In this study, we developed a dual ligand functionalized pluronic-based nanocarrier (NC) for oral delivery of insulin. Chitosan and zonula occludins toxin (ZOT)-derived, tight junction opening peptide were conjugated to NC to increase the permeability of loaded insulin across the small intestine through the paracellular pathway. Surface functionalized NC, either by chitosan or peptide, could modulate the tight junction (TJ) integrity in contrast to no effect of unmodified NC, as evidenced by the change in transepithelial electrical resistance (TEER) and immunostaining of Claudin-4, a tight junction marker, in Caco-2 cell monolayer. On the other hand, dual ligand (chitosan and peptide) functionalized NC significantly further increased the permeation of insulin across Caco-2 cell monolayer. More importantly, insulin loaded, dual ligand functionalized NC could increase the plasma insulin level and efficiently regulate the glycemic response for a prolonged period of time (∼1 day) upon oral administration to diabetic rats, whereas delivery of insulin by single ligand functionalized NCs, either by chitosan or peptide, as well as by unmodified NC and free insulin, could not induce the effective regulation of the blood glucose level. The use of fluorescence dye labeled insulin (FITC-insulin) and Cy5.5 labeled NC revealed that both insulin and dual ligand functionalized NC were adequately penetrated across the whole intestine villi in contrast to limited adsorption of insulin and NC mainly onto the epithelial surface of the intestine for single ligand functionalized NCs. These results suggest that dual conjugation of ZOT-derived peptide and chitosan is a promising approach to functionalize the surface of nanocarrier for oral delivery of protein drugs.
Keywords: Chitosan; Insulin; Oral delivery; Pluronic; Tight junction.
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