International Journal of Pharmaceutics 381 (2009) 205–213 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Pharmaceutical Nanotechnology Melatonin-loaded lecithin/chitosan nanoparticles: Physicochemical characterisation and permeability through Caco-2 cell monolayers Anita Hafner a,∗∗ , Jasmina Lovri ´ c a , Dario Voinovich b , Jelena Filipovi ´ c-Grˇ ci´ c a,∗ a Department of Pharmaceutics, Faculty of Pharmacy & Biochemistry, University of Zagreb, A. Kovacica 1, 10000 Zagreb, Croatia b Department of Pharmaceutical Sciences, University of Trieste, Trieste, Italy article info Article history: Received 30 November 2008 Received in revised form 30 April 2009 Accepted 1 July 2009 Available online 9 July 2009 Keywords: Melatonin Chitosan Lecithin Nanoparticles Transmucosal permeability abstract In this study, the potential of lecithin/chitosan nanoparticles (NPs) as a mucoadhesive colloidal nanosys- tem for transmucosal delivery of melatonin was investigated. The size, zeta potential and melatonin loading of the lecithin/chitosan NPs were investigated as a function of lecithin type (Lipoid S45, S75 and S100) and chitosan content in the preparation. The NPs were characterised by mean diameter and zeta potential ranging between 121.6 and 347.5nm, and 7.5 and 32.7mV, respectively, and increasing with lecithin-negative charge and chitosan content in the preparation. Melatonin loadings were up to 7.1%. All NPs were characterised by prolonged release profiles with an initial burst (approximately 25%), fol- lowed by a slow release phase. Approximately 60–70% of melatonin was released in 4 h. The permeability of melatonin was investigated using Caco-2 cells as an in vitro model of the epithelial barrier. Mela- tonin permeability from an NP suspension prepared with Lipoid S45 lecithin and a lecithin-to-chitosan weight ratio (L/C) of 20:1 (sample C2) was significantly improved compared to the permeability of mela- tonin from the solution (P < 0.001) and from all other NPs investigated (P < 0.05). The results obtained by the cell viability studies (MTT and LDH leakage assays) showed that C2 NP suspension did not induce plasma membrane damage or decrease cell viability and could be safely applied to Caco-2 cells in the concentration range tested (<400 g/ml). © 2009 Elsevier B.V. All rights reserved. 1. Introduction Melatonin is an indole amide neurohormone synthesised mainly in the pineal gland and secreted into the blood in a circadian rhythm. It has been shown to play a critical role in the body’s internal time- keeping system, which regulates the sleep–wake cycle along with other circadian and seasonal rhythms. Melatonin is also known for its immunostimulative and cytoprotective activity (Mao et al., 2004). Exogenous melatonin has been used in the treatment of various circadian rhythm disorders such as jetlag and insomnia. Due to its antioxidative properties, the potential clinical benefit of melatonin can be expected in the treatment of various cancers and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (Schaffazick et al., 2008). Although melatonin is a com- pound that is easily absorbed across the mucosa, its sensitivity to oxidation presents a significant problem for achieving a therapeutic dose. Moreover, its low oral bioavailability, due to variable absorp- tion and extensive first-pass metabolism, indicates the need for new ∗ Corresponding author. Tel.: +385 1 63 94 761; fax: +385 1 46 12 691. ∗∗ Corresponding author. Tel.: +385 1 63 94 765; fax: +385 1 46 12 691. E-mail addresses: ahafner@pharma.hr (A. Hafner), jfilipov@pharma.hr (J. Filipovi ´ c-Grˇ ci´ c). routes of administration and an appropriate delivery system to be developed. Nasal drug delivery, as an alternative route for the administra- tion of systemically acting drugs that have low oral bioavailability, is advantageous due to rapid drug onset and potential for cen- tral nervous system delivery (Costantino et al., 2007; van den Berg et al., 2004). This delivery method, however, is generally restricted to very potent drugs or drugs characterised by rather high water solubility (Ekelund et al., 2005). Another drawback for the nasal administration of drugs is mucociliary clearance, which limits the time allowed for drug absorption to occur. Appropri- ate delivery systems, however, can be developed to overcome the problems related to both poor water solubility and nasal clearance mechanism. An enhanced bioavailability of melatonin was observed in rab- bits after nasal administration of melatonin solution formulated with 40% PEG with or without 1% sodium glycocolate (94% and 55%, respectively) (Bechgaard et al., 1999). Moreover, an 84% absolute melatonin bioavailability from starch microspheres (prepared by an emulsification cross-linking technique) after nasal administra- tion to rabbits was observed (Mao et al., 2004). In addition, nasal clearance studies have shown that more than 80% of the starch microspheres were present in the nasal mucosa 2 h after admin- istration compared to only 30% of starch from the solution. This 0378-5173/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2009.07.001