Linseed Oil Nanoemulsions for treatment of Atopic Dermatitis disease: Formulation, characterization, in vitro and in silico evaluations

Abstract

Atopic Dermatitis (AD) is a chronic skin disease that is characterized by inflammation, dryness and bacterial infection accompanied by immunological changes and develops with the deterioration of the barrier property of the skin. The aim of this study is to prepare and characterize the innovative nanoemulsion (NE) formulations containing Linum usitatissimum seed (linseed) oil (LSO) and to investigate their potential with in vitro and in silico evaluation for the treatment of AD. In this study, LSO-NE formulations were prepared by ultrasonic emulsification method. Initially, the kinetic and thermodynamic stability of the LSO-NE formulations were studied using accelerated stability tests in terms of any physical instability problems such as creaming, sedimentation, coalescence, and phase separation. Following the heating cooling test for 6 cycles, the stable LSO-NE formulations were stored at three different storage conditions (25 ± 2 °C and 60% RH, 40 ± 2 °C and 75% RH, and 5 ± 3 °C) for 60 days to study the physicochemical stability. LSO-NE formulation with the highest stability and suitable physicochemical properties was considered for topical application. In vitro Ames/Salmonella assay was used for determination of the mutagenicity of LSO-NE formulation. Also, by molecular docking analysis, the binding affinities and binding orientations of the active ingredients in LSO to the target (HLA) molecules were determined, and their activities were estimated by means of their interaction mechanisms using in silico modelling.

As a result of the physicochemical stability tests, the formulation encoded with F4P1 was the most durable formulation among them with a mean droplet size of 99.02 ± 1.06 nm, a PdI value of 0.14 ± 0.020, and a zeta potential value of −8.79 ± 0.034 mV. It was determined that the optimum NE formulation released 78.4% of the LSO at the end of 24 h, and 100% of LSO within 48 h. It showed no mutagenic effect on TA98 and TA100 strains of Salmonella typhimurium. In the light of our molecular docking and ADMET calculations, it was determined that LSO will be a potential drug candidate for AD treatment.

Introduction

AD is a chronic skin disease that causes the barrier layer of the skin to deteriorate over time and causes immunological changes, mostly characterized by inflammation, dryness, and bacterial infection [1]. AD is a complex disease that develops as a result of the interaction of both genetic and environmental factors [2]. Skin barrier defects (proteases, filaggrin, loricrin), allergy sources (egg, cow's milk, house dust mite), autoimmune reactions (anti-keratinocyte antibodies) and microorganism colonization (Staphylococcus aureus) play an active role in the development of AD [2]. The prevalence of AD is about 15–30% or 20% in children living in developed countries; It is reported that it is 2–10% or 1–3% in adults [3,4]. Integrity of stratum corneum (SC), the outermost layer of the skin, and the protective characteristics of the skin are dependent on effective AD treatment to recover their properties [5]. Allergens that trigger other diseases should be avoided during the treatment [6]. Current therapeutic applications for the treatment of AD are limited. The treatment depends on the severity of the AD case, primarily topical treatment is conducted but in more severe cases, other methods such as phototherapy and/or systemic treatment might be applied [7].

Agents that restore skin barrier function, have anti-inflammatory effects, or suppress the immune response can be supplemented with topical application in the treatment of AD [8]. The main types of drugs used in the skin treatment of AD disease are topical corticosteroids with anti-inflammatory, antiproliferative, immunosuppressive and vasoconstrictive effects and topical calcineurin inhibitors with anti-inflammatory and immunosuppressive properties [[9], [10], [11]]. In addition, systemic corticosteroids, cyclosporine A, azathioprine, methotrexate, mycophenolate mofetil and alefacept, efalizumab and rituximab are used in the systemic treatment of AD [9,[12], [13], [14], [15]]. The methods used in severe cases are surely beneficial but have considerable side effects which limit their usage. The chemicals used in protecting the activity of drugs and modifying their release pattern are the main cause of the side effects [7]. In addition, common side effects such as erythema, stinging sensation, or less common effects such as itching, headache, sudden pain at the drug administration site can be seen with the current treatment models [16].

As this disease is more common in young children, concerns are raised that long-term use of corticosteroids may suppress the hypothalamic-pituitary-adrenal (HPA) axis, cause growth retardation, and have other side effects such as skin atrophy or acne [17]. Antihistamines may be useful to reduce scratching. However, topical formulations containing antihistamines, such as doxepin cream, can cause sedation if it is used over large areas of the body [17]. Immunomodulatory treatments such as inhibitors of calcineurin have also some side effects and risks. After skin cancer and lymphoma case reports emerged with the use of these agents, the U.S. Food and Drug Administration issued a black box warning stating that these agents should be used with caution, although a causal relationship has not been established [18]. Therefore, new alternative methods are being sought to prevent the risks of drugs and obtain better treatment results [19].

Herbal products have been used therapeutically for a long time all around the world, especially in the treatment of skin diseases, due to the side effects of existing chemical drugs. Herbal products have been successfully used in the treatment of specific skin disorders and also are still being used because their efficacy and safety are superior to conventional treatments and medicines. This has been scientifically proven [20]. For example, fruit acids such as citric, gluconic, gluconolactone, glycolic, acid [21], tannins [22], tea tree essential oil [23] and oral administration of vitex (Vitex agnus-castus) [24] have been reported to be effective in acne treatment due to their exfoliative and antibacterial properties. It has been stated that products such as garlic (Allium sativum) [25] and thyme essential oil [26] have a therapeutic effect in bacterial and fungal skin diseases. In studies on skin cancer, it has been found that topically used red ginseng extracts inhibit chemically induced skin tumors in mice [27]. Propolis is being studied in cancer treatments due to its antimicrobial, anti-inflammatory, analgesic and antitumor effects [28]. Rosemary (Rosmarinus officinalis) [29], thistle (Silybum marianum) [30], and green tea [31] have also been found to have anti-inflammatory and antitumorigenic properties in several mouse models.

In dermatitis, the dried flowers of Arnica montana [32], German chamomile (Matricaria recutita) [33], herbal medicine obtained from traditional Chinese medicine [34], Jewelweed (Impatiens biflora) [35] have been found to be effective due to their anti-inflammatory properties. Heartseases (Viola tricolor), British plantain (Plantago lanceolata), mullein (Verbascum thapsus), and flax (Linum usitatissimum) plants are topically included in eczema treatment because they contain a substance called "mucilage" that is beneficial to soothe and soften the skin [22,24,36].

More than two hundred species of Linum have been identified throughout the world. The species are often grown in parts of the Mediterranean countries, the Balkans and Turkey [37]. The oldest one known among these species is found in Turkey and called Linum usitatissimum. Even more, its oil is easily obtained from the seeds and also the plant is a part of many cultures. The linseed plant has nutritional characteristics and is a rich source of ω-3 fatty acid: α-linolenic acid [38], short chain polyunsaturated fatty acids (PUFA), soluble and insoluble fibers, phytoestrogenic lignans (secoisolariciresinol diglycoside-SDG), proteins and an array of antioxidants [[38], [39], [40], [41], [42]]. The fatty acid content of LSO is given in Table 1. The linolenic acid in LSO contains species of fatty acids that can be converted to eicosapentaenoic acid (EPA) [43] and docosahexsaenoic acid (DHA) [44] by human metabolism.

Studies on the application of EPA on AD cases show positive results for the treatment process. Therefore, the development of NEs containing LSO might be a promising alternative in the AD therapy. Although phytomedicines work better in vitro, their effects are significantly reduced in vivo. This reduction in activity can be attributed to poor solubility and/or unsuitable molecular size [45,46]. Topical NEs might be a convenient system in order to develop the phytomedicines by improving the phytoconstituent stability and increasing the drug solubility, and hence the bioavailability [45,47,48].

NEs are an attractive delivery system in the dermatology field due to their ability to improve the drug release profile and skin penetration [56,57]. NEs designed for the delivery of phytoconstituents are transparent or translucent systems with nanometer-sized droplets [58]. The advantages of NEs could be the high interfacial area related to their nanometer-sized droplets, reduced toxic and irritant side effects, protection of the drug inside the carrier from hydrolysis and oxidation, increased drug penetration and/or permeation, low surfactant concentration and ability to be suitable for both hydrophilic and lipophilic drugs [59,60]. Owing to the many advantages of NEs, several plant bioactives and extracts such as neem oil, eucalyptus oil, zedoary oil, curcumin, citronella oil, capsicum oleoresin etc. have been incorporated into NEs [38,[61], [62], [63], [64], [65], [66], [67], [68]].

In the light of the above information, for the first time, we aimed to develop a topical NE formulation containing LSO for the treatment of AD. NEs were prepared by ultrasonic homogenization method and analyzed in terms of average droplet size, polydispersity index, zeta potential, pH, and conductivity. Accelerated stability studies including centrifugation and thermal stress tests and physicochemical stability studies for a period of 60 days were also performed. In addition, the in vitro release of the optimized NE formulation was conducted by the dialysis membrane method, and the in vitro genotoxic activity of the NE were examined on the S. typhimurium TA98 and TA100 mutant strains. Moreover, molecular docking analysis was performed to determine the most likely binding positions between the active ingredients (α-linolenic acid, oleic acid, and linoleic acid) of LSO and the target Human Leukocyte Antigens (HLAs), which play an important role in immune system activation for AD by presenting antigen peptides to T cells to initiate the skin immune response. Also, by use of the in silico methods, the calculated pharmacokinetic values of the active ingredients (α-linolenic acid, oleic acid and linoleic acid) of LSO contributed an important information in search of its bioactivity [[69], [70], [71]].