多形性膠質母細胞瘤 (Glioblastoma Multiforme, GBM) 到目前為止仍是無法根治的疾病。由於 GBM 本身的抗藥性及血腦屏障 (Blood-Brain-Barrier, BBB)，導致藉由血液運輸的抗癌藥物無法有效通過腦屏障並造成細胞受損凋亡。因此 ， 如何有效通過血腦屏障 (Blood-Brain-Barrier, BBB) 是進行後續研究必須克服的難題之一。在本研究中，我們使用脂質磷酸鈣 (Lipid/Calcium/Phosphate, LCP) 奈米載體 (NPs) 提供庇護以延長體內循環時間，並藉由增加血腦屏障通透性的方式，使奈米載體成功浸潤至腦內。由於 GBM 細胞在其表面過度現趨化因子 受體 (Chemokine receptor 4, CXCR4)，因而可以藉此特性來標靶 GBM。我們在 我們在奈米載體表面修飾 CTCE-9908 (Stromal cell-derived factor 1 analog)，藉由被 CXCR4 辨認以進行腦癌的標靶治療。藉由奈米載體遞送小分子干擾核糖酸 (siRNA) 或小分子核糖酸 (microRNA) 進行腦癌治療。期望藉由具標靶性的載體與遞送基因的方式有效抑制腦瘤生長 。

Nowadays, glioblastoma multiforme (GBM) is still an incurable disease. Due to the development of drug resistant and the present of blood-brain-barrier (BBB), anti-cancer drugs cannot efficiently penetrate BBB and achieve significant cytotoxicity in GBM cells. Thus, there is an urgent clinical need to develop an effective therapeutic strategy to penetrate BBB, target GBM and overcome drug resistance to suppress GBM progression. Thus, in this study, we assembled lipid/calcium/phosphate nanoparticles that could prolong the half-life time during the blood circulation and penetrate BBB to accumulate into the brain. We further modified the surface of NPs with CTCE-9908 peptides, CXCL12 N-terminus derived peptides to target CXCR4-overexpressing GBM cells. The GBM-targeted NPs efficiently delivered the RNA cargoes (siRNA/miRNA) into GBM. We expect that the therapeutic siRNA/miRNA delivered by GBM-targeted NPs may efficiently suppress tumor growth in murine GBM models.

摘要..............................................................2
Abstract.........................................................3
Abbreviation.....................................................4
Table of Contents................................................7
Table of Figures.................................................9
Motivation and aims.............................................10
Introduction....................................................12
2.1 Glioblastoma multiforme.....................................12
2.2 Blood-Brain-Barrier.........................................13
2.3 Potential of nitric oxide on BBB permeability...............14
2.4 Nitric oxide donor..........................................15
2.5 Chemokine receptor 4........................................16
2.6 RNA interference............................................18
2.7 Drug delivery for gene silencing............................21
Materials and Methods...........................................24
3.1 Materials and equipment.....................................24
3.1.1 Materials.................................................24
3.1.2 Equipment.................................................25
3.2 Cell culture................................................26
3.3 Orthotopic tumor model establishment........................27
3.4 Preparation of LCP NPs......................................27
3.5 Characterization of NPs.....................................28
3.6 Cumulative release of NO....................................28
3.7 Cellular uptake.............................................29
3.8 Permeability assay..........................................29
3.9 Quantitative real time polymerase chain reaction............30
3.10 Western Blot Analysis......................................31
3.11 Tissue distribution study..................................32
3.12 Immunofluorescence.........................................33
3.13 Statistics.................................................33
Results.........................................................34
4.1 The gene expression of GBM..................................34
4.2 Cellular uptake of FAM-siRNA transported by CTCE-9908 modified LCP-NO NPs......................................................35
4.4 Enhance the permeability of BBB via NO......................41
4.5 Angiogenesis downregulation of VEGF siRNA-loaded CTCE-LCP NPs in vitro........................................................43
4.6 Biodistribution.............................................45
4.7 Anti-angiogenesis effect and Tumor suppression of VEGF siRNA-loaded CTCE-LCP NPs in vivo.....................................47
Conclusion......................................................50
Discussion and future work......................................52
Reference.......................................................56

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