大腸癌（CRC）是最具破壞性的轉移性腫瘤之一，在台灣癌症發病率中排名第一。在這項研究中，將功能性核-殼立方體用於納米疫苗，該納米疫苗由生物相容的普魯士藍（PB）組成，被基於鐵（III）離子的多層MIL-100（Fe）有機金屬框架（MOF）包覆。通過靶向樹突狀細胞（DC）並以MPLA（單磷酸磷脂A）和AH1胜肽（SPSYVYHQF）抗原的形式刺激免疫反應。通過在PB上構造三層MOF（MIL3 @ PB），有機金屬框架MOF導致的立方體的孔隙率和平均尺寸均增加。表面積為359 m2 / g的疏水性孔洞能夠裝載大量分子藥物，即MPLA和AH1。將甘露糖（man）（一種單醣）偶聯在MIL3 @ PB上來標靶樹突細胞。經由皮下注射將man-MIL3 @ PB注射到小鼠中時，奈米粒子可以通過淋巴引流直接轉運到淋巴器官。本實驗的目的是利用奈米粒子在淋巴結中的積累，透過額外使用免疫刺激劑（如MPLA）和抗原（如AH1）來引起整體免疫反應，最終通過增強T細胞識別能力達到更高的治療效果。另外與免疫檢查點抑制劑結合使用治療添加免疫藥物，抗程序性細胞死亡1（Anti-PD-1）以達到更好的療效。

Colorectal cancer (CRC), one of most devastating metastatic tumor, has ranked first in the incidence of cancer in Taiwan. In this study, the functional core-shell cubes served as nanovaccines composed of a biocompatible Prussian blue (PB) coated by multi-layers of MIL-100(Fe) metal organic frameworks (MOFs) based on iron(III) ions was proposed to elicit immune responses through targeting dendritic cells (DCs) and delivering immune stimulators as MPLA (monophosphoryl lipid A) and antigen as AH1 peptide (SPSYVYHQF). With three layers of MOF constructed on PB (MIL3@PB), both porosity and mean sizes of cubes attributed by MOFs were increased. The hydrophobic pores with surface area of 359 m2/g was able to load the large amounts of molecular drugs, i.e., MPLA and AH1. For DC cell targeting, the mannose (man), a kind of monosaccharide, was conjugated on MIL3@PB. While the man-MIL3@PB was injected into mice through hock immunization, the particles can be transported through the lymphatic drainage directly to the lymphoid organs. The purpose of the experiment is to induce the accumulation of nanoparticles in lymph nodes, arising the overall immune reaction with additional use of immune stimulators as MPLA and antigen as AH1, finally achieving the goal of higher therapeutic effects by means of enhancing T cells recognition with addition of immune drug Anti-programmed cell death-1 (Anti-PD1).

中文摘要 ........................................................................................................................... i
Abstract ............................................................................................................................. ii
List of Tables.................................................................................................................. viii
List of figures ................................................................................................................... ix
Chapter 1 Introduction................................................................................................. 1
Chapter 2 Literature review and theory............................................................................. 2
2.1 Colorectal cancer treatment..................................................................................... 2
2.2 Nanoparticles as drug delivery systems................................................................... 5
2.1.1Metal-organic frameworks (MOFs) as drug delivery system............................ 7
2.3 Immune checkpoint blockade therapy................................................................... 13
2.4 Nanovaccines delivery in cancer immunotherapy............................................. 15
2.5 Biomaterial for Immunotherapy ............................................................................ 20
2.5.1 Implantable biomaterial: Porous scaffolds..................................................... 21
2.5.2 Nanofibrous hydrogel..................................................................................... 22
2.5.3 Metal-organic frameworks (MOFs)................................................................ 24
2.5.4 Gold Nanoparticle .......................................................................................... 25
2.5.5 Microneedle Patch .......................................................................................... 26
Chapter 3 Experimental Section...................................................................................... 28
3.1 Materials................................................................................................................ 28
3.2 Apparatus............................................................................................................... 31
3.3 Methods................................................................................................................. 33
3.3.1 Synthesis of Purussian blue (PB).................................................................... 33
3.3.2 Synthesis of MIL3@PB nanoparticles ........................................................... 33
3.3.3 Synthesis of man-MIL3@PB nanoparticles................................................... 34
3.3.3 Characterizations............................................................................................ 35
3.3.4 Cell culture.................................................................................................... 36
vii
3.3.5 Cell viability assay ......................................................................................... 36
3.3.6 Cellular uptake .............................................................................................. 37
3.3.7 Animal studies................................................................................................ 38
3.3.8 In vivo study of man-MIL3@PB uptaked by DCs ......................................... 38
3.3.9 In vivo animal experiment for biodistribution................................................ 38
3.3.10 In vivo flow cytometry analyses................................................................... 39
3.3.11 Tumor inoculation, combination therapies and tumor volume measurements.
40
3.3.12 Tissue section immunostaining .................................................................... 40
Chapter 4 Results and Discussions.................................................................................. 41
4.1 Characterization of PB, MIL@PB nanoparticles .................................................. 41
4.2 Cytotoxicity of PB, MIL@PB and cell uptake of MIL@PB................................. 50
4.3 In vivo animal experiment for biodistribution ....................................................... 53
4.4 In vivo Immunity and Therapeutic Efficacy .......................................................... 54
Chapter 5 Conclusion ...................................................................................................... 62
Reference......................................................................................................................... 64

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