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  • 學位論文

聚己內酯與聚麩胺酸團聯共聚物之合成與搭載 阿黴素之奈米微胞對人類乳癌細胞之細胞毒性

Synthesis of Poly(ε-caprolactone)-Poly(γ-glutamic acid) (PCL-PGA) Block Copolymer as Doxorubicin Nanomicelle for Human Breast Cancer Cells

指導教授 : 謝明發

摘要


本研究中,以單甲基聚乙二醇-雙聚己內酯聚麩胺酸嵌段共聚合物搭 載抗癌藥物艾黴素餵食野生種人類乳癌細胞(MCF-7/wt)。聚麩胺酸為一生物 可分解且無毒性之高分子聚合物,與聚己內酯共聚合搭載疏水性抗癌藥物 艾黴素,以延長在血液中之半衰期。使用紅外線光譜儀、氫質子核磁共振 儀、示差掃描熱分析儀及動態散射粒徑儀鑑定聚己內酯聚麩胺酸化學結 構;以螢光探針DPH 觀察其微胞形成之行為。微胞的粒徑大小與聚合物組 成、溫度及酸鹼值有關,尺寸介於90 至200 奈米之間。隨著聚麩胺酸鏈長 增加,微胞尺寸隨之下降。相對於高酸鹼質的環境時,在低酸鹼質環境所 形成微胞的尺寸較大,其臨界微胞濃度為6.3 至10.7 重量百分比(63-107 毫 克/升)。單甲基聚乙二醇-雙聚己內酯聚麩胺酸微胞對藥物的承載量及承載 率分別為12.14% 與97.22%。體外細胞實驗結果顯示,單甲基聚乙二醇- 雙聚己內酯聚麩胺酸未搭載藥物之微胞濃度為50 毫克/毫升時並未造成 MCF-7/WT 細胞形態改變,而細胞存活率結果顯示未搭載藥物之微胞具有 極小的毒性。搭載艾黴素之微胞其半致死劑量為6.73 @g/mL,低於單純添 加艾黴素。其藥物釋放速率在酸鹼值為7.4 時高於酸鹼值為5.0 時。添加搭 載藥物微胞與MCF-7/WT 細胞培養後可由螢光顯微鏡發現,搭載藥物微胞 有聚集的現象,證明微胞在溶脢體的酸性環境中。在藥物擴散進入細胞內 之前,藥物累積於細胞質中。初步的動物實驗結果顯示注射雌激素並未影 響BALB/c nu mouse 之體重,但會促進其雌性性徵的表現。卵巢完全切除 IV 之老鼠其腫瘤成長速率在實驗初期不如未切除卵巢之老鼠;然而,完全切 除卵巢之老鼠其腫瘤成長速率會以倍數增加。組織學方面,H&E 染色結果 指出,位於卵巢完全切除老鼠之乳腺脂肪墊下方之細胞為乳癌腫瘤細胞。 本研究至此,成功的合成並分析單甲基聚乙二醇-雙聚己內酯聚麩胺酸共聚 物,並且完成細胞毒性測試。未來預期完成此共聚合物的應用。

並列摘要


In this paper, a poly(γ-glutamic acid)-poly(ε-caprolactone)-poly(γ-glutamic acid) [PEG-(PCL-PGA)2] triblock copolymer was synthesized for the encapsulation of doxorubicin (Dox) drug in the treatment of wild type (MCF-7/wt) human breast cancer cells. Because γ-PGA is a natural food substance, it is non-toxic and readily biodegradable. This was copolymerized with PCL to extend its lifetime when it is circulated in blood pool and to hold the hydrophobic doxorubicin drug. The copolymer PEG-(PCL-PGA)2 was characterized using fourier transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (1H NMR), differential scanning calorimetry (DSC) and dynamic light scattering (DLS). The copolymer micelle diameters were found to be dependent on copolymer composition, pH and time. The particle sizes ranged from 90-200 nm. As the length of the PGA increased, the size of the micelles decreased. In contrast, as the length of the PCL increased, the size of the micelles increased. The micelles formed larger particle sizes at low pH compared to high pH. Changes in the turbidity of the solution which were not reflected as changes in the particle sizes indicated possible pH-dependent morphological changes. The micelles formed at a critical micelle concentration in the range of 6.3-10.7 wt% (63-107 mg/L) with DPH in methanol. In vitro studies involved plasma stability, cytotoxicity of blank micelles, drug loading and release, cytotoxicity of Dox-loaded micelles and fluorescence microscopy. It was found that copolymers which had short length PGA and PCL chains proved to be stable in plasma. Furthermore, the PEG-(PCL-PGA)2 copolymer micelles did not cause any significant morphological changes in MCF-7/wt cells up to an amount of 0.50 mg of PEG-(PCL10-PGA7.5)2. An MTT assay of PEG-(PCL5-PGA8)2 and PEG-(PCL5-PGA2.9)2 showed minimal cytotoxicity of the blank micelles and some degree of membrane disruption. After drug loading, it was found that the DLC and DLE using PEG-(PCL5–PGA2.9)2 were 12.14% and 97.22% respectively. The cytotoxicity of Dox-loaded micelles against MCF-7/wt cells was lower with an IC50 of 1.17 @g/mL, than free dox with an IC50 of 0.065 @g/mL. On II the other hand, the IC50 against MCF-7/Adr was 10 @g/mL and 1.11 @g/mL for Dox-loaded micelles and free Dox, respectively. Drug release was higher at pH 7.4 than at pH 5 but the overall, the system exhibited slow sustained release. Fluorescence microscopy of MCF-7/wt cells incubated with Dox-loaded micelles showed that the micelles can aggregate, indicating contact with the acidic environment of the lysosomes. The drug was also initially present in the cytoplasm before becoming diffuse all throughout the cells. Lastly, preliminary studies in vivo studies were done on a BALB/c nu mouse model showed that estradiol did not have any significant effect on the weight of the mice but that it increased the manifestation of female characteristics. Mice that were ovariectomized grew multiple tumors at a slower rate than mice with intact ovaries. However, the mice that did not undergo overiectomization exhibited a faster growth rate of tumor at the beginning of the study. Histological H and E staining showed that the tumor grown on the lower mammary fat pad of overiectomized mouse is a breast cancer tumor tissue. In conclusion, PEG-(PCL-PGA)2 amphiphilic copolymers were synthesized, characterized and tested for cytotoxicity to target cells. Doxorubicin drug was effectively encapsulated into the micelles. Further studies can be done to characterize the conformational changes, in vivo characteristics and broader applications of this micelle system.

參考文獻


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