以溫和水相法快速封裝酵素及合成鋁基底金屬有機骨架材料用於口服藥物傳輸之研究;A Mild Water-based Approach for Obtaining Encapsulation of Enzyme into Aluminum-based Metal-Organic Frameworks for Oral Drug Delivery Research

Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/92044

Title:	以溫和水相法快速封裝酵素及合成鋁基底金屬有機骨架材料用於口服藥物傳輸之研究;A Mild Water-based Approach for Obtaining Encapsulation of Enzyme into Aluminum-based Metal-Organic Frameworks for Oral Drug Delivery Research
Authors:	黃莉雯;Huang, Li-Wen
Contributors:	化學學系
Keywords:	鋁基底有機骨架材料;NH2-MIL-53(Al);酵素固定化;生物複合性材料;過氧化氫酶
Date:	2023-08-14
Issue Date:	2024-09-19 14:47:57 (UTC+8)
Publisher:	國立中央大學
Abstract:	金屬有機骨架材料相比於傳統無機孔洞材料如微孔的沸石，具高比表面積和優異的熱穩定性。同時且金屬有機骨架材料具有實現孔洞以及結構的多樣性，因此近年來被廣泛應用於分離、催化、氣體儲存和藥物傳遞等領域。鋁是一種便宜、資源充足且其鹽類為無毒之金屬，此外，鋁奈米粒子（Al nanoparticles），具有極佳的生物安全性以及生物相容性，而以鋁為基底的金屬有機骨架材料具出色的熱穩定性和生物相容性，使其成為理想的疫苗佐劑及生物分子載體。酵素在醫學方面有多種運用，可以治療或輔助治療多種疾病，如：代謝缺陷（Metabolic deficiencies）、癌症和心血管疾病等，且具有高效催化和專一的反應能力，但由於酵素在催化完畢後難以與產物在反應溶液中分離，並且在嚴苛的環境下會失去活性，在實際應用上存在許多限制，因此，增加酵素穩定性和實用性的酵素固定化顯得尤其重要。若酵素作為口服藥物，路徑必須先經過胃，因此，若想發展酵素利用口服的方式在小腸中作用，必須要有適當載體，能夠保護酵素，通過胃強酸的環境，同時避免被蛋白酶水解。本論文成功地在溫和水相條件下於20分鐘內快速合成鋁基底金屬有機骨架材料NH2-MIL-53，並成功地封裝過氧化氫酶（Catalase, CAT）於其中，CAT@NH2-MIL-53。該合成過程中沒有使用有機溶劑，不僅可使酵素保持活性，更符合現今對綠色化學的提倡。同時利用X光粉末繞射探討NH2-MIL-53合成最適條件，且藉由跑膠（SDS-PAGE）及等溫氮氣吸/脫附結果證明過氧化氫酶被包覆於NH2-MIL-53中。CAT@NH2-MIL-53利用金屬有機骨架材料的孔洞性質，CAT受到孔洞材料的保護並允許受質（H2O2）進入NH2-MIL-53進行催化反應，防止被大分子蛋白質水解酶（Proteinase K）分解，得到活性（kobs：6.2 ×10-2 s-1），相比於先前本實驗室於CAT@ZIF-90活性（kobs：2.5 ×10-2 s-1），活性增加2.5倍。而我們對CAT的活性趨勢做出推論：隨著合成時間的拉長，CAT的活性隨之下降；隨著合成金屬比例的提高，活性亦會隨之下降。NH2-MIL-53可耐酸性至約pH 1.5，在PBS緩衝溶液 (Phosphate Buffered Saline pH 7.4) 會崩解，而我們模擬CAT@NH2-MIL-53在胃的環境中（浸泡在pH 2鹽酸水溶液含胃蛋白酶），CAT依然保有活性（kobs：1.6 ×10-2 s-1），意即NH2-MIL-53能夠保護CAT抵抗較為嚴苛的環境，提供一種口服藥物之傳遞路徑。 ;Metal-Organic Frameworks (MOFs), recognized for their high surface area and thermal stability, allow for diverse pore structures. This versatility has propelled their use in fields like gas storage and drug delivery. Particularly, aluminum-based MOFs, due to their inherent biocompatibility and the immune-enhancing properties of aluminum, have emerged as promising candidates for vaccine and protein delivery. Enzymes, prized for their high catalytic efficiency and specificity, are key in treating diverse diseases like metabolic deficiencies, cancers, and cardiovascular diseases. However, their effectiveness in oral administration is hampered by deactivation in harsh environments like the stomach′s acidity. This underscores the necessity of enzyme immobilization to boost their stability and practical application. The carrier shields enzymes or proteins from hydrolysis and aids their absorption via the intestinal microvessels, thus enabling their entry into the bloodstream and systemic circulation. In our research, we synthesized the Al-based Metal-Organic Framework (MOF) NH2-MIL-53(Al) under mild aqueous conditions in 20 minutes. We also encapsulated the enzyme catalase (CAT) into the MOF, creating a biocomposite named CAT@NH2-MIL-53. Encapsulation was confirmed through Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and isothermal nitrogen gas adsorption/desorption measurements. Our method preserved enzyme biological activity (kobs of 6.2×10-2 s-1) and provided proteolysis protection against proteases like Proteinase K. This activity which had a kobs of 2.49×10-2 s-1 surpasses our previous CAT@ZIF-90 research at least 2.5 fold times. Conversely, an elongated synthesis time reduces CAT activity, and an increased metal-to-linker synthetic ratio also diminishes activity. Material tolerance assays show that under gastric conditions (pH 2 hydrochloric acid solution with pepsin), the NH2-MIL-53 structure remains stable even at pH 1.5 for 3 hours. The encapsulated catalase (CAT) retains its biological activity (kobs of 1.6×10-2 s-1). Notably, NH2-MIL-53 disintegrates in PBS buffer solution (Phosphate Buffered Saline, mimicking blood conditions) at pH 7.4, indicating potential enzyme release from NH2-MIL-53 in the bloodstream.
Appears in Collections:	[Graduate Institute of Chemistry] Electronic Thesis & Dissertation

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