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研究生:張古明
研究生(外文):Gu-Ming Zhang
論文名稱:開發標靶暨光動力藥物用於誘發體外癌細胞凋亡之研究
論文名稱(外文):Development of a photodynamic therapy with targeted drug delivery system to induce cancer cell apoptosis in vitro
指導教授:侯詠德
指導教授(外文):Yung-Te Hou
口試委員:倪衍玄陳三元
口試委員(外文):Yen-Hsuan NiSan-Yuan Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物機電工程學系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:96
中文關鍵詞:葉酸赤蘚紅幾丁聚醣肝癌細胞(HepG2 cells)光動力療法
外文關鍵詞:Folic acidErythrosin B(Erythrosine)ChitosanHepG2 cellsPhotodynamic therapy(PDT)
DOI:10.6342/NTU202000496
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抗癌光動力療法是透過特定波長的光波照射光敏劑後,產生單重態氧與活性氧物質(ROS),進而觸發ROS/JNK/caspase-3訊號路徑來誘發癌細胞凋亡的治療方法。臨床上若是有:(1)不適合採用手術、化療或放療,與(2)採用手術、化療或放療而失敗的癌症患者,則可以採用此抗癌光動力療法進行治療。目前已核准上市的第一代與第二代光敏劑對癌細胞的靶向性與功能性仍須進一步提升,因此該領域正發展第三代光敏劑,其特色是將功能性分子或具有靶向性的載體與現有的光敏劑進行修飾、改質或交聯,藉此改善現有光敏劑的缺點或彌補其不足之處。基於第三代光敏劑的設計理念與廣義的舊藥新用概念,本研究開發出「以葉酸為標靶分子、赤蘚紅為光敏劑,以及幾丁聚醣為質子海綿效應成分的標靶暨光動力藥物」,其中赤蘚紅原為我國合法的食品添加劑與多國臨床牙科上核准使用的牙菌斑顯示劑,過往的研究中也指出赤蘚紅具有光敏劑的特性,能搭配波長505-533 nm的激發光應用於光動力療法。在藥物製備與細胞實驗方面,我們將葉酸透過交聯劑EDC與NHS交聯於幾丁聚醣來形成葉酸-幾丁聚醣複合物,接著將赤蘚紅-三聚磷酸鈉混合液滴加至上述攪拌中的葉酸-幾丁聚醣複合物來合成葉酸-幾丁聚醣-赤蘚紅複合物(FA-CS-ERY Complex),最後以FA-CS-ERY Complex搭配LED陣列裝置對HepG2肝癌細胞進行體外的抗癌光動力療法誘發細胞凋亡之研究。從實驗的結果可以發現,以FA-CS-ERY Complex (此時赤蘚紅濃度為60 µM)來培養HepG2細胞,並利用平均峰波長507.8 nm、累積光劑量3.17 J/cm2的光線來照射時,其細胞活性僅為不經光動力療法處理之細胞的3.2 %,並有效地誘發凋亡,顯示以FA-CS-ERY Complex進行的抗癌光動力療法具有良好的體外抗癌效果。未來的發展上,由於赤蘚紅不僅為光敏劑,同時還具有聲敏劑與放射增敏劑的特性,因此相信FA-CS-ERY Complex、赤蘚紅與其衍生物可進一步作為以光動力療法為原理基礎之放射動力療法、聲動力療法與X光誘導式(激發式)光動力療法的研發對象。
Anticancer photodynamic therapy is the treatment method which can trigger the ROS/JNK/caspase-3 apoptotic pathway and then induce cancer cells apoptosis via generating singlet oxygens and reactive oxygen species (ROS) from irradiating photosensitizers with the specific optical wavelength. In clinical practice, the patient who is not suitable for surgery, chemotherapy, and radiation therapy, or even failed treatment with the three therapy can be treated with anticancer photodynamic therapy. The functionalities and targetings of first-generation and second-generation anticancer photodynamic therapy photosensitizers which are approved by goverments should be further improved, so the third-generation photosensitizers are under development currently. The features of third-generation photosensitizers are that the photosensitizers are almost existing, and they are modified or cross-linked with functional molecules or targeting carriers to improve the disadvantages of the existing photosensitizers. This study is base on the design principle of third-generation photosensitizers, and the general concept of drug repositioning to develop the photodynamic therapy with targeted drug delivery system. We use folic acid, erythrosine, and chitosan in the system as the cancer targeting molecule, photosensitizer, and ingredient of proton sponge effect, respectively. Erythrosine is originally not only approved food additive in Taiwan but also the approved plaque disclosing agent in clinical dentistry in many countries. Furthermore, previous studies indicate that erythrosine is able to use as photodynamic therapy photosensitizer with the excited wavelength range from 505 nm to 533 nm. In the aspect of medicine preparation and cell experiment, folic acid was conjugated on chitosan with EDC and NHS to form Folic acid-Chitosan complex, and then Erythrosine-Sodium tripolyphosphate mixed solution was added drop by drop into the Folic acid-Chitosan complex solution to synthesize Folic acid-Chitosan-Erythrosine Complex(FA-CS-ERY Complex). The FA-CS-ERY Complex is finally used in HepG2 cancer cell experiments in vitro with LED array device to study on cancer apoptosis induced by anticancer photodynamic therapy. The results of experiments show that the ROS from irradiating FA-CS-ERY Complex with green light can induce apoptosis effectively, and the cell relative viability of photodynamic therapy treatment group with FA-CS-ERY Complex is only 3.2 % of control group, and that the erythrosine concentration of the Complex, average peak wavelength, cumulative light dose, and control group is 60 µM, 507.8 nm, 3.17 J/cm2, and no any treatment, respectively. In summary of the results, the photodynamic therapy treatment with FA-CS-ERY Complex demonstrates a good anticancer curative effect in vitro. Besides photosensitizer, erythrosine is also both sonosensitizer and radiosensitizer, so it could be believed that in future development, FA-CS-ERY Complex, erythrosine, and the derivatives of erythrosine are able to use as research subjects of radiodynamic therapy, sonodynamic therapy, X-ray-induced(or excited) photodynamic therapy that are derived from the mechanism of photodynamic therapy.
口試委員會審定書 i
誌謝 ii
摘要 iii
Abstract iv
圖目錄 ix
表目錄 xiv
符號/縮寫說明 xv
第一章 前言 1
1.1 研究背景 1
1.2 研究動機 5
1.3 研究目的 7
1.4 研究架構 8
第二章 文獻探討 10
2.1 光動力療法(Photodynamic therapy, PDT) 10
2.1.1 光動力作用(Photodynamic action) 10
2.1.2 抗微生物光動力療法(Antimicrobial photodynamic therapy) 11
2.1.3 抗癌光動力療法(Anticancer photodynamic therapy) 14
2.2 赤蘚紅結構與應用特性 21
2.3 葉酸接枝幾丁聚醣 24
2.3.1 葉酸之癌細胞受體標靶特性 24
2.3.2 幾丁聚醣質子海綿效應誘發胞內體逃脫之特性 26
2.3.3 葉酸接枝幾丁聚醣之製備方法 27
2.4 幾丁聚醣應用於光動力療法 28
2.5 葉酸接枝幾丁聚醣應用於光動力治療 33
第三章 研究方法 36
3.1 實驗材料(藥品、耗材、細胞與儀器設備) 36
3.1.1 實驗藥品 36
3.1.2 實驗儀器設備 37
3.1.3 實驗耗材 38
3.1.4 實驗細胞 39
3.2 細胞培養 39
3.2.1 人類肝癌細胞(Hepatocellular carcinoma, Human; HepG2)培養 39
3.3 簡易LED陣列製作 40
3.4 LED燈珠照度與光照穩定度檢測 43
3.5 葉酸-幾丁聚醣-赤蘚紅複合物的合成 44
3.5.1 葉酸-幾丁聚醣複合物的製備 44
3.5.2 葉酸-幾丁聚醣-赤蘚紅複合物的製備與檢測 44
3.6 掃描式電子顯微鏡(SEM)檢測FA-CS-ERY Complex 46
3.7 檢測FA-CS-ERY Complex的Zeta電位 47
3.8 檢測FA-CS-ERY Complex中Erythrosine的包埋率 47
3.9 HepG2黏合/胞吞藥物的螢光顯微鏡檢測 47
3.10 HepG2黏合/胞吞藥物的共軛焦顯微鏡檢測 48
3.11 以HepG2檢測FA-CS-ERY Complex的PDT效果 50
3.12 流式細胞儀檢測FA-CS-ERY Complex的凋亡效果 53
第四章 結果與討論 56
4.1 LED燈珠光照參數與光劑量 56
4.2 FA-CS-ERY Complex的掃描式電子顯微鏡(SEM)結果 60
4.3 FA-CS-ERY Complex的Zeta電位 61
4.4 FA-CS-ERY Complex溶液中赤蘚紅濃度與包埋率 62
4.5 HepG2黏合/胞吞FA-CS-ERY Complex的螢光顯微鏡結果 63
4.6 HepG2黏合/胞吞FA-CS-ERY Complex的Confocal結果 64
4.7 FA-CS-ERY Complex應用於HepG2細胞的PDT效果 66
4.8 流式細胞儀檢測FA-CS-ERY Complex的細胞凋亡結果 73
第五章 結論 75
參考文獻 78
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