研製包覆靛氰綠與阿黴素之標靶氟化奈米乳劑用於乳癌光/化學治療之研究

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姓名

馬韻婷(Yun-Ting Ma) 查詢紙本館藏

畢業系所

生物醫學工程研究所

論文名稱

研製包覆靛氰綠與阿黴素之標靶氟化奈米乳劑用於乳癌光/化學治療之研究
(Synthesis and Characterization of Her-2-targeted Indocyanine Green-Doxorubicin-Loaded Perfluorocarbon Nanodroplets for Photo-Chemo-Therapy of Breast Cancer Cells)

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摘要(中)

本研究為結合光與化學治療，製備包覆靛氰綠與阿黴素之標靶氟化奈米乳劑，目的使乳癌病患能夠利用光治療輔助以降低減少化學藥物使用劑量，進而減緩化學治療時伴隨的副作用與不適，同時又不降低治療效果。製備方法上以二次乳化法合成出包覆靛氰綠(Indocyanine Green；ICG)及阿黴素(Doxorubicin)之全氟溴辛烷(Perfluorooctyl bromide；PFOB)奈米乳劑，隨後以聚乙烯亞胺包覆於奈米乳劑外層。之後我們利用正負電相吸原理將人類表皮生長因子2 (Human Epidermal Growth Factor Receptor 2; HER-2)吸附於奈米粒子表面，完成包覆靛氰綠與阿黴素之標靶氟化奈米乳劑(HER-2 Targeted ICG-DOX-Encapsulated PFOB Double Nano-Emulsions；HIDPDNEs)。經過儀器分析奈米乳劑之平均粒徑與表面電位分別為340  4.5 nm和-57  1.15 mV；ICG與DOX的包覆率分別為95%與60%。此外ICG在模擬人體內溫度下，原本24小時降解至剩餘5成，在有了載體的包覆之下剩餘率還有九成，顯示出載體提供高穩定度，抗癌藥物的藥物釋放，在水溶液中都不到5%。本研究除了對粒子的物理/化學性質，包括粒徑、電性、構形、細胞專一性以及藥品包覆/降解/釋放率測試外，另對於產品在光熱與光動能力上進行分析。我們利用激發波長808 nm搭配強度為6 W/cm2的近紅外光雷射照射HIDPDNEs奈米溶液，結果顯示，溶液光熱能力在粒子包含 ≥ 2 M ICG濃度下下經雷射照射90秒即可超過40oC，而在粒子自由基生成能力方面，在相同雷射條件刺激下，可造成> 6倍單態氧表現量的生成。以上結果顯示本奈米粒子對於癌症治療具有高度潛力。另外，在專一性測試中，使用相同濃度奈米粒子測驗兩小時胞吞作用後細胞肚子內ICG 螢光表現量，結果顯示MDA-MB-453 (HER-2+)的螢光表現量為MCF-7 (HER-2-)的2.4 倍，經過四小時胞吞作用後，更有近4倍的差異值，因此我們可確認HIDPDNEs對HER-2+ 細胞的專一性。最後，我們使用激發波長808 nm且強度為6 W/cm2的近紅外光雷射照射只經過4小時內吞作用HIDPDNEs (內含10 M ICG 及 4.68 M DOX)的MDA-MB-453乳癌細胞5分鐘，和單純的光療(ICG + 近紅外雷射光照射)及化療藥物(DOX)作用相比較之下，HIDPDNEs毒殺能力高於化療藥物1.8倍 (P < 0.05)，與光療藥物相比較毒殺能力則高出2倍之多(P < 0.05)，證明了HIDPDNEs可同時提供光學及化學治療效果使乳癌細胞死亡。本研究結果證明了所設計之複合載體可用於具HER-2受器表現乳癌細胞之治療，並有望在未來發展成為一種高效能標靶治療乳癌的新興材料。

摘要(英)

Breast cancer has long been recognized as one of the most lethal gynecological disease for women due to high drug resistance and serious side effects during treatment. To resolve these issues, a multi-functional human epidermal growth factor receptor 2 (HER-2)-targeted Indocyanine green (ICG)-Doxorubicin (DOX)-loaded perfluorocarbon double-nano-emulsion (HIDPDNE) was developed in this study. The size and surface charge of the HIDPDNE are 340  4.5 nm and -57  1.15 mV, respectively. The encapsulation efficiency for ICG and DOX is 95% and 60%, respectively. Under incubation at 37 oC for 24 h, the amount of entrapped ICG enhanced 5 folds as compared to the freely dissolved ICG and > 80 wt% of DOX remained in the particles, showing that the thermal stability of loaded chemicals in the aqueous solution is improved. Through the detection of ICG-induced fluorescence, we found that the uptake efficiency of HIDPDNEs within 4 h in MDA-MB-453 cells (HER-2+) was 4-fold higher than that in MCF7 cells (HER-2-), showing that the HIDPDNEs indeed have targeting specificity for HER2-expressing cells. In terms of the therapeutic functionality of the nano-agent, our data show that upon NIR laser exposure, the temperature of medium containing HIDPDNEs with ≥ 2-M ICG equivalent enabled to reach > 40oC within 90 sec and its amount of singlet oxygen yielded significantly enhanced ≥ 6 folds after 5-min laser treatment. The capacity of HIDPDNEs in cancer cell killing was further verified by using MDA-MB-453 as the model cell that the motality of the cells which were treated by HIDPDNEs with 10 and 4.68 M of ICG and DOX equivalent, respectively, for 4 h and followed by 5-min NIR light illumination significantly enhanced 1.8- (P < 0.05) and 2 folds (P < 0.05) as compared to the group with DOX alone or ICG + laser, respectively. Overall, the HIDPDNEs which enable to provide both chemo and photo therapies exhibit a high potential for use in destruction of breast tumor.

關鍵字(中)

★ 全氟碳化合物
★ 靛氰綠
★ 阿黴素
★ 標靶性
★ 雙層乳劑
★ 乳癌
★ 光-化學治療

關鍵字(英)

★ Perfluorocarbon
★ Indocyanine green
★ Doxorubicin
★ Brest cancer
★ Double emulsion
★ Photo-Chemo therapy

論文目次

摘要 I
Abstract III
圖目錄 X
表目錄 XIII
第一章緒論 1
第二章研究背景 3
2.1 現今癌症治療方法與研究成果 3
2.2 乳癌治療原理與方法 5
2.2.1乳癌介紹 5
2.2.2乳癌光學診斷 6
2.2.3乳癌治療 7
2.3 研究動機 13
2.4 生物醫藥奈米技術發展 14
2.4.1 製備方法 14
2.4.2 生醫材料特性 15
2.4.3 藥物載體種類 17
2.5 選用材料介紹 19
2.5.1 靛氰綠 (Indocyanine Green；ICG) 19
2.5.2 阿黴素 (Doxorubicin；DOX) 24
2.5.3 全氟碳化合物 (Perfluorocarbons；PFCs) 26
2.5.4 含氟介面活性劑 (Fluorosurfactant) 30
第三章實驗部分 32
3.1 實驗藥品、儀器設備 32
3.1.1 藥品 32
3.1.2 儀器 33
3.1.3 藥物濃度檢量線 35
3.2 實驗整體流程 36
3.3 DOX-ICG全氟碳標靶奈米雙層乳劑之製備 37
3.3.1 F68尾端修飾羧基團 37
3.3.2製備包覆ICG與DOX之PFOB奈米雙層乳劑 38
3.3.3表面修飾聚乙烯亞胺(Polyethyleneimine；PEI) 40
3.3.4表面修飾人表皮生長因子受體2單株抗體(anti-HER-2-mAb) 41
3.4 DOX-ICG全氟碳標靶奈米雙層乳劑之特性分析 42
3.4.1 粒徑分析 42
3.4.2 表面電位分析 42
3.4.3 包覆率分析 42
3.4.4 包藥率分析 42
3.4.5 熱穩定性分析 43
3.4.6 掃描式電子顯微鏡(SEM)拍攝 43
3.5 測定DOX-ICG全氟碳標靶奈米雙層乳劑產品用於光治療之功能 44
3.5.1產品生成光熱之效能(Hyperthermia Effect) 44
3.5.2產品生成單態氧之效能(Generation of Singlet Oxygen) 44
3.6 細胞培養 45
3.7 DOX-ICG全氟碳標靶奈米雙層乳劑對乳癌細胞的毒性實驗 47
3.8 DOX-ICG全氟碳標靶奈米雙層乳劑對乳癌細胞的專一性實驗 48
3.9 DOX-ICG全氟碳標靶奈米雙層乳劑對乳癌細胞的胞殺實驗 49
3.10 統計分析 50
第四章結果與討論 51
4.1 修飾F68尾端羧基化的分析 51
4.2 DOX-ICG全氟碳標靶奈米雙層乳劑的基本分析 52
4.3 DOX-ICG全氟碳標靶奈米雙層乳劑產品的型態分析 53
4.4 DOX-ICG全氟碳標靶奈米雙層乳劑於表面接枝抗體的分析 54
4.5 全氟碳標靶奈米雙層乳劑包覆藥物之熱穩定性分析 56
4.6 DOX-ICG全氟碳標靶奈米雙層乳劑之光熱效能分析 58
4.7 DOX-ICG全氟碳標靶奈米雙層乳劑之光動效能分析 60
4.8 DOX-ICG全氟碳標靶奈米雙層乳劑對乳癌細胞的毒性測試 62
4.9 DOX-ICG全氟碳標靶奈米雙層乳劑對乳癌細胞的專一性分析 63
4.10 DOX-ICG全氟碳標靶奈米雙層乳劑對乳癌細胞的毒殺效能分析 65
4.11 結論 68
第五章未來展望 69
參考文獻 70

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指導教授

李宇翔(Yu-Hsiang Lee)

審核日期

2016-8-25

推文