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1 84 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 汪舒婷等 /D- 甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用 January 25, 2016, 32(1): Chin J Biotech, All rights reserved 医学与免疫生物技术 D- 汪舒婷 1,2, 张权 1,2,3, 叶舟 1,2, 熊永权 1,2, 崔晨宇 1,2, 尹健 ,2,3,,,. D-., 2016, 32(1): Wang ST, Zhang Q, Ye Z, et al. D-mannose-conjugated polymeric micelles for targeted drug delivery. Chin J Biotech, 2016, 32(1): 摘要 : 聚合物胶束作为药物载体具有良好的稳定性和生物相容性, 提高疏水性药物溶解性等优势, 是一类很有应用潜力的药物传输系统 本研究以合成的共价键连 D- 甘露糖的双亲性聚合物分子 (PGMA-Mannose) 为药物载体, 包载抗癌药物阿霉素 (DOX) 制备具有甘露糖受体靶向性和 ph 敏感药物释放特性的新型载药聚合物胶束 利用激光共聚焦显微镜和 MTT 细胞毒性评价方法对载药胶束的细胞内吞摄取和毒性进行评价 实验结果表明, 载药胶束能特异性识别人乳腺癌细胞 MDA-MB-231 表面过度表达的甘露糖受体, 被癌细胞大量摄取并在细胞溶酶体酸性环境内释放药物, 而载药胶束在表面甘露糖受体低表达的 HEK293 细胞中只有少量摄取 与原药 DOX 相比, 该载药胶束对癌细胞的毒性显著提高, 而对正常细胞的毒性较低 因此, 该 PGMA-Mannose 聚合物胶束有望成为一种新型的靶向药物输送系统应用于癌症的治疗 : D- 甘露糖, 聚合物胶束, 靶向药物输送, 受体介导的胞吞, 细胞毒性 Received: February 5, 2015; Accepted: April 3, 2015 Supported by: National Natural Science Foundation of China (No ), Natural Science Foundation of Jiangsu Province (No. BK ). Corresponding authors: Quan Zhang. Tel: ; quanzhang@jiangnan.edu.cn Jian Yin. Tel: ; jianyin@jiangnan.edu.cn (No ) (No. BK )

2 汪舒婷等 /D- 甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用 85 D-mannose-conjugated polymeric micelles for targeted drug delivery Shuting Wang 1,2, Quan Zhang 1,2,3, Zhou Ye 1,2, Yongquan Xiong 1,2, Chenyu Cui 1,2, and Jian Yin 1,2,3 1 The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi , Jiangsu, China 2 School of Biotechnology, Jiangnan University, Wuxi , Jiangsu, China 2 Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi , Jiangsu, China Abstract: Polymeric micelles have exhibited attractive properties as drug carriers, such as high stability in vivo and good biocompatibility, and been successfully used to dissolve various drugs of poor aqueous solubilities. In this study, we developed a new type of polymeric micelles with mannose-mediated targeting and ph-responsive drug release properties for anticancer drug delivery. The polymeric micelles were prepared from an amphiphilic polymer, poly (glycidyl methacrylate)-g-mannose (PGMA-Mannose). An anticancer drug, doxorubicin (DOX), was encapsulated into the micelles during the micellization, and could be released rapidly under acidic condition. The specificity of cellular uptake of the micelles by two different cell lines was studied using confocal laser scanning microscopy and the MTT assay. DOX-loaded micelles were efficiently trapped by mannose-receptor-overexpressing cancer cells MDA-MB-231, whereas mannosereceptor-poor cells HEK293 showed much lower endocytosis towards the micelles under the same conditions. Thus, DOX-loaded micelles displayed higher cytotoxicity to MDA-MB-231 cancer cells as compared with free DOX. The present study demonstrates that PGMA-Mannose micelles are a promising targeted drug delivery system for cancer therapy. Keywords: D-mannose, polymeric micelles, targeted drug delivery, receptor-mediated endocytosis, cytotoxicity [1-3] [4] [5-6] [7] [8-9] [10] (EPR) [11] EPR [12] [13] [14] N- [15-16] cjb@im.ac.cn

3 86 ISSN CN /Q Chin J Biotech January 25, 2016 Vol.32 No.1 D- (PGMA-Mannose) MDA-MB-231 HEK293 MDA-MB 材料与方法 1.1 主要试剂和仪器 (GMA 97%) 2,2 - (Bpy 99%) (98%) (+)-L- (99%) (98%) 2- (MBrP 99%) D- (D-Mannose ) (CuBr 99%) (DOX HCl 99%) ( 1 HNMR) Bruker AVANCE 400M Waters Waters1515 (GPC) (THF) Nicolet Nexus 470 Malvern Nano ZS (DLS) 1 min 3 Dispersion Technology Software version 6.20 (CMC) F JEM (TEM) 200 kv (CLSM) 1.2 炔丙基 -α-d- 吡喃甘露糖的合成 -α-d- [17] 1.3 PGMA-Mannose 的合成 1.5 g GMA 2.5 ml (DMSO) 50 ml 64.4 mg CuBr mg Bpy 81 μl MBrP 40 2 h 10 ml THF (PGMA) mg PGMA 68.5 mg 56.0 mg 7 ml N,N - (DMF) 50 ml h PGMA (PGMA-N 3 ) 60.0 mg PGMA-N 3 15 ml DMF 50 ml 15 min mg -α-d mg 5 ml mg DMF h (MW2 000) PGMA-Mannose

4 汪舒婷等 /D- 甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用 负载抗癌药物阿霉素 10.0 mg PGMA-Mannose 2 ml DMSO 0.45 μm 50.0 mg 10 ml h 20 ml 3 DOX PGMA-Mannose DMSO DOX 10 mmol/l (PBS ph 7.4) 2 ml PBS 1 d (DOX@PGMA- Mannose) 1.5 细胞培养 MDA-MB-231 HEK293 10% 100 U/mL 100 U/mL DMEM 37 5% CO % [18] 1.6 载药胶束的细胞内吞 24 h DOX@PGMA-Mannose (40 μg/ml) ph 7.4 (PBS) 2 4.0% 15 min PBS 2 4, (DAPI 1 μg/ml) [19] 15 min 405/ / 细胞毒性评价 MTT [20] MDA-MB-231 HEK h ph 7.4 PBS 2 DOX@PGMA-Mannose DOX DOX 2 μg/ml ph 7.4 PBS μl MTT (1 mg/ml) 4 h 100 μl DMSO 10 min 490 nm (OD) 2 结果与分析 2.1 PGMA-Mannose 的合成 PGMA-Mannose 1 MBrP GMA PGMA [21] PGMA 1 HNMR ( 2A) M n ( 1 HNMR)=6 030 GPC PGMA PGMA-N 3 PGMA-N 3 -α-d- click PGMA-Mannose 2B PGMA-Mannose 1 HNMR δ=8.1 ppm PGMA-N 3 -α-d- click δ ppm cjb@im.ac.cn

5 88 ISSN CN /Q Chin J Biotech January 25, 2016 Vol.32 No.1 图 1 Fig. 1 PGMA-Mannose 的合成路线图 Synthesis route of the PGMA-Mannose. 图 2 Fig. 2 聚合物的核磁共振氢谱图 1 HNMR spectra of the polymers. (A) PGMA. (B) PGMA-Mannose.

6 汪舒婷等 /D- 甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用 合成聚合物的 FT-IR 分析 3A PGMA cm 1 C=O PGMA cm 1 PGMA-N 3 ( 3B) PGMA-N 3 click cm cm 1 [22] cm 1 click PGMA-Mannose ( 3C) 2.3 PGMA-Mannose 的临界胶束浓度 (CMC) 测定 PGMA-Mannose 334 nm 373 nm 384 nm PGMA-Mannose [23] 4 PGMA-Mannose 图 3 聚合物的红外光谱图 Fig. 3 FT-IR spectra of the polymers. (A) PGMA. (B) PGMA-N 3. (C) PGMA-Mannose. 图 4 PGMA-Mannose 浓度对 I 373 /I 384 比值的影响 Fig. 4 The relationship of fluorescence intensity of I 373 /I 384 and the concentration of PGMA-Mannose. PGMA-Mannose I 373 /I 384 I 373 /I 384 PGMA-Mannose CMC mmol/l 2.4 聚合物胶束在载药前后的 TEM 与 DLS 表征 TEM DLS 5 5A DOX ( 5B) 67.2 nm nm (PDI=0.281) ( 5C 5D) 2.5 药物释放 1 mg 10 ml 1 mg/ml mg/ml cjb@im.ac.cn

7 90 ISSN CN /Q Chin J Biotech January 25, 2016 Vol.32 No.1 图 5 空白胶束和载药胶束的透射电镜照片及在水溶液中的流体力学直径分布图 Fig. 5 TEM images and hydrodynamic size distributions of the micelles. (A) PGMA-Mannose (TEM). (B) DOX@PGMA-Mannose (TEM). (C) PGMA-Mannose (DLS). (D) DOX@PGMA-Mannose (DLS). 478 nm nm (I) (c) I = c R 2 = mg/ml 1 ml DMF 4.8% 20.6% =W 1 /W 0 100% = W 1 /W 2 100% W 0 W 1 W 2 1 ml ph 7.4 ph 5.0 ph ml r/min 1 ml ( ) 3 6 ph h 19.2% ph h 52.0% ph h 50% 48 h 89.6%

8 汪舒婷等 /D- 甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用 91 图 6 在 37 不同 ph 条件下载药胶束的药物释放曲线 Fig. 6 Drug release curves of the DOX-loading micelles at 37 under different ph conditions ( n=3). ph ph ph nm ph nm ph zeta 21.8 mv 5.1 mv ph 2.6 甘露糖受体调节的细胞内吞 7 8 DOX@PGMA-Mannose MDA-MB-231 HEK293 DAPI DOX 7 1 h HEK293 MDA-MB-231 DOX 10 h MDA-MB-231 DOX HEK293 8 MDA-MB-231 HEK 空白胶束和载药胶束的细胞毒性评价 MTT 9 HEK293 MDA-MB μg/ml 图 7 载药胶束 (40 μg/ml) 分别与 MDA-MB-231 细胞和 HEK293 细胞培养 1 h 的激光共聚焦显微镜照片 Fig. 7 CLSM images of the cells incubated for 1 h with DOX@PGMA-Mannose (40 μg/ml) as indicated. (A) MDA-MB-231 cancer cells. (B) HEK293 normal cells cjb@im.ac.cn

9 92 ISSN CN /Q Chin J Biotech January 25, 2016 Vol.32 No.1 图 8 载药胶束 (40 μg/ml) 分别与 MDA-MB-231 细胞和 HEK293 细胞培养 10 h 的激光共聚焦显微镜照片 Fig. 8 CLSM images of the cells incubated for 10 h with DOX@PGMA-Mannose (40 μg/ml) as indicated. (A) MDA-MB-231 cancer cells. (B) HEK293 normal cells A MDA-MB-231 DOX DOX@PGMA-Mannose DOX@PGMA-Mannose DOX DOX@PGMA-Mannose DOX 10B HEK293 图 9 不同浓度的空白胶束分别与两种细胞培养 72 h 的细胞存活率 Fig. 9 Viability of HEK293 cells and MDA-MB-231 cells incubated with PGMA-Mannose at different concentrations for 72 h. Data represent x ±s (n=6). 92% DOX@PGMA- Mannose 图 10 载药胶束 (40 μg/ml) 和原药分别与两种细胞培养不同时间的细胞存活率 Fig. 10 Viability of the cells incubated with DOX@PGMA-Mannose or free DOX at same DOX doses. Data represent x ±s (n=6). (A) MDA-MB-231 cancer cells. (B) HEK293 normal cells.

10 汪舒婷等 /D- 甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用 93 HEK293 HEK293 HEK293 PGMA-Mannose DOX 3 讨论 PGMA PGMA DOX DOX@PGMA-Mannose DOX@PGMA-Mannose DOX@PGMA-Mannose MDA-MB-231 DOX DOX@PGMA-Mannose PGMA-Mannose REFERENCES [1] Rosi NL, Mirkin CA. Nanostructures in biodiagnostics. Chem Rev, 2005, 105(4): [2] Peer D, Karp JM, Hong S, et al. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol, 2007, 2(12): [3] Danhier F, Feron O, Préat V. To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Controlled Release, 2010, 148(2): [4] Al-Jamal WT, Kostarelos K. Liposomes: from a clinically established drug delivery system to a nanoparticle platform for theranostic nanomedicine. Acc Chem Res, 2011, 44(10): [5] Doshi N, Mitragotri S. Designer biomaterials for nanomedicine. Adv Funct Mater, 2009, 19(24): [6] Liu ZJ, Yao P. Versatile injectable supramolecular hydrogels containing drug loaded micelles for delivery of various drugs. Polym Chem, 2014, 5(3): [7] Gillies ER, Fréchet JMJ. Dendrimers and dendritic polymers in drug delivery. Drug Discovery Today, 2005, 10(1): [8] Zhang Q, Liu F, Nguyen KT, et al. Multifunctional mesoporous silica nanoparticles for cancer-targeted and controlled drug delivery. Adv Funct Mater, 2012, 22(24): [9] Zhang Q, Wang X, Li PZ, et al. Biocompatible, uniform, and redispersible mesoporous silica nanoparticles for cancer-targeted drug delivery in vivo. Adv Funct Mater, 2014, 24(17): [10] Su L, Wang CM, Polzer F, et al. Glyco-inside micelles and vesicles directed by protectiondeprotection chemistry. ACS Macro Lett, 2014, 3(5): [11] Liu TJ, Liu S, Sheng SH, et al. EPR effect of amphiphilic copolymer micelles observed by fluorescent imaging. Chem Res Chin Univ, 2011, 27(4): [12] Noh TH, Kook YH, Park CY, et al. Block cjb@im.ac.cn

11 94 ISSN CN /Q Chin J Biotech January 25, 2016 Vol.32 No.1 copolymer micelles conjugated with anti-egfr antibody for targeted delivery of anticancer drug. J Polym Sci, Part A: Polym Chem, 2008, 46(22): [13] Danhier F, LeBreton A, Preat V. RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis. Mol Pharmaceutics, 2012, 9(11): [14] Prabaharan M, Grailer JJ, Pila S, et al. Folate-conjugated amphiphilic hyperbranched block copolymers based on Boltorn H40, poly(l-lactide) and poly(ethylene glycol) for tumor-targeted drug delivery. Biomaterials, 2009, 30(16): [15] Brevet D, Gary-Bobo M, Raehm L, et al. Mannose-targeted mesoposous silica nanoparticles for photodynamic therapy. Chem Commun, 2009, 1(12): [16] Gary-Bobo M, Mir Y, Rouxel C, et al. Mannose-functionalized mesoporous silica nanoparticles for efficient two-photon photodynamic therapy of solid tumors. Angew Chem Int Ed, 2011, 50(48): [17] Zhao JS, Liu YF, Park HJ, et al. Carbohydrate-coated fluorescent silica nanoparticles as probes for the galactose/3-sulfogalactose carbohydrate-carbohydrate interaction using model systems and cellular binding studies. Bioconjugate Chem, 2012, 23(6): [18] Wang Z, Luo J, Wang W, et al. Characterization and culture of isolated primary dairy goat mammary gland epithelial cells. Chin J Biotech, 2010, 26(8): (in Chinese).,,,.., 2010, 26(8): [19] Zhang Q, Luan L, Feng S, et al. Using a bifunctional polymer for the functionalization of Fe 3 O 4 nanoparticles. React Funct Polym, 2012, 72(3): [20] Kong LJ, Ao Q, Xi J, et al. Proliferation and differentiation of MC 3T3-E1 cells cultured on nanohydroxyapatite/chitosan composite scaffolds. Chin J Biotech, 2007, 23(2): (in Chinese).,,,. MC 3T3-E1 /., 2007, 23(2): [21] Li RQ, Niu YL, Zhao NN, et al. Series of new β-cyclodextrin-cored star like carriers for gene delivery. ACS Appl Mater Interfaces, 2014, 6(6): [22] Clavel C, Romuald C, Brabet E, et al. A ph-sensitive lasso-based rotaxane molecular switch. Chem Eur J, 2013, 19(9): [23] Meng QB, Kou YY, Ma X, et al. Tunable self-assembled peptide amphiphile nanostructures. Langmuir, 2012, 28(11): ( 本文责编陈宏宇 )