在現今常見的疫苗療程中，病人需要藉由多次性施打疫苗來引發一個強健的保護性免疫反應。在發展中的國家因為其醫療保健資源本身有限，在接種疫苗的過程中時常面臨許多阻礙。因此，通過控制蛋白質藥物的釋放來開發一個單次施打疫苗，模擬現今醫療上常見的初次免疫/加強免疫的設計方式，將會是一種可行的改善方式。
在本文中，將鄰苯二酚官能化的透明質酸水膠(HA-CA hydrogel)和內部包載卵清蛋白(OVA)之三甲基化幾丁聚醣奈米粒子(TMC/NPs)所製備而成的奈米複合水膠系統(NPs-Gel)，可以作為一個單次施打疫苗。在小鼠皮下接種奈米複合水膠後，原先包裹在水膠中但與水膠間沒有任何共價鍵或其他特異性相互作用的奈米粒子會迅速釋放，被水膠周遭的抗原呈現細胞所吞噬，可做為引發抗原特異性體液免疫反應的初始引發劑量；通過共價鍵或靜電相互作用力而固定在水膠內的奈米粒子，可被募集而來的抗原呈現細胞吞噬後，作為提升免疫反應的加強免疫劑量。
實驗結果顯示，接種單次施打奈米複合水膠能產生顯著的抗原特異性免疫球蛋白。證實本文所開發的奈米複合水膠系統可以做為一個有效的單次施打疫苗，進而引發強效且持久的體液免疫反應。

In developing countries, vaccination is hampered mainly due to the limited access to healthcare to receive multiple doses of vaccine that is necessary for protective immunity. Thus, development of single-injection vaccine that can mimic the prime/boost vaccine regimens by controlling the delivery of protein can be an alternative approach.
Herein, a nanocomposite hydrogel system (NPs-Gel) that is prepared by entrapment of ovalbumin (OVA) loaded N-trimethyl chitosan nanoparticles (TMC/NPs) inside the catechol-functionalized hyaluronic acid hydrogel (CA-HA hydrogel) is proposed as a single-dose auto-booster vaccine. Upon subcutaneous vaccination, the NPs encapsulated in the hydrogel without any covalent linkage or other specific interactions are released rapidly, serving as an initial priming dose for eliciting antigen-specific humoral immune responses. The NPs that are immobilized inside the hydrogel through covalent or electrostatic interactions are ingested by the recruited antigen-presenting cells (APCs), acting as auto-booster doses for sustaining immune response.
The analytical data indicate that a single injection of nanocomposite hydrogel can induce a significantly higher antigen-specific IgG immune response. And these results suggest that nanocomposite hydrogel system developed herein can served as an effective single-injection vaccine for triggering long-lasting humoral responses.

摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 VII
第一章 緒論 1
1.1 傳統疫苗 1
1.2 開發單次施打疫苗的重要性 1
1.3 次單位疫苗(Subunit Vaccine) 2
1.4 顆粒化疫苗(Particulate Vaccine) 3
1.5單次施打疫苗之發展 4
1.6 N-三甲基幾丁聚醣 (N-trimethyl chitosan) 5
1.7 鄰苯二酚官能化透明質酸水膠 7
1.8 研究目的與實驗設計 8
1.9 實驗流程設計圖 11
第二章 實驗材料與方法 12
2.1 實驗材料 12
2.2 N-三甲基幾丁聚醣的製備 12
2.3 N-三甲基幾丁聚醣奈米粒子的製備 13
2.4 接枝鄰苯二酚之透明質酸的製備 14
2.5 奈米複合水膠的製備 15
2.6 藥物釋放檢測 15
2.7 掃描式電子顯微鏡(Scanning Electron Microscope, SEM) 16
2.8 流變儀測量 16
2.9 壓縮楊氏模量(Compressive Young’s Modulus) 16
2.10 澎潤度(Swelling ratios, Qs) 17
2.11 細胞毒性測試 17
2.12 實驗動物 18
2.13 骨髓衍生樹突狀細胞(BMDCs)的培養 18
2.14 骨髓衍生樹突細胞吞噬實驗 19
2.15 共軛焦顯微鏡(Confocal Laser Scanning Microscopy, CLSM) 20
2.16 骨髓衍生樹突細胞活化實驗 21
2.17 細胞浸潤實驗 22
2.18 奈米複合水膠的降解及毒性實驗 22
2.19 抗原特異性抗體反應 23
2.20 統計分析 23
第三章 實驗結果與討論 24
3.1 N-三甲基幾丁聚醣奈米粒子之材料特性及製備 24
3.2 鄰苯二酚官能化透明質酸水膠之材料特性及製備 27
3.3 奈米複合水膠之成分對於N-三甲基幾丁聚醣奈米粒子釋放之影響 30
3.4 奈米複合水膠的特性 33
3.5 奈米複合水膠的細胞毒性實驗 38
3.6 骨髓衍生樹突狀細胞吞噬N-三甲基幾丁聚醣奈米粒子的實驗 39
3.7 奈米複合水膠刺激活化骨髓衍生樹突狀細胞之能力 41
3.8 奈米複合水膠系統在活體試驗中募集細胞浸潤之探討 43
3.9 奈米複合水膠在體內降解的情況及生物體毒性實驗 46
3.10 奈米複合水膠系統引發卵清蛋白抗原特異性抗體免疫反應 48
第四章 結論 51
參考文獻 52

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