以葉酸幾丁聚醣包覆雙光子螢光染劑及阿黴素 聚乙二醇-聚己內酯微胞作為乳癌治療診斷之劑型

Encapsulation of two- photon fluorescent dye and doxorubicin in methoxy poly(ethylene glycol)-poly(caprolactone) micelles decorated with folate-chitosan as theranostic agent

傅國華(Quoc Hue Pho)

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(2016年)

https://doi.org/10.6840/cycu201700044

摘要

奈米藥物已被證明比傳統的癌症化療藥物更有效。奈米藥物可以利用標靶藥物至惡性腫瘤以減少副作用，延長循環時間和增加藥物的生物分佈來提高藥物的治療功效。近年來，奈米醫學診療已發展為結合化療及診斷並已經被證明是治療癌症的有利工具。本研究合成一種新穎的奈米載體，主要利用聚乙烯醇甲醚聚乙二醇-聚己內酯(mPEG-PCL)製備成微胞，並於表面修飾葉酸-幾丁聚醣 (FA-CS)作為一個釋放阿黴素(Doxorubicin，DOX)及雙光子螢光劑 (2QMEH)之載體。首先，合成不同分子量之mPEG-PCL(7500、10000、15000 Da)，並以1H-NMR光譜鑑定mPEG-PCL共聚物的化學結構； PCL的CH2出現在1.3 ppm、1.6 ppm、2.3 ppm、4 ppm；mPEG 的OCH3出現在3.4 ppm及mPEG的CH2基團出現在3.64 ppm。由紅外線光譜儀鑑定結果證明mPEG-PCL官能基團如CH出現在2942.8cm-1位置；PCL的C=O出現在1722.8 cm-1、C-O-C出現在1180.2 cm-1。在差示掃描熱分析儀中看到當分子量由7500 Da增加至15000 Da時，熔點由56.65 ℃下降至54.83 ℃。mPEG5000-PCL2500、mPEG5000-PCL5000及mPEG5000-PCL10000微胞之臨界微胞濃度分別為16.4×〖10〗^(-3)、8.91×〖10〗^(-3)及4.47×〖10〗^(-3) mg/mL。分子量44,220 Da之幾丁聚醣成功合成葉酸接枝的幾丁聚醣。結果顯示經過7小時的反應後，NH2 對葉酸的莫爾比為0.110，葉酸接枝的幾丁聚醣也利用UV-Vis及FT-IR進行檢測。本研究利用葉酸-幾丁聚醣:三聚磷酸鈉(STPP)以1:3之比例成功製備出具有127.1 nm粒徑的葉酸-幾丁聚醣奈米顆粒。結果顯示雙光子螢光於氯仿中的UV-Vis光譜吸收峰在313nm、363nm及458nm。 此外2QMEH在PEG-PCL微胞中以UV光照射可發出亮黃色螢光。mPEG5000-PCL10000為搭載2QMEH和DOX最有效之聚合物。利用ANOVA和EXPERT DESIGN 10.0軟體分析得知mPEG5000-PCL10000在DOX與2QMEH初始濃度分別為1 mg/mL和80 μg/mL下具有最高載藥率為68.6 % 和80.45 %。mPEG5000-PCL10000 微胞對DOX的釋放實驗顯示，釋放240小時後pH5.4比pH7.4的環境下產生更快的DOX釋放速率，其釋放速率為39.4 %。最後，本研究利用mPEG5000-PCL10000之共聚物、FA-CS與STPP比例為1:3、DOX和2QMEH濃度分別為1 mg/mL及80 μg/mL的優化條件下製備以FA-CS修飾DOX-2QMEH-mPEG-PCL且粒徑為289.6 nm之微胞.

關鍵字

奈米顆粒 ； 微胞 ； 藥物遞送 ； 乳腺癌 ； 雙光子螢光劑 ； 阿黴素 ； 聚乙二醇-聚己內酯共聚物 ； 葉酸修飾幾丁聚醣

並列摘要

Nano-medicines have been shown to be more efficacious than traditional cancer chemotherapy drugs. Nano-medicines can deliver drugs in a targeted manner to malignant tumor cells, thus reducing side effects, prolonging circulation time and increasing the bio-distribution of the drug, thereby improving the therapeutic efficacy of drugs. Among therapeutic treatments, theranostic therapy is a combination of chemotherapy and diagnostics and demonstrated to be a powerful tool in cancer treatment. In this study, we synthesized and prepared a novel theranostic nano-carrier based on methoxy poly(ethylene-glycol)-poly(ε-caprolactone) (mPEG-PCL) micelles decorated with folate-chitosan (FA-CS) to co-delivery doxorubicin (DOX) and two-photon fluorescence (2QMEH). Firstly, the mPEG-PCL copolymer was successful synthesized with different molecular weights (7500, 10000, 15000 Da). As the results of characterization, 1H-NMR spectrum proved the chemical structure –OCH3 group of mPEG-PCL such as –CH2 group in PCL (1.3 ppm, 1.6 ppm, 2.3 ppm and 4 ppm), –OCH3 group of mPEG (3.4 ppm) and -CH2 group of mPEG (3.64 ppm). FTIR results proved functional groups of mPEG-PCL such as C-H stretching (2942.8 cm-1), C=O groups of PCL (1722.8 cm-1) and C-O-C stretching (1180.2 cm-1). DSC diagram of mPEG-PCL described that the melting point decreased from 56.65 to 54.83 oC when increasing the molecular weight of increasing from 7500 to 15000 Da. The CMC of mPEG5000-PCL2500, mPEG5000-PCL5000 and mPEG5000-PCL10000 micelles was 16.4×10-3, 8.91×10-3, 4.47×10-3 (mg/mL), respectively. Folate-chitosan conjugate was successfully synthesized from depolymerized chitosan (44,220 Da). As results revealed that the coupling ratio of -NH2 to FA was 0.110 after 7 hours reactions. In addition, FA-CS conjugates were also investigated by UV-Vis and FTIR. Interestingly, FA-CS nanoparticles with particles size of 127.1 nm were successfully prepared at ratio of folate-chitosan to sodium tripolyphosphate (FA-CS: STPP) (1:3). As results, the UV-Vis spectrum of two photon fluorescence (2QMEH) showed three typical absortion peaks at 313 nm, 363 nm and 458 nm in chloroform solvent. In addition, 2QMEH can be dissolved in the core of mPEG-PCL micelles to emit the bright yellow light under UV irradiation (363 nm). The mPEG5000-PCL10000 copolymer was proved as the most efficient polymer to load 2QMEH and DOX. As the results of ANOVA and EXPERT DESIGN 10.0 software, mPEG5000-PCL10000 showed the highest loading efficiency of DOX and 2QMEH of 68.6 % and 80.45 % with initial concentrations of DOX (1 mg/mL) and 2QMEH (80 μg/mL), respectively. The release profile of DOX-2QMEH-mPEG-PCL micelles described that mPEG5000-PCL10000 resulted in a faster rate of DOX release at pH 5.4 as with 39.4% than that of pH 7.4 after 240 hours. Finally, DOX-2QMEH-mPEG-PCL decorated by FA-CS was prepared based on optimized conditions of mPEG5000-PCL10000 copolymer, FA-CS with ratio of 1:3, 1 mg/mL of DOX and 80 μg/m of 2QMEH with particle size of 289.6 nm.

並列關鍵字

nanoparticles ； micelles ； drug delivery ； breast cancer ； two-photon fluorescent agent ； doxorubicin ； polyethylene glycol-block-polycaprolactone ； folate-chitosan.

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