胰腺導管癌（Pancreatic ductal adenocarcinoma，PDAC）是臨床上一種侵襲性高的惡性腫瘤，其起源於胰腺的胰導管上皮細胞。由於胰臟癌難以早期診斷，且現行療法不盡理想，截至目前胰臟癌患者的平均五年存活率仍低於10%；胰臟癌腫瘤微環境（Tumor microenvironment，TME）中過度沉積的纖維化增生基質是造成此不良預後的主要原因之一。目前大多數化療藥物是通過靜脈注射（Intravenous injection）方式給藥，這不僅會給患者帶來不適，同時也造成患者無法在家自行用藥。因此，為了優化胰臟癌治療方式，本研究開發了兩種口服奈米載體投遞系統。第一個投遞系統是一口服膠囊，可同時遞送親水性藥物吉西他濱（Gemcitabine，GEM）和疏水性藥物紫杉醇（Paclitaxel，PTX）；吉西他濱與紫杉醇已被核准用於治療晚期胰臟癌。當此口服膠囊抵達小腸環境，膠囊會溶解並自發地產生二氧化碳氣泡。這些氣泡載體的破裂會產生含紫杉醇的奈米乳化液，並將吉西他濱良好分散於水相中；紫杉醇奈米乳化液通過腸道淋巴系統吸收，而吉西他濱則直接進入血液循環系統，兩者最終都累積在胰臟腫瘤中，發揮其抗腫瘤作用。本研究建立一原位胰臟癌大鼠模型，以評估此口服膠囊之抗腫瘤功效。數據顯示此口服膠囊在抑制腫瘤生長、抑制纖維化基質增生及預防腫瘤轉移方面皆比靜脈注射組別更有效。儘管初步取得良好的治療結果，但根據文獻，奈米藥物載體進入體內後往往很快地被免疫系統清除代謝，並可能對正常組織造成毒性。相較之下，利用內源性細胞作為藥物載體，標靶體內病變部位可作為一種有效的替代策略；動物體內廣泛存在的免疫細胞具有對腫瘤的標靶性，可有效穿透生物屏障，將攜帶之藥物運輸至腫瘤深處。因此，在第二種口服奈米載體投遞系統中，利用阿黴素（Doxorubicin，DOX）和醋酸鋅合成奈米複合物（ZnD）。β-葡聚醣（β-Glucans）是在酵母細胞壁中發現的天然多醣類物質，可作為分散劑並吸附到奈米複合物表面（βGlus-ZnD）。經口服後，βGlus-ZnD可標靶小腸中的微皺摺細胞（Microfold cell，M cell）和腸道淋巴系統中的巨噬細胞（Mϕ）。隨著載體巨噬細胞（βGlus-ZnD@Mϕ）到達腫瘤部位，阿黴素可通過溶酶體胞吐（Lysosomal exocytosis）途徑釋放；同時，巨噬細胞被極化為M1表現型（抗腫瘤），不僅可逆轉腫瘤微環境之免疫抑制性，同時抑制了纖維化基質的增生。將此治療過程與免疫檢查點抑制劑（Immune checkpoint inhibitor）相結合，可進一步提高其抗腫瘤功效。接著建立一自發遠端轉移的胰臟癌小鼠模型，進一步驗證載體巨噬細胞的腫瘤標靶性。最終數據顯示治療後可有效抑制原位胰臟癌腫瘤，並減少遠端轉移結節之數量。本研究中兩種獨特的口服化療方法可望為胰臟癌患者提供更佳的治療策略，提高其生活品質。

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that originates in the exocrine cells in the pancreas. Owing to a lack of early diagnosis and the inadequate response of PDAC to treatments, PDAC has a dismal five-year survival rate (less than 10%). One hallmark of PDAC, which considerably contributes to the unsatisfactory therapeutic outcomes, is excessive deposition of desmoplastic stroma in the tumor microenvironment (TME). Most chemotherapy drugs are administered by intravenous (i.v.) injection, which not only causes discomfort to patients but also cannot be performed at home without a medical professional. Therefore, in order to optimize the PDAC treatment modality, this work develops two oral nanocarrier delivery systems. The first an oral capsule system that enables the concurrent delivery of gemcitabine (GEM, hydrophilic) and paclitaxel (PTX, hydrophobic). GEM plus PTX is an effective combination treatment for advanced PDAC. The as-prepared oral capsules spontaneously initiate effervescence to produce bubble carriers in the small intestine. The bursting of these bubble carriers generates nanoemulsions that encapsulate PTX and disperses GEM in the aqueous phase, promoting intestinal absorption. Hydrophobic PTX is absorbed through the intestinal lymphatic system, while hydrophilic GEM directly enters the blood circulatory system; both ultimately accumulate in the pancreatic tumor, exerting their antitumor effect. The antitumor efficacy is evaluated in an orthotopic pancreatic tumor rat model. Data reveal that the as-proposed oral formulation is more effective in tumor inhibition, stroma resolution, and metastases prevention than the i.v. formulation. Despite this promising outcome, the literature demonstrates that such nanosized drug delivery systems can barely escape from rapid immunological clearance and may induce off-target toxicity. As an alternative strategy, endogenous cells are used as camouflaged drug carrier that target diseased sites. The ubiquitous immune cells that respond to (tumoral) chemotactic cues and penetrate biological barriers, are ideal for delivering concealed drug cargoes deep into the tumor. Therefore, for the second oral nanocarrier delivery system, doxorubicin (DOX) and zinc acetate are used to construct nanocomplexes (ZnD). β-Glucans, which are natural polysaccharides that are found in the cell walls of yeasts, are added as dispersants and adsorbed onto the nanocomplexes (βGlus-ZnD). After oral administration, βGlus-ZnD target microfold cells (M cell) in Peyer’s patches (to be absorbed) and macrophages (Mϕ) in the intestinal lymphatic system (to be hitchhiked). As the carrier macrophages (βGlus-ZnD@Mϕ) arrive at the tumor site, DOX is released via the lysosomal exocytosis pathway; simultaneously, Mϕ are repolarized to the M1-like (antitumor) phenotype, which not only reverses immunosuppressive TME but also resolves the desmoplastic stroma. Combining this process with immune checkpoint inhibitors further improves its antitumor efficacy. The tumor-homing effect of βGlus-ZnD@Mϕ is proven in a spontaneous metastatic PDAC mouse model. The data indicate that the primary pancreatic tumor is shrunk, while the number of metastatic nodules are decreased. The aforementioned unique approaches to oral chemotherapy provide opportunities for treating outpatients with PDAC, improving their quality of life.

摘要----------I
Abstract----------III
Table of Contents----------V
List of Figures----------VIII
List of Tables----------XIV
Chapter 1: Introduction----------1
1-1. Pancreas and pancreatic ductal adenocarcinoma (PDAC)----------1
1-2. Oral chemotherapy----------2
1-3. Gemcitabine (GEM) and Paclitaxel (PTX)----------3
1-4. Macrophage-laden drug delivery----------4
Chapter 2: A Bubble Bursting-Mediated Oral Drug Delivery of Lipophilic and Hydrophilic Chemotherapeutics for Treating Pancreatic Tumors in Rats----------6
2-1. Introduction----------7
2-2. Results and discussion----------10
2-2.1. Optimization of formulation----------10
2-2.2. Effervescent activity----------12
2-2.3. In vivo dose optimization----------14
2-2.4. Routes of absorption and biodistributions of drugs----------16
2-2.5. Pharmacokinetics of PTX and GEM----------19
2-2.6. Antitumor efficacy----------22
2-2.7. Animal positron emission tomography (PET) imaging----------24
2-2.8. Histological analyses----------26
2-3. Conclusions----------27
2-4. Materials and methods----------27
2-4.1. Materials----------27
2-4.2. Formulation optimization----------28
2-4.3. Effervescent activity----------28
2-4.4. Animal study----------29
2-4.5. Preparation of enteric-coated capsules----------29
2-4.6. Orthotopic pancreatic tumor model----------29
2-4.7. Routes of absorption and biodistributions of drugs----------30
2-4.8. Pharmacokinetics of PTX and GEM----------31
2-4.9. Antitumor efficacy----------31
2-4.10. Animal PET imaging----------32
2-4.11. Statistical analysis----------33
Chapter 3: Macrophage-Hitchhiked Orally Administered β-Glucans-Functionalized Nanoparticles as “Precision-Guided Stealth Missiles” for Targeted Pancreatic Cancer Therapy----------34
3-1. Introduction----------35
3-2. Results and discussion----------37
3-2.1. Characteristics of ZnD/βGlus-ZnD NPs----------37
3-2.2. Cellular uptake and trafficking of ZnD/βGlus-ZnD NPs----------40
3-2.3. Degradation of ZnD/βGlus-ZnD NPs in lysosomal environment----------41
3-2.4. Lysosomal efflux of DOX----------41
3-2.5. Cytotoxicity of ZnD/βGlus-ZnD NPs toward Mϕ----------44
3-2.6. Potential of βGlus-ZnD NPs in activation of Mϕ----------45
3-2.7. Biodistribution of ZnD/βGlus-ZnD NPs----------45
3-2.8. Antitumor efficacy of βGlus-ZnD NPs----------48
3-2.9. Modulation of TME----------51
3-2.10. Role of endogenous Mϕ----------52
3-2.11. Combination therapy----------54
3-2.12. In vivo safety----------55
3-2.13. Efficacy in late-stage treatment----------55
3-3. Conclusions----------59
3-4. Materials and methods----------60
3-4.1. Materials----------60
3-4.2. Preparation of yeast-derived βGlus----------60
3-4.3. Preparation and characterization of ZnD/βGlus-ZnD NPs----------61
3-4.4. Cellular uptake and trafficking of ZnD/βGlus-ZnD NPs----------62
3-4.5. Degradation of ZnD/βGlus-ZnD NPs in lysosomal environment----------63
3-4.6. Lysosomal Efflux of DOX----------63
3-4.7. Cytotoxicity of ZnD/βGlus-ZnD NPs toward Mϕ----------64
3-4.8. Potential of βGlus-ZnD NPs to Activate Mϕ----------64
3-4.9. Animal Study----------65
3-4.10. Biodistribution of ZnD/βGlus-ZnD NPs----------65
3-4.11. Transport route----------65
3-4.12. Antitumor efficacy----------66
3-4.13. Histological analysis----------67
3-4.14. Flow cytometry----------68
3-4.15. Statistical analysis----------68
Chapter 4: Reference----------69
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