Pharmaceutical Nanotechnology
Pharmacological activity of peroral chitosan–insulin nanoparticles in diabetic rats

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Abstract

The objective of the present study was to evaluate the effects of formulation parameters on the in vivo pharmacological activity of the chitosan–insulin nanoparticles. Chitosan–insulin nanoparticles were prepared by ionotropic gelation at pH 5.3 and 6.1 and denoted as F5.3np and F6.1np, respectively. F5.3np and F6.1np administered orally at insulin doses of 50 U/kg and/or 100 U/kg were effective at lowering the serum glucose level of streptozotocin-induced diabetic rats. The 100 U/kg-dose F5.3np sustained the serum glucose at pre-diabetic levels for at least 11 h. In comparison, F6.1np had a faster onset of action (2 h versus 10 h) but lower efficiency. The effectiveness of peroral F5.3np and F6.1np in lowering the serum glucose level of streptozotocin-induced diabetic rats was ascribed to the local effect of insulin in intestine. Confocal micrographs showed strong interaction between rat intestinal epithelium and chitosan nanoparticles 3 h post-oral administration.

Introduction

The oral route is the preferred route of drug administration for patients on chronic therapy. However, the oral delivery of many therapeutic peptides and proteins remain an unresolved challenge mainly because of the large size, hydrophilicity, and instability of these macromolecules (Lee and Yamamoto, 1990). In this study, we proposed to use chitosan nanoparticles as a carrier for the oral delivery of insulin.

Chitosan is a mucoadhesive, polycationic polymer that can facilitate drug absorption by localizing drug concentration around absorptive cells (Lehr et al., 1992) and prolonging drug residence in the gut (Leung et al., 1991). It is also an effective permeability enhancer because of its depolymerizing action on cellular F-actin and the tight junction protein ZO-1 (Schipper et al., 1997). Co-administered chitosan has been shown to enhance the transport of 14C-mannitol (Schipper et al., 1996), buserelin (Kotéz et al., 1997), vasopressin (Kotéz et al., 1997, Lueßen et al., 1997), and insulin (Kotéz et al., 1997) across the Caco-2 monolayers. Chitosan, in the form of glutamate solution and nanoparticles, is also reported to promote the transport of insulin through the nasal epithelium of sheep and rabbits, respectively (Illum et al., 1994, Fernandez-Urrusuno et al., 1999).

Oral delivery of insulin via polymer nanoparticles has also met with some success. The first report in 1988 suggested that insulin encapsulated in poly(isobutylcyanoacrylate) (PIBCA) nanocapsules had a long-term (up to 20 days) hypoglycemic effect in diabetic rats after oral administration (Démage et al., 1988). Peroral administration of insulin-loaded nanoparticles of poly(lactide-co-glycolide) (Barichello et al., 1999, Carino et al., 2000) and poly(fumaric-co-sebacic) anhydride (Mathiowitz et al., 1997) also controlled the plasma glucose levels of rats against an initial glucose load.

A recent study has shown that peroral chitosan–insulin nanoparticles could significantly lower the serum glucose levels of alloxan-induced diabetic rats (Pan et al., 2002). However, it is not known if the pharmacological activity was accompanied by increased plasma insulin levels. Neither did the authors control the pH at which the nanoparticles were prepared. In a previous study, we have found that the association and in vitro release of insulin were strongly influenced by the pH of the chitosan–insulin nanoparticle dispersion (Ma et al., 2002). Insulin association to chitosan nanoparticles at pH 5.3 was 50% less efficient than that at pH 6.1. However, the insulin association at pH 5.3 was stronger, where more than 75% of the associated insulin remained intact in vitro release studies. In contrast, insulin association at pH 6.1 was highly labile, the insulin rapidly and completely dissociating when the chitosan nanoparticles were diluted with aqueous media. These differences between the pH 5.3 and 6.1 formulations could have an impact on the in vivo pharmacological activity of the chitosan–insulin nanoparticles.

The objective of the present study was to evaluate the effects of formulation parameters on the in vivo pharmacological activity of the chitosan–insulin nanoparticles. In addition to formulation pH, we determined the effects of cross-flow filtration on the in vivo efficacy of chitosan–insulin nanoparticles prepared at pH 5.3 and 6.1. Both the serum glucose and serum insulin levels of the diabetic rats following the treatment were monitored in the study. Interaction between fluorescein isothiocyanate (FITC)-labeled chitosan nanoparticles and rat intestinal epithelium was also studied using confocal laser scanning microscopy.

Section snippets

Materials

Chitosan, from Aldrich Chemical Co. (Milwaukee, WI), was determined by dilute solution viscometry (Wang et al., 1991) to have a molecular weight of 186 kDa, and by the first derivative method of UV spectrometry (Tan et al., 1998) to have a degree of deacetylation of 85%. Fluorescein isothiocyanate (FITC), streptozotocin (SZ) and insulin (from porcine pancreas, 1 mg was equivalent to 27.8 USP units) were from Sigma Chemical Co., St. Louis, MO, USA; pentasodium tripolyphosphate (TPP) was from

Characterization of chitosan–insulin nanoparticles

The F6.1np had a mean size about 70 nm larger than the F5.3np (Table 1), but this size difference might not be important in view of the relatively large polydispersity of the dispersions (∼0.5). The larger mean size of the F6.1np was related to its lower zeta potential, which resulted in the formation of fewer crosslinks via the TPP ions. The removal of free TPP and insulin by cross-flow filtration caused significant increases in the mean size, polydispersity and zeta potential of the F5.3np and

Discussion

The F5.3np and F6.1np were effective in lowering the serum glucose levels of streptozotocin-induced diabetic rats when administered orally at insulin doses of 50 and/or 100 U/kg to the rats. Of particular interest was the activity of the higher dose F5.3np, which maintained the serum glucose level of the treated rats at pre-diabetic levels (60% of baseline) for at least 11 h post-administration. Results generated from the control dosing samples suggest that the pharmacological response of the

Conclusions

Chitosan–insulin nanoparticles formulated with an insulin loading concentration of 4.28 U/mL at pH 5.3 and 6.1 were effective in lowering the serum glucose level of streptozotocin-induced diabetic rats when given at insulin doses of 50 and/or 100 U/kg to the rats. In particular, F5.3np administered at the insulin dose of 100 U/kg was able to maintain the rat serum glucose level at pre-diabetic levels for at least 11 h. The pharmacological activity of the nanoparticles was not accompanied by

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