攜帶simvastatin之控制徐放型藥物載體促進骨缺損修復之研究

Development of control-released simvastatin drug carriers for bone repair

陳建勳(Jian-Xun Chen)

指導教授 ： 何美泠

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2010年)

摘要

中文摘要
前言:
Simvastatin，是hydroxy-3-methylglutaryl coenzyme A(H- MG-CoA)還原酶抑制劑，為臨床上常用的降血脂藥物。近年來，一些活體內與活體外研究指出simvastatin具有促進骨生成的效果。而本實驗室過去活體外研究發現simvastatin可以促進骨母細胞的增生，並誘導人類骨髓幹細胞走向骨化作用。但是，許多研究指出simvastatin以全身性地給予方式對於促進骨生成的效果仍無一致的結果，原因在於無法長時間的維持simvastatin在骨骼週圍的有效促骨化濃度。因此，本研究擬利用適合量產的技術製成具有骨誘導性，可控制釋放simvastatin的藥物載體，以局部性的給予方式應用於骨缺損之修復。
材料與方法:
本研究使用藥物載體製備技術:噴霧造粒(spray drying)，製造微小球劑型simvastatin/PLGA/HAp載體，分成兩個組別[S-0(0mg HAp)、S-100(100mg HAp)]。外一方面，由新加坡大學利用電紡織(eletrospining)製造的纖維劑型simvastatin/PLGA/HAp載體，分成兩個組別[F-0(0mg HAp)、F-200(200mg HAp)]，纖維劑型與微小球劑型均個別進行體外釋放測試，並利用高效能液相層析儀偵測藥物載體的simvastatin包覆率、藥物載體釋放simvastatin的趨勢與濃度。微小球劑型藥物載體須進行粒徑分析，與利用掃描式電子顯微鏡觀察材料表面型態。體外活性測試:使用D1細胞(BALB/c mouse bone marrow mesenchymal stem cell) ，檢測載體之藥物釋放對骨髓間質幹細胞活性之影響，以及對於骨髓間質幹細胞的促骨化(osteogensis)程度。最後，在活體內測試中，我們使用BALB/c mouse，大範圍骨缺損模式(2 mm defect)，手術後二、四週，以放射影像觀察骨頭癒合情形，並以組織學評估骨缺損部位的骨痂生成，細胞生長於lacunae之比率，血管新生的情形。
結果與討論:
利用噴霧造粒製造的simvastatin/PLGA微小球，其粒徑分佈為:S-0組的粒徑範圍是111±20 um、S-100組的粒徑範圍是126±14 um，且SEM影像顯示微小球表面平滑。根據體外釋放的結果顯示，每毫克的藥物載體每日釋放劑量範圍，S-0:0.88~2.3uM，S-100:0.77~0.32uM，F0:1.2 ~ 4.1 uM，F-200:0.8~3.9uM且均能控制釋放simvastatin持續至少兩週。在細胞活性測試中，給予F-0、F-200的組別與控制組相比，顯著性地降低細胞活性，與1uM simvastatin組相比並無顯著性差異。S-0(2mg)、S-100(10mg) 與控制組相比，顯著地降低細胞活性。在促進細胞之骨礦質化測試 給予纖維劑型與微小球劑型的組別均能顯著地增加D1細胞之骨礦質化的程度。動物實驗結果指出，手術後二、四週，給予藥物載體的兩個組別(F-0、F-200)，其骨基質生成、骨細胞新生、血管新生都有顯著性的增加。而給予藥物載體的兩個組別(F-0、F-200)之間並無顯著性的差異。我們的研究結果證實了利用電紡織製作的simvastatin/PLGA纖維劑型載體，具有控制釋放simvastatin的特性，且所釋放的simvastatin仍具有促骨化活性並達到促進骨缺損修復的作用。而simvasta- tin/PLGA微小球劑型載體，亦具有控制釋放simvastatin的特性，且在活體外活性測試中證明，所釋放的simvastatin仍具有促骨化活性並達到促進骨化的作用。

關鍵字

控制釋放 ； 藥物載體 ； 骨修復

並列摘要

Abstract
Introduction:
Simvastatin, a hydroxy-3-methylglutaryl coenzyme A (HMG -CoA) reductase inhibitor, is a common clinical use cholesterol lowering drug. Recent reports indicated that simvastatin had a potent stimulating effect on osteogensis in vitro and in vivo. Our previous study also demonstrated that simvastatin stim- ulates osteoblast proliferation and enhances osteogenesis of hBMSCs. However, the systemic administration of simvastatin showed diverse and controversial effect on osteogensis because it’s difficult to maintain the therapeutic concentration of simva- statin for a long time around the defect site. Therefore, this study is to develop the drug loaded carriers with the properties of controlled releasing simvastatin and with osteogensis effect
Materials and Methods:
Simvastatin encapsulated PLGA were prepared by electrospining (fibrous carrier) and spray drying (microsphere). Fibrous carrier included two groups: [F-0(simvasatatin /PLGA /0 mg HAp),F-200 (simvasatatin/PLGA /200 mg HAp)]. Micro-sphere included two groups: [S-0(simvasatatin/PLGA/0mgHAp), S-100 (simvasatatin/PLGA/ 100mg HAp)].Microspheres was additionally characterized the surface morphology by a scanning electron microscope(SEM) and the size distribution by a particle size analyzer. The releasing profile and encapsulate efficiency of carriers were analyzed by HPLC (High performance liquid chromat- ography). To elevate the effect of carriers in vitro, we tested cell viability by MTT assay and mineralization by Alizarin red staining on D1 cells (mouse bone marrow mesenchymal stem cell). In vivo study, we tested the effect of carriers on bone repair with a mouse model of bone defect grafted with a necrotic bone. We evaluated the bone repair by using radiography and histology (H&E staining for callus formation, osteocyte/ necrotic-bone area and vWF staining for neo-vasculization ).
Results and conclusion:
Results from particle size analysis showed that the size distribution of microspheres are following: S-0:111±20 um、S-100:26±14 um. The SEM images showed that the surface morphology of microsphere is smooth. The results from in vitro releasing test showed daily release concentrations of simva- statin from each mg of the carrier were following : S-0:0.88~2.3uM, S-100:0.77~0.32uM, F-0:0.2~4.1uM, F-20- 0:0.8~2.9uM and sustained release of simvastatin is for 2 weeks. The results of cell vialibity test showed F-0, F-200, S-0(2mg) and S-100(10mg) significantly decrease the cell viability. The bioactivity test showed that simvastatin/PLGA fibrous carrier and microsphere significantly enhanced mineralization of D1 cell. The results of in vivo test further showed that simvastatin /PLGA fibrous carriers (F-0 and, F-200) increased callus formation, the rate of cell grown in the grafted and neo-vasculization. Our results illustrated the feasibility of using simvastatin/PLGA fibrous carriers with controlled-release property not only sustainedly release the constant concentration of simvastatin ,but also enhanced osteogensis in vitro and in vivo. Simvastatin/ PLGA microspheres with contro- lled-release property that can sustained release the constant concentration of simvastatin and enhanced osteogensis in vitro.

並列關鍵字

control-released ； simvastatin ； drug carriers ； bone repair

參考文獻

參考文獻

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