多形性膠質母細胞瘤(Glioblastoma multiforme, GBM) 被視為中央神經系統中最具侵略性的惡性癌症，五年存活率僅不到5%。目前臨床測試證實以免疫療法治療多形性膠質母細胞瘤確實能提升存活率，然而僅能達到有限的治療效果，可歸因於腦微血管中的緊密組織—血腦屏障(blood brain barrier, BBB)，血腦屏障會限制藥物的遞送效果並降低其穿透腫瘤的能力。一氧化氮(nitric oxide, NO)不僅在血管擴張中扮演了重要的角色，也能夠阻斷血腦屏障，進而促進血腦屏障的通透性。許多研究指出同時遞送抗癌藥物以及一氧化氮載體能誘導瓦解血腦屏障並增加藥物累積於腫瘤中，進而提升治療的效率，因此我們開發一種靶向脂質磷酸鈣(lipid-calcium-phosphate, LCP)奈米粒子，並在奈米粒子中搭載一氧化氮載體以及細胞程式死亡配體-1 (programmed cell death-ligand 1, PD-L1)小分子干擾核糖核酸(small interfering RNA, siRNA)，並於奈米載體表面修飾能辨識CXCR4的胜肽以提高標靶效率，透過一氧化氮來調控血腦屏障以及腫瘤內的血管，並透過此奈米載體遞送免疫檢查點抑制劑PD-L1 siRNA至腫瘤，抑制PD-L1的表現，進而活化胞毒性T細胞並增加胞毒性T細胞浸潤於腫瘤，藉此達到抑制腫瘤的增長。本研究所開發之新型能協同遞送一氧化氮及PD-L1 siRNA之CXCR4靶向脂質磷酸鈣奈米粒子，將有望做為多形性膠質母細胞瘤的高潛力臨床免疫療法策略。

Glioblastoma multiforme (GBM) is one of the most aggressive malignant cancers of the central nervous system. The five-year survival rate was less than 5%. Immunotherapy is considered as the promising strategy to treat GBM; however, only the limited therapeutic effect can be achieved. The limited effect can be attributed to the presence of blood-brain barrier (BBB). Blood-brain barrier is the tight junction structure of the brain, which can protect the brain from unnecessary or toxic compounds. Unfortunately, BBB hinders the penetration of most of drugs. Nitric oxide (NO) not only plays an important role in vasodilation but modulates the BBB. NO can help open the BBB and improve the penetration of cargoes into GBM tumors. Therefore, we developed a CXCR4-targeted lipid-calcium-phosphate nanoparticle with NO donors (LCP-NO NPs) as an immunotherapeutic strategy. We also conjugated with CXCR4-targeted antagonist, CTCE-9908 peptide, to enhance targeting ability of nanoparticles. The delivery of NO resulted in increasing BBB permeability and enhanced gene delivery through BBB. CXCR4-targeted LCP-NO NPs could efficiently deliver gene into cancer cells. Moreover, we also encapsulated the immune checkpoint inhibitor, PD-L1 siRNA, which could silence PD-L1 expression, stimulate the immune system, activate the cytotoxic T cells, promote the infiltration of T cells into GBM cells, and suppress GBM progression. We proved that CXCR4-targeted LCP-NO NPs had significant ability for delivering siRNA into tumor cells in vivo and suppressing the tumor growth. Also, our treatment strategy could activate the immune system, promote T cells infiltration, and reduce the tumor growth. Thus, CXCR4-targeted NPs may serve as a potential immunotherapy for GBM by codelivery of NO and PD-L1 siRNA.

摘要 I
Abstract II
致謝 III
目錄 IV
圖目錄 VI
表目錄 VIII
縮寫表 IX
第一章、研究動機 1
第二章、文獻探討 3
2.1 腦癌 3
2.2 血腦屏障 4
2.3 一氧化氮 4
2.4 腫瘤逃脫免疫系統 6
2.5 免疫檢查點抑制劑療法 8
2.6 核糖核酸干擾(RNA interference, RNAi) 11
2.7 藥物遞送系統 12
第三章、實驗材料與方法 14
3.1 使用材料 14
3.2 細胞培養 14
3.3 動物實驗與腦癌模型建立 15
3.4 製備搭載siRNA的LCP-NO奈米粒子 15
3.5 LCP性質分析 16
3.6 藥物釋放測試 16
3.7 DNIC分解動力學分析 17
3.8 藥物通透性試驗 17
3.9 細胞攝取分析 17
3.10 mRNA表現量分析 18
3.11 免疫螢光染色 20
3.12 西方墨點法分析 (Western blot) 20
3.13 TUNEL細胞凋亡檢測 21
3.14 流式細胞儀(Flow cytometry)分析 22
3.15 蘇木精-伊紅染色 (Hematoxylin and Eosin 22
staining, H&E staining) 22
3.16 TEER測量 23
3.17 毒性測試 23
3.18 統計資料 23
第四章、實驗結果與討論 25
4.1 LCP以及LCP-NO奈米載體物理性質分析 25
4.2 修飾CXCR4-targated胜肽於LCP-NO奈米載體於腦癌細胞(ALTS1C1)之攝取量體外實驗 28
4.3 一氧化氮調控血腦屏障機制之體外實驗 30
4.4 PD-L1 siRNA搭載於CXCR4-targeted LCP-NO NPs對原位腦癌小鼠之組織攝取量分析 35
4.5 PD-L1 siRNA搭載於CXCR4-targeted LCP-NO NPs對原位腦癌小鼠之療效測試 38
4.6 PD-L1 siRNA搭載於CXCR4-targeted LCP-NO NPs的安全性測試 40
第五章、結論 42
第六章、討論與未來展望 43
第七章、參考文獻 45

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