免疫抗原呈現細胞（APCs）活化後會遷移至淋巴結，誘導下游效應 T 淋巴
細胞的生成。這些 T 淋巴細胞會滲透到癌細胞中，通過相互作用抑制轉移性腫
瘤。然而，目前尚無能夠精準靶向樹突狀細胞（DCs）的奈米顆粒，且抗原利用
率低。此外，腫瘤的免疫特權和異質性使其能夠抵抗免疫攻擊，限制了免疫治療
的效果。
在本研究中，我們設計了一種奈米催化儲庫（CN）。該儲庫以平板六角形的
三氧化二鐵（α-Fe2O3）為核心載體，內含兒茶酚作為抗原儲庫和二氧化錳作為催
化劑，用於引發整體免疫反應。靜脈注射的奈米顆粒通過其扁平形狀和負電荷特
性，經血液輸送至脾臟免疫器官。在肺部腫瘤處，鐵和錳離子協同觸發的類芬頓
反應釋放 Fe2+/Mn2+，降低癌細胞中高表達的谷胱甘肽（GSH）水平，從而增強化
學動力療法的效果。同時，氧化還原過程催化內源性 H2O2 分解產生 O2，緩解腫
瘤微環境（TME）的缺氧問題。另一方面，CN@ antigen 通過 CN 載體在細胞內
生成劇毒氫氧自由基（•OH），誘發免疫原性細胞死亡（ICD），並在體外捕獲損
傷相關分子模式（DAMPs）和腫瘤特異性抗原（TSA）。這些抗原被脾臟的樹突
狀細胞吞噬後，促進其成熟，並遷移至淋巴結，誘導 T 細胞浸潤，提高免疫細胞
的識別能力。
最後，加入預防性療程以提前防止腫瘤生成並徹底抑制腫瘤轉移。並且通過
使用免疫檢查點抑制劑（aPD-1）阻斷腫瘤細胞與 T 細胞的配體結合，並輔以免
疫激活劑（R848）來提高樹突細胞的成熟率，予以實現良好的抗腫瘤免疫反應。

After activation, antigen-presenting cells (APCs) migrate to the lymph nodes, where they induce the generation of downstream effector T lymphocytes. These T lymphocytes infiltrate cancer cells and interact with them to inhibit metastatic tumors.
However, currently, there are no nanoparticles that can precisely target dendritic cells（DCs）, and the antigen utilization rate is low. Additionally, the immune privilege and heterogeneity of tumors allow them to resist immune attacks, limiting the effectiveness
of immunotherapy.
In this study, we designed a nanocatalytic reservoir (CN). This reservoir uses flat hexagonal hematite (α-Fe2O3) as a core delivery vehicle, containing catechol as an antigen repository and manganese dioxide as an effective catalytic stimulus to induce a
comprehensive immune response. Intravenously injected nanoparticles, due to their flat shape and negative charge, are transported via the bloodstream to immune organs such as the spleen. At the tumor site in the lungs, the Fenton-like reaction triggered by iron and manganese ions releases Fe2+/Mn2+, reducing the high levels of glutathione (GSH)
expressed in cancer cells, thereby enhancing the level of chemodynamic therapy.
Simultaneously, the redox process catalyzes the decomposition of endogenous H2O2 to produce O2, alleviating the hypoxia in the tumor microenvironment (TME). On the other hand, CN@ antigen uses the CN carrier to generate highly toxic hydroxyl radicals (•OH) inside the cells, inducing immunogenic cell death (ICD). It captures damage
associated molecular patterns (DAMPs) and tumor-specific antigens (TSAs) in vitro.
These antigens are then phagocytosed by DCs in the spleen, promoting their maturation and migration to the lymph nodes, inducing T cell infiltration and improving the immune cells' recognition capability.
Finally, add preventive treatments to proactively prevent tumor formation and thoroughly suppress tumor metastasis. Use immune checkpoint inhibitors (aPD-1) to block the binding of tumor cell ligands to T cells, and complement this with immune activators (R848) to enhance the maturation of DCs to achieve a good anti-tumor immune response.

Table of contents
中文摘要 ...................................................................................................................II
ABSTRACT...............................................................................................................III
致謝 ...................................................................................................................V
TABLE OF CONTENTS.......................................................................................... VI
LIST OF SCHEMES.................................................................................................IX
LIST OF FIGURES ....................................................................................................X
CHAPTER 1 INTRODUCTION................................................................................1
CHAPTER 2 LITERATURE REVIEW AND THEORY.........................................3
2.1 INTRODUCTION OF LUNG CANCER .........................................................................3
2.1.1 A summary of the present condition of lung cancer .....................................3
2.1.2 Mechanism in the tumor microenvironment.................................................5
2.1.3 Advancements in the Treatment of Lung Cancer..........................................9
2.2 OVERVIEW OF THE CANCER IMMUNOTHERAPY ....................................................11
2.2.1 Types and development of immunotherapy................................................11
2.2.2 Immune checkpoint inhibitors therapy .......................................................15
2.2.3 Mechanism of the immunogenic cell death (ICD)......................................18
2.2.4 The dying cells express damage-associated molecular patterns.................21
2.3 SPLEEN-SELECTIVE DELIVERY SYSTEM................................................................24
2.3.1 System of spleen accumulation...................................................................24
2.3.2 Nanoparticles boost immunity by delivering antigens to immune organs..30
2.3.3 The antigen is delivered by dendritic cells and induces their maturation...33
2.3.4 Activation of antigen-presenting cells stimulates the immune system. ......35
2.4 INTRODUCTION OF CATALYTIC NANOSERVOIR......................................................38
2.4.1 Advantages of nanoparticle-mediated delivery in cancer immunotherapy.38
2.4.2 The applications of Iron oxide (Fe2O3) nanoparticles in biomedicine........40
2.4.3 The significance of polydopamine-embedded nanoparticles in immune
stimulation.................................................................................................43
2.4.4 Introduction of chemodynamic therapy (CDT) effect ................................44
2.4.5 Advantages of manganese ion nanomaterials in immunotherapy...............47
CHAPTER 3 EXPERIMENTAL SECTION...........................................................50
3.1 MATERIALS .........................................................................................................50
3.2 APPARATUS .........................................................................................................55
3.3 METHOD .............................................................................................................58
VII
3.3.1 Synthesis of hexagonal nanoplates α-Fe2O3 ...............................................58
3.3.2 Polydopamine coating of α-Fe2O3 ..............................................................58
3.3.3 Synthesis of Catalytic Nanoreservoir (CN) ................................................59
3.3.4 Characterizations of nanoparticles..............................................................60
3.3.5 Characterizations of the materials functionality .........................................61
3.3.6 Cell culture for B16F10/DC2.4/RAW264.7 ...............................................62
3.3.7 Live & Dead with nanoparticles.................................................................64
3.3.8 Cellular viability test...................................................................................64
3.3.9 Cellular uptake assay ..................................................................................65
3.3.10 Preparation of nanomaterials with attached proteins................................67
3.3.11 Characterizations of antigen-captured nanoparticles................................67
3.3.12 Dendritic cell maturation ..........................................................................68
3.3.13 Macrophage polarization ..........................................................................70
3.3.14 DCFH-DA for hydroxyl radical ROS detections......................................71
3.3.15 Nanoparticles targeting test and ROS detection in Mitochondria ............72
3.3.16 In vivo biodistribution study.....................................................................73
3.3.17 In vivo nanoparticles colocalization and metabolism study .....................76
3.3.18 Using nanoparticles loaded with immune checkpoint and adjuvants in
flow cytometry ..........................................................................................77
CHAPTER 4 RESULTS AND DISCUSSION .........................................................79
4.1 PROPERTY STUDY ................................................................................................79
4.1.1 Synthesis and characterization of nanoparticles.........................................80
4.1.2 The functionality of nanoparticles ..............................................................93
4.2 IN VITRO STUDY ..................................................................................................99
4.2.1 The differences in cytotoxicity between different cells and nanoparticles
.................................................................................................................100
4.2.2 DC2.4 cellular uptake following the treatment.........................................101
4.2.3 Analyzing dendritic cell maturation in in vitro experiments using relevant
markers....................................................................................................105
4.2.4 RAW264.7cellular uptake following the treatment ..................................110
4.2.5 Analyzing macrophage polarization in in vitro experiments using relevant
markers....................................................................................................114
4.2.6 B16F10 cellular uptake following the treatment ......................................119
4.2.7 The intracellular catalysis assay for testing the generation of ROS in
nanoparticles ...........................................................................................122
4.2.8 Antigen capture study ...............................................................................126
4.3 IN VIVO STUDY ..................................................................................................133
4.3.1 Accumulation tests of CN .........................................................................133
VIII
4.3.2 Colocalization of nanoparticles in organs.................................................139
4.3.3 Analysis of the anti-tumor immune response following treatment with CNbased nanoparticle therapy......................................................................142
CHAPTER 5 CONCLUSION.................................................................................158
CHAPTER 6 REFERENCE ...................................................................................159

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