Figure 2Stratum corneum structure and organization: A) “Bricks and mortar” Two- model (adapted from (Prausnitz et al., 2012)); B) Stratum corneumstructure: cornified envelope (blue); corneocyte lipid envelope (CLE, black); corneodesmosome (brown). C)Lamellar membrane structure with intercellular lipids. Insert, electron micrograph: Murine skin was fixed in Karnovsky’s fixative, and post-fixed with 1 % aqueous osmium tetroxide, containing 1.5 % potassium ferrocyanide (adapted from Uchida (Uchida and Park, 2016)).
The interior of the corneocytes is filled with keratin filaments embedded in a matrix mainly consisted of filaggrin (filament aggregating histidine-rich protein) and its breakdown products. These amino acids together with certain ions, such as chloride, sodium, lactate and urate form the natural moisturizing factors (Ovaere et al., 2009). These substances are endogenous humectants that keep the hydration of the SC on an adequate level. It is important for skin elasticity and permits regularly function for hydrolytic enzymes of desquamation (Fowler, 2012). Reduced levels of filaggrin and properly natural moisturizing factors are caused by mutations of the filaggrin gene connected with atopic dermatitis, ichthyosis vulgaris, psoriatic skin, ichthyosis and general xerosis (dry skin) (Uchida and Park, 2016, Fowler, 2012). The cornified envelope (CE) that encloses the Corneocytes is formed by structural proteins crosslinked by transglutaminases (Prausnitz et al., 2012). CE has a stable stiff property to withstand mechanical barrier stress (Figure 2B) and is surrounded by a layer of corneocyte lipid envelope (CLE) (Uchida and Park, 2016). This CLE is a support to form lamellar membrane structures and controls exit of hydrophilic agents from corneocytes, but the roles of CLE in the SC remain totally understood (Uchida and Park, 2016).
The extracellular space in the SC is filled by lipid-enriched layers (Uchida and Park, 2016) which are composed by ceramides (45–50%), cholesterol (25%), cholesterol sulphate (5%) and free fatty acids (FFAs) (10–15%) (Ng and Lau, 2015, Uchida and Park, 2016, Madison, 2003). The lipids form lamellar membranes that stretch out in a horizontal direction parallel to the corneocytes (Figure 2C). In addition, corneodesmosomes are found in the lamellar membrane structure. They are modified desmosomes including corneodesmosin, a structural protein that determines corneocyte adhesion (Ovaere et al., 2009). Corneodesmosomes consist of desmoglein-1 and desmocollin-1 connected to other corneocytes, intensify cohesion and regulate desquamation by their pH-dependent degradation (Uchida and Park, 2016).
Ceramides are mainly responsible for the skin renewal process (Mizutani et al., 2009) and play a critical role for the lamellar organization of this layer barrier (Prausnitz et al., 2012). At least 14 different classes of ceramides have been isolated from the human epidermis (Meckfessel and Brandt, 2014, Jurek et al., 2019, Sofia A. Costa Lima., 2018, Mojumdar et al., 2016). Each ceramide molecule contains a polar head group from sphingoid nature (sphingosine, phytosphingosine, 6-hydroxysphingosine or dihydrosphingosine) and two hydrocarbon chains derived from a fatty acid, a sphingoid or fatty acid ester moiety.
Cholesterol is the second most prevalent lipid by weight in theSC (Zbytovská et al., 2008, Pouillot et al., 2008, Prausnitz et al., 2012). The increment of cholesterol concentration in the lipid bilayers correlates with the diminution of the membrane thickness and density, and at the same time with an extension of the membrane surface area. Cholesterol fluidizes the SC lipid bilayers at skin temperature (Ng and Lau, 2015, Zbytovská et al., 2008).
The content of FFAs is 10 – 15% of the SC lipids and they consist mainly of very-long-chain species with ≥18 carbon atom and are mostly saturated, like ceramides.
In addition to lipid species, cholesterol sulphate is typically at 2–5 % weight ratio. Its potential functions are to help in the formation of the lipid lamellae and stabilization of the SC by inhibiting enzymatic degradation of corneodesmosomes (Prausnitz et al., 2012, Ng and Lau, 2015).
The majority of lipids is synthetized by keratinocytes in stratum granulosum and packed in lamellar bodies. These organelles deliver their lipid content in the extracellular space by fusion with the plasma membrane of keratinocytes of the stratum granulosum (Pouillot et al., 2008). Any changes in the concentrations of these main lipid components can damage barrier integrity that mediates normal barrier function.
These lipid structures prevent the excessive loss of water from the organism and block the entry of most topically administered drugs that have high molecular weight and low lipophilicity. Thereby, this barrier represents a great challenge for drug delivery across the skin, intended either for local effects or for systemic therapy (Groen et al., 2008).
Concerning the functions of SC , it is established that this layer is responsible for the barrier and immune functions, namely preventing excessive water loss, maintaining body temperature, preventing the entry of xenotoxic chemicals and allergens as well as the invasion of microbes. Its functions also comprise the protection of the epidermis from oxidative stress and from mechanical stress.
The epidermal permeability barrier prevents intake and uptake of compounds. The SC barrier functions may be partially connected to its low hydration of 15%–20% and its very high density (1.4 g/cm3 in the dry state) (Walters and Roberts, 2002). The body temperatures maintenance and water balance are regulated by blocking surplus evaporation of water from nucleated layers. One of the basic properties of this barrier is the prevention of penetration of allergens, microbial pathogens and xenotoxic chemicals (Uchida and Park, 2016). As a general rule, a compound with molecular weight larger than 500 Dalton cannot pass through the SC (Bos and Meinardi, 2000).
The epidermis has a pH gradient: the extracellular pH stays neutral between the stratum granulosum and SC , where it becomes more acidic to ca. 4.5 at the outer SC layer. Acidification enables antimicrobial activity and regulates, by enzymatic activity performed by proteases, the formation and desquamation of the epidermal barrier. The use of alkaline or neutral soaps lead to the increase of the SC pH, which conducts to untimely cleavage of corneodesmosomes, decline in SC cohesion and following impairment of the epidermal barrier. The pH changes are reported to matter in the pathogenesis of skin diseases such as atopic dermatitis, acne vulgaris and Candida albicans infections (Ovaere et al., 2009). For example, with pH below 5.5, the growth of Pseudomonasacne , Staphylococcus epidermidis and a problematic microbial pathogen, Staphylococcus aureus , are inhibited (Uchida and Park, 2016). The permanent bacterial flora in the skin presents a comprehensive ecosystem. Staphylococcus and Micrococcusstrains and Diphtheroid bacilli are the main part of nonpathogenic microflora. They consume the sebum like basic nutrient and confine skin colonization by potentially pathogenic organisms (Pouillot et al., 2008).
SC plays an important role in the innate immunity, which is related with the presence of antimicrobial peptides such as cathelicidin, dermcidin, RNase7, S100A7/psoriasin and defensins (Uchida and Park, 2016). They have revealed potent antimicrobial activities against a wide spectrum of microbes, including gram-negative and gram-positive bacteria, fungi and some viruses. Cathelicidin antimicrobial peptide (CAMP) is inducible with infection, injury or inflammatory response and stimulates the production of a signal lipid, sphingosine-1-phosphate (S1P) under stress conditions and activate vitamin D receptor (VDR). Also, defensins are classified in three subfamilies, α-, β-, and θ-defensin. They are inducible peptides in epidermis in response to microbial infection, inflammation. and differentiation (Uchida and Park, 2016). Adaptive immunity in theSC is associated with the availability of urocanic acid. Thetrans isomer of urocanic acid is generated from histidine (principally from NMF) by histidase but can be converted to thecis isomer through exposure to ultraviolet (UV) radiation.Cis -urocanic acid binds to the serotonin [5-hydroxytryptamine (5-HT)] receptor to eliminate immune function (Egawa et al., 2010). Urocanic acid is an epidermal major chromophore, which works as a powerful endogenic UV absorbent. Most lower UV wavelengths (UVB = 280–315 nm) are absorbed in the epidermis, but longer wavelengths (UVA = 315–340 nm) get to the dermis. The bulk amounts of proteins, lipids, and nucleotides has individually low potent chromophores, but they can form the UV barrier together (Uchida and Park, 2016). The SC is constantly exposed to oxidants, including UV light, chemical oxidants and air pollutants. α- and γ-Tocopherol, ascorbic acid and glutathione are the chief hydrophobic anti­oxidants of SC , providing the lipid bilayers stability and safeguarding from lipid peroxidation (Pouillot et al., 2008).
Dermis
The dermis is the bigger layer of the skin, with a thickness of approximately 1–2 mm and provides important physical properties, namely flexibility, elasticity and tensile strength. It is an integrated system of fibrous and connective tissue, composed by collagenous and elastin fibbers, accommodating epidermally derived appendages (hair follicles, nails, sebaceous glands and sweat glands) and sensory nerve endings, lymphatic vessels and blood capillaries. Thus, metabolic exchanges between the epidermis and the blood systems may occur as well as clearance of cell metabolic products and penetrated foreign agents. The dermis contains resident cells (e.g., fibroblasts and mast cells) and cells from the immune system, including macrophages and dermal dendritic cells.
The thermal barrier, energy depositary and protection from physical stroke are mainly connected with adipose tissue associated with collagen fibbers, found in the lower reticular dermis layer. The water content reaches 70%, favouring hydrophilic drug uptake. Below the reticular dermis layer, the fibrous connective tissue transitions to the adipose tissue of the hypodermis. This is mainly constituted of adipocytes interconnected by collagen fibbers, forming a thermal barrier able to store energy and protect from physical shock (Sofia A. Costa Lima., 2018, Forster et al., 2009).
Hypodermis
The subcutaneous layer or hypodermis is the innermost layer of the skin and consists of fat cells. However, this layer can be absent in some thin skin, for example on the eyelid. The hypodermis is between the skin and the subjacent tissues of the body, such as muscles and bones. Larger lymphatic and blood vessels are standing in this layer. Consequently, the major functions of the hypodermis are insulation, mechanical integrity and support and conductance of the vascular and neural signals of the skin (Alkilani et al., 2015, Ng and Lau, 2015).
Drug skin penetration routes
The skin is an attractive site for delivery of drugs and cosmetics. But normal skin is a serious barrier to drug absorption, which is why pharmacologists and cosmetologists became interested in the development of new drugs, formulations and ways of delivery.
Drugs can be administrated through the skin providing a local action (topical administration) or a systemic effect, reaching the bloodstream. The types of administration of drugs through the skin can be organized in many ways, depending of the criteria selected. A possible classification can be performed considering the invasive and non-invasive nature of the different routes. Regarding invasive routes of administration, they can be categorized as the routes in which the drugs can enter through the skin by needle injections (subcutaneous, intramuscular or intravenous routes) and those that consist in the implantation of a device. In the subcutaneous route, the needle is inserted into fatty tissue thus reaching the bloodstream. This type of administration is usually used for the administration of many protein drugs (eg. insulin) as they are destroyed in the digestive tract. Intramuscular route is considered preferably over subcutaneous route for the administration of larger volumes of drugs. In the intravenous route, the drug is administrated directly into a vein and is used to give a drug in a rapid and in a well-controlled manner the drug is delivered immediately to the bloodstream. Drugs can also be administrated by implantation of a device which is inserted under the skin. This type of administration is probably the less commonly used and is usually considered for the delivery of the drugs by controlled release along the time for longer periods .
Considering the non-invasive methods, there are four possible routes of drug penetration across the skin: intercellular, intrafollicular, transcellular and polar (Pouillot et al., 2008), as depicted in Figure 3A. Sometimes, the diffusion through the skin appendages (e.g., fair follicles, sebaceous glands and sweat glands) is classified as appendageal route (Prausnitz et al., 2004, Ng and Lau, 2015). Intercellular and transcellular ways are considered transepidermal pathways.