biology of the skin study

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Skin is the largest organ of the human body. It is a continuous membrane or sheet covering the entire body surface which composed of two main layers; the epidermis and the dermis. The thickness of the two layers varies over different regions of the body in which the epidermis is thickest on the palms and soles of the feet whereas the dermis is thickest on the back and thinnest on the palms. The epidermis is the uppermost layer that generates the stratum corneum which provides the skin's protective barrier. This membrane prevents extraneous materials getting into the body as well as controls and prevents the loss of materials from within the skin. The dermis is the thick, fibrous layer beneath the epidermis that contains blood vessels, hair follicles and glands that produce sweat, which helps regulate body temperature and sebum, an oily substance that helps keep the skin from drying out. It also contains several connective tissue proteins which are collagens, elastin and proteoglycans. Collagen covers the body, elastin provides elasticity and strength while proteoglycans are involved in damage repair. Figure 2.1 shows the cross section of epidermis and dermis.

The epidermis is composed of four distinct layers which are the stratum corneum (horny layer), stratum granulosum (granular layer), stratum spinosum (spiny layer) and stratum basale (basal layer). It is in a continual state of renewal as the keratinocytes, the major cell type within the epidermis are formed, mature and die. Keratinocytes which are formed by cell division from the stem cells in the basal layer of the epidermis move upward through the epidermis, continually maturing and changing structure via a process called differentiation. As the newly formed keratinocytes move upward, they flatten, lose their nucleus and die and then begin to produce increasing amount of keratins so that the cells of stratum corneum are completely filled with keratins. This process of cell maturation and increased keratin production is known as keratinization in which the stratum corneum is made up from from dead cells called corneocytes. The highly keratinized cells of the stratum corneum are packed tightly together and are surrounded by a multilayered lipid structure which is often compared to bricks and mortar. Structural strength is provided through the keratin-filled bricks and waterproofing through the layer of lipid mortar (Rhein and Babajanyan, 2006).

Figure 2.1: Cross section of epidermis and dermis. (Rhein and Babajanyan, 2006)

The stratum corneum is composed of lipids and proteins. The upper layer lipids contain predominantly cholesterols, ceramides and free fatty acids but do not contain phospholipids, which are typically found in other biological membranes. On the skin surface, the sebaceous lipids or sebum originating from the sebaceous glands will become an important entity, along with the interactions and mixing with the sebaceous lipids with the epidermis lipids (Rhein and Babajanyan, 2006). The lipids are organized in a multilayered structure and are attached hydrophobically to the corneocyte envelope on the surface of the corneocyte. The presence of these lipids along with the coating of sebum resulted in the expose surface of skin or hair having a hydrophobic nature. At pH levels of 5 to 8, the skin surface contains negatively charged hydrophilic sites. This combination of hydrophobicity and hydrophilicity affects the nature of soils attracted and the cleansing efficiency (Rhein, 2007).

The cells of the horny layer are constantly worn away and replaced by newly keratinized cells, in a continual cycle. The entire stratum corneum is shed in approximately 14 days so that means a new cell takes about 28 days to differentiate and reach the top of the stratum corneum. The cycle or rate balance of these cell renewals is important in order to maintain a healthy skin barrier. Many factors could contribute to the disturbance of the cycle and balance including exposure to substances such as cosmetics, low humidity, cold weather and hormones or drugs. Variety of skin diseases including psoriasis, atopic eczema and acne involves the disturbance of the normal sequence. Therefore, it is actually important to maintain a consistent skin care regimen in order to ensure the continuing health and vitality of this important organ.

2.1.1 Skin penetration pathways

Dermatological and cosmetic preparations contain active principles which can only act when they penetrate at least the outermost layer of the skin. However, the efficacy of topically applied actives is often suboptimal because the transport into the skin is slow due to the resistance of the outermost layer of the skin, the stratum corneum. In all cases, the actives penetrate into the skin by using two dffusional routes which are appendageal route and transepidermal route. The appendageal route comprises transport via the sweat glands and the hair follicles with their associated sebaceous glands. These appendages can act as low resistance, shunt pathways and general alternative penetration routes for the penetration of actives. It is true for ionic or non-ionic hydrophilic permeants that do not readily penetrate through bulk stratum corneum. However, they were believed to play only a minor role in transport processes since they represented such a minor part (< 0.1%) of the total skin area (Michniak-Kohn, 2005).

Transepidermal route which means through the stratum corneum exists in two different pathways which are the intercellular pathway or the intracellular (transcellular) pathway. Intercellular refers to permeation through the intercellular lipid matrices while trancellular refers to permeation through both the keratin-filled corneocytes and the intercellular lipid matrices. Traditionally, it was known that water-soluble molecules permeate through the intracellular keratin filaments while lipid soluble molecules diffuse through the lipid matrix region between the filaments. However, based on more recent data, it is deduced that the intercellular pathway is the major permeation route for most molecules, hydrophilic and lipophilic (Michniak-Kohn, 2005). For enhancement of penetration, several techniques can be used including chemical enhancers, physical enhancers, the control of skin's water hydration, supersaturation of the actives as well as the formulation approaches. It is important to understand the potential routes for actives penetration because the changing of actives molecule, modifying the penetration enhancers and choosing a delivery system can produce an optimal penetration of actives through a desired penetration pathway.

Skin cleansing

Personal cleansing is important to achieve cleanliness and freshness by removing oily soils from the face and the body. For this purpose, surfactants make up the highest amount of the ingredients in most personal cleansing products thus gives the product the ability to remove dirt from human skin surfaces. This ability is based on surfactants molecules that consist of hydrophilic groups that are attracted to water molecules and hydrophobic groups that are attached to oils and grease in dirt. When incorporated into diluted aqueous solution, together with mechanical action while applying the cleanser, the hydrophobic part dissolves lipids and keeps the soils suspended until they are rinsed away. Repulsive forces between the negatively charged skin surface and anionic surfactants too can help keep suspended soils from redepositing on skin (Ertel, 2006).

The interaction of surfactants with skin is important in order to enhance the cleansing process, the compatibility with the skin and avoiding the possibility of skin irritation. Harsh surfactants and exposure of the stratum corneum to a relatively high amount of surfactants (5-20%) tend to damage the protein and lipid structure in the stratum corneum, leading to irritation and dryness. The extent of damage will depend upon the nature of the surfactant and the cleansing conditions such as water temperature and hardness as well as the pH. Stratum corneum's swelling is significantly higher at higher pH compared to low pH values (Subramanyan and Ananthapadmanabhan, 2007). As a result from the swelling, surfactants and other harsh ingredients can penetrate deeper into the skin possibly leading to irritation and itchiness. Approaches to minimize this cleansing damage include the use of mild surfactants or lower levels of surfactants as well as the addition of emollients in the cleanser. With the growing interest of achieving skin functional benefits, especially moisturisation from wash-off systems, surfactant damage to skin need to be minimized in order to deposit and deliver the beneficial ingredients effectively. Alternative solution to avoid possible surfactant interaction with the skin or the ingredients is to encapsulate or incorporate the active ingredients in a specific appropriate delivery system.

2.2 Kacip Fatimah/ Labisia Pumila

Kacip Fatimah is a small woody and leafy plant that grows and can be found widely in the shade of forest floors. The leaves are about 20 meters long and they are traditionally used as a kind of tea for women as an herbal medicine to induce and facilitate childbirth as well as a post-partum medicine. The plants are usually boiled and the water soluble extracts are taken as drinks. Other traditional uses of the plants include treatments of dysentery, rheumatism, gonorrhea and to drive away the formation of gas. It is also known to avoid painful or difficult menstruation and to help tone and firm the abdominal muscles. All these properties are due to the presence of phytoestrogens that is naturally found in the plant.

Recent research has found out that Kacip Fatimah has the potential features to be developed as an anti-aging ingredient in cosmetics. Choi et al (2009) has reported that Labisia Pumila's extract has the potential protection of skin cells from photoaging by UV radiation. Skin aging analysis is based on the principle of reactive oxygen species (ROS) generation that is known to be one of the primary causes of aging. It is proved that Labisia Pumila's extract has a strong protective effect against cell damages and prevents increasing level of ROS generation in skin. This is supported with works by Norhaiza et al (2009) that have reported the antioxidative properties of Kacip Fatimah's plant which is believed to play a role in protection against a variety of diseases and delaying ageing process. Analysis from this work proved the high antioxidant activity in Kacip Fatimah due to β-carotene and flavonoids that might scavenge the reactive oxygen species (ROS) generated by UV irradiation leading to protective efficacy against UV. Since these properties leads to the potential of Kacip Fatimah use as a novel anti-aging ingredient in cosmetics, further studies are needed for incorporation into formulations such as encapsulation or designing an appropriate delivery system for transporting Kacip Fatimah to the deeper layers of the skin.

2.3 Delivery system in cosmetics

Most cosmetic products formulation has a substance or substances that provide the beneficial properties to the skin and hair which are called 'active' ingredients. These active ingredients such as anti-aging, anti-acne and moisturisation normally have a specific delivery system in order to be specifically delivered at targeted location. Therefore, a delivery system is defined as the way to carry or transport the actives to a substrate. It acts as carriers where the active ingredients are either absorbed, adsorbed, volatilized or remains on the surface of the skin. Choice of delivery system is important as it may influence the active's behavior upon application. Important properties that may influence delivery of the active include the percentage of active entrapment, particle size, shape, charge (zeta potential) and elasticity. Encapsulation with delivery systems can potentially protect active molecules from degradation by direct exposure to severe environments. In other words, encapsulation can reduce the loss of activity of the active compounds. An encapsulant, or shell, frequently plays an important role as a carrier for delivery of the molecules to the target organs. In addition, the shell performs a release mechanism to control the diffusion level of active molecules under specific conditions.

Various cosmetic delivery systems are currently used and reported as well as new developments on delivery systems currently being studied. This includes the traditional oil-in-water (o/w) and water-in-oil (w/o) emulsions that are commonly used due to the fact that cosmetic products normally contain both water-soluble products and oil components. Other than that, liposomes have shown a great potential as a delivery system for a variety of drug substances. In skincare products, it has been formulated in different kinds of cosmetics range such as lotions and creams but is not stable in the presence of surfactants. Other delivery systems include micro or nanocapsules; a core material surrounded by a coating layer and particulate system which are composed of various types of particles in the size range of submicrometers (nanoparticles), micrometers (microspheres) and even millimeters (beads) which could be formed from variety of polymers (Magdasi, 1996).

2.4 Chitosan

Chitosan which have been used in pharmaceutical, cosmetics, food, and other industries is derived from chitin by alkaline deacetylation, and it is the second most abundant biopolymer after cellulose. Chitin can be obtained from exoskeletons or hard shells of crustaceans such as crabs, shrimps, prawns, lobsters and cell walls of some fungi such as aspergillus and mucor. Chitosan has one primary amino and two free hydroxyl groups for each C6 building unit. Due to the easy available of free amino groups in chitosan, it carries a positive charge and thus in turn reacts with many negatively charged surfaces/polymers also undergoes chelation with metal ions. However, chitosan is a weak base and is insoluble in water or organic solvents but it is soluble in dilute aqueous acidic solution (pH <6.5) which can convert the glucosamine units into a soluble form R-NH3+. It gets precipitated in alkaline solution or with polyanions and forms gel at lower pH. Therefore, several factors should be considered when designing a formulation. Figure 2.2 shows the structures of cellulose, chitin and chitosan and the deacetylation process of chitin is showed in Figure 2.3.

Chitosan is a natural, bioadhesive, biocompatible, and biodegradable polymer that has been used as the vesicle to encapsulate drugs or active ingredients. It is unique in that it possesses both nonionic hydrophobic functionality as well as a hydrophilic cationic charge. Chitosan, through its cationic glucosamine groups, interacts with keratin, an anionic protein found in the skin. This interaction confers bioadhesive characteristics to systems that employ it. In addition, when not deacetylated, the acetamino groups of chitosan are an interesting target for hydrophobic interaction with the skin lipids (Cattaneo, 2005). Biological activity of chitosan is attributable to its polycationic nature. When they are protonated, the amine groups of chitosan interact with the electronegative sites at the surface of the substrate, in this case; the skin.

The physical and chemical properties of chitosan depend upon the source of the shells and the various treatments applied. One of the application is the degree of deacetylation (DD) which corresponds to the quantity of amine groups released so that the higher it is, the more effective chitosan may be. It varies according to the duration, the temperature and the concentration of the alkaline used in the process. The value gives the proportion of monomeric units of which the acetylic groups that has been removed, indicating the proportion of free amino groups that could be reactive after dissolution in weak acid. It could vary from 70% to 95% depending on the method. It is important to know this parameter to indicate the cationic charge of the molecule after dissolution in a weak acid.

Figure 2.2: Structures of cellulose, chitin and chitosan (Kumar, 2000)

Molecular weight of the native chitin is reported to be as high as many millions Daltons. However, enzymatic or chemical methods can bring down and produce a lower molecular weight of chitosan. The chain length of chitosan polymer can influence the behaviour or the changes in its biological activity. When the chain becomes shorter, it could be dissolved directly in water without the need of an acid but when it's too short, the local charge density is low and prevents the polymer from desirably interacting with the negative sites of the skin surface. Chitosan with high molecular weight or large size too show low biological activity. The change of molecular configuration of the chitosan from a simple, random coil to a more compact almost globular structure assumed to be the reason for the effect. The electrostatic interaction between the chitosan and the surface cells could be limited due to the fact that the majority of active ingredients would be confined to the interior of the molecular structure and would be sterically inaccessible.

Figure 2.3: Deacetylation process of chitin (Kumar, 2000)

Chitosan's biological activity can also be influenced by its purity, viscosity, solubility and also pH. Purity is quantified as the remaining ashes, proteins, insolubles, and also bio-burden (microbes, yeasts and moulds). This is a factor because the remains tend to block active sites, the amine grouping. Chitosan's viscosity value which is depending on its molecular weight is suitable for characterizing a chitosan solution useable in a formulation. The higher the molecular weight, the more viscous is the chitosan solution. Solubility of a chitosan solution is more active than in powder form. It may be improved by ultrasonication and/or by freeze-drying after centrifugation. Ultrasonication produces rearrangement of the chitosan's structure to make it more soluble and increasing its biological activity. In addition, the chitosan must preferably be in its polycationic form which is at a pH below its pKa which is 6.2 in order to be biologically active. For a high positive charge density, the chitosan solution preferably must be in a pH value range from 5.0 to 5.5 (Leuba et al, 1991).

Surface charge of chitosan

The surface charge or bioadhesive properties of chitosan are influenced by

several factors including the origin and nature of chitosan, physical structure, pH,

chemical nature, concentrations, solution conditions but most important factor to consider of all is degree of deacetylation (DD). Chitosan have different degree of deacetylation depending on the chitin source and the methods of hydrolysis. Increasing the DD will increase the relative proportion of amine groups, which were able to be protonated, favouring the adsorption of chitosan on anionic substrates. When diluted in aqueous acids (pH ~ 5.5), chitosan produces protonated amine groups along the chain and this facilitates the electrostatic interaction between polymer chains and the negatively charged skin. DD value is important since it indicates the cationic charge of chitosan after dissolution in a weak acid.

Works by Guo et al (2003) has reported the zeta potential measurements of chitosan coated liposomes which showed that all values were positive (9.1 to 43.9mV). Since the chitosan carried a high positive charge, the adsoption of chitosan increased the density of positive charge and made the zeta potential positive. Higher positive value is observed with the interaction of chitosan with negatively charged liposomes. Similar works have been done by Phetdee et al (2009) which has observed that the zeta potential of chitosan coated liposomes was positive (6-29mV). Increased concentration or amounts of chitosan resulted in a significantly higher value of vesicle charge. At a concentration of 0.1% chitosan, the amount of chitosan is just in a magnitude to result in a weak positive charge (+8mV) but when amounts are increased further, stronger positive charge is observed in the order of +23mV to +25mV, which is the normal positivity of chitosan. These works demonstrated that a lot of different factors contributing to the surface charge of chitosan as both chitosan used in these works are with the similar DD value of more than 90%.

2.4.2 Chitosan nanoparticles

The ability of controlling the degree of deacetylation and the molecular weight of chitosan gives the benefits to develop micro/nanoparticles. Chitosan has many advantages in becoming the polymeric carriers in nanoparticles including the ability to control the release of active agents, avoids the use of hazardous organic solvents since it is soluble in aqueous acidic solution, its free amine groups that are ready for crosslinking, its cationic nature and its mucoadhesive character (Agnihotri et al, 2004) Chitosan nanoparticles have been extensively developed and explored for pharmaceutical application in the last two decades and now more has been explored for cosmetics application.

The preparation of chitosan nanoparticles has been developed based on chitosan microparticles technology. As reported by Tiyaboonchai (2003) there are many methods available including the ionotopic gelation, microemulsion, emulsification solvent diffusion and polyelectrolyte complex. For example of active agents encapsulation, Yoksan et al (2009) has reported the encapsulation of ascorbyl palmitate in chitosan nanoparticles by oil-in-water emulsion and ionic gelation processes. Briefly, the process was carried out by droplet formation via an oil-in-water emulsion, followed by droplet solidification via ionic gelation using sodium triphosphate pentabasic (TPP) as a crosslinking agent. Encapsulation efficiency of ascorbyl palmitate in the nanoparticles was about 39%-77% respectively and the amount of released ascorbyl palmitate from nanoparticles was enhanced at high pH due to the deprotonation of chitosan. Ascorbyl palmitate was released very quickly due to the weakened or disappeared electrostatic interaction between ammonium ions on chitosan chains and phosphoric groups of TPP molecules. This could be the way of release of active ingredients from chitosan nanoparticles when applied on skin that is yet to be studied.

In the study of Kim et al (2006), retinol-encapsulated chitosan nanoparticles were prepared for application of cosmetic and pharmaceutical applications. For this preparation, water soluble chitosan with the molecular weight of 18000 and degree of deacetylation of 96% was used. The water soluble chitosan is used to overcome the limits of application to bioactive agents such as gene delivery carriers, peptide carriers and drug carriers in acidic solution. Chitosan was dissolved in deionized water and added with retinol solution with ultrasonication. Characterization of chitosan nanoparticles using Fourier Transform Infrared (FITR) spectroscopy suggested that the retinol was encapsulated into chitosan nanoparticles by ion complex. X-ray diffraction pattern and High Performance Liquid Chromatography (HPLC) analysis showed that the retinol was stably and efficiently encapsulated into chitosan nanoparticles with the efficiency of more than 60% for all formulations with different amounts of retinol respectively.

Cattaneo (2004) has patented a high viscosity chitosan-based topical compositions for the delivery of water insoluble active agents (such as retinoic acid) in which the active agent is entrapped either dissolved or in the form of suspended particles in a suitable dispersing agent in a chitosan matrix and then mixed with anionic polymers. Such particles are able to deliver compounds to skin with little associated irritation without the addition of surfactants or emulsifiers. In certain embodiments, the particles include an inner core containing the active agent and an outer coating formed from a matrix comprising cationic and an anionic polymer. Skin permeability test has been done and it showed that the amount of active agents penetrated percutaneously from the chitosan matrix formulation was 40% lower that that obtained when using the free drug formula. These results indicate that the chitosan-based delivery system was able to reduce systemic absorption by 40%. However, it did not interfere with the amount of retinoic acid taken up by the skin at the site of action.

This patent by Cattaneo (2004) could be a good reference for this research as it involves the formation of chitosan nanoparticles incorporating an active agent to be delivered to the skin. From the patent, high viscosity of chitosan matrix used reduces systemic absorption of active agent. Since this research focuses on deposition on skin as well as absorption through the skin, a lower viscosity of chitosan will be used as the controlled release of active agents to make the actives penetrate and release easier into the skin, It has the similarity with this research except the fact that this work involves cleanser which has a higher amount of surfactants which could induce higher skin irritation. Principles from all these works mentioned above could be referred to as a guideline in order to develop the desired chitosan nanoparticles.