Thiol functionalized polymethacrylic acid-based hydrogel microparticles for oral insulin delivery

Abstract

In the present study thiol functionalized polymethacrylic acid–polyethylene glycol–chitosan (PCP)-based hydrogel microparticles were utilized to develop an oral insulin delivery system. Thiol modification was achieved by grafting cysteine to the activated surface carboxyl groups of PCP hydrogels (Cys-PCP). Swelling and insulin loading/release experiments were conducted on these particles. The ability of these particles to inhibit protease enzymes was evaluated under in vitro experimental conditions. Insulin transport experiments were performed on Caco-2 cell monolayers and excised intestinal tissue with an Ussing chamber set-up. Finally, the efficacy of insulin-loaded particles in reducing the blood glucose level in streptozotocin-induced diabetic rats was investigated. Thiolated hydrogel microparticles showed less swelling and had a lower insulin encapsulation efficiency as compared with unmodified PCP particles. PCP and Cys-PCP microparticles were able to inhibit protease enzymes under in vitro conditions. Thiolation was an effective strategy to improve insulin absorption across Caco-2 cell monolayers, however, the effect was reduced in the experiments using excised rat intestinal tissue. Nevertheless, functionalized microparticles were more effective in eliciting a pharmacological response in diabetic animal, as compared with unmodified PCP microparticles. From these studies thiolation of hydrogel microparticles seems to be a promising approach to improve oral delivery of proteins/peptides.

Introduction

Nano/microparticles composed of bioadhesive polymers are promising systems for oral drug delivery applications [1]. They can extend the residence time of dosage forms in the gastrointestinal tract (GIT), and hence improve the bioavailability of orally administered drugs. Hydrogels composed of hydrophilic polyacrylates such as poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) exhibit mucoadhesive properties [2], [3]. PAA/PMAA-based hydrogels are also capable of chelating divalent ions (e.g. calcium and zinc), which in turn affects the thermodynamic stability and the biological activity of calcium-dependent proteolytic enzymes like trypsin and chymotrypsin [4]. A reduction in extracellular divalent ion concentration can also result in the opening of epithelial tight junctions, allowing paracellular drug transport across the intestinal epithelium [5].

Surface modification was suggested as an interesting approach in further improving the efficacy of mucoadhesive delivery systems [6], [7]. Attachment of specific functional entities (such as lectins, thiols, etc.) to the polymer surface has proved effective in enhancing their mucoadhesion behaviour [8], [9]. In general, bioadhesive polymers display non-specific interactions with mucus layers. In contrast, lectins/thiol groups mediate mucoadhesion through specific interactions to promote adherence with the mucosal layer. Lectins have the ability to bind glycoproteins of the mucus in a specific manner, thereby improving interaction with mucosal surfaces [10], [11]. On the other hand, addition of thiol groups to polymers, often referred as ‘thiomers’, can significantly enhance polymer–mucus interaction by the formation of disulphide linkages [12].

Interestingly, the introduction of thiol functional groups has also improved the permeation enhancing capability of mucoadhesive polymers [13]. Surface thiolation of hydrogel systems seems to be an interesting approach in this respect, assuming that thiol groups may help in anchoring the delivery system to the mucus layer surface, whereas the hydrogel character of the system may help its diffusion across the mucus layer to reach absorption sites on the epithelium. PCP hydrogel particles developed by the ionic gelation method are attractive in developing oral insulin delivery systems [14]. An attempt has been made to improve the efficacy of PCP hydrogel carriers by the attachment of surface thiol groups [7]. Mucoadhesion experiments were conducted and the ability of these systems to improve the paracellular permeability of hydrophilic macromolecules was studied with the help of Caco-2 cells and excised intestinal tissue. Thiol groups at varying concentrations were attached to the PCP particles and, based on the results obtained, an optimized system was selected for this investigation.

In the present study in vitro and in vivo studies were performed to evaluate the efficacy of thiolated PCP microparticles for oral insulin delivery. Detailed in vitro experiments were carried out to evaluate the efficacy of modified particles in terms of swelling, insulin loading/release, protease inhibition and insulin transport properties. Finally, an in vivo experiment was conducted in a diabetic animal model to evaluate the potential of this oral insulin delivery system.