利用幾丁聚醣支架與聚乳酸微球體進行軟骨素分解酵素之控制釋放

Abstract

脊髓受傷時，被活化的星狀細胞會移形至受傷處並分泌細胞外基質，這些細胞外基質被稱為疤痕組織，會阻礙新生的神經細胞與軸突的生長並阻礙神經修復。軟骨速分解酵素(Chondroitinase ABC, ChABC) 已被證實可分解疤痕組織中醣蛋白結構的醣鏈，有助於神經細胞與軸突的向外生長，產生功能性的回覆並幫助神經修復。有鑒於ChABC的不穩定，並且需要長時間使用ChABC才可有效分解醣鏈，因此探討以固定化酵素以及雙層乳化法維持並延長ChABC活性並將設計孔徑大小不同的神經導管作為固定化之材料。 本研究利用幾丁聚醣以及明膠設計出孔隙大小不同的神經導管，幾丁聚醣神經導管的孔徑大小為100~160μm，而幾丁聚醣/明膠神經導管孔徑大小為20~40μm。以此二種神經導管做為固定化之材料，並將ChABC以離子鍵結以及共價鍵結進行固定化。實驗結果顯示，ChABC以幾丁聚醣神經導管為材料透過離子鍵結進行固定化可得0.07 U/mg，並且48小時後仍可維持48% 的活性。顯示固定化酵素確實有助於維持ChABC活性。以雙層乳化法將ChABC包覆於PDLLA製作微球體並進行控制釋放。7天內所釋放出的ChABC活性均小於0.0026 U/ml。以nanogold(10 nm)、polylysine (Mw:500-2000)與polylysine (Mw:20000 -30000)為安定劑均可提高ChABC活性，其中以polylysine (Mw:500-2000)保護ChABC之效果最佳，7天後活性可達到0.0162 U/ml。顯示利用雙層乳化法並搭配適當的保護劑所製作之微球體，確實可達到控制釋放的效果，使ChABC活性得以延長。

In spinal cord injury, activated astrocytes migrated to the injury site and secreted the extracellular matrix (ECM), called scar tissue. The scar tissue impeded the re-growth of neurons and axon and inhibited nerve regeneration. It had proved that Chondroitinase ABC can digest the polysaccharide of proteoglycan in scar tissue, promoted the re-growth of neuron, axon and behavior recovery. In order to improve the stability of chondroitinase ABC, and maintain the activity of chondroitinase ABC, the feasibility of using immobilized chondroitinase ABC on nerve conduit and encapsulated chondroitinase ABC in PDLLA microsphere were studied further in this work. Chitosan and gelatin were used to fabricate the conduit with different pore diameter. The pore diameter of chitosan conduit was 100-160 μm, and the pore diameter of chitosan/gelatin conduit was 20-40μm. Chondroitinase ABC was immobilized on surface of the conduit by ionic binding and covalent binding. The activity of chondroitinase ABC immobilized on chitosan conduit by ionic binding was 0.07 U/mg, and the activity recovery was 48% after 48 hours. The stability of the immobilized chondroitinase ABC was significantly improved. PDLLA microsphere was fabricated through double emulsion and controlled release of chondroitinase ABC. Activity of chondroitinas ABC released from microsphere without stabilizer was lower than 0.0026 U/ml during seven days. The results indicated that the microsphere made by double emulsion and added the optimal stabizer can increase the efficacy of the control release and the duration of chondroitinase ABC activity.