本論文為研發一種可撓式快速溶解微針 (MN) 貼片應用於 DNA 疫苗經皮輸送和增強穩定性之微針系統，是由可快速溶解、生物相容性、成膜性極好的聚乙烯醇 (Polyvinyl alcohol, PVA) 作為微針的主材料，此微針穿刺皮膚後，能夠藉由體內的組織液將微針結構快速溶解釋放出疫苗，通過改變 PDMS 親疏水性質，可以讓疫苗更均勻的塗佈在 PDMS 模具上，微針包覆 PCV2 疫苗，藉由微針製程上的不同，其包覆率最高可到達 87.8 %，包覆量最高可達 22.4 μg，我們由穩定性試驗得知，疫苗包覆在微針中儲存在37 ℃ 下可維持 111 天而不會有任何損壞。由體外試驗證實我們所設計之微針型態具有足夠之機械強度穿刺於角質層，且微針在 10 分鐘內即可溶解於皮膚中讓疫苗釋放在富含抗原呈現細胞之表皮層，且微針穿刺皮膚之痕跡可在 40 分鐘內迅速癒合，避免傷口感染之風險。動物試驗證實在接種後 3 週，微針相較於肌肉注射 (IM) 產生之抗體效價量相差 10 倍，此結果證實微針在人體中可誘發高抗體濃度反應，引發免疫後的最強免疫反應

This study developed a flexible and rapidly-dissolvable microneedle (MN) patch for transdermal delivery and stability enhancement of DNA vaccine. The MN patch is made by polyvinyl alcohol (PVA) with the properties of rapid dissolution, biocompatibility and film-forming. Besides, the vaccine encapsulated in MN would be rapidly released after insertion of the MN patch to the skin. We were able to encapsulate 22.4 μg of PCV2 plasmid DNA vaccine in MNs per patch with a loading rate of 87.8 wt % when casting the PCV2 plasmid DNA vaccination the PDMS mode surface. We also done the stability study, the activity of vaccine encapsulated in MNs can be maintained exceed 111 days without any damage at 37 ℃ of storage. The ex vivo study demonstrated that the MNs have excellent mechanical strength to puncture the stratum corneum and needles can be dissolved within 10 minutes to release the vaccine in epidermis then the hole can be quickly healed within 40 minutes to avoid the infection risk. The Animals studies showed that the induced antibody titers via MN patch administration were about 10-time higher than intramuscular (IM) administration after 3 weeks of vaccination. The results confirmed that the MNs can induce high antibody concentration in body to induce the strongest immune responses after immunization.

論文審定書……………………………………………………………i
誌謝……………………………………………………………………ii
中文摘要………………………………………………………………iii
英文摘要………………………………………………………………iv
目錄……………………………………………………………………v
圖目錄…………………………………………………………………ix
表目錄…………………………………………………………………xii
第一章 介紹……………………………………………………………1
1.1.一般藥物輸送劑型……………………………………………1
1.1.1.口服劑型………………………………………………1
1.1.2.注射劑型………………………………………………3
1.1.3.經皮輸送劑型…………………………………………5
1.2.經皮藥物輸送與微針貼片……………………………………6
1.2.1.經皮藥物輸送…………………………………………6
1.2.2.微針貼片………………………………………………8
1.3.疫苗……………………………………………………………13
1.3.1.疫苗起源………………………………………………13
1.3.2.不活化疫苗……………………………………………13
1.3.3.減毒疫苗………………………………………………14
1.3.4.DNA疫苗………………………………………………14
1.3.5.VLP 疫苗………………………………………………15
1.4. PCV2 介紹…………………………………………………15
1.5.經皮免疫機制………………………………………………16
1.7.研究目的……………………………………………………17
第二章 實驗材料與方法……………………………………………19
2.1.實驗藥品、耗材與動物……………………………………19
2.2.材料…………………………………………………………20
2.2.1.聚乳酸………………………………………………20
2.2.2.聚乙烯醇……………………………………………21
2.2.3.螢光染劑羅丹明 B (Rhodamine B) ………………21
2.2.4.蔗糖 (Sucrose) …………………………………22
2.3.儀器設備……………………………………………………22
2.4.可溶式微針貼片……………………………………………24
2.4.1.聚乙烯醇微針貼片之製備…………………………24
2.5.包覆 Rhodamine B 染劑之可溶式微針貼片………………26
2.5.1. 聚乙烯醇 / Rhodamine B 溶液配製………………26
2.5.2.包覆 Rhodamine B 微針貼片之製備………………26
2.5.3.包覆率測試……………………………………………27
2.6.包覆 DNA 疫苗之可溶式微針貼片…………………………28
2.6.1.GFP-plasmid DNA 之製備……………………………28
2.6.2.包覆 GFP-plasmid DNA 之可溶式微針貼片製備…29
2.6.3.包覆 GFP-plasmid DNA 之可溶式微針貼片包覆率
測試…………………………………………………………30
2.6.4.包覆 GFP-plasmid DNA之可溶式微針貼片生物
活性…………………………………………………………31
2.6.5.細胞培養與後續將 GFP-plasmid DNA 轉染進細胞
表現…………………………………………………………32
2.6.6.包覆GFP-plasmid DNA可溶式微針貼片儲存穩定性
測試…………………………………………………………33
2.6.7.PCV2-plasmid DNA 疫苗之製備……………………34
2.6.8.包覆DNA疫苗之聚乙烯醇微針貼片之製備…………35
2.6.9.包覆DNA疫苗之聚乙烯醇微針貼片包覆率實………35
2.6.10.包覆DNA疫苗之聚乙烯醇微針貼片生物活性……37
2.6.11.包覆DNA疫苗之聚乙烯醇微針貼片穩定性測試…37
2.6.12.Western blotting…………………………………38
2.7.微針穿刺能力測試……………………………………………39
2.7.1.微針機械強度試驗……………………………………39
2.8.皮膚穿刺試驗及後續組織切片染色…………………………40
2.8.1.聚乙烯醇微針針尖溶解時間與百分比測試…………40
2.8.2.體外豬皮穿刺…………………………………………40
2.8.3.活體小鼠穿刺…………………………………………41
2.8.4.皮膚組織切片…………………………………………42
2.8.5.固定組織與染H&E (Hematoxylin & Eosin) …………42
2.9.皮膚穿刺傷口癒合測試………………………………………43
2.10.小鼠免疫試驗………………………………………………44
第三章 結果與討論……………………………………………………46
3.1. PDMS 模具改性實驗…………………………………………46
3.2.可溶式聚乙烯醇微針貼片………………………………47
3.3.微針機械性質…………………………………………………49
3.4.微針針尖溶解時間與百分比分析……………………………50
3.5.微針穿刺能力分析……………………………………………52
3.6.皮膚穿刺傷口癒合分析…………………………………53
3.7.包覆染劑之可溶式微針貼片…………………………………54
3.7.1.包覆 Rhodamine B 染劑微針貼片…………………54
3.7.2.包覆率分析……………………………………………56
3.8.裝載 Plasmid DNA 之可溶式微針貼片製備………………56
3.8.1.包覆 GFP-plasmid DNA 之可溶式微針貼片…………56
3.8.2.包覆率分析……………………………………………58
3.8.3. 生物活性分析…………………………………………59
3.8.4.儲存穩定性分析………………………………………62
3.9.裝載 PCV2 疫苗之可溶式微針貼片製備…………………65
3.9.1.包覆 PCV2-plasmid DNA 疫苗之可溶式微針貼片…65
3.9.2.包覆率分析……………………………………………66
3.9.3.生物活性與儲存穩定性………………………………67
3.10.小鼠免疫試驗結果…………………………………………69
第四章 結論…………………………………………………………72
參考文獻……………………………………………………………74
圖目錄
圖1-1皮膚構造示意圖………………………………………………7
圖1-2固體與塗佈型微針……………………………………………9
圖1-3中空型微針……………………………………………………10
圖1-4高分子聚合物微針……………………………………………12
圖1-5應變性免疫反應的發展階段簡圖……………………………16
圖1-6可溶式微針製作示意圖………………………………………17
圖1-7可溶式微針應用示意圖………………………………………18
圖2-1聚乳酸化學結構圖……………………………………………20
圖2-2聚乙烯醇化學結構圖…………………………………………21
圖2-3 Rhodamine B 化學結構圖……………………………………21
圖2-4蔗糖 (Sucrose) 化學結構圖…………………………………22
圖2-5Rhodamine B 微針包覆率試驗簡圖……………………………27
圖2-6包覆DNA疫苗之聚乙烯醇微針貼片包覆率實驗簡圖…………36
圖2-7微針機械強度試驗簡圖………………………………………39
圖2-8體外豬皮穿刺簡圖……………………………………………41
圖2-9皮膚穿刺傷口癒合測試簡圖…………………………………43
圖2-10小鼠免疫試驗流程簡圖………………………………………45
圖3-1利用聚乙烯醇來改善 PDMS 表面的疏水性…………………47
圖3-2聚乙烯醇可溶式微針及不同倍率之 SEM 影像……………48
圖3-3機械強度測試…………………………………………………49
圖3-4微針針尖溶解影像圖、溶解時間與百分比……………………51
圖3-5包覆 Rhodamine B 聚乙烯醇微針穿刺豬皮之組織切片……53
圖3-6皮膚穿刺傷口癒合影像圖……………………………………54
圖3-7包覆 Rhodamine B 之聚乙烯醇微針陣列在立體顯微鏡下使用白光拍攝…………………55
圖3-8包覆 Rhodamine B 染劑之聚乙烯醇微針貼片包覆效率……57
圖3-9包覆 GFP plasmid DNA 聚乙烯醇微針陣列在立體顯微鏡下使用白光拍攝………………57
圖3-10包覆 GFP-plasmid DNA 之聚乙烯醇微針貼片包覆效率…58
圖3-11 GFP-plasmid DNA 包覆在聚乙烯醇微針與 Naked GFP-plasmid DNA 放置前與放置在 37 ℃ 烘箱中 6 天之凝膠電泳圖………………………………………………………………………60
圖3-12 GFP-plasmid DNA 包覆在聚乙烯醇微針與 Naked GFP-plasmid DNA 放置前與放置在 37 ℃ 烘箱中 6 天，觀察兩者轉染進細胞 (HEK-293) 之 GFP-plasmid DNA 之生物活性…………61
圖3-13 GFP-plasmid DNA 包覆在聚乙烯醇微針中放置在 37 ℃ 烘箱中進行儲存穩定性試驗……………………63
圖3-14包覆 PCV2-plasmid DNA 之聚乙烯醇微針陣列在
立體顯微鏡下使用白光拍攝…………………………………………65
圖3-15包覆 PCV2-plasmid DNA 之聚乙烯醇微針貼片包覆率……………………………………………66
圖3-16微針裝載 PCV2-plasmid DNA 與 Naked PCV2 plasmid DNA 放置於 37 ℃ 烘箱不同天數，藉由 Western blotting 觀察 PCV2 plasmid DNA 之儲存穩定性…………………………………………68
圖3-17小鼠以 PCV2-plasmid DNA 疫苗免疫試驗產生抗體之結果…………………………71
表目錄
表3-1小鼠免疫試驗之結果……………………71

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