Calcium depletion-mediated protease inhibition and apical-junctional-complex disassembly via an EGTA-conjugated carrier for oral insulin delivery

Calcium (Ca2+) has a crucial role in maintaining the intestinal protease activity and in forming the apical junctional complex (AJC) that preserves epithelial barrier function. Ethylene glycol tetraacetic acid (EGTA) is a Ca2+-specific chelating agent. To maintain the concentration of this chelator in areas where enzyme inhibition and paracellular permeation enhancement are needed, this study synthesized a poly(gamma-glutamic acid)-EGTA conjugate (gamma PGA-EGTA) to form nanoparticles (NPs) with chitosan (CS) for oral insulin delivery. The results of our molecular dynamic (MD) simulations indicate that Ca2+ ions could be specifically chelated to the nitrogen atoms, ether oxygen atoms, and carboxylate oxygen atoms in [Ca(EGTA)](2-) anions. By chelating Ca2+, gamma PGA-EGTA conferred a significant insulin protection effect against proteases in intestinal tracts isolated from rats. Additionally, calcium depletion by gamma PGA-EGTA could stimulate the endocytosis of AJC components in Caco-2 cell monolayers, which led to a reversible opening of AJCs and thus increased their paracellular permeability. Single-photon emission computed tomography images performed in the biodistribution study clearly show the I-123-insulin orally delivered by CS/gamma PGA-EGTA NPs in the heart, aorta, renal cortex, renal pelvis and liver, which ultimately produced a significant and prolonged hypoglycemic effect in diabetic rats. The above results confirm that this gamma PGA-EGTA conjugate is a promising candidate for oral insulin delivery. (C) 2012 Elsevier B. V. All rights reserved.