臺灣博碩士論文加值系統

本研究目的為開發智慧靶向型奈米微粒，針對乳癌骨轉移腫瘤進行化學治療。將具骨骼高親和性與葉酸受器靶向之兩種標靶配體(targeting ligand)做為製備奈米微粒的材料，賦予奈米微粒雙重標靶的功能進而發揮主動標的骨轉移乳癌細胞4T1的能力，同時結合化療藥物Paclitaxel (PTX)能有效抑制乳癌細胞於骨內的生長及轉移。
以生物可降解高分子poly(lactic-co-glycolic acid) (PLGA) 利用乳化法製備搭載化療藥物PTX的奈米微粒(Nanoparticles, NPs)，將葉酸、Alendronate (Ad)修飾的聚乙二醇琥珀酸酯 (TPGS-Folate、TPGS-Ad)作為介面活性劑，使標靶配體暴露於奈米微粒表層。此載藥奈米微粒的水合直徑約125.9 nm，PTX的搭載量約為3.78 wt%。採用人體骨骼組織主成分羥基磷灰石(Hydroxyapatite, HA)評估奈米微粒的吸附親和性，發現帶有Ad的載藥奈米微粒可有效提升對HA的吸附情形，優於未具骨標靶功能奈米微粒約三倍之多。將葉酸受器過度表現的4T1乳癌細胞進行體外細胞實驗，結果顯示與未修飾葉酸分子的載藥奈米微粒相比，含有葉酸的載藥奈米微粒可大幅提高被4T1癌細胞的攝取量高達三倍。
利用乳癌細胞移植於脛骨模擬癌症骨轉移之小鼠，進行載藥奈米微粒的尾靜脈注射給藥與腫瘤抑制評估，結果顯示具雙標靶功能之載藥奈米微粒可有效抑制骨內腫瘤生長，使小鼠維持50%以上的存活率達50天，且無明顯的癌細胞轉移情形與副作用產生。不僅如此，Alendronate除了具備骨環境標靶功能之外，也能抑制蝕骨細胞內重要酵素farnesyl pyrophosphate synthase的作用，加速蝕骨細胞凋亡達到減緩骨侵蝕的效果，因此雙標靶功能之載藥奈米微粒的給予也使腫瘤患部之骨結構可維持良好構型，有效降低骨侵蝕的作用。故此智慧靶向型載藥奈米微粒可望作為化學治療用於癌症骨轉移之良好藥物傳輸系統。

Bone is a most favorable microenvironment for tumor growth and an invariable site for metastatic breast cancer. Widely methods applied to treat bone metastasis are radiotherapy, chemotherapy and surgery. However, these therapeutic approaches are severely limited due to the poor selectivity and low permeation in the bone metastasis region. The aim of this study is to develop a dual targeting drug delivery system carrying therapeutic payloads of paclitaxel (PTX) for improved therapeutic efficacy in bone metastases. High-affinity bone targeting ligands, Alendronate (Ad) and folate (FA) were conjugated with D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) as a surfactant of the biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating PTX by emulsion method. The folate/Alendronate-modified nanoparticles (FA/Ad (3/2) NPs) with hydrodynamic diameter of 125.9 nm have drug loading content 3.78 wt%. Compared to the non-targeted NPs, the folate-modified NPs were significantly internalized in 4T1 cancer cells with overexpressed folate receptor, confirmed by the increase in the intracellular DiI fluorescence intensity by 3-folds. Besides, FA/Ad (3/2) NPs showed much higher affinity for hydroxyapatite in vitro and bone in vivo than non-targeted NPs. Finally, not only the tumor growth and lung metastasis of 4T1 breast tumor was significantly inhibited, but also the osteolytic bone destruction were reduced by the chemotherapy of FA/Ad (3/2) NPs. In conclusion, these results strongly demonstrate that the developed FA/Ad (3/2) NPs as a dual targeting drug delivery system is a promising therapeutic strategy for enhancing chemotherapeutic efficacy of bone metastatic breast cancer.

目錄 2
圖目錄 6
表目錄 9
Abstract 10
摘要 11
一、研究動機 12
二、文獻探討 14
2.1 惡性腫瘤 14
2.2 乳癌骨轉移 14
2.2.1 癌症骨轉移之微環境介紹 15
2.2.2 癌症骨轉移治療現況 17
2.3 奈米微粒藥物載體 17
2.3.1 奈米微粒藥物傳輸載體用於癌症治療 17
2.3.2 高分子奈米微粒 (Polymeric nanoparticles) 19
2.3.4 主動標靶及被動標靶 21
2.4 Enhanced permeation and retention (EPR) effect 22
2.5 葉酸Folate (folic acid)主動標靶 23
2.6 化合物Alendronate sodium trihydrate (Ad)主動標靶 24
2.7化學治療藥物 Paclitaxel (PTX)介紹 25
2.7.1 研究背景 25
2.7.2 藥物作用機制 26
三、實驗方法與步驟 28
3.1 實驗藥品 28
3.2 實驗儀器 29
3.3 高分子合成與鑑定 30
3.3.1 Succinic anhydride活化之聚乙二醇琥珀酸酯(TPGS-SA)合成步驟 30
3.3.2 Alendronate sodium trihydrate修飾之聚乙二醇琥珀酸酯(TPGS-Ad)合成步驟 31
3.3.3 葉酸修飾之聚乙二醇琥珀酸酯(TPGS-FA)合成步驟 32
3.3.4 羅丹明B修飾之PLGA (PLGA-Rhodamine)合成步驟 33
3.4 奈米微粒製備與性質探討 33
3.4.1 搭載化療藥物PTX之高分子奈米微粒製備 33
3.4.2 載藥高分子奈米微粒的粒徑分布與表面電荷分析 34
3.4.3 奈米載體之穩定性測試 36
3.4.4 載藥高分子奈米微粒之藥物包覆效率與包覆含量測定 36
3.4.5 高分子載藥奈米微粒體外(in vitro)釋放實驗與分析 37
3.4.6 PTX-loaded Ad NPs對骨主成分Hydroxylapatite (HA)之吸附情形測試 37
3.5 體外細胞實驗 38
3.5.1細胞來源 38
3.5.2細胞繼代 38
3.5.3細胞計數 39
3.5.4細胞對奈米微粒吞噬情形分析 39
3.5.5以共聚焦激光掃描顯微鏡(CLSM)觀察細胞對奈米微粒吞噬情形 40
3.5.6細胞毒性分析 40
3.6 小鼠體內動物實驗 41
3.6.1動物來源 41
3.6.2腫瘤模型建立 41
3.6.3奈米微粒於臟器及腿骨之分佈 41
3.6.4血液生化分析 42
3.6.5腫瘤抑制評估 42
3.6.6 Micro-CT電腦斷層掃描觀察腫瘤患部骨侵蝕之情況 42
3.6.7動物犧牲與組織包埋 43
3.6.8組織切片 43
3.6.9組織切片Hematoxylin and eosin (H&E)染色 43
3.7 數據統計 44
四、結果與討論 45
4.1 高分子鑑定及分析 45
4.1.1 TPGS-Ad組成分析鑑定 45
4.1.2 TPGS-FA組成分析鑑定 47
4.2 高分子載藥奈米微粒特性分析 48
4.2.1 高分子載藥奈米微粒製備極粒徑分析 48
4.2.2 高分子載藥奈米微粒之粒子穩定性評估 51
4.2.3 高分子載藥奈米微粒PTX裝載含量及藥物釋放評估 52
4.2.4 高分子載藥奈米微粒對Hydroxylapatite (HA)吸附效率評估 53
4.3 細胞實驗 57
4.3.1 葉酸主動標靶之細胞吞噬評估 57
4.3.2 各組NPs之細胞毒性分析 61
4.4 動物實驗 63
4.4.1 藥物載體於小鼠體內累積分布 63
4.4.2 腫瘤抑制評估 65
五、結論 73
六、參考文獻 74

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