**1. Introduction**

Eczema or atopic dermatitis (AD) and psoriasis are the most common chronic inflammatory skin diseases. AD is characterized by pruritus, dry skin, eczematous injuries, and lichenification [1]. The onset of AD commonly occurs during childhood, and it is associated with a significant morbidity and reduced quality of life. The prevalence of AD has increased over the past 30 years, and its incidence has increased 2- to 3-fold in recent years. Approximately, 20% of children and 1–3% of adults are affected by this disorder [2]. Although the pathogenesis of AD is not completely understood, it has been associated with various factors: alteration of the skin barrier function, immune dysregulation, infections, and environmental processes. The alterations in the skin barrier function may be caused by deficient levels of ceramides and proteins involved in keratinocyte differentiation and prevention of transepidermal water loss [3,4]. Several genetic factors, such as mutations in filaggrin genes, could cause this situation [5]. The defective barrier function allows the penetration of allergens and irritant agents that trigger inflammation through Th2

responses (with increased interleukins: IL-4, IL-5, IL-13 cytokines) in the acute phase and Th1 responses (with increased Interferon (IFN)-gamma and IL-12) in chronic injuries [6]. In addition, the usual scratching stimulates keratinocytes to release other inflammatory cytokines like Tumoral Necrosis Factor (TNF)-alpha, IL-1, and IL-6, which perpetuate chronic inflammation. Ultimately, the modified state of the skin contributes to bacteria colonization, which further worsens the disorder [6].

Likewise, psoriasis is a chronic and inflammatory skin disease with a marked immune component. Its most typical skin manifestation consists in erythematous plaques, often spread over large areas of the body. The worldwide prevalence varies from 1% to 8%, depending on the geographical areas. Although it can begin at any age, its onset frequently occurs in adulthood, with one out of three cases during childhood [7]. A major characteristic in psoriasis pathogenesis is the inappropriate activation of the immune system, which leads to keratinocyte hyperplasia, altered T cell function and angiogenesis, among others [8–10]. Besides, oxidative stress and increased expression of insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF) also participate in the pathogenesis [11].

The main goals of the treatment of these pathologies include restoring skin barrier, limiting itching, decreasing inflammation, and controlling immune alterations. To achieve them, a wide plethora of drugs may be used, with systemic immunosuppressive agents, topical corticosteroids, and topical calcineurin inhibitors (TCIs) being the first-line drugs in the pharmacological management of AD and psoriasis [12]. However, systemic treatments are not exempt of potential serious adverse side effects, such as kidney and liver disfunction or myelosuppression, which complicate the management of severe cases [13–15]. Local side effects associated with long-term use of topical steroids and TCIs are relatively common, such as skin atrophy, ecchymosis, erosions, striae, delayed wound healing, purpura, easy bruising, and acne [16]. In addition, the Food and Drug Administration (FDA) has reported warnings for the use of some TCIs due to the potential risk of skin cancer and lymphomas [17].

The proinflammatory cytokines involved in AD and psoriasis stimulate the expression of inducible nitric oxide synthase (iNOS) in keratinocytes. In consequence, skin lesions of AD and psoriasis present higher levels of nitric oxide, which has been implicated in the pathogenesis of atopic eczema and psoriasis [18]. Oral and topical cyanocobalamin (B12) have been used as an alternative for preventing or ameliorating AD and psoriasis [19]. Although the first trials dated from the 1960s, not many studies have been carried out recently [20]. Januchowski et al. compared the effect of B12 cream (0.07%) with a moisturizer base for the treatment of childhood eczema in a double-blinded randomized trial using the SCORAD scale (Scoring of Atopic Dermatitis). Intraindividual comparisons showed significant differences in reduction of SCORAD values between B12 cream and placebo. Particularly, topical B12 significantly improved treated skin more than placebo at 2 and 4 weeks. Recently, mild-to-moderate plaque psoriasis has been treated with topical B12 ointments for 12 weeks in two clinical randomized trials [21]. Strücker et al. compared the effect of B12 with calcipotriol (vitamin D<sup>3</sup> analogue), obtaining similar decreases in the PASI score (Psoriasis Area Severity Index) after the studied period. Moreover, there was a marked diminution of the efficacy of calcipotriol after 4 weeks of therapy, whereas the efficacy of B12 remained largely constant over the whole observation period, making it more suitable for long-term use [22]. Similarly, a positive outcome was obtained when comparing B12 and emollient cream in the study done by Del Luca et al. The differences in PASI reduction were noticeable at week 2 and increased during the following 10 weeks. Two weeks after the end of the treatment, the psoriatic lesions evolved negatively (especially the ones treated with B12), proving the potential role of cyanocobalamin-based formulation in the treatment of epidermal disorders [23].

Despite these promising results, the molecular weight (1355 Da) and hydrophilicity of B12 limit its diffusion through intact human skin, given that one of the main functions of the skin is to prevent the penetration of exogenous substances to the body [24]. As the barrier function is recovered during treatment, B12 permeability might be gradually reduced.

Research on cutaneous permeability has shown that skin diffusion may be improved by using a suitable formulation, and it can even be used for systemic administration [25]. The strategies to overcome the limitations for drug permeation through the skin are addressed to disrupt the barrier function of the stratum corneum or to change the drug physicochemical properties. In this sense, although water-based vehicles have been reported to be suitable for B12 topical delivery, other studies suggest that the use of chemical enhancers, such as ethanolic solutions and oleic acid/propyleneglycol-based formulations, provide a better diffusion through the skin [26,27]. In addition, other active strategies, such as iontophoresis and microneedeling, showed higher penetration rates for B12 as well [27,28].

Nanosystems are another strategy to enhance drug delivery through the skin, consisting of the design of different types of nanocarriers, such as lipid nanoparticles, polymeric micelles, and poly (β-amino ester) [29–34]. Among those, liposomal-type systems have shown promising results in transdermal purposes [35,36]. Liposomes are microscopic vesicles with an aqueous core surrounded by one or more outer layers composed of non-toxic and biodegradable phospholipids, which are commonly used for their ability to encapsulate hydrophilic and lipophilic drugs [37]. Biomedical research has led to the development of modified lipid vesicles that include surfactants (transfersomes) or ethanol (ethosomes) in their composition, which provide greater flexibility, causing different carrier–skin interactions and thus enhancing skin drug delivery [38,39]. Cyanocobalamin liposomes have already been prepared by Arsalan et al. to improve its photostability and by Vitetta et al. to achieve systemic absorption after oral administration [40,41].

Based on these criteria, the aim of this study was to prepare and characterize stable lipid vesicles—liposomes, transfersomes, and ethosomes—to improve cyanocobalamin skin penetration.
