

The percentages of some ceramides in the lipid matrix are 29.9% of ceramide 6, 21.7% of ceramide 2, 14.8% of ceramide 4, 13.9% of ceramide 5, 11.9% of ceramide 3 and 7.8% of ceramide 1 [33] (**Figure 4**).

The permeability of this membrane can be increased by decreasing the concentration of cholesterol and increasing the amount of short-tailed fatty acids and unsaturated fatty acids. A decrease of ceramides also decreases the barrier function of the skin. Ceramides form a multilayer lamellar structure with other lipids. Ceramides also act as a water modulator and their decrease also decreases the water-holding capacity [34]. The majority of ceramides of the stratum corneum has an even number of carbons and the most abundant has 44 and 46 carbons. Ceramides with number of carbons higher than 60 were also observed in a non-negligible concentration [35]. The permeability of the synthetic lipid membrane was not changed with the length of the ceramides. The changes in head groups of ceramides also did not change the permeability [36]. Other research showed that the chain length of ceramides has a huge influence on the barrier permeability. It was also observed that a synthetic barrier composed of a limited amount of ceramides (3 in this case) has some effect on barrier properties. A pig skin containing lamellae with a wider distribution of ceramides has higher permeability than synthetic lamellae with three ceramides. This can be explained by mismatches between various lengths in the lipid lamellae. The wider distribution of ceramide chains affects lamellar

**Figure 4.** Structure of some ceramides fatty acid and cholesterol in the lipid matrix [33].

and also lateral organization. The wider distribution leads to a hexagonal organization, and synthetic ceramides with a low ceramide chain distribution leads to an orthorhombic organization [37]. A long-periodic phase is formed in the presence of a certain level of unsaturated ceramides, and a long-periodic phase is associated with fluid domain inside. A saturated ceramide EOS-S is important for the stabilization of the orthorhombic phase [38].

threshold energy is relatively high (in range 10–100 eV) depending on the target/impinging ion combination. The sputtered yield changes with the angle of incident, the substrate material or the roughness of surface. Eroded species consist of atoms or small clusters of target material. Investigation of metallic targets with impinging metallic ions showed that the mechanism of sputtering depends on the ratio of impinging ion mass (M1) and the mass of the atom in the sample (M2) [41]. In the case of M1 < M2, the threshold energy does not depend on the mass M1 or M2. As the mass M2 increases, the probability of reflection of ion increases.

If M2 ≥ M1, the threshold energy for sputtering is higher than in the first case. The threshold energy also does not depend on the mass of M1 and M2, and sputtering follows the

The threshold is higher in the second case because atoms of the target are sputtered by secondary atoms which were released by ion bombardment. Pure physical sputtering can be achieved mostly in discharges with inert gases. Bombardment of lipids inside the stratum corneum will follow mostly the second mechanism when argon is used and the first one in the case of helium. Physical sputtering is also present in nitrogen, oxygen or air plasma, but

Argon is one of the simplest media for treatment of biological material. Excited states of Ar

states have a relatively long lifetime equal to 38 s [43] in vacuum. However, this time is reduced in atmospheric pressure. The energy difference between the ground state and the metastable state of Ar is 11.55 eV, which can be released by collision. A molecular dynamics simulation demonstrated the sputtering of a lipid-like material by argon ions (**Figure 5A** and **B**). The sputtering threshold energy was between 10 and 20 eV. The yield of sputtered particles increases to four carbon atoms per ion impacting the surface at an energy of 50 eV and increases more slowly up to 10 at 100 eV [44]. If we suppose a similar behavior of lipids and polymers during bombardment, argon ion bombardment of the polymers can lead to decreas-

, CO, CO2

polymer structure [45]. The sputtering of polymer surface by Ar ions leads to graphite-like

, Ar2 +

) [42] can be produced in any electrical dis-

and H2

, and disordered

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is 15.8 and 15.5 eV, respectively. Argon metastable

, HCOOCH3

**1.** Ion penetrates through the surface layer and creates vacancy, releasing atom.

**2.** Impinging ion and also released atom deform structure of material.

 and Ar2 +

The mechanism of sputtering follows the next steps:

**3.** Atom of the first layer is sputtered by impinging ion.

**3.** Atom of the first layer is sputtered by released atom.

chemical sputtering can be much more dominant.

*4.1.1.1. Argon bombardment/argon plasma*

charges. Ionization energy of Ar+

(Ar\*), metastable states (Arm) and Ar ions (Ar+

ing of side chains of the polymer, volatile CH4

**2.** Ion is implanted in sublayer and deform structure of material.

**1.** Ion penetrates through the surface layer.

mechanism:

#### **3.3. Role of fatty acids**

The reduction of free fatty acid (FFA) chains was observed in skin diseases which are known by impaired skin barrier function. The reduction of the fatty acid chain can be caused by an increase in the amount of shorter FFA chains such as with 16 or 18 carbons. A shorter chain allows an increase in vibrations followed by conformational disordering. An increase in the number of unsaturated FFA reduces the packing density of the lipid organization [36]. An analysis of the composition of free fatty acids of human stratum corneum showed a dominant presence of saturated free fatty acids with carbon chain lengths from 16 to 30. The most abundant were FFAs with 24 and 26 carbons (50% of all FFAs). The content of unsaturated FFAs appeared to be around 2% of all FFAs; mostly with 18 carbons and with traces of FFAs with 16, 17 and 20 carbons; 1% of di-unsaturated FFAs (chain length of 18 carbons) [35].

#### **3.4. Role of cholesterol**

Cholesterol is situated near the ester bond of ceramides and this position allows the formation of a hydrogen bond between the cholesterol ─OH group and the carbonyl group C═O. The presence of cholesterol has an influence on the formation of the long periodicity phase of ceramides (13 nm). There is a minimal amount of required cholesterol for the formation of the long periodicity phase of ceramides (without cholesterol, long periodicity phase is not formed). Cholesterol does not influence only the presence of long periodicity phase but also the packing density in the long periodicity phase [39]. Cholesterol is very important for correct skin barrier function [40].
