搭載艾黴素之葉酸接枝高分子微胞對腫瘤細胞的標靶性及細胞吞噬路徑探討

Tumor Cell Targeting and Endocytosis Pathways of Doxorubicin Loaded Folate-Conjugated Polymeric Micelles

李毓倫(Yu-Lun Li)

指導教授 ： 謝明發

中原大學/工學院/生物醫學工程研究所/碩士(2012年)

https://doi.org/10.6840/cycu201200264

摘要

傳統的化學療法缺乏對正常細胞與癌細胞的辨識性，癌細胞的表面根據其生理功能會表現不同的受器，葉酸受器會過表現在特定癌細胞膜表面，因此本研究將葉酸接枝到星狀聚己內酯-聚乙二醇共聚合物上，做為葉酸受器表現的人類口腔癌細胞(KB)與乳癌細胞(MCF-7)的標靶藥物載體。經傅立葉轉換紅外線光譜儀鑑定結構，並利用核磁共振光譜儀的分析計算分子量，得到4SPCL50-PEG-Folate、4SPCL100-PEG-Folate、4SPCL150-PEG-Folate分子量分別為17168、23361和28680 g/mol，並成功製備出搭載艾黴素的微胞(DMC50F、DMC100F、DMC150F)，由動態光散射分析結果得到微胞粒徑分別為126.2、128.7和123.1奈米。經紫外光光譜儀分析藥物的包覆率分別為76、88.5和33.5 %。因為DMC100F包覆率與載藥率為最好的組別，因此選擇DMC100F做為細胞實驗的藥物載體。DMC100F在pH 5 的酸性磷酸鹽緩衝液中經過 60 小時後的藥物釋放量為84 %，而在 pH 7.4 的中性磷酸鹽緩衝液下釋放量為29 %。從半致死劑量(IC50)比較DMC100與DMC100F的毒殺效果，在MCF-7分別為9.144與6.701 μg/mL，在KB分別為8.306與7.501 μg/mL，顯微鏡螢光影像顯示DMC100F與MCF-7及KB細胞培養一小時後細胞內皆有藥物的螢光影像，利用流式細胞儀分析微胞的吞噬路徑，結果顯示，癌細胞表面的葉酸受器被葉酸抑制其介導功能後，DMC100F在 KB細胞株被抑制48-57 %，MCF-7細胞株被抑制26-39 %。另外我們也發現以甲基環糊精做為脂筏蛋白介導的胞吞作用抑制劑，可以使KB細胞株被抑制69 %，MCF-7細胞株被抑制56 %。在本研究中可以發現DMC100F可以藉由幾種路徑進入細胞，使更多藥物累積在細胞內，並達到毒殺的效果，且在短時間內都具有明顯的標靶效應，因此DMC100F為具有發展潛力的藥物載體。

關鍵字

葉酸 ； 艾黴素 ； 葉酸受器 ； 微胞 ； 星狀聚己內酯-聚乙二醇

並列摘要

The specificity between normal cells and cancer cells in conventional chemotherapy is in-sufficient. The plasma surface of cancer cells is expressed various receptors depending on the physiological functions of the cells. Folate receptor (FR) is overexpressed in the apical membrane surface of certain cancer cells making a potential target of tumor therapeutics. Hence, in this study, folate-decorated micelles based on the star-shaped PCL-PEG copolymer were prepared for targeting to the folate receptor overexpressing in human oral cavity carcinoma cells (KB) and human breast cancer cells (MCF-7). The structure and molecular weight of micelle materials was characterized by FT-IR and 1H NMR. The of 4SPCL50-PEG-Folate、4SPCL100-PEG-Folate and 4SPCL150-PEG-Folate are 17168、23361 and 28680 g/mol, respectively. The doxorubicin-loaded micelles were prepared using dialysis method (DMC50F、DMC100F、DMC150F). The particle size of the DOX-loaded micelle was 126.2、128.7 and 123.1 nm, respectively. The drug loading efficiency were 76、88.5 and 33.5 %, respectively. The total release of DOX in a period of 60 h at pH 7.4 and 5.4 account for 29 % and 84 % of total DOX concentration loaded; respectively. The IC50 of DMC100 and DMC100F are 9.144 and 6.701 μg/mL in MCF-7, 8.306 and 7.501 μg/mL in KB cell. The fluorescent images revealed that micelles were uptake into KB cells and MCF-7 cells after one hour. Furthermore, flow cytometric analysis was used to assay the different uptake mechanisms and trafficking pathways. The result showed that the 48-57 % DMC100F was in KB cells and 26-39 % in MCF-7 cells inhibited by folic acid, the interaction with expressed folate receptors on the surface of cancer cells. In addition, we found the cellular uptake was 69 % in KB cells and 56 % in MCF-7 reduced by methyl-β-cyclodextrin, an inhibitor of the caveolae-mediated endocytosis pathway. These finding suggest that DMC100F enter cells via multiple pathways and might be a potential carrier for cancer treatment.

並列關鍵字

Star-shaped PCL-PEG ； Folate receptor ； Doxorubicin ； Folic acid ； micelle

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