發展以葡聚醣修飾之金奈米粒子為長效胰島素之基材

Abstract

糖尿病是一種糖分代謝異常的疾病，糖尿病病患無法調控自身的血糖濃度，而持續高血糖的狀態會對人體組織產生許多不良的影響並造成一些併發症，例如:中風、心臟病、腎衰竭，甚至是失明。根據世界衛生組織於2106年提出的統計報告，全球約有4.42億成年人患有糖尿病，而每年約有3百萬人死於糖尿病及其併發症。現今最被廣泛用於糖尿病治療的方法為胰島素注射，然而每日頻繁的注射卻病患會有疼痛的問題，因此糖尿病醫療是個需要研究與改進的課題。近年來，奈米材料已經被應用在胰島素攜帶材料的研究上，例如:有機巨分子、金屬奈米粒子以及奈米團簇，雖然這些研究展現了奈米材料攜帶胰島素進入人體的可行性以及延長胰島素活性的能力，然而這些奈米胰島素載台需要長時間的合成過程。在此一論文的研究中，我們合成一種葡聚醣修飾之金奈米粒子，其對於胰島素具有親合能力，最大的胰島素吸附量約為 103.38 µg/mg (17.8 nmol/mg)，而其與胰島素的辨認位置位於胰島素B鏈上編號1-22號胺基酸序列，換言之，奈米粒子可以跟胰島素產生具有特異性結合的共聚物，而其解離常數也在論文中被推算為4.02  10-6 M左右。此外，胰島素受器對於胰島素之解離常數約為42 pM，遠小於共聚物之解離常數，因此我們相信胰島素受器與奈米粒子的之間競爭作用可以讓胰島素從奈米粒子表面慢慢釋放，進而達到長效胰島素釋放的功能。小鼠的前脂肪細胞3T3-L1被選為此一研究之模型細胞，由於3T3-L1細胞經過分化過程後會展現相較於其他細胞株更為靈敏的胰島素反應，因此可以用來估算胰島素的活性。結果顯示未與奈米粒子形成共聚物的胰島素會在加入細胞環境後4個小時失去活性，而共聚物可以將胰島素的作用時間延長至12小時。總而言之，本論文發展之胰島素載台可以藉由與胰島素生成共聚物之方式來有效延遲胰島素之作用時間，是一種具有潛力成為新型攜帶胰島素的基材，而為了探索此共聚物更進一步的臨床應用，活體動物實驗是不可或缺的下一階段。

Diabetes mellitus is a metabolic disorder disease. Diabetes patients usually have trouble in controlling their blood glucose level. As a consequence, several serious health issues such as blindness, kidney injury, and heart disease may occur in these patients. According to the report of World Health Organization in 2016, there are ~442 million adults suffering with diabetes, and over 3 million people die because of diabetes every year. Insulin injection is a commonly used in medical treatment for diabetes patients. Nevertheless, it is painful to have frequent insulin injections daily. Hence, such medical treatments are needed to be improved. Recently, nanomaterials including organic polymers and metal nanoparticles/nanoclusters (NPs/NCs) have been used as alternative delivery agents to develop the insulin carrier. Although several reports have demonstrated the effectiveness of NP-based delivery agents for lasting insulin activity, time-consuming synthesis steps are required to generate insulin immobilized NPs. In this study, we discovered that dextran encapsulated gold NPs (AuNPs@Dextran) generated from one-pot reactions have certain binding affinity toward insulin. The maximum binding amount of insulin onto AuNPs@Dextran was estimated to be ~ 103.38 µg/mg (17.8 nmol/mg). The binding site of insulin towards AuNP@Dextran is explored at the sequence number of 1-22 on B-chain. That is, insulin can bind onto the surface of AuNPs@Dextran through molecular recognition with a dissociation constant as low as ~ 4.02  10-6 M. It has been known that the Kd between insulin and insulin receptor is ~42 pM, which is much lower than the Kd between insulin and AuNPs@Dextran. In this study, we proposed that the conjugates of AuNPs@Dextran and insulin can be used as a long insulin lasting agents. That is, insulin can be gradually released from the conjugates via the competition between AuNPs@Dextran and insulin receptor on the cell membrane of host cells. Mouse 3T3-L1 cells that can differentiate to adipocyte cells and have sensitive response to the presence of insulin were selected as the model cells in this study. Our experimental result shows that AuNP@Dextran-insulin conjugates could last insulin activity up to 12 h, while free form insulin lost its activity after 4 h. These results demonstrated that AuNP@Dextran-insulin conjugates can be potentially used as insulin controlled-release agents and therefore the activity of insulin can be prolonged. Nevertheless, further in vivo studies are required to demonstrate the practice of this approach as alternative insulin medicine.