胰島素插入晶格間位之氫氧基磷灰石作為蛋白質藥物傳遞釋放模型應用於糖尿病治療

Abstract

蛋白質等大分子藥物是未來醫藥的重點發展方向，目前此類藥物皆以注射為主。能夠以病人更易接受的方式給藥，將是大分子藥物能否成為未來藥物傳輸系統主流趨勢的要件。本研究目的在於發展出新穎的材料載藥模型—胰島素插入晶格間位之氫氧基磷灰石(insHAP)，以陶瓷本身晶體結構載蛋白質藥物，並探討氫氧基磷灰石作為胰島素載體材料之特性。實驗主要分為材料研發、材料特性測試、載藥量測試、藥物釋放測試、體外細胞實驗與初步之動物實驗。為了避免氫氧基磷灰石合成之高溫環境造成胰島素變性，本研究發展出在室溫操作條件下利用單一步驟之二合水磷酸氫鈣水解法製備insHAP。insHAP粒徑分佈於865~1797 nm。透過insHAP之XRD分析、FTIR分析、SEM與TEM觀察，證實胰島素確實插入氫氧基磷灰石晶格間位。以TGA分析得到insHAP因胰島素分解所損失之重量為11.25%。BCA計算得insHAP載藥量為13.33%，有效包覆率為86.08%。藥物釋放測試顯示insHAP於鹼性環境模擬一般體液情況下僅釋放少量胰島素，於酸性環境模擬細胞溶小體內情況下氫氧基磷酸石很快被降解，胰島素大量被釋出。由WST-1與LDH試驗結果顯示insHAP不影響細胞增生與存活，也不會對細胞造成顯著毒性。TEM也觀察到含有insHAP之細胞溶小體，證實細胞會攝入insHAP。動物實驗顯示insHAP以高劑量肌肉注射於STZ誘導糖尿病之Wistar大鼠，血糖緩慢下降並能維持4日正常血糖。綜合以上結果， insHAP可作為胰島素傳遞釋放模型，此新型的藥物傳遞系統模型於未來將有潛力應用於插層螢光蛋白、抗體、生長因子等生物製劑，或插層標靶治療癌症藥物達到靶向效果，有機會成為新的藥物遞送系統選擇。

Protein and other macromolecular drugs have great potentials for the future development of medicine. However, most of these drugs are dosed by injection. It would become main trend if way of dosing macromolecular drugs can be more receptive to the patient. The purpose of this study is to develop a novel model of insulin-inserted hydroxyapatite (insHAP) and to prove whether crystal structure of hydroxtapatite could carry protein drugs. The experiment was divided into five parts: material synthesis and property testing, drug loading test, drug release test, in vitro studies and preliminary animal study. In order to avoid insulin denaturation, we develop a single step insHAP synthesis by hydrolysis of brushite (DCPD). insHAP size distribution is in the range of 865~1797 nm. Through XRD analysis, FTIR, SEM and TEM observations, we confirm that insulin is indeed inserted into hydroxyapatite lattice. TGA analysis shows the weight loss due to insulin decomposition is about 11.25%. BCA essay calculated drug loading of insHAP is 13.33% and drug entrapment efficiency is 86.08%. The drug release tests show insHAP only releases a small amount of insulin in the alkaline environment, which simulated the environment of general body fluids. On the other hand, hydroxyapatite was quickly degraded and had a great number of insulin release in the acidic environment, which simulated the environment inside lysosomes. In in vitro studies, WST-1 test shows there is no negative effect on cell viability and cell proliferation. The relatively low cytotoxicity of insHAP is proven by LDH test. Furthermore, we confirm by Cell TEM that insHAP could go through cellular uptake and get into lysosomes. Animal study indicates the blood glucose of STZ-induced diabetic Wistar rats after intramuscular injection of insHAP decreases slowly. It can maintain normal blood sugar for about 4-day period. Based on the above results, insHAP as insulin delivery model is a breakthrough for diabetes treatment. This novel drug delivery model will be potential in the future for carrying not only fluorescent protein, antibody, growth factor and other biological agents, but also cancer drugs for targeted therapy. This study offer s a new choice for protein drug delivery.