線性與分支性聚己內酯兩性高分子之艾黴素奈米載體對乳癌治療之體外及體內研究

In vitro and In vivo Studies of Linear and Branched Amphiphilic Block Copolymers Bearing Poly(ε-Caprolactone) Block as Doxorubicin Nanocarriers for Breast Cancer Treatments

阮文強(Nguyen-Van Cuong)

指導教授 ： 謝明發

中原大學/工學院/生物醫學工程研究所/博士(2011年)

https://doi.org/10.6840/CYCU.2011.00064

摘要

近年來奈米藥物遞送技術對於標靶藥物到腫瘤細胞扮演著重要的角色；即減少副作用，延長藥物在體內循環時間及增加藥物在人體內的擴散程度因而增加藥物的療效。本研究之目的是發展對於野生種與抗藥性人類乳腺癌細胞可以增加療效的奈米化艾黴素高分子微胞。本研究設計一系列聚己内酯-單甲基聚乙二醇為主的團聯共聚合物微胞。首先，利用單甲基聚乙二醇與 ε-己内酯製備不同鏈長的聚己內酯和固定鏈長單甲基聚乙二醇的三團聯共聚合物(單甲基聚乙二醇-聚己内酯-單甲基聚乙二醇共聚合物)，並利用氫核磁共振光譜儀，凝膠透層析儀，傅立葉轉換紅外線光譜及視差掃描卡計鑑定合成的共聚合物化學結構。這些兩性共聚合物可以在水溶液中自組裝成奈米微胞，其疏水性的內核可搭載艾黴素。微胞粒徑介於40-92奈米，包覆艾黴素的微胞，其載藥率與包覆率分別為3.7-7.4 %和 26.4-49.2 %。由於微胞的性質受pH值影響，從艾黴素在不同酸鹼溶液的釋放曲線可發現，藥物在pH 5.4比pH 7.4釋放快。此外，包覆艾黴素的微胞與單純艾黴素對抗藥性人類乳腺癌細胞的細胞毒性相似，抗藥性人類乳腺癌細胞可以更快且大量的吸收包覆艾黴素的微胞。在ICR小鼠體內，包覆艾黴素的微胞在血漿中循環的時間比純艾黴素長。在SD大鼠體內的生物分布實驗顯示，注射微胞劑型4小時後發現艾黴素在腎跟脾臟有顯著的外滲。比較多次注射包覆艾黴素微胞和只有單純艾黴素時(5毫克/公斤，3次分別在第0、7和14天注射)，種植在裸鼠的野生種人類乳腺癌细胞之腫瘤生長會被抑制。在第二部分合成星狀的聚己内酯-聚乙基乙烯磷酸己内酯。此高分子可以自組裝成奈米尺寸的微胞，平均粒徑133.9-150.0奈米。在水溶液中，疏水性的微胞內核搭載艾黴素，包覆艾黴素的包覆率為 55.2 %。在細胞實驗中，包覆艾黴素的微胞受到pH值影響，包覆艾黴素的微胞對於野生種與抗藥性人類乳腺癌細胞皆具有明顯的細胞毒性。從共軛焦顯微鏡跟流式細胞儀的結果顯示，包覆艾黴素的微胞可做為抗藥性人類乳腺癌細胞藥物遞送系統的載體。此外，利用氫核磁共振光譜儀，凝膠透層析儀，傅立葉轉換紅外線光譜及視差掃描卡計系統性的鑑定合成的共聚合物化學結構。微胞經由巨噬細胞分泌一氧化氮測試，紅血球溶血測試和人類乳腺癌細胞毒性測試都顯示為安全無毒性的。被動藥物遞送系統的奈米載體會有全身毒性且生物可利用率低。因此本研究的第三部分，藉由葉酸標靶到人類乳癌細胞，以主動和被動運輸將藥物遞送到細胞內來克服這些限制。由於星狀聚乙二醇-聚己內酯共聚合物具有較佳的載藥量，且為了標定到具有過表現葉酸受器的人類乳腺癌細胞，本研究將葉酸接枝到星狀聚乙二醇-聚己內酯共聚合物上，利用氫核磁共振光譜儀，凝膠透層析儀，傅立葉轉換紅外線光譜及視差掃描卡計鑑定合成的共聚合物。包覆率和載藥率分別為 90 ％和13 ％。包覆艾黴素的微胞粒徑是148.2奈米。從細胞吸收艾黴素的實驗結果可發現，對野生種人類乳腺癌細胞，包覆艾黴素的葉酸-聚乙二醇-聚己內酯微胞與單純艾黴素的細胞吸收結果相似。但是，對抗藥性人類乳腺癌細胞，包覆艾黴素的葉酸-聚乙二醇-聚己內酯微胞比單純艾黴素的細胞吸收好。本研究證明，包覆艾黴素的葉酸-聚乙二醇-聚己內酯微胞在癌症治療上為一個有潛力的載體。

關鍵字

單甲基聚乙二醇 ； 藥物遞送系統聚己内酯 ； 微胞 ； 乳癌

並列摘要

Recently, nanosized drug delivery system plays a pivotal role by delivering drugs in a targeted manner to the malignant tumor cells, e.g. reducing side effects; prolonging circulation time and increasing biodistribution of drugs, therefore increase the therapeutic efficacy of drugs. The purpose of this study was to develop polymeric nanocarriers of doxorubicin (DOX) that can increase the therapeutic efficacy of DOX for both sensitive and resistant breast cancer cell lines (MCF-7 and MCF-7/ADR). Toward this goal, a series of nanosized micelles composed of block copolymers have been developed, for which the design was based on the use of poly(ε-caprolactone) (PCL) block polymer decorated with poly(ethylene glycol) block (PEG). In the first formulation, triblock copolymers of monomethoxy poly(ethylene glycol) (mPEG) and ε-caprolactone were prepared with various lengths of PCL compositions and a fixed length of mPEG segment, named as EC220E, EC80E and EC36E. Additionally, the structures of the synthesized copolymers were systematically characterized with proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC), respectively. These amphiphilic linear copolymers can self assemble into nanoscopic micelles with their hydrophobic cores, encapsulated doxorubicin in an aqueous solution. The particle size of prepared micelles were around 40-92 nm. The DOX loading content and DOX loading efficiency were 3.7-7.4% and 26.4-49.2%, respectively. Release profile of DOX-was pH-dependent and faster at pH 5.4 than that at pH 7.4. Additionally, the cytotoxicity of DOX-loaded micelles was found to be similar with free DOX in drug-resistant cells. The greater amounts of DOX and fast uptake into the MCF-7/ADR cells from DOX-loaded micelles suggest a potential application in cancer chemotherapy. Moreover, the micelle composed of triblock copolymer EC220E was injected in mice for evaluations of possible toxicities of polymeric carriers. Importantly, the circulation time of DOX-loaded micelle in the plasma significantly increased compared to that of free DOX in rats. Biodistribution study displayed that plasma extravasation of DOX in liver and spleen occurred in first 4 h. Lastly, the tumor growth of human breast cancer cells in nude mice was suppressed by multiple injections (5 mg/kg, 3 times at day 0, 7 and 14) of DOX-loaded micelles as compared to that with multiple administrations of free DOX. In the second formulation, the star-shaped hydrophobic poly(ε-caprolactone) segment was conjugated to the biodegradable, biocompatible and water soluble polymer; polyphosphoester (PPE); poly(ε-caprolactone)-poly(ethyl ethylene phosphate) (PCL-PEEP). For extending study, the micelles composed of the star-shaped copolymer were prepared and DOX was encapsulated in the PCL core. The star-shape copolymer can self assemble into nanoscopic micelles with a mean diameter of 133.9-150.0 nm and a spherical shape. The hydrophobic micellar cores encapsulated DOX in an aqueous solution with a loading efficiency of 55.2%. The in vitro release of DOX from DOX-loaded micelles was pH-dependent. DOX-loaded micelles present significantly enhanced cytotoxicity to both MCF-7 and MCF-7/ADR cells after second incubation of cell with DOX and DOX-loaded micelles. Moreover, results of confocal microscopy and flow cytometry of DOX-loaded micelles demonstrate the feasibility of this delivery system for effective therapy of drug-resistant tumors. Additionally, the structures of the synthesized copolymers were systematically characterized with proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC), respectively. The prepared empty micelles (placebo) show safe behaviors based on in-vitro nitric oxide, hemolytic tests and MTT assays. In the third formulation, the drug delivery systems using conventional nanocarriers are associated with the systemic toxicity and poor bioavailability of drug due to lack of its specificity. The objective of this study is to overcome these limitations by introduction of folic targeting ligand in drug delivery system to deliver drug within tumor cells via passive and active-mediated endocytosis. Hence, in this study, folate-decorated micelles based on the star-shaped folate-PEG-PCL copolymeric were prepared for targeting to the folate receptor overexpressing in human breast cancer cells. The structure was characterized by 1H NMR, FT-IR and DSC. The self assembly of amphiphilic copolymer and DOX delivery application were investigated. The drug loading efficiency and drug loading content are 90% and 13%, respectively. The particle size of the DOX-loaded micelle was 148.2 nm. The results obtained in the intracellular uptake showed that the similar cellular uptake of DOX and DOX-loaded FOL-PEG-PCL micelle were observed in MCF-7 cells. However, the uptake of DOX-loaded FOL-PEG-PCL micelle was higher than that of free DOX in MCF-7/ADR cells. This study demonstrates the feasibility of DOX-loaded FOL-PEG-PCL micelle as a potential carrier for cancer treatments.

並列關鍵字

Micelle ； poly(ethylene glycol) and breast cancer. ； poly(ε-caprolactone) ； drug delivery system

參考文獻

[181] Hsieh, M.F.; Lin, T.Y.; Gau, R.J.; Chang, H.T.; Lo, Y.L.; Lai, C.H. Biodegradable polymeric nanoparticles bearing stealth PEG shell and lipophilic polyester core. J. Chin. Inst. Chem. Engrs. 2005, 36, 609-615.

[1] Benson, J.R. An alternative to initial axillary-lymph-node dissection. The Lancet Oncology. 2010, 11(10), 908-909.

[2] Team, N.L.S.T.R. The National Lung Screening Trial: Overview and Study Design. Radiology. 2010.

[4] Malandrino, P.; Al Ghuzlan, A.; Castaing, M.; Young, J.; Caillou, B.; Travagli, J.-P.; Elias, D.; de Baere, T.; Dromain, C.; Paci, A.; Chanson, P.; Schlumberger, M.; Leboulleux, S.; Baudin, E. Prognostic markers of survival after combined mitotane- and platinum-based chemotherapy in metastatic adrenocortical carcinoma. Endocr Relat Cancer. 2010, 17(3), 797-807.

[6] Radu, A.; Pichon, C.; Camparo, P.; Antoine, M.; Allory, Y.; Couvelard, A.; Fromont, G.l.; Hai, M.T.V.; Ghinea, N. Expression of Follicle-Stimulating Hormone Receptor in Tumor Blood Vessels. N Engl J Med. 2010, 363(17), 1621-1630.