開發癌細胞膜包覆的靛氰綠-喜樹鹼-全氟化奈米乳劑用於皮膚黑色素癌光化學治療

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陳采昕(Cai-Sin Chen) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

開發癌細胞膜包覆的靛氰綠-喜樹鹼-全氟化奈米乳劑用於皮膚黑色素癌光化學治療
(Development of Cancerous Membrane-Covered Indocyanine Green-Camptothecin-Loaded Perfluorinated Emulsions for Photochemotherapy of Melanoma)

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至系統瀏覽論文 (2028-7-1以後開放)

摘要(中)

細胞膜包覆奈米粒子(或奈米乳劑)的技術正越來越多地被提及，而塗有細胞膜的奈米乳劑被預期會有一些優勢，首先利用細胞膜的外塗層作為天然靶點，可以解決外源性物質容易被免疫系統清除的問題，其次利用細胞膜偽裝奈米乳劑的目的是欺騙癌細胞，使其認為同樣是癌細胞，如此一來包裹在癌細胞膜中的奈米乳劑就可以聚集在癌細胞中，實現天然藥物靶向的功能。
首先合成靛氰綠（Indocyanine green, ICG）-喜樹鹼（Camptothecin, CPT）-負載的全氟化奈米乳劑（Indocyanine green-camptothecin-loaded perfluorinated emulsions, ICPEs），結合化療和光療的協同治療，此外細胞膜是從小鼠黑色素瘤（B16F10）細胞中提取出來，再利用共擠壓的方式將ICPEs和癌細胞膜（Cancerous membranes, CMs）結合，最後形成外層癌細胞膜包覆內核奈米乳劑(Cancerous membrane-covered indocyanine green-camptothecin-loaded perfluorinated emulsions, CMICPEs)。
ICPEs的尺寸和電位測量值為230.43 ± 2.96 nm和-22.24 ± 1.48 mV，CMICPEs的尺寸和電位測量值為248.41 ± 8.33 nm和-32.78 ± 0.29 mV，結果顯示包覆細胞膜後尺寸增大。ICG和CPT在奈米乳劑中的包覆率分別為96.64 ± 2.81%和61.11 ± 11.86%。藥物釋放的評估試驗中可見CMICPEs在照射近紅外光（808 nm，6 W cm-2）下快速釋放藥物。ICPEs和CMICPEs在37℃下的ICG穩定性比游離的ICG提升了約1.5倍。在近紅外光（808 nm，6 W cm-2）照射下可以觀察到CMICPEs的光熱效應，並且CMICPEs的光動力效應優於游離的ICG。體外細胞毒性試驗結果可見CMICPEs照射NIR與相同藥量的free CPT相比更具細胞毒性，存活率分別為6.06%和64.70%，有顯著差異，在體內的腫瘤療效試驗中，證實CMICPEs有抑制腫瘤的效果，並且在體內螢光成像試驗中，觀察到CMICPEs具有良好腫瘤滯留效果與腫瘤的靶向能力。從以上的實驗評估中，我們可以期望這項技術的發展，並且預期它作為一種新興的癌症治療材料的潛力。

摘要(英)

Cell membrane-encapsulated nanoparticle (or nanoemulsion) technology has attracted significant attention in recent times. Nanoemulsions coated with cell membranes exhibit some advantages. First, using the outer coating of the cell membrane as a natural target may solve the problem of exogenous material being targeted and cleared by the immune system. Second, the nanoemulsions disguised by cell membranes can be misrecognized by cancer cells as their own; hence, nanoemulsions encased in cancer-cell membranes can reach other cancer cells and achieve natural drug targeting.
Herein, indocyanine green (ICG)-camptothecin (CPT)-loaded perfluorinated emulsions (ICPEs) were synthesized. Cell membranes were extracted from melanoma (B16F10), and coextrusion was employed to combine the ICPEs with cancerous membranes (CMs). Finally, CM-covered ICPEs (CMICPEs) were formed.
The size and potential measurements of the ICPEs and CMICPEs were 230.43±2.96 nm and –22.24±1.48 mV and 248.41±8.33 nm and –32.78±0.29 mV, respectively. The sizes increased after coating the cell membrane. The encapsulation rates of the ICG and CPT in the nanoemulsions were 96.64±2.81 and 61.11±11.86%, respectively. CMICPEs can achieve rapid drug release under near-infrared irradiation (NIR, 808 nm, 6 W cm-2). The ICG stability of the ICPEs and CMICPEs at 37 °C increased by approximately 1.5 times the stability exhibited by free ICG. The photothermal effect of the CMICPEs was observed under NIR irradiation (808 nm, 6 W cm-2). The photodynamic effect of the CMICPEs was determined to be better than that of the free ICG. In vitro cytotoxicity test results indicated that the CMICPEs under NIR were more cytotoxic than free CPT at the same dose, with significant differences in their survival rates (6.06 and 64.70%, respectively). In an in vivo tumor therapy test, CMICPEs were shown to have tumor suppressive effects, and good tumor retention and tumor targeting ability were observed in an in vivo fluorescence imaging test. We expect this technology to be further developed using these experimental results, and we anticipate its potential as an emerging material for cancer therapy.

關鍵字(中)

★ 黑色素瘤
★ 仿生奈米乳劑
★ 靛氰綠
★ 免疫逃逸
★ 同源靶向
★ 光熱療法
★ 光動力療法

關鍵字(英)

★ melanoma
★ biomimetic nanoemulsions
★ indocyanine green
★ immune escape
★ homologous targeting
★ photothermal therapy
★ photodynamic therapy

論文目次

摘要 i
Abstract iii
致謝 v
目錄 vi
圖目錄 xi
表目錄 xiv
縮寫符號說明 xv
第一章緒論 1
1-1 研究動機 1
1-2 研究目的 2
第二章文獻回顧 3
2-1 皮膚癌 3
2-1-1 皮膚癌綜述 3
2-1-2 黑色素瘤(Melanoma) 4
2-1-3 黑色素瘤分期 6
2-1-4 黑色素瘤發展進程 10
2-1-5 黑色素瘤致病因素 11
2-1-6 黑色素瘤治療方法 13
2-2 奈米粒子 15
2-2-1 仿生奈米粒子的出現 15
2-2-2 癌細胞膜包覆奈米粒子 16
2-2-3 合成細胞膜包覆奈米粒子 19
2-3 光治療(Phototherapy) 21
2-3-1 光熱療法（Photothermal therapy, PTT） 21
2-3-2 光動力療法（Photodynamic therapy, PDT） 21
2-4 藥品 23
2-4-1 靛氰綠(Indocyanine green, ICG) 23
2-4-2 喜樹鹼(Camptothecin, CPT) 24
2-4-3 全氟碳化物(Perfluorocarbons, PFC) 25
第三章實驗步驟及材料 26
3-1 實驗藥品及儀器設備 26
3-1-1 實驗藥品 26
3-1-2 儀器設備 29
3-2 實驗流程 30
3-3 製備包覆ICG及CPT全氟碳雙層奈米乳劑 31
3-4 癌細胞膜 33
3-4-1 細胞培養 33
3-4-2 癌細胞膜提取 34
3-4-3 擠壓癌細胞膜 35
3-5 合成癌細胞膜包覆全氟碳雙層奈米乳劑 36
3-6 物理性質分析 37
3-6-1 包覆率分析(Encapsulation Efficiency, EE) 37
3-6-2 載藥率分析(Loading Capacity, LC) 38
3-6-3 粒徑分析 39
3-6-4 表面電位分析 39
3-6-5 超高解析冷場發射掃描式電子顯微鏡 (CFE-SEM) 40
3-6-6 西方墨點法(Western Blotting) 40
3-7 ICPEs & CMICPEs 藥物釋放測定 45
3-7-1 ICPEs & CMICPEs 靜態下藥物累積釋放 45
3-7-2 CMICPEs 照光後藥物釋放 46
3-8 Free ICG & ICPEs & CMICPEs藥物熱穩定測試 46
3-9 Free ICG & ICPEs & CMICPEs光療效果試驗 47
3-9-1 光熱力效果試驗 47
3-9-2 光動力效果試驗 48
3-10 奈米乳劑進入細胞試驗 49
3-11 體外試驗 49
3-11-1 體外細胞毒性測試 49
3-11-2 螢光顯微鏡成像 51
3-12 動物試驗 52
3-12-1體內抗癌療效試驗 52
3-12-2 體內化療藥物殘留試驗 53
3-12-3 體內螢光成像 54
3-12-4 器官組織染色 55
3-13 統計分析 56
第四章實驗結果與討論 57
4-1 基礎物理性質 57
4-1-1 ICPEs & CMICPEs 之外觀 57
4-1-2 ICPEs之包覆率及載藥率 57
4-1-3 ICPEs & CM & CMICPEs之粒徑與表面電位 58
4-1-4 表面型態分析 59
4-1-5 西方墨點法 60
4-2 ICPEs & CMICPEs 藥物釋放測定 61
4-2-1 ICPEs & CMICPEs 靜態下藥物累積釋放 61
4-2-2 CMICPEs 照光後藥物釋放 62
4-3 Free ICG & ICPEs & CMICPEs藥物熱穩定測試 63
4-4 Free ICG & ICPEs & CMICPEs光療效果試驗 65
4-4-1 光熱力效果試驗 65
4-4-2 光動力效果試驗 67
4-5 奈米乳劑進入細胞試驗評估 69
4-6 體外試驗 70
4-6-1體外細胞毒性測試 70
4-6-2 螢光顯微鏡成像 71
4-7 動物試驗 73
4-7-1體內腫瘤治療效果 73
4-7-2 化療藥物體內分佈 76
4-7-3 生化及血球數分析 77
4-7-4 體內螢光成像圖 79
4-7-5 器官組織學分析 81
第五章結論與未來展望 85
參考文獻 86

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指導教授

李宇翔

審核日期

2023-7-25

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