Wu Yang, Zhang Wei, Zhang Jian, Mao Zhi-Xiang, Ding Li, Li Hao, Ma Rong, Tang Jin-Hai
Research Center of Clinical Oncology, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & Nanjing Medical University Affiliated Cancer Hospital.
Department of General Surgery, The First Affiliated Hospital with Nanjing Medical University.
J Vis Exp. 2019 Aug 6(150). doi: 10.3791/58527.
Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This work presents the preparation of methionine functionalized biocompatible block copolymers via RAFT polymerization. Firstly, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide (BNHEMA-b-APMA, BA) was synthesized via RAFT polymerization using 4,4'-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent. Subsequently, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-guanidinopropyl)methacrylamide (methionine grafted BNHEMA-b-GPMA, mBG) was prepared by modifying amine groups in APMA with methionine and guanidine groups. Three kinds of block polymers, mBG1, mBG2, and mBG3, were synthesized for comparison. A ninhydrin reaction was used to quantify the APMA content; mBG1, mBG2, and mBG3 had 21%, 37%, and 52% of APMA, respectively. Gel permeation chromatography (GPC) results showed that BA copolymers possess molecular weights of 16,200 (BA1), 20,900(BA2), and 27,200(BA3) g/mol. The plasmid DNA (pDNA) complexing ability of the obtained block copolymer gene carriers was also investigated. The charge ratios (N/P) were 8, 16, and 4 when pDNA was complexed completely with mBG1, mBG2, mBG3, respectively. When the N/P ratio of mBG/pDNA polyplexes was higher than 1, the Zeta potential of mBG was positive. At an N/P ratio between 16 and 32, the average particle size of mBG/pDNA polyplexes was between 100-200 nm. Overall, this work illustrates a simple and convenient protocol for the block copolymer carrier synthesis.
可逆加成-断裂链转移（RAFT）聚合结合了自由基聚合和活性聚合的优点。本工作介绍了通过RAFT聚合制备蛋氨酸功能化生物相容性嵌段共聚物的方法。首先，以4,4'-偶氮双（4-氰基戊酸）（ACVA）为引发剂、4-氰基戊酸二硫代苯甲酸酯（CTP）为链转移剂，通过RAFT聚合合成了N,N-双（2-羟乙基）甲基丙烯酰胺-b-N-（3-氨基丙基）甲基丙烯酰胺（BNHEMA-b-APMA，BA）。随后，通过用蛋氨酸和胍基修饰APMA中的胺基，制备了N,N-双（2-羟乙基）甲基丙烯酰胺-b-N-（3-胍基丙基）甲基丙烯酰胺（蛋氨酸接枝的BNHEMA-b-GPMA，mBG）。合成了三种嵌段聚合物mBG1、mBG2和mBG3用于比较。采用茚三酮反应定量APMA含量；mBG1、mBG2和mBG3的APMA含量分别为21%、37%和52%。凝胶渗透色谱（GPC）结果表明，BA共聚物的分子量分别为16,200（BA1）、20,900（BA2）和27,200（BA3）g/mol。还研究了所得嵌段共聚物基因载体与质粒DNA（pDNA）的复合能力。当pDNA分别与mBG1、mBG2、mBG3完全复合时，电荷比（N/P）分别为8、16和4。当mBG/pDNA多聚体的N/P比高于1时，mBG的Zeta电位为正。在N/P比为16至32之间时，mBG/pDNA多聚体的平均粒径在100-200nm之间。总体而言，本工作展示了一种简单便捷的嵌段共聚物载体合成方案。
