包覆靛氰綠與喜樹鹼之標靶全氟碳奈米乳劑 研製於強化乳癌螢光擴散光學影像暨 光/化學治療之研究

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姓名

郭柏緯(Po-Wei Kuo) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

包覆靛氰綠與喜樹鹼之標靶全氟碳奈米乳劑研製於強化乳癌螢光擴散光學影像暨光/化學治療之研究
(Fabrication of HER2 Target Indocyanine Green-Camptothecin-Loaded Perfluorocarbon Nanodroplets for Photochemotherapy and Fluorescence Diffuse Optical Tomography of Breast Cancer)

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摘要(中)

HER2過度表達型乳腺癌由於其高發生率、抗藥性和預後差，長期以來一直是女性最致命的乳癌亞型之一。而初期診斷出乳腺癌後的立即治療能夠使病患的5年存活率顯著提高到> 90 %，表明除了有效的治療策略外，成功的早期診斷能有效改善HER2 +乳腺癌的存活率。在本研究中，我們以二次乳化法並進行表面修飾合成出包覆靛氰綠(Indocyanine Green；ICG)及喜樹鹼(Camptothecin；CPT)之標靶人類表皮生長因子2 (Human Epidermal Growth Factor Receptor 2 (HER-2))之全氟碳化物奈米乳劑(HER2 Target ICG-CPT-Encapsulated PFC Double-Nano-Emulsions；HICPDNEs)的全氟化碳雙層納米乳液，用於光化學治療和螢光擴散光學斷層掃描（FDOT）診斷之顯影劑。根據結果，HICPDNEs的粒徑大小和表面電位分別為292.2 ± 5.6 nm和-13.5 ± 3.1 mV。藥物CPT和ICG的包覆效率分別為40.31 ± 7.6 %和99.12 ± 0.33 %。在體外模擬人體溫度37 ℃下經過48小時之後，HICPDNEs的降解率與游離的ICG水溶液相比多延長了73 %，而CPT之藥物釋放率僅為4.29 %，顯示載體的良好穩定性。此外，HICPDNEs在808 nm近紅外光波段，強度為6 W/cm2的雷射照射5分鐘後的升溫效果，20 μM的HICPDNEs可以達到42 ℃對癌細胞造成損害。在單態氧生成量試驗中，相同濃度下的HICPDNEs均遠高於游離的ICG水溶液，顯示本藥物載體在光動力具有極佳潛力。在體外細胞試驗中，將HICPDNEs與接枝抗體前的載體(ICPDNEs)進行比較，利用與HER2+之乳癌細胞(MDA-MB-453)證實HICPDNEs對HER2過度表現型細胞具專一性；細胞毒性試驗中，HICPDNEs在與MDA-MB-453細胞共同培養16小時後洗掉以激發波長為808 nm，強度為6 W/cm2的雷射光源照射5分鐘後，經過24小時之細胞存活率，和單純以CPT作用的細胞組別相比，HICPDNEs的細胞毒殺效果在高濃度(> 40 μM)下皆有顯著效果。在FDOT影像診斷中，以乳房組織與腫瘤仿體模擬實際狀況之量測，可偵測到含ICG之腫瘤仿體內之螢光且穿透深度達到45 mm，且根據本研究定義之「腫瘤切除品質指標」評估影像之結果，1 μM的HICPDNEs的值為24.89，具有作為後續臨床上診斷之潛力。本研究證實HICPDNEs具有良好的潛力成為乳腺癌治療和早期診斷的藥物載體，然而更多的研究需要在未來進一步驗證並持續的優化。

摘要(英)

HER2-expressing breast cancer has long been recognized as one of the most lethal gynecological disease for women due to high incidence, its drug resistance and poor prognosis. On the other hand, treatment of breast cancer in its early stage may dramatically enhance the 5-year survival rate to > 90 %, indicating that in addition to development of an effective therapeutic strategy, successful early detection plays a crucial role in the improvement of survival rate of HER2+ breast cancer. In this study, a type of theranostic agent named human epidermal growth factor receptor 2 (HER2)-target Indocyanine green (ICG)-Camptothecin (CPT)-loaded perfluorocarbon double-nanoemulsions (HICPDNEs) was developed for use in photochemotherapy and fluorescence diffuse optical tomography (FDOT) diagnostics. According to our result, the size and zeta potential of HICPDNEs is 292.2 ± 5.6 nm and -13.5 ± 3.1 mV, respectively. The encapsulation efficiency for CPT and ICG is 40.31 ± 7.6 % and 99.12 ± 0.33 %, respectively. Under incubated at 37 ℃ for 48 hr, The degradation of HICPDNEs almost prolong 73 % compared with Free ICG, and only 4.29 % drug release. Hyperthermia effect of HICPDNEs under 808 nm laser exposure with intensity of 6 W/cm2 for 5 min, 20 μM of HICPDNEs can achieve 42 ℃. To generation of singlet oxygen, HICPDNEs are higher than the free ICG enormously at the same concentration, showed the potential of the phototherapy. Compare to the ICPDNEs, HICPDNEs are more effective to combine the HER2+ breast cancer cell (MDA-MB-453) due to its target mechanism, indicated HICPDNEs has specification on HER2 positive cancer cell. In cytotoxicity, HICPDNEs incubated with cell for 16 hr and expose the 808 nm laser (6 W/cm2) for 5 min. its cell viability indicated good effective than the free CPT in high concentration (> 40 μM). In diagnosis, Tumor-like inclusions containing HICPDNEs in breast phantoms could be detected up to a depth of 4.5 cm using a FDOT system, according to 「Tumor resection quality index」, the best value is 24.89 in 1μM of HICPDNEs. Above of all, HICPDNEs has a good potential to be a theranostic agent for treatment and early diagnosis in breast cancer. More studies that we need to further validate in the future.

關鍵字(中)

★ 全氟碳化合物
★ 靛氰綠
★ 喜樹鹼
★ 乳癌
★ 雙層乳劑
★ 螢光擴散光學斷層掃描

關鍵字(英)

論文目次

目錄
第一章緒論 1
1-1 研究背景與動機 1
1-2 研究目的 4
第二章文獻探討 5
2-1 乳癌 5
2-1-1 乳癌分期與分類 5
2-1-2 診斷方法 8
2-1-3 治療方法 11
2-1-4 光療 12
2-1-5 標靶治療 15
2-2 螢光擴散光學斷層掃描 18
2-2-1 光學特性 18
2-2-2 乳房與腫瘤組織光學特性 19
2-2-3 擴散光學量測技術 21
2-2-4 螢光擴散光學斷層掃描檢測技術 23
2-2-5 螢光標靶顯影劑 23
2-3 靛氰綠 26
2-4 喜樹鹼 29
2-5 全氟碳化物 31
第三章實驗材料與方法 34
3-1 實驗藥品及儀器 34
3-1-1 藥品 34
3-1-2 儀器 35
3-2 實驗流程 37
3-3 全氟碳標靶奈米雙層乳劑之製備 38
3-3-1 Pluronic F68 羧酸化改質 38
3-3-2 製備包覆 ICG/CPT 奈米雙層乳劑 39
3-3-3 表面接枝人類表皮生長因子受體 2 單株抗體 40
3-4 HICPDNEs 物理特性分析 41
3-4-1 粒徑分析 41
3-4-2 表面電位分析 41
3-4-3 HICPDNEs 表面接枝抗體之測定 41
3-4-4 包覆率分析 41
3-4-5 包藥率分析 42
3-4-6 掃描式電子顯微鏡(SEM)分析 42
3-4-7 熱穩定性分析 42
3-5 HICPDNEs 於光治療功能之試驗 43
3-5-1 HICPDNEs 之升溫之效能 43
3-5-2 HICPDNEs 生成單態氧之效能 43
3-6 細胞體外實驗 43
3-6-1 細胞培養 43
3-6-2 HICPDNEs 對 HER2 過度表現乳癌細胞的專一性試驗 44
3-6-3 HICPDNEs 體外細胞毒性試驗 44
3-7 HICPNDEs 於 FDOT 系統之顯影功能試驗 46
3-7-1 液態仿體之製作 46
3-7-2 HICPDNEs 與純 ICG 水溶液最佳螢光強度之濃度測定 47
3-7-3 HICPDNEs 及 ICG 水溶液於乳房組織仿體之 FDOT 影像效果48
3-7-4 FDOT 影像重建之影像面積量化分析 51
3-8 統計分析 52
第四章實驗結果與討論 54
4-1 修飾 Pluronic F68 尾端羧基化的分析 54
4-2 HICPDNEs 的物性化性分析 55
4-2-1 HICPDNEs 粒徑及表面電位分析 55
4-2-2 HICPDNEs 之表面形態分析 56
4-2-3 HICPDNEs 於表面接枝抗體分析 57
4-2-4 HICPDNEs 內 CPT 與 ICG 的包覆、包藥率分析 59
4-2-5 HICPDNEs 熱穩定性分析 59
4-3 HICPDNEs 於光治療功能之試驗分析 62
4-3-1 HICPDNEs 之升溫效果分析 62
4-3-2 HICPDNEs 生成單態氧之效能分析 64
4-4 HICPDNEs 體外細胞實驗之分析 65
4-4-1 HICPDNEs 對 HER2 過度表現乳癌細胞的專一性試驗分析 65
4-4-2 HICPDNEs 體外細胞毒性試驗 67
4-5 HICPDNEs 於 FDOT 系統之顯影功能試驗分析 70
4-5-1 HICPDNEs 與 ICG 水溶液最佳螢光強度之濃度測定分析 70
4-5-2 HICPDNEs 與 ICG 水溶液於乳房組織仿體之 FDOT 影像分析71
第五章結論與未來展望 84
參考文獻 86
附錄一藥物濃度與吸收值之檢量線 93
附錄二 FDOT 與 DOT 之影像重建 94

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指導教授

李宇翔(Yu-Hsiang Lee)

審核日期

2018-1-24

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