Development of methotrexate loaded fucoidan/chitosan nanoparticles with anti-inflammatory potential and enhanced skin permeation

https://doi.org/10.1016/j.ijbiomac.2018.12.014Get rights and content

Abstract

In this study, fucoidan/chitosan nanoparticles were developed for the topical delivery of methotrexate towards the treatment of skin-related inflammatory diseases. Based on the fucoidan/chitosan (F/C) weight ratio, three different nanoparticles (1F/1C; 3F/1C; 5F/1C) were produced and characterized. Methotrexate was loaded in these polymeric nanoparticles achieving a drug loading of ca. 14% and an entrapment efficiency of 96, 87 and 80% for 1F/1C; 3F/1C and 5F/1C nanoparticles, respectively. Methotrexate-loaded fucoidan/chitosan nanoparticles exhibited size within the 300–500 nm range, positive zeta potential for 1F/1C nanoparticles (+60 mV) and negative surface charge for the 3F/1C and 5F/1C nanoparticles (−40 and −45 mV, respectively). Methotrexate loaded in 3F/1C and 5F/1C nanoparticles did not affect cells viability and presented lower cytotoxicity than free methotrexate, in fibroblasts and human keratinocytes. MTX-loaded fucoidan/chitosan nanoparticles lead to a significant reduction of pro-inflammatory cytokines produced by activated human monocytes. Skin permeation studies showed that methotrexate-loaded nanoparticles permeated the pig ear skin barrier reaching after 6 h, a 2.7- and 3.3-fold increase for 3F/1C and 5F/1C nanoparticles, relative to free methotrexate. In conclusion, fucoidan/chitosan nanoparticles, in particular the ratio 5F/1C, is safe, exerts an anti-inflammatory effect and increase skin permeation thus can potentially be used for methotrexate topical delivery.

Introduction

Research in marine sources is emerging in the nanobiotechnological field and has proven to be valuable in different areas, such as biomedicine, cosmetic, and food [1]. Marine polysaccharide-based materials have recently attracted attention due to their good biocompatibility, biodegradability, low cost, and abundance [2]. These materials can be found in different biological origins, such as marine algae, animals and microorganisms, and consist of a large complex group of different macromolecules with diverse biological applications and interesting features to apply in the design of a drug delivery system [3]. Fucoidan is a fucose-rich sulfated polysaccharide obtained primarily from brown seaweeds. It possesses a wide range of biological activities already reported as anti-coagulant, anti-thrombotic, anti-angiogenic, anti-proliferative and anti-cancer [1,[4], [5], [6], [7]]. In the nanomedicine field, this marine polysaccharide has been applied in the development of imaging agents, protein delivery, small drug delivery, gene delivery, regenerative medicine, therapeutic and diagnostic as recently reviewed [8]. Chitosan is a naturally occurring biopolymer isolated by the N‑deacetylation of chitin, present in the exoskeletons of marine crustaceans, including shrimp and crab. This polysaccharide has the characteristic of mucoadhesion and enhances paracellular drug transport via the transient opening of the tight junctions between epithelial cells [[9], [10], [11]]. Therefore, chitosan has been widely applied for mucosal drug delivery, particularly as oral and nasal delivery systems [11,12]. The oppositely charged chitosan and fucoidan were explored, and their electrostatic interactions resulted in nanoparticle-based delivery systems [13,14]. In the literature, most of the research is based on a higher chitosan amount in relation to fucoidan. Given the biological activities of this sulphated polysaccharide, the present work intends to explore the predominant presence of fucoidan in the nanoparticles, thus called fucoidan/chitosan.

The topical route is considered the most convenient and comfortable method of drug administration for patients. The skin is an exceptionally effective barrier and it prevents the permeation of most of the drugs applied for therapeutic purposes [15]. In fact, most of the commercially available topical dosage forms have poor penetration, resulting in poor therapeutic benefit [16]. Hence a delivery system that makes the skin more permeable and that enters the skin by multiple mechanisms to enhance drug delivery is of great formulation interest. Skin-related diseases are an emerging health problem and the use of available systemic drugs reveals a high incidence of side effects. In order to attain a maximum efficacy through topical administration, the drugs in use should be retained at skin target site for the longest time possible. However, conventional topical anti-inflammatory drugs present limitations as poor penetration capability and rapid clearance from the skin [17]. Drug retention within the skin can be extended by encapsulating the drugs in nanoparticles and/or blended into hydrogels. Therefore, a good delivery system that can enhance both the retention time and the distribution of topical drugs is needed [18]. The various carriers majorly used for topical delivery include liposomes, niosomes, lipid nanoparticles [19]. Development of lipid nanoparticles for dermal application takes advantage of their occlusive properties and lipid interaction to improve skin penetration and follicular penetration as well [20]. However their limited drug loading capacity and leakage during storage may hamper a consensus topical administration for lipid nanoparticles, motivating the search for other skin-delivery systems [21]. Fucoidan/chitosan assembled as nanoparticles have not yet been mentioned for topical use, but their inherent physicochemical and biological properties are very promising. In fact, fucoidan and chitosan have been combined in hydrogel forms aiming some applications in tissue engineering and wound healing [22,23]. Also, the topical applications of chitosan nanoparticles have already been reviewed, highlighting the special features of this polysaccharide [24]. Due to its anti-inflammatory properties, fucoidan was also reported in the treatment of inflamed skin, seeking management of atopic dermatitis by IgE suppression in blood mononuclear cells [25]. It was proved that fucoidan exerts similar effect to dexamethasone, enabling a long term treatment [26].

Methotrexate (MTX) is a well-known drug used to treat and manage inflammatory skin-related diseases like rheumatoid arthritis, atopic dermatitis and psoriasis [27]. Considering the difficult management of the severe side effects of MTX, a topical formulation with a high degree of skin penetration could be an useful alternative for the treatment of locally inflamed skin. There are two major barriers in MTX bioavailability and clinical efficacy: its poor solubility in aqueous solution; and its rapid degradation in the intestinal tract [18]. Studies suggest that MTX incorporation in nanoparticles can overcome these shortages as well as improve stability and bioactivity issues [[28], [29], [30]].

The aim of this study was to prepare fucoidan/chitosan nanoparticles for the topical administration of MTX. Three fucoidan/chitosan weight ratios were chosen and the developed nanoparticles (1F/1C; 3F/1C; 5F/1C) were physicochemically characterized using dynamic light scattering (DLS) and cryo-scanning electron microscopy (cryo-SEM). The effect of fucoidan/chitosan nanoparticles on fibroblasts and keratinocytes was assessed by the mitochondrial viability assay. Following a 18 h in vitro treatment with the F/C nanoparticles, their anti-inflammatory activity was assessed using representative pro-inflammatory cytokines interleukin-1 β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [31]. Moreover, the ability of MTX to permeate the pig ear skin when incorporated in the marine-based polysaccharide nanoparticles was also evaluated. Results demonstrate that 3F/1C and 5F/1C nanoparticles are safe for biological application and both allow MTX skin delivery. These findings confirm that the fucoidan/chitosan nanoparticles represent a promising platform for skin-related inflammatory diseases, by combining methotrexate and fucoidan/chitosan nanoparticles for a highly effective therapy.

Section snippets

Materials

Fucoidan (from Fucus vesiculosus, MW 50.000–190.000 Da), chitosan (MW 190.000–310.000 Da), dimethyl sulfoxide and sodium chloride were supplied from Sigma-Aldrich (St Louis, MO, USA). Methotrexate was a gift from Excella (Feucht, Germany). Acetic glacial acid was obtained from VWR (Radnor, PA, USA). Double-deionized water was provided by an ultra pure water system (Arium Pro, Sartorius AG, Göttingen, Germany). pH measurements were achieved using a Crison pH meter GLP 22 with a Crison 52-02 tip

Characterization of fucoidan/chitosan nanoparticles

The fucoidan/chitosan nanoparticles preparation method was based on electrostatic interactions. As these marine polysaccharides are weak polyelectrolytes, the solution's pH value determines their dissociation degree. Thus, fucoidan solution exhibited a maximum negative charge (ca. −40 mV) at neutral pH, while chitosan solution exhibited a maximum positive charge (ca. +65 mV) at pH 3. The nanoparticles were prepared based on the ionization state of fucoidan and chitosan, negative sulfate and

Conclusions

In this work, fucoidan/chitosan nanoparticles were produced based on their electrostatic interactions, optimized and characterized for methotrexate topical delivery. The three weight ratios of fucoidan/chitosan showed a good capacity for the incorporation of MTX. Unloaded and drug-loaded 3F/1C and 5F/1C nanoparticles demonstrated to be biocompatible with fibroblasts and keratinocytes. The 5F/1C nanoparticles exhibited a significant inhibition of pro-inflammatory cytokines (IL1-β, IL-6 and

Acknowledgments

This research was partially supported through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF) and COMPETE under the Partnership Agreement PT2020 UID/QUI/50006/2013-POCI/01/0145/FEDER/007265 and the projects PEst-C/MAR/LA0015/2013, PTDC/MAR-BIO/4694/2014. This work also received financial support from the European Union (FEDER funds through COMPETE POCI-01-0145-FEDER-016790). AB acknowledges her fellowship under project

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