Plasma Devices for Cosmetic and Aesthetic Treatment

Abstract

Plasma medicine is a relatively new scientific discipline that employs nonthermal atmospheric plasmas for a variety of applications including sterilization, dental care, cosmetics and skin disorders. The development of skin treatments is also very important for effective cosmetic and medical applications. Recently, it has been issued that radicals generated by plasma can be a solution for this treatment. Plasma-induced RONS production provides biocompatibility by inducing skin cell differentiation and proliferation. The biocompatibility of nonthermal plasma has opened up new research field and market for cosmetics and aesthetics in skin.

7.1 Plasma Devices

Plasma medicine is a relatively new scientific discipline that employs nonthermal atmospheric plasmas for a variety of applications including sterilization, dental care, cosmetics and skin disorders, wound therapy, blood coagulation, and, more recently, cancer treatment. Plasma sources for plasma medicine are commonly operated in air, argon, or helium with little air contaminants. As a result, they produce reactive oxygen and nitrogen species (RONS), such as OH, O₂•, O, NO, H₂O₂, NO₂, NO₃, ONOO, NO₂, ONOO, which are thought to be responsible for plasma's biological effects. Although CAP appears to be a potential anti-cancer drug, direct irradiation of cancer cells or tissue by CAP has significant limitations, including the necessity for a consistent plasma supply and the possibility of a rapid temperature rise during therapy. Furthermore, it is inconvenient for many cancer kinds throughout the body.

There are four types of plasma discharge: floating electrode technique with ground electrode, non-floating electrode method without ground electrode, jet type, and DBD type. A plasma jet is a basic plasma source that uses a hollow cathode construction. The driving frequency is roughly 10 kHz, while the high frequency ranges from 10 to 100 MHz, and microwave plasma jet stations are categorized based on frequency. The DBD technique involves covering both electrodes with a dielectric substance and creating a discharge in the gap between the two electrodes. The discharge at the surface DBD is constructed using two electrodes on a substrate and a high current delivered to the electrodes to create plasma discharge. The floating electrode technique is a discharge method that uses skin as a ground electrode instead of the ground electrode mentioned above.

7.2 Opportunities for Plasma Devices in Cosmetics/Aesthetics Applications

Nonthermal atmospheric pressure plasma is an innovative technique that has opened up new research avenues in cancer therapy and other medical sectors such as dental brightening, wound treatments, and skin care. The plasma device produces plasma by combining various gases or by employing a high voltage and current. Ionized gas molecules, electrons, excited atoms, ultraviolet (UV) radiation, electromagnetic fields, and other particles are among the plasma particles released. Plasma devices caused reactive atoms to connect with one another, as well as reactive oxygen and nitrogen species (RONS). Plasma-generated reactive species have physiological features that may encourage therapeutic uses for skin therapy.

Since the 1990s, plasma has been employed as one of the sterilizing procedures for medical devices. Plasma sterilization has the benefit of providing safe and non-toxic dry pasteurization. Following sterilization, the charged particles produce active oxygen via ion reactions while producing water and oxygen. When free oxygen is provided to microorganisms, bacteria, viruses, and fungus can perish effectively. Plasma has recently been shown to have anticancer properties in several tumors such as the brain, breast, prostate, ovaries, and lungs. Several medical gadgets that utilize plasma needles have also been reported.

A technique of processing plasma particles under three-dimensional settings may be constructed with appropriate physical components and mechanical cavity elements in the case of cosmetics and aesthetics. Skin treatment method development is also critical for effective beauty and medicinal applications. It was recently suggested that plasma might be to blame. Plasma-induced RONS production causes skin cell differentiation and proliferation. There is, however, no evidence to support plasma therapy for skin cell differentiation.

The skin’s barrier system not only protects against antigens and hazardous chemicals, but it also prevents medications and cosmetics from penetrating the dermis. Several technologies, including the use of chemicals and skin ablation devices, have been developed to improve the absorption capacity of skin for cosmetics. The cost and inconvenience of these measures, on the other hand, underline the need for an unique and safe means of enhancing skin absorption.

The influence of low temperature atmospheric pressure plasma (LTAPP) on drug penetration through the skin and its mode of action [1]. HaCaT human keratinocytes and hairless mice were treated with LTAPP, and cellular and tissue gene expression, as well as morphological alterations, were studied [1]. They discovered that LTAPP exposure decreased E-cadherin expression in skin cells, resulting in the loss of cell-cell connections. LTAPP also inhibited E-cadherin expression and impeded intercellular connection formation inside the tissue, resulting in increased absorption of hydrophilic substances, eosin, and epidermal growth factor. Within 3 h of LTAPP exposure, the drop in E-cadherin expression and epidermal barrier function had entirely restored. These findings suggest that LTAPP can cause a transient reduction in skin barrier function by altering E-cadherin-mediated intercellular contacts, resulting in improved transdermal medication and cosmetic delivery.

The combination of nonthermal atmospheric pressure plasma with 15% CP is more effective for teeth bleaching than traditional light sources. The temperature of the tooth surface was kept about 37 °C, showing that the plasma did not cause any thermal damage to the tooth. The use of plasma had no structural effects on the bleached surface. Nonthermal atmospheric pressure plasma has been shown to be harmless to bleached enamel. As a result, plasma tooth bleaching treatments constitute a cosmetic dentistry technology with several potential practical uses [2].

Foster presented the first commercial plasma system, the Portrait_ PSR, and reviewed early in vivo therapy outcomes. The Portrait_ PSR is essentially a nitrogen-generated radio frequency plasma jet. A high energy therapy (3–4 J) resulted in regulated skin damage. After 10 days, the epidermis had entirely recovered. They also demonstrated continued collagen formation, elastosis decrease, and gradual skin rejuvenation one year following the therapy. Patients experienced a 60% improvement in skin texture, including wrinkle reduction and skin tone enhancement [3].

The application of plasma to the nail surface via a specially constructed prototype enables the longer-lasting nail polish touted by many traditional nail varnish makers in commercials. This innovative, working prototype is simple to use and low-cost; it is also portable due to its battery power. The goal of this paper is to investigate the changes in fingernails caused by plasma utilizing surface analysis methodologies. This article also looks into the benefits of plasma therapy for nail varnish adherence and drying times [4].

Through plasma pulses generated by radiofrequency energy being applied to nitrogen gas, plasma skin regeneration provides energy to the skin. High-energy, single-pass treatments have been shown to produce positive outcomes with a great safety record. A total of three full-face treatments with energy settings ranging from 1.2 to 1.8 J were given to eight individuals every three weeks. The quality of the regenerated epidermis, the length of downtime, and erythema were noted prior to each successive treatment. Six individuals had full-thickness skin biopsy samples taken both before and 90 days after their final treatment. Four days after each treatment, 30 days after the second, and 90 days after the third, patients were seen for follow-up visits.

Researchers discovered a 37% decrease in face rhytides three months following treatment, and trial participants reported a 68% improvement in overall facial attractiveness. In 4 days, re-epithelialization was finished. Patients reported that the erythema persisted for an average of 6 days following therapy. The first treatment's duration of epidermal regeneration was greater than that of the subsequent treatments (9 vs 4 and 5 days, respectively). Following the initial treatment, one patient experienced localized hyperpigmentation, which disappeared by the follow-up appointment on day 30. There was no hypopigmentation or scarring. A histopathological assessment A ring of new collagen at the dermo epidermal junction and less thick elastin in the upper dermis were visible 3 months after therapy. The new collagen was 72.3 m deep on average. With little recovery time, photodamaged facial skin can be successfully treated using plasma skin regeneration and the multiple low-energy treatment technique. Results are similar to one high-energy treatment, but recovery takes less time [5].

Plasma is designed to sculpt the pigments and tones of the skin. This non-invasive form provides high utilization for clinical applications, the behavior of plasma is multi-directional stimulating, uses low levels of energy to directly irritate the skin, while using custom options to explain in a distinct way. This can be compared to existing technologies such as laser irradiation or LED irradiation, or the main differentiation between emerging technologies is the size of the treatment area.

In addition to the use of cosmetics, new physical technologies have recently emerged in the field of skin treatment, and various devices have been proposed for skin care, anti-aging and rejuvenation applications (mechanical cleansing devices, massage devices, ultrasonic waves, light, radio frequency, and cooling). Among these innovations, plasma devices were developed mainly for medical use [6] to treat skin, but recently for cosmetic use. In addition, some small plasma devices can be found mainly in the cosmetics/esthetic market for skin rejuvenation, but so far most of them lack a scientific foundation. As a result, problems arise not only in the user but also in the effectiveness and safety of these devices when managing the skin after repeated procedures.

It is required to specify the region enclosing the cosmetics in order to deal with the possibilities of using cold plasma in cosmetics. It is challenging to precisely draw the line between aesthetics and medical (cortical) (including reconstruction molding and non-surgical procedures). Typically, aesthetics and medicine refer to modifications to the body or its function. Because they simply need to take care of their look without altering their body or function, cosmetics appear to be different from medicine or aesthetics.

7.3 Trends of Plasma Technology in Cosmetics and Marketing

Plasma is designed to sculpt the pigments and tones of the skin. This non-invasive form provides high utilization for clinical applications, the behavior of plasma is multi-directional stimulating, uses low levels of energy to directly irritate the skin, while using custom options to explain in a distinct way. This can be compared to existing technologies such as laser irradiation or LED irradiation, or the main differentiation between emerging technologies is the size of the treatment area.

Besides the use of cosmetic products, new physical technologies have recently emerged in the field of skin treatment and various devices are proposed for skin care, anti-aging and rejuvenation applications (mechanical cleansing devices, massager instruments, ultrasounds, light, radiofrequencies, cool sculpting,). Among these innovations, plasma devices have been developed to treat skin, mainly for medical use [8] but more recently for cosmetics applications.

Moreover, some small plasma devices can now be found on the market for cosmetics/aesthetics applications, mainly for skin rejuvenation, but, up to now, mostly with a lack of scientific fundament. This raises the problem of the effectiveness of such devices in the care of the skin and their safety of use, both for the user but also for the skin after repeated treatments.

To address the possibilities of using cold plasma in cosmetics, it is necessary to define which area covers the cosmetics. The boundaries between medicine (dermatology), aesthetic medicine (including reconstructive and plastic surgery and non-surgical procedures) and cosmetics are difficult to define precisely. Usually, medicine and aesthetic medicine imply a modification of the body or of its functions. Cosmetics seems to be different from medicine and aesthetic medicine as it should not modify the body or its functions but only improve its external aspect.

According to the European Union Cosmetics Directive, a cosmetic “any substance or preparation designed to be applied to the skin, hair, nails, lips, external genital organs, teeth, and mucous membranes of the oral cavity with the sole or primary goals of cleaning, perfuming, altering appearance, reducing body odor, protecting, or maintaining the health of the various external parts of the human body (epidermis, hair system, nails, lips, and external genital organs). When used under typical or fairly anticipated situations, they must not harm human health.” (Regulation of the European Community, CE No. 1223/2009; available at: http://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX%3A32009R1223) Cosmetics are defined as “materials intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body...for washing, beautifying, enhancing attractiveness, or altering the look” under the US Federal Food, Drug & Cosmetic Act (FD&C Act).

The definition is less clear when it comes to cosmetic gadgets. According to the FDA (https://www.fda.gov/medical-devices/products-and-medical-procedures/cosmetic-devices), cosmetic devices are used in the US “to improve appearance and do not impart any health benefits.” The situation is changing in Europe, where the majority of medical and cosmetic/aesthetic devices will be regulated more strictly under one group (EU Regulation 2017/745; available at https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32017R0745).

So, it remains to debate whether plasma devices can be used for skin care and whether they can be considered as cosmetic or medical devices according to their effects on skin and the respective regulations of various countries.

In 2017, the world’s cosmetics exports scale was about 94.2 billion and especially in France cosmetics exports scale was amounted to 15.2 billion dollar which accounting for 16% of the world’s cosmetic export market and taking first place. In 2017, France's Trade Specification Index (TSI) appears at 0.6 and followed by Korea (0.5) and Italy (0.4).

In Korea, skin care equipment is classified managed as beauty device and industrial products. As for medical device items, low-frequency stimulation, ultraviolet irradiator, infrared irradiator, light irradiator, ultra-high frequency stimulation, high-frequency stimulation, ultrasonic stimulation, paraffin bath, treatment steam machine, treatment heater medical treatment, limb circulation, treatment laser irradiator, medicine inhaler, and vibrators for medical treatment etc.

According to the medical equipment, the skin care equipment also needs approval from the Korea Food and Drug Administration (KFDA). Skin care and aesthetic equipment are categorized under one of four medical device classification systems used in Korea, which is most similar to the EU system. Despite efforts to create a market for the sale of plasma cosmetics/aesthetics devices, there is currently no item category or management regulation for atmospheric plasma devices. Cosmetic devices that use plasma technology must be divided into groups based on whether they use high-pressure, atmospheric-pressure, or low-pressure plasma with or without a thermal effect.

It is recommended that the skin care devices in Korea be presented in the “Guideline for Evaluation Equipment for Clinical Treatment for Wound healing Using Plasma” published by KFED. The guidelines include measurements of plasma density (electron density), ozone, nitrogen monoxide, nitrogen dioxide production, OH radical optical emission production, ultraviolet radiation production, photo-biological safety, plasma degree, etc. of plasma equipment.

Floating electrode plasma sources are currently used by the majority of plasma beauty devices in Korea. Human skin serves as the ground electrode in a floating-electrode plasma discharge, and discharge takes place between the electrode of the device and the skin. Based on the non-floating electrode jet plasma whitening machine, this device was created.

Recently it has been reported that plasma devices have functions mainly for wrinkles, acne, wound healing, wound removal, and skin regeneration. This product can helps rejuvenate the skin to promotes collagen formation and can cure the acne and atopy to kills microbes on skin.

Target	Plasma source	Species	Feed gas	References
Skin decontamination	Jet and dielectric barrier discharge (DBD)	Human	Argon	Daeschlein et al. [96]
Wound healing	Jet	Rat	Helium	Nastuta et al. [125]
Wound healing	Jet	Mouse	Argon	Xu et al. [126]
Root canal of tooth	Jet	Human	Argon	Bussiahn et al. [127]
Wound healing	Jet	Human	Argon	Heinlin et al. [128]
Chronic wound	Torch	Human	Argon	Isbary et al. [8]
Wound healing	Jet	Rat	Argon	Salehi et al. [129]
Skin decontamination	Jet	Human	Argon	Daeschlein et al. [98]
Wound healing	Jet	Human	Argon	Isbary et al. [8]
Wound healing	Jet	Mouse	Argon, Helium	García-Alcantara et al. [130]
Sterilization of surface wound	Floating Electrode (FE)-DBD	Mouse Human	Room air	Fridman et al. [131]
Tooth whitening	Micro-jet	Human	Air	Pan et al. [132]
Wound healing	Jet	Mouse	Argon	Schmidt et al. [133]
Bactericidal effect in biofilm of infected wounds	Torch	Rat	Argon	Ermolaeva et al. [134]
Wound healing in diabetes	Jet	Rat	Helium	Fathollah et al. [135]
Wound healing	DBD	Rat	Argon, oxygen mixture	Hung et al. [60]
Acne	DBD	Human	Room air	Chutsirimongkol et al. [136]
Rejuvenation	Jet	Human	Helium	Heinlin et al. [13]
Transdermal drug delivery	DBD	Human	Argon	Shimizu et al. (2015)
Actinic keratoses	Jet	Human	Argon	Wirtz et al. [137]
Transdermal delivery	DBD	Porcine	Room air	Kalghatgi et al. [138]
Skin penetration	Jet	Mouse	Air	Liu et al. [139]

The plasma discharge can be distinguished into a floating electrode method with a ground electrode, a non-floating electrode method without a ground electrode, a jet and a DBD according to the form of the electrode. The plasma jet is a hollow cathode electrode structure. A driving frequency of 10 kHz, a high frequency of 10–100 MHz, and microwave plasma jet stations of GHz are classified by frequency. The DBD method involves discharging in the area between two electrodes that are separated by a dielectric layer. The surface eruption Two electrodes are created on a substrate for the DBD, and after a high electric current is delivered to the electrodes to discharge plasma, a dielectric is created on the surface of the dielectric. The floating electrode method is a discharge technique in which the ground electrode in the non-floating electrode approach previously mentioned is replaced by the skin or a similar material [7].

7.4 Optimization of Plasma Dose for Wound Healing and Cancer Treatment

Several studies investigated the effectiveness of non-thermal bio-compatible plasma (NBP) to heal acute and chronic wounds. However, Initial studies addressed the safety concerns and bacterial load decrease in protracted wounds [8,9,10,11]. How can a single application express two opposite characters? “Sola dosis facit venenum” according to Paracelsus in 1538, “means just the dose makes the poison. The case is the same for plasma, the dose determines its outcomes which means a low dose can be beneficial, however, a high dose may cause destruction. Thus, for wound healing low dose is recommended however, a low dose may not have required killing impact while treating cancer. Moreover, the term dose is not uniform or well defined because there are numerous plasma devices with various operating characteristics. Therefore, one of the key issues requiring future clinical studies to pay attention is the optimization of doses in relation to an application or clinical trials, it will help to understand the mechanism of NBP at the molecular level. Although research on NBP mechanisms in cancer treatment and wound healing has progressed well, however, there is still, a lot that needs to be explored. Thus, the molecular mechanism of NBP has been under investigation to find the optimum procedure for clinical practices. Although, the main mechanism of NBP in wound and cancer treatment is explained briefly (Fig. 7.1).

Fig. 7.1

Non-thermal bio-compatible plasma (NBP) for the treatment of cancer and wounds treatment. Low or optimal NBP therapy promotes tissue growth, wound healing, and purification. Reactive oxygen and nitrogen species acting separately or combined are thought to have an impact on the reported results. Long-term cancer therapy increases oxidative stress, which causes cancer cells to die as a result

Additionally, reactive oxygen and nitrogen species (RONS), electromagnetic fields, and ultraviolet radiations are all linked to the biological effectiveness of NBP. Chemical reactive oxygen and nitrogen species play a part in enhancing biological applications and are directly related to the ability of wound healing and tumor inhibition. The impact of UV light during NBP administration is most likely minimal. Compared to its natural sun exposure, the UV intensity during NBP treatment for wound healing is significantly reduced [12,13,14,15].

7.5 NBP Anti-Cancer Effect During in Vivo and in Vitro Application

NBP’s anti-cancer mechanism in vivo treatment is debatable. Phosphate-buffered saline (PBS) after plasma exposure is associated with the production of H₂O₂, ${\text{NO}}_{2}^-$, and OH, which are covered by a 1 mm gelatin film [16]. This indicates that ROS diffusion across the skin is possible. Another possibility is to control tumors by NBP through immune response activation [17,18,19]. The uniform nanosecond pulsed DBD (nspDBD) has been shown to activate macrophages and improve healing in artificial wounds [19]. Thus, nspDBD is shown to augment the anti-tumor efficacy through both, tumor cell death and activation of macrophage function [18].

In the NBP treatment, a layer of cell culture media was used in melanoma cells in in vitro studies [20,21,22]. The natural phenomena of metabolism engage in the generation of long-lived and short-lived reactive species which is congruent to the production of RONS during NBP. The role of RONS has already been reported in the propagation of physiological and pathological mechanisms for example wound regeneration, cell propagation, apoptosis, and immunogenic responsive element [23]. A schematic example of the interaction of NBP with cells in vitro and in vivo is provided illustration is shown in (Fig. 7.2).

Fig. 7.2

NBP and cell interaction in vitro and in vivo are schematically illustrated. The primary causes of cancer cells dying in vitro have been identified as reactive oxygen and nitrogen species in the culture media. Furthermore, it remains a perplexing mystery in plasma medicine how tumor tissues internal growth might be prevented by the administration of NBP above the skin

7.6 Human Skin Anatomy

The human body consists of an external layer, skin that collects approximately, 1/3 of circulating blood. On the basis of its morphological and physiological features categorized for instance protective, homeostatic, or thermo and osmoregulation, etc. The epidermis, dermis, and hypodermis are the three primary integuments that makeup skin as shown in Fig. 7.3 [24, 25].

Fig. 7.3

Human skin anatomy. Structure of skin layer which has three layers, outermost is epidermis, middle layer dermis and inner layer hypodermis also called subcutaneous layer

Epidermis

Depending on the number of layers and cell size, the thickness of the epidermis varies from one person to the next, as well as from one region to the next. The epidermis is comprised of numerous layered and distinguished into sections: (i) non-viability of the epidermis and (ii) viability of the epidermis.

Non-viability of epidermis

Non-viability of the epidermis of human skin also known as the horny layer and increases many times in thickness when hydrated which is roughly 10 mm thick in dry conditions [26, 27]. The non–viable epidermis consists of Corneocytes, keratinized cells organized in numerous lipid bilayer structures. In a structure where corneocytes are represented as bricks and bilipid layers are thought to be mortar used for the perception of drugs work as an amalgamation hurdle with its specific ‘brick and mortar, just as it is like a wall in between [28, 29].

Viability of epidermis

Underneath the SC, the viability of the epidermis is divided into four layers, viz., stratum lucidum, stratum granulosum, stratum spinosum, and stratum germinativum [26]. Keratinocytes that are flat, clear, and dead make up stratum lucidum. Two to four keratinocyte cell layers with a 3 m thickness make up the stratum granulosum. The desmosomes group the stratum spinosum also known as the prickle cell layer, which has a thickness of 50–150 m and is composed of 8–10 layers of polygonal keratinocytes. It is located right above the basal cell layer. The innermost layer of the epidermis, known as the stratum basale is made up entirely of basal cells. It aids in hydration retention, but as it ages, it loses this function. [30].

Dermis

The 3–4 mm thick dermis is a matrix of connective tissues, blood vessels, sweat glands, hair follicles, lymph vessels, and nerves in a matrix, its thickness of 3–4 mm. Collagen and elastic fibers are connective tissues that provide skin strength and flexibility, respectively [30].

Hypodermis/subcutaneous layer

The dermis and epidermis are supported by the larger blood and lymph vessels in the hypodermis, which also function as a zone for storing fat. It contributes to mechanical strength and controls body temperature. Additionally, this layer is made up of blood vessels, skin nerves, and pressure- sensitive organs [28].

7.7 Methods of Enhancing Skin Permeability

Various methods are used to control the SCs barrier-like nature. There are divided into passive/chemical or active/physical approaches [31, 32]. The following is a brief demonstration of the many methodologies and ways based on their principles and mechanisms of action.

Passive/chemical strategies

Penetration enhancers, supersaturated systems, prodrugs, liposomes, and other vesicles are all products of passive methods. Chemical penetration enhancers could serve through one or greater of the subsequent mechanisms [33]: (i) drug operation across the intercellular pathway is increased by interfering with the SCs highly ordered bilipid structure (e.g. terpenes and azones), (ii) Through an intracellular pathway, interacting with protein shape of corneocytes (e.g. pyrrolidones, dimethylformamide, dimethyl sulphoxide) (iii) Improving the distribution of the solute throughout the SC (e.g. propylene glycol, polyethylene glycol) [30].

Active/physical techniques

The active physical techniques mainly describe the thermal ablation technique. Large MW (>500 Da) hydrophilic molecules, such as proteins and peptides have been attained via active methods. However, Active/assisted strategies are under progress for the active transport of larger biomolecules. Many physical and active methods have been demonstrated across the formation of biomolecules.

Thermal ablation

The thermal ablation procedure, also known as thermophoresis increases the drug perception across the skin and involves the depletion or elimination of the SC [34, 35]. Additionally, the thermal exposure rate should be shorter to sustain a significant skin surface temperature compared to the underneath viable epidermis [36]. It would be attained by two methods: (i) For an extended period of time at a moderate temperature (100 °C), and (ii) during a shorter time at a relatively high temperature (100 °C). The following procedures can be used to achieve thermal ablation: (i) chemical heating based on thermal ablation (ii) Miroporation based on thermal ablation

Chemical heating based on thermal ablation

Chemical methods have been employed to promote medication penetration through the skin. The local body temperature rose as a result of chemical substances. They determined the intensity of heat production. The majority of commercial transdermal patches use two initiators, such as oxygen and water, are used [37, 38]. The patch comprises an iron powder based on heat generating chemical together with 70 mg of lidocaine and tetracaine. An alternative method known as Eutectic Mixture Local Anaesthetics (EMLA^®)–based cream, which contained 2.5% of lidocaine and prilocaine. In a double-blind, randomized study with 82 adult human subjects, the EMLA^® cream was applied to one antecubital surface prior to a vascular access procedure and the Synera^® patch was applied to the other [39].

Miroporation based on thermal ablation

Thermoporation, commonly referred to as microporation, is a method for forming aqueous channels across the SC to improve the permeability of active substances passing through the skin into the systemic circulation. In this method, a variety of metallic filaments are briefly kept in contact with the skin surface. The passage of electric current along these filaments causes them to heat up, resulting in localized disintegration and vaporization of the SC. Resultantly, microchannels formed on the skin's surface. Afterward, the use of transdermal formulations such as gels, creams, patches, or vaccinations will increase the permeability of the medications that have been included [40, 41]. Due to the use of sterile and disposable metal filaments, the microporation device offers the advantage of reducing the danger of the transfer of blood-borne pathogens [42].

Thermoporation is a technique that uses controlled thermal radiation to increase drug absorption through the skin. passport and Tixel, two FDA-approved devices, can create a corporation. With their patented patch method, known as PassPort™, Altea Therapeutics Corporation has made an significant advancement in the delivery of drugs and vaccine via the skin (Altea Therapeutics Corp., Atlanta, GA). Aqueous micropores are used for the ablation of the SC by heat. The micropore/microchannels are reported to have a width of 50–200 μm and a depth of 30–50 μm. This technology permits the non-invasive, economical, and regulated delivery of drugs of numerous therapeutic types. This method has the advantage of avoiding the usage of needles, pumps, and costly devices which are employed in other techniques [36, 43]. Furthermore, the patient’s application of the patch can be recorded by this device along with the date and time [44].

7.8 Skin and Its Microenvironment

Skin is made up of numerous layers of defense. It has a built-in defense system. Hair and hair follicles, as well as a sebaceous gland and a sweat gland, are distinctive skin appendages. It is possible to experience external stimuli due to some sensory organs, such as the Pacini corpuscle. The layers of epidermis are made up of keratinocyte cells. The palms of the hands and the soles of the feet have specific types of skin layers called stratum corneum and stratum lucidum. The lamina basalis separates the epidermis from the dermis. This epidermis also contains a variety of other cell types, including Merkel, Langerhans, and melanocytes. The extracellular matrix fibers and a small number of cell types, including fibroblasts and mast cells, make up the vascularized dermis layer that regulates skin moisture levels. The skin's stratum corneum, which has an acidic surface layer, and the accompanying bacteria that live there and in hair follicles. For the transportation of oxygenation by oxygen coming on one side from the atmosphere and on the other side from dermis blood vessels, the skin layer maintains an oxygen gradient [45] (Fig. 7.4).

Fig. 7.4

a Experimental setup of NBP device for chitosan treatment. b Diagrammatic representation of a dielectric barrier discharge (DBD) plasma generator

7.9 Chitosan Biocompatible Material as Skin Rejuvenation

Chitin is converted into chitosan, a natural, new polyheterosaccharide copolymer, using an alkaline deacetylation process. It has been demonstrated that chitosan and its derivatives are efficient sources for boosting musical and transmucosal deliveries [46]. Chitosan's biological characteristics, such as biocompatibility, non-toxicity, and biodegradability, have opened up new possibilities for the treatment of skin conditions and bone regeneration. The improved biomaterial properties of chitosan, such as mucosal adherence and absorption, are mostly due to its surface chemistry. Chitosan's positive charge and ability to adhere to various epithelial surfaces open up new possibilities for medication interactions with mucus layer. Future skin treatments might derive from these features. Due to its stimulation of osteoblast cell proliferation and attachment as well as the production of mineralized bone matrix, chitosan is used as a bone scaffold material and may open up new therapy options for skin in the future [47, 48]. A promising method to improve affectivity toward cells, NBP treatment of 3D chitosan scaffolds have the potential to augment biological effects. The 3D chitosan scaffolds treated with NBP, however, primarily have a sporadic effect [49,50,51,52] (Fig. 7.5).

Fig. 7.5

Chitosan morphology was examined using scanning electron microscopy (SEM) following plasma treatment [53]. Chitosan pore size demonstrates and gives significance enhancement of the cell’s attachment on scaffold

7.10 Skin Treatment by Using Nonthermal Plasma

Reactive species production plays major role inside body by metabolic activity. For maintained of metabolic activity inside cells it is necessary to absorb reactive species after its generation because it causes many side effects while it interrupts in other normal cells function. So, in our body there are some antioxidant enzymes are present to absorb these free radicals like catalase. Therefore, stem cell self-renewal and differentiation are greatly influenced by the balance of intracellular reduction-oxidation (redox) homeostasis [54,55,56]. The most effective method for cell rejuvenation is the introduction of non-thermal biocompatible plasma. Plasma contains reactive species that are crucial for activating certain pathways that are highly beneficial for cells [57, 58]. Plasma produces reactive oxygen and reactive nitrogen species, which could indicate a crucial role for the second a participant in the cell's active antioxidant system and signaling network. These reactive species are important for both treating and rejuvenating the skin. The current understanding of the processes by which NBP regulates cell proliferation and differentiation through redox change is provided in this review [58]. Understanding the role of redox homeostasis in stem cell differentiation control and carefully elaborating the underlying molecular mechanisms would offer important new approaches that NBP stimulated stem cell differentiation for skin treatment [59, 60]. Non-thermal plasma also has certain antibacterial properties that can be used as a therapeutic technique for the management of skin diseases and chronic wounds. This study [61, 62] evaluated the effects of plasma on the healing of a rat model of a full-thickness acute skin wound. Skin wounds from a study were analyzed by histological and gene expression analyses three, seven, and fourteen days after the wounding. The wounds were exposed to three daily plasma treatments for one or two minutes [63]. When compared to untreated wounds, plasma therapy effectively increased epithelization and wound contraction on day 7. In conclusion, plasma treatment improved acute skin wound healing effectiveness while minimizing side effects. Since it is the largest organ in the human body and occupies a unique position, the skin plays a significant role in many different ways. It acts as a bridge between the interior of the organism and the external world [64, 65]. The entire body is protected from external aggressions and significant water loss by its keratinized tegument [66,67,68]. The skin serves as a significant physical barrier against pathogens and the environment, but it also produces vitamin D, regulates temperature, senses humidity and mechanically, absorbs, excretes, and secretes molecules, among other things [69, 70]. Maintaining its integrity is crucial to stop the loss of function [71,72,73]. Ancient civilizations used a variety of techniques to conduct cosmetic skin care. With an increase in life expectancy in the 21st century, people are putting more emphasis on skin care to seem younger [74, 75]. Because of this, there is an increasing need for skin care products on a global scale to meet consumer demand. The global market for cosmetics was worth 508 billion dollars in 2018. By 2025, the market is anticipated to be worth roughly 758 billion US dollars. The variety of modern skin care options includes both physical and chemical treatments. While skin peeling treatments are frequently prescribed by licensed beauticians, creams, serum, and oils are frequently utilized as DIY home remedies for skin maintenance [76, 77]. Some of the equipment is also available and used commercially for skin care. One of them that is frequently used for rejuvenation is lasers and LED lights. By physically removing the outer layers of the skin and triggering skin cell metabolism for a more thorough skin rejuvenation, these light sources interact with skin cells to enhance their ability for renewal. For skin treatment, more intrusive and pricey methods like cosmetic surgery may occasionally be needed. Ionized gases are currently used in revolutionary technology-based physicochemical methods for non-surgical skin treatments [78, 79]. Dermatology has long employed cold atmospheric pressure plasma-mediated skin treatments to promote wound healing. It is demonstrating quicker healing abilities and increased cell renewal capabilities. Today, plasma offers a fresh approach to the beauty industry. Several studies are currently being conducted to apply plasma with various materials, such as microneedles. One method for allowing plasma to easily penetrate the skin is the use of microneedles. As plasma has a variety of reactive properties, these properties play a significant role when skin cells are penetrated [80,81,82]. However, research is ongoing to identify the precise mechanisms of action of cold plasma effects on skin as well as their scientific underpinnings at the cellular and molecular levels. Plasma offers new directions in the field of study and is a well-known skin-treatment technology [83,84,85].

7.11 Plasma Activated Water Play Important Role in Skin Rejuvenation

Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer, accounting for 85% of all cases. In addition to being extremely difficult to treat, there is currently no advanced approach for treating lung cancer. Nonthermal plasma opens up new therapeutic possibilities in medicine. This study's results have shown that oral administration of plasma-treated water (PTW) is effective in treating NSCLC [86, 87]. Oral administration of this mixture to mice demonstrated no toxicities even at the highest dose of PTW, after a single dose and repeated doses for 28 d in mice [88,89,90]. Cold plasma in water produces a variety of reactive species. PTW shown promising anticancer effects on chemo-resistant lung cancer cells, according to in vivo investigations. There are several plasma treated liquids which are also useful for medication purposes. Plasma activated water one of the promising tools for skin treatment purpose. As it contains high number of reactive species and it is easy to store water for longer time [91,92,93]. The PTW anticancer strategy appears to be complex in preventing angiogenesis and proliferation of cancer cells while promoting apoptosis. Oral administration of plasma-activated water may prove to be a promising kind of therapy for skin conditions. As a result, plasma activated liquids may present a unique approach to treating skin conditions and promoting skin cell renewal in the future [94, 95].

7.12 Plasma Skin Regeneration Treatment in the Dermo-Cosmetic Application

There are various skin diseases and the severity of skin diseases can vary from benign, over-disturbing (i.e. minor eczema or ichthyosis), and painful (i.e. infected chronic wounds) to lethal (i.e. malignant melanoma). Mild inflammatory skin diseases are usually treated with topical cremes made up of disinfected substances and steroids. In addition, severe forms of antibiotic treatment are inevitable, whether it is topical application as a component of cream or consistently. In particular, in the case of chronic skin diseases superinfected patients suffer from painful treatments and side effects of medications.

7.12.1 Epithelialized Skin Diseases that Are Highly Contaminated with Germs

7.12.1.1 Atopic Eczema Treatment

Atopic eczema is a highly prevalent form of eczema (3–5% of the population is affected). Rash, inflammation, and dry and itchy skin are some of the signs and symptoms. Patients are normally treated with a moisturizer followed by anti-inflammatory and topical antimicrobial treatments. An anti-inflammatory and topical microbial medication is typically given to patients after a moisturizer. Mertens and his colleagues reported a case study in 2009 about atopic eczema in a patient. In this study, the patient's left arm was exposed to plasma, while the right arm was treated with hydrating cream. They used DBD device with an output of 0.2 W. The plasma treatment time was 1 min per day for 30 days. The energy density per day can be calculated at around 1 J/cm².

After 30 days, a decrease in some symptoms such as swelling and redness on the upper arm, which was uncovered by the plasma, can be observed. In addition, the patient reported a reduction in itching from 8 to 3 points. The point scale ranged from 0 to 10, with 10 being the most severe itch. Eczema improved by two points overall (scale of −5 to 0–5 points, indicating complete cure and severe worsening of eczema). No side effects were observed during and after the study [96]. Moreover, agar plates were pressed onto the skin of the patients and the results showed a reduction of 1 log level in the bacterial load of the plasma-treated skin after two days (Staphylococcus aureus).

Daeschlein and others also reported high inactivation rates for plasma treatment using a plasma jet in another study. The plasma jet was made of argon gas and operated at a frequency of 1.5 MHz and voltage supply of 1–5 kV [97]. A decline in Staphylococcus aureus colonies grown on agar media was measured by 2.7 log levels after plasma treatment for 2 min (RF 2.7). The efficiency of plasma inactivation varied among the five different species tested in this study, indicating that plasma doses are appropriate for the efficient eradication of wound harmful microbes. While the lowest reduction factor, 1.9 log steps, was found for Enterococcus faecium, this still represents a pronounced inactivation of bacteria, which seems to be suitable for inactivating nearly all types of realistic microbial contamination, colonization, and also infection.in vivo. Pseudomonas aeruginosa, extended-spectrum beta-lactamases (ESBLs) multidrug resistance pathogens, E.coli, Staphylococcus aureus, Staphylococcus epidermidis, hygienic disinfection, preoperative skin antisepsis, and highly gentamicin-resistant enterococci (HLGR), which offer new perspectives on skin disinfection and wound decontamination, were also shown to be significantly reduced by plasma in vitro [98]. In addition, plasma therapy may be effective in curing dermatological conditions involving parasites, such as dermatitis, as a strong killing effect against Demodex folliculorum has been observed [99].

7.12.2 Wounded Epidermis and Germ-Contaminated Skin Diseases Treatment

7.12.2.1 Chronic Wounds with Plasma Treatment

Many people have wound infections on their feet and legs, which are caused by aortic illnesses (15%), hyperglycemia (5%), and other arteriolar disorders. Venous ulcers affect the elderly in particular, and their treatment consumes a significant percentage of the healthcare budget necessitating the development of cost-effective alternative ulcer remedies [100, 101]. Plasma has the potential to be such an alternative, not only because it can be produced at a low cost, but also because it is easy to handle. In addition, plasma treatment combines several mechanisms of action that are beneficial to the wound healing process including (i) the potent antibacterial impact might reduce the number of bacteria present in the injured areas, preventing the recovery process from being slowed down by invasive colonization [102]; (ii) plasma is known to stimulate the proliferation of endothelial cells [103]; and (iii) plasma treatment leads to a decrease in pH, which would also support the healing process since the hyperacidity of wounds is also a natural response of the body [104].

In vivo tests, Isbary and colleagues investigated the effects of argon plasma on infected wounds [105]. In 2010, they published the results of the first clinical trial using the indirect plasma technology MicroPlaSter. Additionally, for routine wound care, 36 patients with 38 ulcers (mainly venous, traumatic, arterial, and diabetic causes) were treated with plasma for 5 min daily.

Patients acted as their controls in the control areas, which were merely treated with normal wound care and were around 3 cm2 in diameter. There were 291 treatments carried out, with the findings revealing a 34% reduction in bacterial load in the wounds (p < 10–6) [106]. A year later, another study by Isbary and co-workers found that plasma applications could help people with the genetic disorder Hailey-Hailey disease [107]. Severe outbreaks of this disease often cause blisters and rashes that often lead to chronic, infected sores when they burst. One patient with Hailey-Hailey was treated using MicroPlaSter, a newer version of the MicroPlaSter device that is more convenient to use due to its smaller size and flexible treatment arm with four joints. The torch was held at a distance of 2 cm from the target region for 5 min. As a result, the plasma treatments significantly improved the healing process in both the patient's right axilla and groin. After plasma treatment a sustained positive effect was also observed; the patient remained symptom-free for several months. In the recent clinical trial on chronic wounds conducted by the same research group, two MicroPlaster devices were evaluated using two minutes of treatment time with the same experimental conditions (except for the period of the therapy session) [108].

A considerable decrease in microbial contamination was observed in the patients who received plasma treatments for the injuries. A large decrease in microbial infections of 40% (p < 0.016) was noted in the wounds and a decrease of 23.5% (p < 0.008) with the use of MicroPlaSter β. Overall, no side effects occurred in these studies by Isbary, and the applications were also well tolerated by patients. MicroPlaSter α, a major reduction in bacterial load of 40% (p < 0.016) and a reduction of 23.5% was observed in wounds. It is safe to suppose that in all of this research on chronic wounds, the plasma and tissues of deeper skin layers, such as keratinocytes in the proliferative basal layer or fibroblasts in the dermis, were given access to wound pathogens. Although not entirely appreciated, the direct microbiological actions by the plasma over eukaryotic cells are of tremendous interest. To utterly make sure that no long-term damage or side effects will occur, possible genotoxic effects of plasma treatment must be investigated genetically.

To this end, a risk analysis is going to be performed for each plasma device (kinpen MED and our DBD device), including the investigation of cell damage at the DNA and cell membrane levels. For this purpose, common genotoxicity and cytotoxicity tests such as the Ames test and various host cell reactivation tests are performed. The Ames test takes a look at may be used to decide the mutagenic capacity of chemical substances (in our case this will be plasma). Different strains of Salmonella typhimurium with a mutation in histidine biosynthesis are used for the test so that the auxotrophic mutants require the addition of histidine for their growth. Treatment with the mutagenic substance can generate revertants, which can grow on a histidine-free medium.

This method has been successfully used to detect the mutagenicity of various substances metabolized by the cytochrome P450 enzyme system [109]. To replace animal testing two plasmid DNA vector assay systems are planned as methods and the infected tissues renewal tests use gene sequences to quantitatively analyze the recovery of DNA rate of cells [107, 110]. Plasma is used to treat a non-replicating reporter gene plasmid that codes for an enzyme before it is transfected into host cells. The expression level of the reporter gene would be reflected in the plasma treatment causing DNA lesions that are repaired by the host cells.

Consequently, the enzymatic expression would be an indirect indicator of plasma's mutagenic potential. The Plasmid-Shuttle-Vector-Mutagenesis-Assay is another test system that has been successfully used to detect age-related DNA repair capacity in various cells. [111]. This assay is based on a plasmid that has E. coli microbial repressor tRNA genes (supF genes) which serve as a mutation indicator [112]. Plasma will be used to treat the plasmid DNA (pSP189), which will then be transfected into host cells, isolated after a few days, and transformed into bacteria. Light blue or white colonies indicate a mutation in the supF gene, and the number of these colonies indicates the mutation frequency.

The mutant plasmids may also be used for mutation spectra and sequence analysis. Finally, these assays are useful for the characterization of the two different plasma sources as well as the standardization of experimental parameters and criteria for medical applications.

7.12.3 The Effect of Plasma on the Skin Surface

7.12.3.1 CAP Treatment Restores the Physiological pH Barrier

RONS generated by CAP also induced acidification in the target as well as oxidizing and stimulating effects. It is common to observe decreasing the initial pH in moist three-dimensional structures and semi or poorly buffered fluids [113]. It might be explained by the existence of acidic substances in liquids that develop from the progenitor NO•, which results in the production of nitrous (HNO₂) and nitric (HNO₃) acids [114]. The plasma exposure time is proportional to its acidification. The rapid pH decline and seems stable the pH values around 3.5 and 2.5 as a result of the temporary development of the HONO/ONO buffer and the production of nitrous acid [113,114,115]. Human triglycerides and porcine epidermal sebaceous may both become much more acidic, after exposure to CAP.

The medical experiments using healthy human epidermis confirmed CAP-induced acidification. [116, 117]. Due to its acidifying characteristics, CAP therapy may help in protecting healthy skin. Cold plasma may enhance and improve skin rejuvenation by reducing the pH. Physiological acidification has been demonstrated to boost potency as well as enhance fibroblast growth in chronic wound infections [118].

Physiological values can cause pathologies when skin pH is greater, and strong acidic pH might damage the outer tissues. Skin contact with CAP must be strictly regulated to prevent chemical burns [117]. Chemical peels, also known as chemexfoliation, are used in cosmetics to gently exfoliate the epidermis's outer layer and force skin renewal. To decrease pH and remove the outer layers of the epidermis, organic acids are frequently utilized in this technique. The successful plasma application might provide a comparable non-invasive peeling effect. Furthermore, CAP therapies may also be able to restore the physiological pH barrier and promote the aged skin because the rise of alkaline pH with age weakens the threshold [119].

7.12.3.2 The Plasma Effect Improves Skin Hydration and Acidification

The proper amount of water is required for healthy and functional skin. Strong water-absorbing GAGs like hyaluronic acid keep the dermis hydrated. The epidermal has a relative humidity that ranges from 15 to 30% in the outer skin to 70% in the vital portion. The inner layer can detect humidity in the environment and adjust the metabolic processes [120]. The highly porous chemicals that make up Organic Hydration components and keratinocytes, the dying cells that make up skin barriers called corneocytes retain moisture [121, 122]. Strong adhesion between corneocytes prevents significant moisture loss. In addition, ceramides and other intercellular lipids further provide hydrophilic barriers that prevent dehydration [123]. Skin moisture may be influenced in two different ways by cold plasma therapies. In the beginning, CAP might weaken the layer of the skin and stop flowing the epidermal outer layer. A brief, temporary moisture depletion was noticed inside the human skin surface following plasma treatment. [124]. The goal of plasma skin rejuvenation is to preserve the thermal wounded tissues during the healing process with the non-ablated, dry skin [124]. The skin may attract more water molecules following plasma therapy because CAP can release ions on the surface layer. Within the first few seconds of plasma therapy, the human epithelial tissue becomes much more wettable [124]. The plasma therapy enhances the adherence of nail polish for aesthetic purposes, and fingernail hydrophilicity has also been found to improve [124].