開發降冰片烯酸酐改質膠原蛋白水膠之藥物釋放系統

Development of a drug delivery system based on carbic anhydride modified collagen hydrogels

邱梓曄(Zi-Ye Qiu)

指導教授 ： 蔡偉博

國立臺灣大學/工學院/化學工程學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202203307

摘要

近幾十年來，膠原蛋白是生物醫學應用中用途最廣泛的生物材料之一，主要是由於其在細胞外基質中的仿生和結構組成。且由於其水凝膠的成本低、免疫原性低、多功能性、生物相容性，孔洞性以及與天然細胞外基質的相似，膠原蛋白水凝膠被廣泛用於作爲藥物釋放系統。
但由於通過化學交聯或物理交聯的膠原蛋白水凝膠存在需要使用大量交聯使時間變短，機械性質較弱，交聯劑殘留而產生細胞毒性的問題，不能夠很好的作爲藥物釋放系統。因此本研究诣在開發一種基於膠原蛋白且能夠快速光交聯，機械性質較強以及低細胞毒性的水凝膠作爲藥物釋放系統。
在此研究中，通過降冰片烯二酸酐對第一型膠原蛋白進行改質。第一型膠原蛋白上的氨基在堿性環境下與降冰片烯二酸酐上的羧基進行反應，從而達到對第一型膠原蛋白的改質。其降低等電點后能夠在中性環境下溶解便於後續應用並具有降冰片烯二酸酐上的碳碳雙鍵以進行後續的光交聯。
在完成改質后，首先對降冰片烯二酸酐改質之第一型膠原蛋白進行鑒定，通過不同的鑒定方法證明了降冰片烯二酸酐成功對第一型膠原蛋白進行改質并且擁有高改質率。接著在後續水凝膠的設計中，加入二硫蘇糖醇作為交聯劑通過硫醇-烯反應來改善降冰片烯二酸酐改質之第一型膠原蛋白水凝膠的性質。後續的水凝膠性質測定中，我們使用了降冰片烯改質之第一型膠原蛋白水凝膠作爲對照組進行比較。實驗結果顯示，加入二硫蘇糖醇作爲交聯劑的水凝膠具有更小的溶脹率，更强的機械性質以及更強的抗酶降解力。
在確定加入二硫蘇糖醇作爲交聯劑的水凝膠相比純降冰片烯二酸酐改質之第一型膠原蛋白水凝膠有著更好的性能后，對加入二硫蘇糖醇的水凝膠進行作爲藥物釋放系統的能力進行了測試。通過搭載抗生素，對加入不同濃度的二硫蘇糖醇作爲交聯劑的水凝膠進行藥物釋放測試。結果顯示，有兩組水凝膠的藥物釋放速率可以有效的減緩，可以達到更長效的抗菌效果。之後選取這兩組水凝膠進行抗菌實驗。結果顯示，搭載此藥物的水凝膠能夠很好的發揮抗菌作用。
最後我們對此種水凝膠的細胞毒性進行測試，結果顯示加入二毫摩爾每升二硫蘇糖醇的降冰片烯改質之第一型膠原蛋白水凝膠具有良好的生物相容性。因此，此研究認爲降冰片烯二酸酐改質之第一型膠原蛋白之水凝膠具有作爲藥物釋放系統的潛力并且可以在後續的研究中通過改變交聯劑和搭載的藥物對此種水凝膠作爲藥物釋放系統的潛力進行進一步的研究。

關鍵字

膠原蛋白 ； 降冰片烯二酸酐改質之膠原蛋白 ； 二硫蘇糖醇 ； 硫醇-烯反應 ； 水凝膠之藥物釋放系統

並列摘要

Collagen has been one of the most versatile biomaterials for biomedical applications in recent decades, mainly due to its biomimetic and structural composition in the extracellular matrix. Collagen hydrogels are widely used as drug delivery systems due to their low cost, low immunogenicity, versatility, biocompatibility, porosity, and similarity to natural extracellular matrices.
However, due to the problems of chemical cross-linking or physical cross-linking of collagen hydrogels: a large amount of cross-linking agent is required to shorten the cross-linking time, the mechanical properties are weak, and the cross-linking agent remains and causes cytotoxicity, so it not suitable for used as a drug release system. Therefore, this research aims to develop a collagen-based hydrogel with fast photo-crosslinking, strong mechanical properties and low cytotoxicity as a drug delivery system.
In this study, collagen type I was modified by carbic anhydride. The amino group on the collagen type I reacts with the carboxyl group on the carbic anhydride in a alkaline environment, so as to achieve the modification of the collagen type I. After lowering the isoelectric point, it can be dissolved in a neutral environment for followed application and has a vinyl group on carbic anhydride for followed photo-crosslinking.
After completing the modification, the collagen type I modified by carbic anhydride was characterized first. Different detection methods proved that carbic anhydride successfully modified the collagen type I and had a high degree of substitution. Then, in the design of the subsequent hydrogel, dithiothreitol was added as a cross-linking agent to improve the properties of the carbic anhydride modified collagen type I hydrogel through thiol-ene reaction. In the subsequent determination of hydrogel properties, we used the carbic anhydride-modified collagen type I hydrogel as a control group for comparison. The experimental results show that the hydrogel with dithiothreitol as a cross-linking agent has a smaller swelling ratio, stronger mechanical properties and stronger resistance to enzymatic degradation.
After confirming that the hydrogel added with dithiothreitol as a cross-linking agent has better performance than the pure carbic anhydride-modified collagen type I hydrogel, the performance of the hydrogel added with dithiothreitol was determined. The ability of the hydrogel to perform as a drug delivery system was tested. By carrying antibiotics, the hydrogels with different concentrations of dithiothreitol as cross-linking agents were tested for drug release. The results show that the drug release rate of two groups of hydrogels can be effectively slowed down, and a longer-lasting antibacterial effect can be achieved. Then these two groups of hydrogels were selected for antibacterial experiments. The results show that the hydrogel loaded with this drug can show a good antibacterial effect.
Finally, we tested the cytotoxicity of this hydrogel, and the results showed that the norbornene-modified type I collagen hydrogel with 2 mM dithiothreitol had good biocompatibility. Therefore, this study shows that the carbic anhydride modified collagen type I hydrogel has the potential as a drug delivery system and can try to change the cross-linking agent or the loaded drug for further investigation of the potential of carbic anhydride modified collagen type I hydrogel as the drug delivery system.

並列關鍵字

Collagen ； Carbic anhydride modified collagen ； Dithiothreitol ； Thiol-ene reaction ； Hydrogel drug delivery system

參考文獻

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