研製包覆靛氰綠與絲裂黴素C之標靶全氟碳奈米乳劑應用於膀胱癌光-化學治療之研究

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林?君(Yu-Chun Lin) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

研製包覆靛氰綠與絲裂黴素C之標靶全氟碳奈米乳劑應用於膀胱癌光-化學治療之研究
(Fabrication and Characterization of EGFR-Targeted Indocyanine Green- Mitomycin C-Incorporated Perfluorocarbon Nano-Agents for Photochemotherapy of Bladder Cancer Cells)

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摘要(中)

膀胱癌是泌尿系統中最致命的疾病之一，根據世界衛生組織統計，膀胱癌好發於男性，其發生率在男性癌症中排名第九，死亡率排在全球第十三。目前絲裂黴素C（Mitomycin C；MMC）是最常用的抗膀胱癌藥物之一，然而，高劑量引起的嚴重副作用嚴重妨礙了其應用，為了解決上述問題達到其治療目的，本研究發展製備包覆靛氰綠（Indocyanine Green；ICG）與絲裂黴素C標靶表皮生長因子（Epidermal Growth Factor Receptor；EGFR）之全氟碳化物(Perfluorocarbon；PFC)雙層奈米乳劑（EGFR Target ICG–MMC- Encapsulated PFC Double Nanoemulsion；EIMPNEs），我們預期該試劑能發揮光與化學治療的功能以減緩傳統化學治療時伴隨的副作用與不適，同時又不犧牲治療效果。經過儀器分析EIMPNEs之平均粒徑與表面電位分別為分別為304.22 ± 18.65 nm和-12.67 ± 0.4 mV；ICG與MMC的包覆率分別為98.22 ± 0.86%與41.72 ± 6.86%。在體外模擬人體溫度 37°C下經過 48 小時之後，EIMPNEs所包覆ICG的降解率與游離的 ICG 水溶液相比減少了51%，顯示ICG在有了載體的包覆能夠獲得較佳之穩定度，並以此解決該物質易於水溶液中降解的問題。MMC在37oC避光環境下之藥物釋放率為11.61%，而在近紅外光照射（808 nm; 6 W/cm2）下之藥物釋放率為38.14%。此外，在近紅外光照射（808 nm; 6 W/cm2）下，EIMPNE含有? 40 μM ICG濃度的情況下其溫度在30秒內能夠達到> 40°C ，且5分鐘近紅外光照射後產生的單態氧量與相同濃度之游離ICG溶液相比增加11.2倍。此一結果證明了EIMPNEs對於光熱力和光動力療法的應用具有高度潛力。在細胞專一性試驗中，我們藉由比較EIMPNEs與接枝抗體前的載體─包覆靛氰綠與絲裂黴素C 之全氟碳化物奈米乳劑（ICG–MMC-Encapsulated PFC Double Nanoemulsion；IMPNEs）對EGFR表現型之膀胱癌細胞（T24）的結合效率，結果顯示EIMPNEs對T24的結合效率高出IMPNEs 1.6倍，證實EIMPNEs對EGFR表現型細胞具專一性。細胞體外試驗中（in vitro），EIMPNEs在與T24 細胞共同培養 4小時經近紅外光照射（808 nm ; 6W/cm 2）5 分鐘，再經過 24 小時培養後測量其細胞存活率，結果顯示EIMPNEs對膀胱癌細胞的毒殺效果顯著，僅需使用內含相當濃度18 μM MMC的EIMPNEs即可達到單獨使用90 μM MMC所造成的細胞死亡率。總結來說，本研究證明了EIMPNEs可同時提供EGFR+膀胱癌細胞標靶性以及綜合光及化學治療的效果，因此具有高度的潛力在未來發展成為一種高效能標靶治療膀胱癌的新興材料。

摘要(英)

Bladder cancer is one of the most lethal urinary diseases. According to the statistics of World Health Organization, bladder cancer ranks as the ninth most frequently-diagnosed cancer with a higher incidence rate in men, and is the 13th leading death of cancer worldwide. Currently mitomycin C (MMC) is one of the most commonly used anti-bladder cancer drugs. However, serious side effects resulted from high dose severely hamper its application. To meet therapeutic purpose without aforementioned issues, we aim to manufacture anti-EGFR
indocyanine green (ICG) mitomycin C (MMC) encapsulated perfluorocarbon double nanoemulsions (EIMPNEs), and explore their photochemotherapeutic efficacy on EGFR-expressing bladder cancer cells in vitro. Based on the DLS analysis, the size and surface charge of the EIMPNEs are 304.22 ± 18.65 nm and -12.67 ± 0.4 mV, respectively. The encapsulation efficiencies of ICG and MMC are 98.22 ± 0.86% and 41.72 ± 6.86%, respectively. Under incubated at 37°C for 48 hours, The degradation of EIMPNEs almost prolong 51 % compared with Free ICG, and only 11.61 % drug release. In terms of the phototherapeutic efficacy of the agents, our data show that upon near infrared laser (NIR) exposure (808 nm; 6W/cm2), the temperature of the EIMPNEs with ? equivalent 40-μM ICG concentration was able to reach > 40oC within 30 sec and compare with the free ICG the amount of singlet oxygen generated significantly enhanced ? 10 folds after 5-min NIR laser treatment, demonstrating their potential availability for applications of photothermal and photodynamic therapy. Compare to the IMPNEs, EIMPNEs are more effective to combine the EGFR bladder cancer cell (T24) due to its target mechanism, indicated EIMPNEs has specification on EGFR-expressing bladder cancer cells. In cytotoxicity, EIMPNEs incubated with cell for 4 hr and upon NIR expose (808 nm; 6W/cm2) for 5 mins, our data showed that the EIMPNEs were effective in bladder cancer cells eradication ,and the resulting cell death rate was even higher than that caused by five-fold enhanced amount of encapsulated MMC alone. With the merits of improved ICG stability, EGFR binding specificity, and effective cancer cell eradication, the EIMPNEs exhibit potential for use in EGFR-expressing bladder cancer therapy with lower chemotoxicity.

關鍵字(中)

★ 膀胱癌
★ 光-化學治療
★ 靛氰綠
★ 絲裂黴素C
★ 全氟碳化合物
★ 雙層乳劑

關鍵字(英)

★ Bladder cancer
★ Photochemotherapy
★ Indocyanine green
★ Mitomycin C
★ Perfluorocarbon
★ Double emulsion

論文目次

摘要.................................................I
Abstract...............................................III
誌謝.....................................................V
第一章緒論.............................................1
1.1 研究背景..............................................1
1.2 研究動機與目的........................................2
第二章文獻回顧..........................................4
2.1 膀胱癌概述............................................4
2.1.1 膀胱癌病理種類......................................4
2.1.2 膀胱癌致病因素......................................7
2.1.3膀胱癌治療方式及副作用................................8
2.2 光療.................................................9
2.2.1光熱力治療（Photothermal therapy；PTT）.............10
2.2.2光動力治療 (Photodynamic therapy；PDT)..............11
2.3 靛氰綠（Indocyanine Green；ICG）.....................13
2.4 絲裂黴素C（Mitomycin C；MMC）........................16
2.5 全氟碳化合物（Perfluorocarbon；PFC）..................18
2.6 聚乙二醇（PluronicsR）...............................20
第三章實驗部分.........................................23
3.1 實驗藥品、儀器設備....................................23
3.1.1 藥品..............................................23
3.1.2 儀器..............................................24
3.1.3 藥物濃度檢量線.....................................26
3.2 實驗整體流程.........................................27
3.3 MMC-ICG全氟碳標靶奈米雙層乳劑之製備...................28
3.3.1製備包覆ICG與MMC之PFOB奈米雙層乳劑...................28
3.3.2表面修飾人表皮生長因子受體單株抗體（Anti–EGFR–mAb）...29
3.4 EIMPNEs物理特性分析.................................31
3.4.1粒徑分析............................................31
3.4.2表面電位分析........................................31
3.4.3 EIMPNEs表面接枝抗體之測定...........................31
3.4.4 掃描式電子顯微鏡（SEM）拍攝.........................32
3.4.5 包覆率分析.........................................32
3.4.6 包藥率分析.........................................32
3.4.7 熱穩定性分析.......................................32
3.5 測定EIMPNEs用於光治療功能之試驗.......................33
3.5.1產品照光後升溫之效能（Hyperthermia Effect)...........33
3.5.2產品生成單態氧之效能（Generation of Singlet Oxygen）.34
3.6　細胞培養............................................34
3.7　EIMPNEs對EGFR表現型膀胱癌細胞的專一性試驗.............34
3.8　EIMPNEs體外細胞胞殺試驗..............................35
3.9　統計分析............................................36
第四章結果與討論.......................................37
4.1　EIMPNEs的基本物性化性分析............................37
4.2　EIMPNEs產品表面型態分析..............................38
4.3　EIMPNEs產品表面型態分析..............................39
4.4　EIMPNEs內之MMC與ICG的包覆、包藥率分析................41
4.5　EIMPNEs之熱穩定性分析...............................41
4.6　EIMPNEs之照光升溫效能分析............................45
4.7　EIMPNEs之單態氧生成效能分析..........................47
4.8　於NIR照射下MMC釋放率分析.............................50
4.9　EIMPNEs對EGFR表現型之膀胱癌細胞的專一性分析...........52
4.10　EIMPNEs對膀胱癌細胞之胞殺效能分析....................54
第五章結論.............................................58
第六章未來展望.........................................59
參考文獻.................................................60

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指導教授

李宇翔(Yu-Hsiang Lee)

審核日期

2018-7-24

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