多功能奈米微粒做為降血糖蛋白質藥物之 口服釋放載體的研究

Multifunctional Nanoparticles for Oral Delivery of Anti-hyperglycemia Protein Drugs

莊爾元

指導教授 ： 宋信文

國立清華大學/工學院/化學工程學系/博士(2013年)

摘要

Calcium (Ca2+) plays a crucial role in maintaining intestinal protease activity and forming the apical junctional complex (AJC) that conserves 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, the first part of this study synthesized a poly(γ-glutamic acid)–EGTA conjugate (γPGA–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. Through chelating Ca2+, γPGA–EGTA conferred a significant insulin protection effect against proteases in intestinal tracts isolated from rats. Additionally, calcium depletion by γPGA–EGTA could stimulate endocytosis of AJC components in Caco–2 cell monolayers, which led to reversible opening of AJCs and thus increased their paracellular permeability. Single-photon emission computed tomography images performed in the biodistribution study clearly show the 123I–insulin orally delivered by CS/γPGA–EGTA NPs in the heart, aorta, renal cortex, renal pelvis and liver, which ultimately exerted a significant and prolonged hypoglycemic effect in diabetic rats. These experimental results confirm that the γPGA–EGTA conjugate is a promising candidate for oral insulin delivery.
The second part of this study examined the feasibility of preparing above-mentioned pH–responsive NP system composed of CS and γPGA–EGTA for oral insulin delivery in diabetic rats during an oral glucose tolerance test (OGTT). OGTT has been used largely as a model to mimic the period that comprises and follows a meal, which is often associated with postprandial hyperglycemia. Based on Förster resonance energy transfer (FRET), this work also demonstrated the ability of γPGA–EGTA to preserve insulin from an intestinal proteolytic attack in living rats, owing to its ability to deprive the environmental calcium from the intestinal lumen. Additionally, EGTA–conjugated NPs were effective in disrupting the epithelial tight junctions, accordingly facilitating the paracellular permeation of insulin throughout the entire rat small intestine. Furthermore, results of positron emission tomography (PET) and computer tomography (CT) verified the effective absorption of the permeated insulin into the systemic circulation as well as promotion of the glucose utilization in the myocardium, and skeletal muscles of the chest wall, forelimbs and hindlimbs, resulting in a significant glucose–lowering effect. The above consequences imply that as-prepared EGTA–conjugated NPs are a promising oral insulin delivery system to control postprandial hyperglycemia and thus may prevent the related diabetic complications.
Current insulin therapy via subcutaneous administration can lead to occasional hypoglycemia and peripheral hyperinsulinemia, owing its inadequate glycemic control and nonphysiological route. The third part of this study evaluates the feasibility of using bovine insulin and exendin-4 in a form of combination therapy, as orally delivered by nanoparticles composed of CS and γPGA (CS/γPGA NPs), to control blood glucose levels in type 2 diabetes mellitus (T2DM) rats. Experimental results indicate that CS/γPGA NPs could enhance the intestinal paracellular permeation; consequently, the exogenous bovine insulin and exendin-4 could be delivered into the liver and pancreas, where they could elicit their glucoregulatory activities. In response to the stimulus of exogenously delivered bovine insulin and the endogenously secreted rat insulin stimulated by the ingested exendin–4, significant glucose utilization was found in the cardiac and skeletal muscles, resulting in the glucose–lowing effect. Owing to its synergic stimulation effects, the hypoglycemic effect of oral ingestion of NPs containing bovine insulin and exendin–4 was significantly greater than that of the group solely treated with insulin NPs. The above results demonstrate that oral combination therapy with bovine insulin and exendin–4 improves the modulation of blood glucose levels in T2DM rats, making it highly promising for treating those T2DM patients not adequately controlled by the current insulin therapy.

關鍵字

糖尿病 ； 胰島素 ； 藥物釋放 ； 奈米微粒

參考文獻

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