半乳糖胺修饰阳离子型刷形嵌段共聚物的合成与表征

doi:10.6023/A14030185

化学学报 ›› 2014, Vol. 72 ›› Issue (5): 569-576.DOI: 10.6023/A14030185 上一篇下一篇

研究论文

半乳糖胺修饰阳离子型刷形嵌段共聚物的合成与表征

郝莹, 张洋, 何金林, 尚修娟, 张明祖, 倪沛红

苏州大学材料与化学化工学部江苏省先进功能高分子材料设计及应用重点实验室苏州 215123

投稿日期:2014-03-15 发布日期:2014-04-17
通讯作者: 倪沛红 E-mail:phni@suda.edu.cn
基金资助:
受国家自然科学基金（Nos. 21374066，21074078）、江苏省基础研究计划（自然科学基金）——滚动资助项目（No. BK2011045）、江苏高校优势学科建设项目资助.

Synthesis and Characterization of PEGylated Brush-type Polycation Modified with Galactosamine

Hao Ying, Zhang Yang, He Jinlin, Shang Xiujuan, Zhang Mingzu, Ni Peihong

College of Chemistry, Chemical Engineering and Materials Science, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Soochow University, Suzhou 215123

Received:2014-03-15 Published:2014-04-17
Supported by:
Projects supported by the National Natural Science Foundation of China (Nos. 21374066 and 21074078), the Natural Science Foundation of Jiangsu Province for Rolling Support (No. BK2011045), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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摘要/Abstract

利用两步原子转移自由基聚合（ATRP）和聚合物后修饰反应，制备半乳糖胺修饰的阳离子型刷形嵌段共聚物P（PEGMEMA-co-PEGMA_-Gal）-b-PDMAEMA. 首先，通过ATRP法引发单体聚乙二醇单甲醚甲基丙烯酸酯（PEGMEMA）和聚乙二醇甲基丙烯酸酯（PEGMA）聚合，得到末端含氯基的无规共聚物P（PEGMEMA-co-PEGMA）；再利用其作为大分子引发剂，引发水溶性单体甲基丙烯酸-2-（N，N-二甲氨基）乙酯（DMAEMA）进行ATRP反应，获得三组分两嵌段刷形共聚物P（PEGMEMA-co-PEGMA）-b-PDMAEMA；最后，利用PEGMA结构单元中的侧链羟基，经聚合物反应，键合具有肝靶向功能的半乳糖分子（Gal），成功获得P（PEGMEMA-co-PEGMA_-Gal）-b-PDMAEMA. 其中，PDMAEMA侧基的二甲氨基[—N（CH₃）₂]在中性和弱酸性介质中可发生质子化，形成聚阳离子链段，能够缩合DNA，用作基因载体. 通过核磁共振氢谱（¹H NMR）、红外光谱（FT-IR）和凝胶渗透色谱（GPC），对聚合物的化学结构、分子量及分子量分布进行表征. 利用凝胶阻滞电泳研究阳离子型嵌段共聚物与DNA的结合能力、利用动态激光光散射仪（DLS）测试聚阳离子/DNA复合物的表面zeta电位值、粒径及粒径分布. 通过细胞毒性试验（MTT法）表征载体的生物相容性，并分别研究复合物对宫颈癌（HeLa）细胞和肝癌（HepG2）细胞的转染能力. 实验结果表明，这种半乳糖胺修饰的阳离子型刷形共聚物具有较低的细胞毒性，在肝靶向基因输送中具有潜在的应用.

关键词: 半乳糖胺, 刷形共聚物, 原子转移自由基聚合, 基因载体

Gene therapy has been generally regarded as a promising treatment for numerous hard curable diseases, such as cancer, genetic and infectious diseases. Seeking a safe and efficient vector plays the most challenging role in gene therapy. In the past decades, non-viral polycation has attracted much more attentions as a promising gene vector due to their good biocompatibility, high condensation ability, as well as easily-adjustable and controlled structures. In the present work, a series of galactosamine (Gal)-conjugated brush-type cationic copolymer P(PEGMEMA-co-PEGMA_-Gal)-b-PDMAEMA were prepared via a combination of two-step atom transfer radical polymerization (ATRP) technique and polymer reaction. Firstly, random copolymerization of poly(ethylene glycol)methyl ether methacrylate (PEGMEMA) and poly(ethylene glycol) methacrylate (PEGMA) was carried out to yield the copolymer P(PEGMEMA-co-PEGMA), which was further used as a macroinitiator to polymerize 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA), producing the cationic copolymer P(PEGMEMA-co-PEGMA)-b-PDMAEMA. Finally, the brush-type copolymer P(PEGMEMA-co-PEGMA_-Gal)-b- PDMAEMA was obtained by the modification of pendent hydroxyl groups with galactosamine using N,N'-carbonyldiimi-dazole (CDI) as the coupling agent. PDMAEMA is a well-known pH-sensitive polycation and has been widely applied for non-viral gene delivery, while the brush-type hydrophilic chains provide the carrier with favorable biocompatibility, prolonged blood circulation time, and reduced non-specific adsorption of proteins. PDMAEMA block would be partially protonated to afford some hydrophobic domains and positive charges at neutral condition, which can bind with DNA via electrostatic interaction to form polycation/DNA polyplex. The Gal moiety on the surface made the polyplex easily recognized by asialoglycoprotein receptors (ASGPRs) over-expressing hepatoma cells and internalized via a receptor-mediated endocytosis process. The chemical structures, molecular weights and molecular weight distributions of P(PEGMEMA-co-PEGMA_-Gal)-b-PDMAEMA copolymer were characterized by ¹H NMR, FT-IR, and GPC measurements. The DNA binding capacity of this copolymer was investigated by agarose gel electrophoresis and zeta potential measurements, which indicated that DNA migration could be completely retarded when the N/P ratio was higher than 1.5. The DLS analysis demonstrated that the polyplex held an average particle size of about 106 nm. In addition, in vitro cytotoxicity (MTT assay) and transfection assays were also examined, the results showed that the copolymer possessed relatively lower cytotoxicity than branched PEI (25 kDa), and the transfection of polyplex into HepG2 cells was more efficient than that of HeLa cells studied by live cell imaging system owing to specific ligand-receptor interactions between Gal and ASGPRs on the surface of HepG2 cells. This study presents a facile strategy for the preparation of biocompatible polymeric micelles that will act as a promising platform for hepatoma-targeting delivery of genes.

Key words: galactosamine, brush-type copolymer, ATRP, gene delivery

引用此文

郝莹, 张洋, 何金林, 尚修娟, 张明祖, 倪沛红. 半乳糖胺修饰阳离子型刷形嵌段共聚物的合成与表征[J]. 化学学报, 2014, 72(5): 569-576.

Hao Ying, Zhang Yang, He Jinlin, Shang Xiujuan, Zhang Mingzu, Ni Peihong. Synthesis and Characterization of PEGylated Brush-type Polycation Modified with Galactosamine[J]. Acta Chimica Sinica, 2014, 72(5): 569-576.

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半乳糖胺修饰阳离子型刷形嵌段共聚物的合成与表征

Synthesis and Characterization of PEGylated Brush-type Polycation Modified with Galactosamine

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