Oral Bioavailability of Insulin Contained in Polysaccharide Nanoparticles Bruno Sarmento,* ,² Anto ´ nio Ribeiro, ‡ Francisco Veiga, ‡ Domingos Ferreira, ² and Ronald Neufeld § Department of Pharmaceutical Technology, Faculty of Pharmacy of the University of Porto, 4050-047 Porto, Portugal, Department of Pharmaceutical Technology, Faculty of Pharmacy of the University of Coimbra, Coimbra, Portugal, and Chemical Engineering Department, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Received April 7, 2007; Revised Manuscript Received July 21, 2007 The pharmacological activity of insulin-loaded dextran sulfate/chitosan nanoparticles was evaluated following oral dosage in diabetic rats. Nanoparticles were mucoadhesive and negatively charged with a mean size of 500 nm, suitable for uptake within the gastrointestinal tract. Insulin association efficiency was over 70% and was released in a pH-dependent manner under simulated gastrointestinal conditions. Orally delivered nanoparticles lowered basal serum glucose levels in diabetic rats around 35% with 50 and 100 IU/kg doses sustaining hypoglycemia over 24 h. Pharmacological availability was 5.6 and 3.4% for the 50 and 100 IU/kg doses, respectively, a significant increase over 1.6%, determined for oral insulin alone in solution. Confocal microscopic examinations of FITC-labeled insulin nanoparticles showed adhesion to rat intestinal epithelium, and internalization of insulin within the intestinal mucosa. Encapsulation of insulin into dextran sulfate/chitosan nanoparticles was a key factor in the improvement of the bioavailability of its oral delivery over insulin solution. Introduction Among the controlled release formulations, polymeric col- loidal systems have shown a certain degree of success for the oral delivery of therapeutic proteins like insulin, 1,2 calcitonin, 3 octeotride, 4 and cyclosporine, 5 among others. Nanoparticles formulated from natural polymers like alginate, agar, agarose, chitosan, or synthetic polymers such as poly(lactic acid), poly- (lactic-co-glycolic acid), poly(ǫ-caprolactone), and poly(phos- phoesters) have attracted significant interest as protein carriers. 6-8 The gastrointestinal (GI) uptake of proteins like insulin can be improved by association to nanoparticles, mainly to protect insulin from degradation in the GI tract. Low pH and protease hydrolysis have demonstrated to be an important limitation to intestinal absorption of intact insulin 9 and transport into systemic circulation. 6 These carriers have been improving oral peptide delivery due to their prolonged retention in the GI tract and excellent penetration into the mucus layer. 10 Further, they are able to be taken up by the M cells of the Peyer’s patches, a type of lymphatic island within the intestinal tract that represents the major gateway through which nanoparticles may be ab- sorbed. 11 However, the mechanisms by which nanoparticles based on natural polysaccharides have improved the oral absorption of proteins still needs further elucidation. 12 Polysaccharides are natural biodegradable hydrophilic poly- mers, which exhibit enzymatic degradation behavior and good biocompatibility. They can also form polyelectrolyte complexes by electrostatic interactions between oppositely charged groups resulting in ion-pairing without altering the integrity of the polymer. 13 Nanoparticle delivery systems that have been developed by employing natural polysaccharides have the potential to retain protein stability, increase the duration of the therapeutic effect of proteins as well as the possibility of administration through nonparenteral routes. 14 Such polymers can be easily modified chemically and biochemically, and are highly stable, safe, nontoxic, with gel forming properties, suggesting their suitability to be used for oral protein delivery. 15 Chitosan (Chit), the most widely employed natural polysaccha- ride, is able to reduce the transepithelial electrical resistance, promoting the passage of small electrolytes and transiently opening tight conjunction between epithelial cells 16 and to combine with anionic sialic acid residues of the intestinal mucosa 17 due to mucoadhesive properties. The adhesion of Chit at the site of insulin GI absorption may offer various advantages for its uptake. 18,19 Previous work with Chit coated alginate nanoparticles 20 and other Chit-based nanoparticles 2,21 demon- strated their potential to orally administer insulin, but those formulations also showed premature gastric insulin release, exposing insulin to undesired GI degradation. Dextran sulfate (DS) is a biodegradable and biocompatible branched negatively charged polyanion able to strongly interact with positively charged proteins. Chit and DS have been complexed to formulate different drug delivery systems, 22-25 but their application to oral delivery of proteins has not been explored. They are intrinsically hydrophilic, which may con- tribute to the encapsulation of water-soluble proteins and to promote longer circulation times in ViVo. Recently, we formu- lated insulin into DS/Chit nanoparticles, which fully retained insulin in simulated gastric medium, and sustained the release up to 24 h in simulated intestinal medium, while preserving its bioactivity. 26 The main objective of the present study was to determine whether insulin encapsulated in DS/Chit nanoparticles induced a hypoglycemic effect after oral administration to diabetic rats. The use of a single nanoparticle administration is the most * Corresponding author. E-mail: bruno.sarmento@ff.up.pt. Tel: +351 222078949. Fax: +351 222073977. ² University of Porto. ‡ University of Coimbra. § Queen’s University. 3054 Biomacromolecules 2007, 8, 3054-3060 10.1021/bm0703923 CCC: $37.00 © 2007 American Chemical Society Published on Web 09/18/2007 Downloaded by PORTUGAL CONSORTIA MASTER on July 8, 2009 Published on September 18, 2007 on http://pubs.acs.org | doi: 10.1021/bm0703923