The role of chitosan as coating material for nanostructured lipid carriers for skin delivery of fucoxanthin

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Abstract

Fucoxanthin (FUCO) is a marine carotenoid characterized by antiproliferative properties against hyperproliferative cells. The aim of this work was to design and develop nanostructured lipidic carriers (NLCs) based on bacuri butter and tucumã oil and loaded with FUCO, intended for skin application to prevent skin hyperproliferative diseases and in particular psoriasis. The presence of FUCO should control the hyperproliferation of skin diseased cells and the lipids forming the NLC core, rich in antioxidants and characterized by wound healing properties, should favor the restoring of skin integrity. NLCs were coated with chitosan (CS) to improve their biopharmaceutical properties (bio/mucoadhesion and wound healing) and to combine the advantages of lipidic nanoparticles with the biological properties of CS. Chitosan coated and non-coated NLC were prepared by means of high shear homogenization and characterized for chemico-physical and biopharmaceutical properties (in vitro biocompatibility and cell uptake towards normal dermal human fibroblasts). Moreover, the pharmacological activity of FUCO loaded in NLCs was assessed in psoriatic-like cellular model. NLCs were characterized by dimensions ranging from about 250 to 400 nm. Moreover, the CS coating and FUCO loading determined an increase of size. Moreover, TEM and zeta potential analysis confirmed the presence of CS coating on nanoparticle surface, thus conferring to nanoparticle good bioadhesion properties. NLCs uptake in fibroblasts was observed and NLC-FUCO-CS caused a reduction of cell viability with a less marked effect in fibroblasts rather than in psoriatic cells, highlighting the capability of this system to control skin hyperproliferation and inflammation. The loading of NLC-FUCO-CS in pullulan film should render NLCs application easy, without impair prompt interaction of the drug with the skin. Considering the overall results skin application of CS coated NLCs loaded with FUCO seems a promising approach to control skin hyperproliferation and to preserve skin integrity in psoriatic skin.

Introduction

Fucoxanthin (FUCO) is a marine carotenoid found in numerous classes of microalgae (such as bacillariophytes, bolidophytes, chrysophytes, silicoflagellates, and pinguiophytes) and it is present in edible brown seaweeds (macroalgae such as phaeophytes). It is known that certain long-lived populations have a regular consumption of brown algae (major source of FUCO) and this seems related to carotenoid role in prevention and treatment of cell hyperproliferation. In particular, FUCO is a bioactive compound which has several proven biological properties, including anticancer, antioxidant, anti-inflammatory, antimicrobial, and anti-angiogenic properties (Miyashita et al., 2011, Kumar et al., 2013).

FUCO antiproliferative activity is mainly related to the activation of caspase pathway. Moreover, FUCO presents a down regulation of Bcl-2 proteins with a pro-apoptotic effect, it is able to interfere with MAPK and GADD45 proteins that regulate mitosis and cell proliferation, it downregulates DNA binding activities of NF-kB and pIkBα, and upregulates IkBα. Furthermore, it is able to reduce expression of N-Myc oncogene and survivin with antiangiogenic activity (Hosokawa et al., 2004, Liu et al., 2009, Chung et al., 2013, Martin, 2015).

Moreover, FUCO is able to inhibit the expression and the secretion of matrix metalloproteinase-9 (MMP-9) and it has an antityrosinase activity, decreasing melanogenesis. On the contrary, no effect has been proved in normal cells (Moghadamtousi et al., 2014).

Among hyperproliferative skin diseases, psoriasis and in particular chronic plaque psoriasis (psoriasis vulgaris) is the most common form accounting for about 90% of cases. Typical lesions are monomorphic with sharply demarcated erythematous plaques covered by silvery lamellar scales. Psoriasis causes the thickening of epidermal layer, hyperkeratosis (thickened cornified layer), and parakeratosis (cell nuclei present in the cornified layer), alteration in dermal papillae and in the associated blood vessels. Moreover, dermis and epidermis are characterized by an inflammatory infiltrate containing T-lymphocytes, macrophages, mast cells, and neutrophilic granulocytes. Bleeding is one of the symptoms related to psoriasis due to scratching caused by itching.

Lipids are key factors in stratum corneum layer and horny cells (dead keratinocytes). These are fundamental for maintaining skin moisture and integrity.

Starting from 90s, lipidic nanoparticles have been extensively studied as carriers in skin delivery. In fact natural lipids have been considered as enabling excipients thanks to their unsoapable fractions, rich in eutrophic factors such as vitamins and antioxidants (Mandawgade and Patravale, 2008, Hajj Ali et al., 2015). Bacuri butter (Platonia insignis Mart., Clusiaceae) and tucumã oil (Astrocaryum vulgare, Arecaceae) are lipids from Amazonian region.

Bacuri butter, rich of fatty acid, is mainly composed of 50–55% palmitic acid (C16:0), 5–10% palmitoleic acid (C16:1), 10–20% oleic acid (C18:1). It has a high amount of unsaturated fatty acids: the ratio saturated / unsaturated is 3:1. The unsoapable fraction is up to 5% and it represents a rich source of polyisoprenylated xanthones and benzophenones, which are responsible for biological activities, as antioxidant and wound healing enhancer (Kumar et al., 2013, Lustosa et al., 2016). Tucumã oil is composed of 30% palmitic acid (C16:0), 65% oleic acid (C18:1), 3,5% linoleic acid (C18:2) and 5% linolenic acid (C18:3). The ratio of saturated / unsaturated is 1:3. The unsoapable fraction is up to 1.5–2% and the oil contains high amount of vitamin B2 (riboflavin) and bioactive compounds. In particular, it is rich of polyphenols such as catechin and quercetin characterized by antioxidant and wound healing properties (Bora et al., 2011, de Rosso and Mercadante, 2007).

Moreover, chitosan (CS), a cationic polysaccharide based on glucosamine and N-acetylglucosamine obtained by partial deacetylation of chitin (Duan et al., 2010, Sailaja et al., 2010, Zhang and Kawakami, 2010), possesses several properties fundamental in skin diseases: it is able to enhance coagulation, by both hemagglutination and promoting platelet activation, it prolongs its retention to target substrates by means of bio/mucoadhesion, it is able to support extracellular matrix regeneration during skin reconstruction and wound healing in its early phases (Sandri et al., 2011, Jayakumar et al., 2010, Manivasagan et al., 2017). Furthermore, it enhances remodeling process increasing also angiogenesis.

Given these premises, the aim of this work was the design and the development of Nanostructured Lipid Carriers (NLCs) based on bacuri butter and tucumã oil. These were loaded with FUCO, and they were intended for skin application to prevent/treat skin hyperproliferative diseases, in particular psoriasis. The presence of FUCO should control the hyperproliferation of skin cells and the lipids forming the NLC core, rich in antioxidant and characterized by wound healing properties, should favor the restoring of skin integrity. NLCs were coated with chitosan (CS) to improve their biopharmaceutical properties, thus combining the advantages of lipidic nanoparticles with the biological properties of CS. In particular they should enhance the residence time in the application area, via bioadhesive interaction to allow an intimate contact between FUCO and skin lesions in the treatment of hyperproliferative skin diseases. For this purpose, the NLCs were loaded in pullulan film to allow an easily application and to achieve a prompt release of NLC in order to favor the FUCO activity towards the lesion. The nanoparticles were prepared by means of high shear homogenization and characterized for chemico-physical and biopharmaceutical properties (in vitro biocompatibility and cell uptake towards normal dermal human fibroblasts). Moreover, the pharmacological activity of FUCO loaded in NLCs was assessed in psoriatic-like cellular model.

Section snippets

Materials

For the NLCs the following materials have been used:

Fucoxanthin (FUCO): analytical standard (C42H58O6) with purity of >95% (Cayman Chemical Company® (Ann Arbor, MI, USA)); active ingredient in NLC ≥ 95% purity (Sigma Aldrich, Italy) (logP = 10 (Delbrut et al., 2018); solubility in water 1,211*10−10% w/w).

Lipids: Bacuri butter (melting point 35 °C) and tucumã oil (kindly provided by Amazon Oil Industry).

Surfactants: polysorbate 80 and sorbitan monooleate (Sigma Aldrich, Italy);

Chitosan (CS) (MW:

Particle size, zeta potential and transmission electron microscopy

Fucoxanthin loaded NLCs were successfully prepared using high shear homogenization method and the procedure was suitable to prepare CS associated nanocarriers using bacuri butter and tucumã oil as natural lipids. Previous studies (Sandri et al., 2010 and Sandri et al, 2017) evidenced that the protocol used allowed to obtain a stronger CS coupling at NLC surface with respect to coating by mixing.

Table 1 reports the physicochemical properties of NLCs: blank NLCs (NLC), FUCO loaded NLCs (NLC-FUCO)

Conclusions

High shear homogenization was proved as suitable method to prepare CS coated NLCs, loaded with FUCO. The systems were characterized by dimensions ranging from about 250 to 400 nm with a narrow distribution. CS coating and FUCO loading determined a nanoparticle size increase, confirmed by TEM. Moreover, TEM and zeta potential analysis confirmed the presence of CS coating on nanoparticle surface and evidenced difference in particle structure attributable to drug loading which could rearrange the

Acknowledgements

The authors wish to thank Dr. P. Vaghi for CLSM, Dr. M. Boiocchi for TEM (Centro Grandi Strumenti, University of Pavia).

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