硫酸化軟骨素-聚己內酯共聚物之製備與分析

聚已內酯（Polycaprolactone，PCL）為聚酯類，結構中具有酯鍵，因此能被酵素分解，在人體中也可被循環代謝，同時也被證明是具有良好的生物相容性的材料之ㄧ，對於藥物通透性良好，我們期望能以聚己內酯作為包覆藥物的載體材料之ㄧ。由於聚己內酯疏水性高，而且結構具有較高的結晶性，為了修飾聚己內酯的疏水性質和結晶性質以利於藥物釋放過程，我們利用硫酸化軟骨素（Chondroitin sulfate，CS）加以修飾，硫酸化軟骨素為天然高分子單體，分布於哺乳類動物的細胞間質之間，具有相當良好的親水性質以及生物相容性，作為藥物載體的基質材料可以期望會增加微胞的親水性和避免被體內的免疫系統辨識。我們將聚己內酯和硫酸化軟骨素分別用雙鍵修飾後，透過自由基聚合反應組成共聚物，命名為CXPY，X及Y分別表示CS以及PCL之進料重量比例。共聚物以紅外線光譜儀（FT-IR）、核磁共振光譜儀（NMR）、凝膠滲透層析儀（GPC）及熱差示掃描分析儀（DSC）分析，並透過一次乳化（simple emulsion）的方式讓共聚物自組裝成為微胞並以螢光試劑進行臨界微胞濃度（Critical micelle concentration，CMC）測量，CMC值為1.17×10-4-8.86×10-3 mg/ml。以穿透式電子顯微鏡（TEM）來觀察微胞型態，微胞呈現為均勻的球形，而組成的微胞溶液經由動態光散射法（DLS）測量其粒徑大小都在300 nm以下；於C1P10於生理緩衝之下測試微胞在15天內粒徑也沒有產生變大的情形。　　微胞對於蛋白質藥物的包覆和釋放情形則是以小牛血清蛋白（Bovine serum albumin，BSA）作為模型蛋白藥物；包覆率（Entrapment efficiency）可達80 %以上；在藥物釋放實驗中，未添加酵素的情況下，被微胞包覆的BSA不會被釋放出來；而在添加Esterase與Chondroitinase ABC之下，於50天內則有近80 %的釋放量。在細胞實驗中，以MTT assay進行細胞毒性測試，結果顯示材料並無顯著毒性。利用FITC做為螢光基團來修飾BSA（FITC-BSA），用來追蹤微胞包覆FITC-BSA體外細胞攝取探討，並與只投入FITC-BSA作為對照組，透過雷射共軛焦顯微鏡（Laser Scan Confocal Microscope）觀察，結果顯示以微胞乘載的實驗組其細胞有較多的攝取量。

關鍵字

微胞；藥物載體；硫酸化軟骨素；聚己內酯

並列摘要

Polycaprolactone (PCL) is one of the polyesters, which are biodegradable due to the ester bonds in the structure that can be degraded by enzymes in human body. PCL has been proven to be a biocompatible material and high permeability for drugs. Thus we used it in our drug delivery system. PCL is highly hydrophobic and crystalline. In order to promote the drug-release efficiency, we use chondroitin sulfate (CS) to compensate these characters. CS, a natural polymer in the mammals’ extracellular matrix (ECM), is highly hydrophilic and performs a good biocompatibility. The drug carriers made of CS is expected to increase the hydrophilicity and avoiding to be recognized by the immune system in human body. First, we modified the PCL and CS structure with double bond, and copolymerized the two materials through the free radical reaction. The copolymers were named as CXPY, X was the feed weight ratio of CS and Y was the feed weight ratio of PCL. The copolymers were analyzed with Fourier transform infrared spectrophotometer (FT-IR), 1H-NMR, gel permeation chromatography (GPC), differential scanning calorimeter (DSC). Then we prepared the micelles with copolymers through a simple emulsion. With the fluorescence probe techniques, we calculated the critical micelle concentration (CMC) was in the range of 1.17×10-4-8.86×10-3 mg/ml. Under transmission electron microscope (TEM), the profiles of the micelles were spherical. By dynamic light scattering (DLS), the size of micelles were all under 300 nm, and didn’t aggregate nor change in 15 days in double deionized. water. In the drug-loading test, we used bovine serum albumin (BSA) as a model protein. The entrapment efficiency was above 80 %. In the drug-release test, the micelles released a few BSA content with adding esterase or without any enzymes. In contrast, the micelles released 80 % BSA in 50 days in the presence of both esterase and chondroitinase ABC. In cell experiment, we chose the MTT assay to examine biocompatibility of the copolymers, which didn’t show remarkable cytotoxicity. The BSA was labeled with FITC (FITC-BSA) in order to examine the cellular uptake of the FITC-BSA-loaded micelles. We used the FITC-BSA as a control. With the use of confocal microscopy, the internalized micelles were more than the control.