大腸癌是最常見的癌症形式 ，由於不健康的生活方式、不衛生及不規則的飲食習慣，可能導致正常大腸黏膜表面轉變為腺瘤性息肉。目前主要的治療方法包括手術切除，放射療法和化學療法，但其術後容易復發及有些許的副作用。而免疫療作為近年來治療癌症的新方法，由於其副作用少、對治療腫瘤有長期的效果及低毒性。因此有許多研究透過奈米藥物載體結合免疫療法來提高在腫瘤的累積及治療。在本研究中，我們以3D列印的技術用於微針翻模的製程，並以快速溶解、高生物相容性及生物可降解性材料聚乙烯醇 (polyvinyl alcohol, PVA) 作為微針材料，在扎入皮膚後，藉由體內的組織液將微針快速溶解釋放出奈米粒子，來達到治療效果。
此微針系統是以經皮吸收的方式傳遞奈米粒子並結合靜脈注射的方式注射免疫檢查點抑制劑（aPD1）在透過高週波磁場以磁熱的方式來治療大腸癌。經皮吸收系統傳遞奈米粒子不僅方便，還具有克服血液中藥物濃度的波動變化的潛力。另外，氧化鐵奈米粒子被作為Resiquimod（R848）免疫佐劑的載體，其中氧化鐵奈米粒子被巨噬細胞膜（M1型）包覆，目的是模擬白細胞的生物學特性。透過SEM及TEM分析證實了奈米粒子確實被細胞膜包覆住。在細胞的實驗中也證實，經修飾後的奈米粒子不但可增加在腫瘤細胞的累積外還能逃避被免疫細胞攝取，接著將奈米顆粒進一步嵌入微針中以形成有效的遞送系統並進行體內的實驗。由結果表明氧化鐵奈米粒子被巨噬細胞膜包覆後透過高週波加熱及注射免疫抑制劑有較好的免疫反應。

Colon cancer (CC) is the most common cancer due to unhealthy lifestyle, unhygienic and irregular dietary habits which may in turn lead to the change in normal colonic epithelium to an adenomatous polyp. The current treatments against this disease involve surgical removal of the tumor, radiotherapy, and chemotherapy, all of which are inefficient that bring several side effects and high risks such as postoperative recurring and metastasis. Immunotherapy is a novel therapeutic strategy for cancer. Its advantage including few side effect, long-term anti-tumor effect and minimal toxicity. Various researches are being conducted using nanoparticle for the therapeutics of innumerable diseases across the globe, including drug delivery, chemotherapy, hyperthermia and immunotherapy, etc. The subcutaneous administration of nanoparticle is not only convenient but it also has the potential to overcome the most common problem of fluctuating concentration of drug in the plasma.
In this study, we used 3D printing technology for the microneedle remolding process, and polyvinyl alcohol (PVA), a rapidly dissolving, highly biocompatible and biodegradable polymer, is used as the microneedle material. After puncturing into the skin, the microneedles were quickly dissolved by the tissue fluid in the body, releasing nanoparticles to achieve the therapeutic effect. Our treatment system involved the administration of microneedle transdermal delivery of nanoparticles followed by a high-frequency magnetic field (HFMF) operation as well as intravenous injection of immune checkpoint inhibitors (anti-PD1), and was implemented on mice with colon cancer. In this current study, iron oxide nanoparticles were used as carrier of an immune adjuvant, Resiquimod (R848), and coated with macrophage membrane (M1 type) in order to mimic the biological properties of white blood cells. In SEM and TEM analysis, it was confirmed that the nanoparticles are indeed coated by cell membranes. The facts that modified nanoparticles can not only increase the accumulation of tumor cells but also has immune evasion property had been verified through In vitro studies. Afterwards, the nanoparticles were further embedded into the microneedle as the effective delivery system conducted on in vivo experiments. The results were shown that iron oxide nanoparticles are coated with macrophage membranes and through HFMF heating and intravenous injection of aPD1 stimulate the immune response.

中文摘要 I
Abstract II
誌謝 IV
Table of contents I
List of schemes III
List of figure IV
Chapter 1 Introduction 1
Chapter 2 Literature Review and Theory 1
2.1. Treatment of Colon Cancer 1
2.2. The Properties of Magnetic Mesoporous Nanoparticles and Cancer 1
2.3. Application of Cell Membrane - Coated Nanoparticles 2
2.4. Immunotherapy of Cancer 4
2.5. Microneedles 8
2.5.1 Transdermal Drug Delivery System 8
2.5.2 Types of Microneedles 10
2.5.3 Applications of microneedles 16
Chapter 3 Experimental Section 28
3.1 Materials 28
3.2 Apparatus 31
3.3 Method 33
3.3.1 Synthesis of Mesoporous Iron Oxide Nanoparticles 33
3.3.2 M1 Macrophage Membrane Isolation 34
3.3.3 IO@MM NP Preparation by Sonication 34
3.3.4 Formula of Microneedle 36
3.3.5 Two-Step Fabrication Process of Microneedles 36
3.3.6 Characterizations 38
3.3.7 Microneedle patch mechanical properties 39
3.3.8 Skin insertion of microneedle patches ex vivo 40
3.3.9 Cell culture 40
3.3.10 In Vitro Macrophage Polarization by M1 Treatment 41
3.3.11 Cellular uptake 42
3.3.12 Targeting ability of the IO/IO@MM was quantified by flow cytometry 42
3.3.13 Cell viability assay 43
3.3.14 In vivo experiments 44
3.3.15 Ex vivo analysis of different groups of T cells 45
Chapter 4 Results and Discussions 49
4.1 Synthesis and characterization of IO@MM 49
4.2 Fabrication and Characterization of Microneedles 55
4.3 Microneedle Mechanical Strength Test 58
4.4 Microneedle In Vitro Puncture Test 59
4.5 Characterization of M1 Macrophage 62
4.6 Cell uptake and cytotoxicity of IO@MM 63
4.7 In vivo animal experiment 67
Chapter 5 Conclusions 73
Reference 74

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