Chitosan-Coated Solid Lipid Nanoparticles for Insulin Delivery

Abstract

The delivery of therapeutic proteins like insulin, exploiting routes of administration different from the traditional injectable forms, has been investigated extensively, taking advantage of the nanotechnology tools available nowadays in the massive drug delivery system pipeline. In this chapter, we describe in detail the preparation of solid lipid nanoparticles (SLN), further coated with the mucoadhesive polymer chitosan, intended for intestinal absorption of insulin after oral administration. We give special focus on the characterization of the SLN and of the biomacromolecule by itself after encapsulation, because of the intrinsic labile properties of insulin during the manufacturing process. We also describe methods to determine the in vitro intestinal permeability of insulin that solid lipid and chitosan-coated SLN can afford, as well as in vivo models to evaluate the hypoglycemic effect in diabetic animals.

Introduction

Insulin has been usually administered subcutaneously for the treatment of diabetes mellitus. Several attempts have been made to find alternative routes of insulin administration that improve patient compliance and avoid injections (Cefalu, 2004, Ghilzai, 2003, Owens, 2002). Among all routes, oral delivery is the most desirable one since it is noninvasive thus avoiding the contamination/infection risks associated with injectables, and is physiologically advantageous, because it better mimics the normal insulin pathway after endogenous secretion (Hoffman and Ziv, 1997, Owens, 2002). However, insulin, as other polypeptides, presents formulation challenges such as poor chemical and physical stability and short plasma half-time. This often leads to low bioavailability, mainly resulting from degradation in the stomach, digestion and inactivation in the intestinal cavity by proteolytic enzymes, and poor permeability through the intestinal epithelium because of its lack of lipophilicity and high molecular weight (Carino and Mathiowitz, 1999).

Solid lipid nanoparticles (SLN) are submicron colloidal carriers (usually 50–1000 nm), composed of lipids that are solid at body and room temperature, and dispersed either in an aqueous surfactant solution or water (Rawat et al., 2006). The lipids that compose SLN can be complex acylglycerol mixtures, highly purified triacylglycerols or waxes. SLN gained increased interest among nanoparticulate systems in the past years mainly due to their biocompatibility, good tolerability, biodegradation (Almeida and Souto, 2007, Sarmento et al., 2007b), and even by the possibility of large industrial scale production (Muller et al., 2000). It has been reported that lipid nanoparticles encapsulating therapeutic proteins can improve their bioavailability, prolong their blood residence time, and/or modify their biodistribution (Garcia-Fuentes et al., 2005). In fact, SLN are able to protect proteins from enzymatic degradation in the intestinal environment and enhance their selective uptake, resulting in prolonged therapeutic effect (Sarmento et al., 2007b). Therefore, SLN have been proposed for the oral delivery of insulin for the treatment of diabetes mellitus (Liu et al., 2007, Sarmento et al., 2007b, Trotta et al., 2005, Zhang et al., 2006). Actually, SLN and other lipid-based drug delivery systems are known to enhance the oral absorption of many drugs (Charman, 2000, Charman et al., 1997). When SLN are administered orally, they can be absorbed either through the membranous epithelial cells (M-cells) of the Peyer's patches in the gut-associated lymphoid tissue (GALT) or transcellularly (Damge et al., 2008).

Surface modification of nanoparticles with chitosan, a natural cationic polysaccharide derived from chitin, is a promising strategy to enhance the penetration of encapsulated macromolecules, such as insulin, through mucosal surfaces. This biopolymer has been used for developing drug delivery systems because of its good biocompatibility, biodegradability, low toxicity, antimicrobial, mucoadhesive, and absorption enhancing properties (Braz et al., 2011). Insulin-loaded chitosan nanoparticles have been shown to yield a significant hypoglycemic effect when administered orally to diabetic animal models (Cui et al., 2009, Reis et al., 2008, Sarmento et al., 2007c, Sarmento et al., 2007d). In fact, the mucoadhesive properties of chitosan may enhance drug uptake, because the contact with the intestinal epithelium is maintained for longer periods and the penetration of the active drug is improved because of the prolonged concentration gradient between the intestinal membrane and nanoparticles. Further, chitosan is an effective permeability enhancer as it reversibly changes tight junctions (Cano-Cebrian et al., 2005, Smith et al., 2005).

Recently, our group demonstrated the ability of chitosan-coated SLN to enhance the intestinal uptake of insulin by showing its physiologic effect after oral administration to rats (Fonte et al., 2011). Comparatively to uncoated SLN, we were able to reach a significant improvement of the hypoglycemic effect. This is probably due to chitosan mucoadhesive properties which promote the intestinal insulin uptake while overcoming the degradation of insulin in the gastrointestinal tract. Nevertheless, there is a major limitation in the oral delivery of insulin-loaded nanoparticles, besides the rate of permeation of nanoparticles through the intestinal epithelium, which is the elimination of nanoparticles by the mononuclear phagocyte system (MPS) (Champion et al., 2008, Yin et al., 2007). Macrophages present in tissues such as the lymph nodes, liver, spleen, and bone marrow are also an additional factor in the elimination of nanoparticles. The role of PEG (Owens and Peppas, 2006) and some hydrophilic polysaccharide (Lemarchand et al., 2004) coating in avoiding phagocytosis of nanoparticles by macrophages is reported in the literature. Recently, we have demonstrated that chitosan-coated SLN were not internalized by the murine macrophage cell line, RAW 264.7, while uncoated SLN were taken up by these cells (Sarmento et al., 2011). Chitosan was able to provide stealth properties to SLN, resulting in the absence of phagocytosis. All these findings open perspectives for the optimization of long-time blood circulating chitosan nanoparticles.