腫瘤治療方法日益多元，除了傳統的化療，手術切除等，更發展出被認為是下個世紀主要且有效克服癌症腫瘤的新療法，免疫療法，而近年更有許多結合療法研究興起，藉由免疫療法改變腫瘤免疫微環境的能力，增強現有療法的治療效果，如使用臨床化療藥物Doxorubicin (DOX) 結合免疫檢查點抑制劑1‐methyltryptophan (1MT)，加強腫瘤抑制效果[2]，或以免疫佐劑Imiquimod (R837) 與免疫檢查點抑制劑 (anti-CTLA-4) 結合放射線治療，引起的協同作用克服癌症[3]。
在本研究中，開發出利用乳化法製成合成粒徑約150 nm之固態脂質奈米粒子，同時包覆TLR7 agonist免疫佐劑 (R837) 活化樹突細胞與adenosine receptor antagonist (Caffeine)，載體中包覆之R837及Caffeine含量分別約為6.3與5.9 wt%，本奈米載藥系統更具有低細胞毒性之特性，利用EPR effect (Enhanced Permeability and Retention effect) 累積至腫瘤區及周邊淋巴結處，此奈米載體具強化腫瘤區免疫細胞以及全身性免疫系統的能力，引起腫瘤區癌細胞進行免疫式凋亡，促使癌細胞抗原產生並且釋放，另外，本研究中使用之固態脂質奈米粒子在細胞內可被細胞本身之脂質分解脢 (Lipase) 所分解，經放射線照射後細胞凋亡後，細胞內奈米載體瓦解後累積的R837及Caffeine得以釋放，釋放之藥物活化抗原呈現細胞，如樹突細胞、巨嗜細胞等，進一步吸引T細胞浸潤至腫瘤區，改變腫瘤免疫微環境，達到免疫治療腫瘤抑制的療效。

Immunotherapy is a rising strategy against cancer in recent years, comparing to traditional therapy, such as chemotherapy or surgery, immunotherapy has less side effects. To overcome tumor immunosuppressive microenvironment, combining traditional clinical drug with check point blockade[2] has positive results.
In this study, we developed a solid lipid nanoparticles (SLNs), simultaneously loaded imiquimod and caffeine in the hydrophobic core. Imiquimod (R837) is one kind of toll-like receptor 7 (TLR7) agonist and Caffeine is an adenosine receptor (AR) antagonist. Our SLNs system could be degraded by lipase in cells, we also use x-ray as a method not only to induce immunogenic cell death (ICD) but also help the drug released from cells after cell death, it is important whether cancer cells could release enough tumor antigen in immunotherapy, Imiquimod and tumor antigen could activate antigen presenting cells (APC) synergistically in our delivery system, T cells stimulated by APC and infiltrate into tumor region. However, caffeine can activate systemic immune system, improving tumor immunosuppressive microenvironment. We hope to develop a platform for immunotherapy.

摘要 1
Abstract 2
致謝 3
目錄 4
圖目錄 8
表目錄 10
一、 研究動機 11
二. 文獻回顧 13
2.1. 惡性腫瘤 13
2.2. 乳癌 13
2.3. 免疫系統 14
2.4. 癌症免疫治療現況 15
2.5. 腫瘤微環境 16
2.5.1. 腫瘤免疫微環境 16
2.5.2. Enhanced Permeation and Retention effect (EPR effect) 16
2.6. 免疫式細胞凋亡 (Immunogenic cell death ，ICD) 17
2.7. 放射線治療 18
2.8. 固態脂質奈米粒子 20
2.8.1. 固態脂質奈米粒子製程 20
2.9. Adenosine receptor antagonist咖啡因 (Caffeine) 21
2.10. Toll-like receptor agonist咪喹莫特 (Imiquimod) 22
三. 研究方法 24
1. 奈米粒子的製備 24
1.1. 搭載 R837與Caffeine之固態脂質奈米粒子製備 24
1.2. 固態脂質奈米粒子之粒徑分布與電性測試 24
1.3. 奈米粒子之藥物裝載效率與包覆率 25
1.4. 奈米粒子穩定性測試 25
1.5. 奈米粒子體外藥物釋放實驗與分析 26
2. 體外細胞實驗 26
2.1. 細胞來源 26
2.2. 細胞培養液配製與磷酸鹽緩衝溶液 26
2.2.1. 配製DMEM 細胞培養液 (For RAW264.7) 26
2.2.2. 配製DMEM/Pyruvate細胞培養液 (For 4T1細胞) 27
2.3. 細胞繼代 27
2.4. 細胞計數 28
2.5. 細胞對奈米粒子吞噬情形評估 28
2.6. 奈米粒子對細胞毒性分析 29
2.7. 利用螢光顯微鏡觀察放射線引起乳癌細胞免疫式凋亡情形 30
2.8. 以Annexin V與PI觀察低劑量放射線對細胞凋亡之影響 31
2.9. Wound healing assay 31
2.10. Macrophage polarization 31
2.11. 以transwell實驗討論放射線造成癌細胞產生ICD結合奈米粒子對巨噬細胞活化之影響 32
3. 動物實驗 33
3.1. 動物來源 33
3.2. 腫瘤模型建立 33
3.3. 腫瘤內載體累積時間分布 33
3.4. 腫瘤抑制評估 34
3.5. 血液T細胞分析 34
3.6. 小鼠淋巴結之免疫細胞染色 35
3.7. 小鼠腫瘤之免疫細胞染色 35
3.8. cAMP ELISA之腫瘤樣品製作 36
3.9. Rechallenge 36
四. 結果與討論 37
1. 奈米粒子特性分析 37
1.1. 固態脂質奈米粒子特性分析 37
1.2. 固態脂質奈米粒子穩定度評估 38
1.3. 載體之體外藥物累積釋放行為 38
1. 體外細胞實驗 39
1.2. 載體生物相容性評估 39
1.3. 細胞對奈米粒子吞噬情形分析 40
1.4. 不同劑量放射線刺激乳癌細胞進行免疫式凋亡情形分析 42
1.5. 以Annexin V與PI觀察放射線造成細胞凋亡情形 44
1.6. Wound healing assay 45
1.7. 奈米粒子對Macrophage極化程度分析 46
1.8. 放射線結合奈米粒子對Raw264.7之MHC I表現量之影響 47
3.1. 載體於小鼠腫瘤累積分布 48
3.2. 放射線照射劑量與空間分布關係圖 49
3.3. 腫瘤治療抑制曲線 51
3.4. 腫瘤重量 52
3.5. 脾臟重量 52
3.7. 淋巴結免疫細胞分析 53
3.7.1. 樹突細胞 (Dendritic cells) 53
3.7.2. T 細胞 54
3.8. 腫瘤區免疫細胞分析 56
3.8.1. T細胞 56
3.8.2. myeloid-derived suppressor cells (MDSC) 58
3.9. 腫瘤區cyclic-AMP表現量 58
3.10. 小鼠體重變化 59
3.11. 腫瘤Rechallenge實驗 60
3.11.1. Systemic immune response 60
3.11.2. Primary tumor 抑制曲線圖 62
3.12.3. Secondary tumor抑制實驗 62
五. 結論 63
六. 參考文獻 64

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