多型性膠質母細胞瘤是目前臨床上最具侵略性的腦瘤。由於這種腫瘤具有非常高的浸潤性和侵略性，導致病患即使在接受治療後仍然有非常高的機率復發，也導致了這種癌症的死亡率非常的高。手術切除是目前在臨床上最常使用於治療腫瘤的方法之一，透過手術的切除除了能清除大部分的腫瘤也能減緩腫瘤生長造成的過高的腦壓。但由於多型性膠質母細胞瘤具有相當高的浸潤性，使得要在不過度傷害周邊正常功能腦區的情況下要完全移除腫瘤細胞是非常困難的。因此大部分的病患在1到1.5年內腫瘤都會復發。
本研究中，我們結合了免疫、氫氣及磁熱治療來進行多型性膠質母細胞瘤的術後治療。首先，我們透過將PEGDA和dextran打入微流體晶片製造出了大小約在150微米且性質均一的微球。透過這種可注射式的新月形微球可以將攜帶的免疫檢查點抑制劑anti-PD1和含有免疫調節功能的佐劑R848帶到手術後腫瘤移除的空洞並原位釋放。此外結合多孔性奈米氧化鐵奈米粒子並利用多孔洞的結構附載硼烷氨並給予高週波磁場照射，產生的氫氣及磁熱治療能更進一步的清除殘存的腫瘤細胞，同時放出更多腫瘤相關抗原來增強微球所誘發的免疫反應。藉由結合上述的兩種載體，免疫細胞能大量的被吸引到腫瘤被移除後的空腔並清除殘存的腫瘤細胞來達到更好的術後治療效果。
為了減少動物試驗的誤差與實驗動物數量我們透過3D腫瘤微球來模擬載體在腫瘤區的治療效果，在進行磁熱治療後雖然可以殺死44%的腫瘤細胞但腫瘤微球的整理結構依然保持完整，但如果將磁熱治療結合氫氣治療不僅可以殺死腫瘤細胞還能使腫瘤微球的結構崩塌。在動物實驗的結果我們也發現，結合磁熱、氫氣及免疫治療能有效的抑制腫瘤生長並延長小鼠的中位存活期到39天。

Glioblastoma, the most-invasive brain tumor, is characterized by the highest mortality rate, short lifetime and high invasive with a great tendency of recurrence. Surgical resection is the primary option in clinical treatment, however the infiltrating tumor cells inside the normal brain parenchyma cannot be completely removed because of the damage to the healthy region. As the result, most patients recur within 1 to 1.5 years.
In this study, we combine immunotherapy, hydrogen therapy and hyperthermia therapy for postoperative therapy of glioblastoma (GBM). First, microfluidic system was used to fabricate microspheres (MPs) which were made of PEGDA and dextran, the size of microspheres is 150 μm. The injectable moon-like microspheres can assemble in the restriction cavity where the solid tumor be removed and be adaptable to the cavity.
By encapsulating immune modulator (R848) and immune checkpoint blockade antibody (anti-PD1) in the microspheres, immune response would be activated and attack the rest of tumor cells. Moreover, the treatment also uses the iron oxide nanoparticles encapsulating ammonia borane that could generate hydrogen gas by applying an external magnetic field can further increase the effect of postoperative therapy by selective eliminating reactive oxygen species (ROS), inhibiting basal respiration of mitochondria and activate CD8+ T cells. By combining microspheres and iron oxide nanoparticles, immune cells will be recruited to the resection cavity and activated. At the same time, the infiltrating tumor cells treated for hyperthermia and hydrogen therapy will release tumor- associated antigen that can further increase the activity of immune cells to scavenge the tumor cell. The results of tumor spheres indicate that the hyperthermia treatment could kill 44% cells in tumor sphere but the structure of tumor sphere is still completely. However, combining with hydrogen therapy can not only kill the tumor cells but make the structure of tumor spheres collapse. The in vivo results also show that the group which applied with the combination treatment of hyperthermia, hydrogen and immune therapy can prolong the median survival rate to 39 days.

中文摘要 I
ABSTRACT III
致謝 V
Chapter 1 Introduction 1
Chapter 2 Literature review and theory 3
2.1 Nowadays brain cancer 3
2.2 Postoperative therapy 5
2.3.1 Injectable hydrogel application and property 9
2.3.2 Injectable microsphere hydrogel 13
2.4 Magnetic particle application and property 17
2.5 Hydrogen therapy 20
Chapter 3 Experimental section 23
3.1 Materials 23
3.2 Apparatus 24
3.3 Method 26
3.3.1 Fabrication of microfluidic chip 26
3.3.2 Fabrication of microspheres by microfluidic chip 27
3.3.3 Synthesis of mesoporous iron oxide nanoparticles 28
3.3.4 Release study 28
3.3.5 Cell culture 29
3.3.6 Cellular uptake 29
3.3.7 Cell viability assay 30
3.3.8 Co-culture of microspheres and tumor sphere 31
3.3.9 Penetration of the nanoparticles in ALTS1C1 spheroids 32
3.3.10 Immunohistochemistry staining 33
3.3.11 In vivo experiments 34
Chapter 4 Results and Discussions 36
4.1 Characterization of microspheres 36
4.2 Drug release test of microspheres 38
4.3 Synthesis and characterization of Fe3O4 nanoparticles 40
4.4 Hydrogen generation 42
4.5 Cytotoxicity of materials 43
4.6 Co-culture of microspheres and tumor spheres 46
4.7 Cell uptake of Fe3O4 nanoparticles 49
4.8 Penetration of Fe3O4 nanoparticles into tumor spheres 51
4.9 Hydrogen therapy and hyperthermia effect to tumor sphere 52
4.10 In vivo animal therapy and analysis 55
4.10.1 Tumor recurrence analysis and immune response after the postoperative therapy 55
4.10.2 In vivo therapy 60
Chapter 5 Conclusions 62
Reference 63

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