Figure 3Skin Drug Delivery. A) Pathways into the skin for transdermal drug delivery: a) Transcellular pathway (penetration through the corneocytes); b) Intercellular pathway (penetration between the corneocytes through the intercellular lipids); c) Intrafollicular pathway (penetration through the hair follicles); d) Polar pathway (penetration through the polar pores); adapted from (Sofia A. Costa Lima., 2018); B) Types of drugs entrance routes through the skin.
The intercellular pathway involves the passage of the drugs through the lipid matrix that occupies the intercellular spaces of the corneocytes and is usually the preferred route for lipophilic substances. Otherwise, the transcellular pathway, also known as the intracellular pathway, occurs through the successive skin layers and dead cells and allows the transport of hydrophilic or polar substances. The transappendageal pathway uses the different skin appendages to enter through the skin. Various sweat glands, hair follicles and pores opening to the outer surface of the skin via their ducts can be used as a possible way for the entrance of drugs. These polar pores are located between cells and encircled by polar lipids, which make small holes in SC (Alkilani et al., 2015, Sofia A. Costa Lima., 2018). Hence, it was considered an inessential pathway for drug penetration but nowadays, current research suggests that hair follicles and sweat glands may present an alternate pathway for a diffusing molecule (Uchechi et al., 2014). In the polar pathway, the penetration of the drugs occurs through the polar pores available in the skin.
When the drugs are able to penetrate deep in the skin, from the surface through the various layers, this type of penetration is called “transdermal drug delivery” (TDD). The drug firstly pass through theSC and then permeates via the viable epidermis and dermis by diffusion. After reaching the dermal layer, the drug becomes available for the uptake into the systemic circulation (Alkilani et al., 2015). TDD has advantages namely over hypodermic injections as the drugs (usually administrated in patches) can be applied only one time and released for a longer period of time (with no need of additional applications), is almost pain-free and doesn’t lead to the generation of dangerous medical waste such as needles and syringes. Furthermore, transdermal devices can be self-administered, and the administration can be easily stopped in case of need by removal of the patch (Van Gele et al., 2011).
The different types of drugs entrance routes through the skin and their classification are summarized in Figure 3B.
Healthy skin mimetic models
Despite de fact that the in vivo human skin is the most realistic and gold standard experimental model for the investigation of drugs interaction with the skin, the use of this model is not always possible mainly because of the ethical concerns, regulatory issues, laboratory facilities and the potential risk associated to the eventual toxic effects of the drugs (Van Gele et al., 2011). Moreover, the results obtained by the use of human ex vivo models present significant variability because samples are usually obtained from different anatomical places of the same donor, different donors and have unpredictable character depending on the different subjects or different age groups (Flaten et al., 2015). These facts reinforce the need of alternative models able to better mimic the real scenario of drug interaction with the skin and concomitantly allowing reproducible results (Abd et al., 2016).
In this section, an overview of the existent ex vivo and in vitro mimetic skin models will be given.
Ex vivo human and animal models
During long time, the main way for the preclinical research of new drugs and for the optimization of topical drug formulations was the investigation considering the use of ex vivo skin mimetic models. The literatures describe two main groups of ex vivo models obtained from human or animal organisms (see references (Abd et al., 2016, Flaten et al., 2015) for reviews).
Human skin is absolutely the most suitable model for study TDD (Ruela et al., 2016). The skin samples used in ex vivo permeation assays can be obtained from different origins namely from plastic surgeries, amputations or cadavers and in generally the skin excerpts can be collected from different organs, such as the abdomen, back, leg or breast (Schaefer et al., 2008). Different membrane types can be obtained by using human skin excerpts for further use in drug permeation studies. Full-thickness skin models, in which the excisions containing connective tissue and subcutaneous fat and consists of all layers below, including the dermis, are reported as useful model to test different drugs and formulations (Abd et al., 2016, Cross et al., 2003, Manca et al., 2014, Junyaprasert et al., 2012, Dragicevic-Curic et al., 2008, Dragicevic-Curic et al., 2010, Elmoslemany et al., 2012, Bragagni et al., 2012, Cal, 2006, Sahle et al., 2014, Gaur et al., 2013, Marimuthu et al., 2012).
Ex vivo epidermal membranes models are also used for permeation experiments and those models are obtained from thermal treatment of full-thickness skin (immersion in hot water) (Junyaprasert et al., 2012, Kligman and Christophers, 1963) or by chemical action namely by the use of different reagents such as ethylenediaminetetraacetic acid, ammonia and enzymes (Cross et al., 2003) in order to separate the membrane at the dermal–epidermal junction. Other methodologies using human dermatomed skin (Dragicevic-Curic et al., 2010, Dubey et al., 2007, Clares et al., 2014, Marepally et al., 2013) or dermatopharmacokinetic method in which tape stripping is used to remove SC layers have been described (reviewed in (Abd et al., 2016)).
More recently, abdomen skin samples from patients who underwent abdominoplasty are used as skin models (Ternullo et al., 2018). Many examples report the use of human ex vivo skin models (reviewed in references (Flaten et al., 2015, Abd et al., 2016)), as the investigation of the dermal uptake and percutaneous penetration of some organophosphate esters in a human skin ex vivo model (Frederiksen et al., 2018). In another study, the effect of some nanoemulsions containing alpha-tocopherol was evaluated in skin wounds either in cell lines and using ex vivo human biopsies samples (Bonferoni et al., 2018).
The use of skin perfusion models, a surgically prepared portion of skin including subcutaneous fatty tissue with assured continuous vascular circulation is reported (Ternullo et al., 2017a, Ternullo et al., 2017b), to test different drugs, namely nanoparticle formulations. The use of this model is considered a promising strategy since they present benefits over in vitro models, as they overcome the existence of only epidermis and part of the dermis and the lack of a vascular system as verified in the most commonly used in vitro models (Ternullo et al., 2017a, Ternullo et al., 2017b).
Regarding animal ex vivo models, pig skin models are the most relevant because of the multiple anatomical, physiological and histological similarities with the human skin such as the dermal/ epidermal thickness ratio, epidermal thickness, similarity in hair follicle and blood vessel density in the skin and content of SCceramides, dermal collagen and elastin (Abd et al., 2016). The pig skin is easily obtained as a waste from animals slaughtered for food. Amongst the different parts of the pig body, the central outside part of the porcine ear has been the mostly recommended due to the analogy with human skin layers (Meyer et al., 2006). Variability of permeability in different samples of pig skin also takes place. The pig ear skin permeability is comparable with human skin. In fact, studies showed a good correlation especially for lipophilic substances. Furthermore, the age of the animal influences the permeability of the drugs, however most of literature does not specify the age of animal (reviewed in (Flaten et al., 2015)).
Many different drugs and formulations such as liposomes, nanoparticles and microemulsions have been studied using ex vivo pig skin models. Amongst the number of studies available, some reports describe the evaluation of the permeation of liposomes containing different drugs in excised pig ear (Scognamiglio et al., 2013, Knudsen et al., 2012, Gillet et al., 2011). Other studies tested the permeation of different nanoparticles in pig ear models (Gomes et al., 2014, Pople and Singh, 2011, Şenyiğit et al., 2010). Most recently, new formulations including a transferosomal gel were tested using pig ear skin as an ex vivomodel for the study of the transdermal permeation and delivery of the drug (Das et al., 2017). The use of excerpts from other pig skin regions, namely from abdomen (Nagelreiter et al., 2013) and dorsum (Hathout et al., 2010) is also described. Furthermore, newborn pig skin excisions are used as skin models for evaluation of topical drug formulations (Cilurzo et al., 2007).
In addition to the pig skin models, several other animals are used namely primates, mice, rats, guinea pigs, rabbits, bovines (udder) and snakes (shed skin). However, these models require ethical permissions. Since 2009, the use of animals for collection of toxicological data for cosmetic ingredients has been prohibited in the EU (76/768/EEC, February 2003) (Van Gele et al., 2011).
Mainly due to the fact that primate research is highly restricted and very expensive, skin of rodents (mice, rat and guinea pigs) is sometimes considered for permeation studies, due to its high availability, small size and quite low price. There are available many hairless strains which can be advantageous for this type of studies (Abd et al., 2016). Amongst rodents, rat skin is most like human skin however many studies pointed out the fact that its skin is more permeable than human skin (Barber et al., 1992, Chowhan and Pritchard, 1978, Hughes and Edwards, 2010, Schmook et al., 2001, van Ravenzwaay and Leibold, 2004). Yet, a study has shown that hairless mouse skin is an inadequate model for assessing the effects of the skin penetration enhancers (Bond and Barry, 1988).
Shed snake skin was considering as well as a useful model to mimic human skin and it can be obtained without killing the animal however it lacks hair follicles (Rigg and Barry, 1990, Itoh et al., 1990, Wonglertnirant et al., 2012, Kumpugdee-Vollrath et al., 2013). Additionally, udders from slaughtered cows are used as an ex vivo animal model and studies involving the comparison of this model with porcine skin has confirmed that both models are well correlated, thus enabling its use for studies regarding topical administration of drugs (Netzlaff et al., 2006).
The several ex vivo animal models mainly differ in the thickness of SC , hair density, number of corneocyte layers, hydration, lipid profile and morphology which may constitutes several advantages and limitations of each model. The most relevant features are summarized in Table 1 (Flaten et al., 2015).