α-CD-PAMAM抗血清阳离子聚合物的构建及其作为基因载体的性能评价

Construction of serum-resistant cationic polymer α-CD-PAMAM and evaluation of its performances as gene delivery vector

本文合成了一种α-环糊精(α-cyclodextrin,α-CD)与聚酰胺-胺(polyamidoamine,PAMAM)的接枝聚合物(Cy D-G1)。~1H核磁测试结果表明,每个环糊精分子上平均接枝了6.4个PAMAM-G1分子。凝胶电泳结果显示,Cy D-G1可以有效结合DNA,并保护DNA免于核酸酶DNase I的降解。当载体与DNA复合物的N/P比为40时,可以压缩DNA形成平均粒径为120 nm左右的粒子,复合物表面的zeta电位约为+21 m V。该复合物可以在血清存在的条件下保持粒子的完整性并在360 min内稳定性良好。与对照品PEI-25K载体相比,Cy D-G1在高浓度时仍表现出较低的细胞毒性。将Cy D-G1与市售Lipofectamine 2000和PEI-25K对比转染发现,Cy D-G1/DNA复合物在多种细胞系中具有较高的转染率,而且转染水平不受血清影响。通过激光共聚焦观察并结合流式细胞分析表明,该阳离子聚合物介导DNA可以在4 h内有效进入细胞核内。上述结果证明,该阳离子聚合物作为一种非病毒型基因传递系统具有优良的性能以及体内给药应用的潜在可行性。

Polyamidoamine （PAMAM） dendrimers as synthetic gene vectors are efficient gene delivery systems. In this study, a kind of a-cyclodextrin-PAMAM conjugates polymer （CyD-G1） was synthesized as a gene delivery vector. Based on ^1H NMR detectation, about 6.4 PAMAM-G1 molecules was grafted onto an a-CD core. Agarose gel electrophoresis revealed that CyD-G1 could efficiently bind with DNA to condense them into nano-scale particles, which showed a similar binding capacity of PEI-25K. Besides, it could protect DNA from DNase I degradation in a low N/P ratio. When N/P ratio in the CyD-G1/DNA polyplex was 40, the average particle size of CyD-G1/DNA polyplex was about 120 nm, and zeta potential was ＋21 mV. This polyplex could maintain its particle size in serum-containing solution within 360 min. In comparison with PEI-25K carrier, CyD-G1 showed low cytotoxicity in various cell lines. Cell transfection results showed that CyD-GI efficiently delivered DNA into cells at N/P = 80 compared with Lipofectamine 2000 and PEI-25K.Unlike Lipofectamine 2000 and PEI-25K, in serum-containing test condition, CyD-G1/DNA polyplex could maintain the transgene activities. The results of confocal laser scanning microscopy indicated that most DNA entered into cell nuclei within 4 h, and this phenomenon was consistent with the results calculated by flow cytometry. Taken together, CyD-G1 showed good transgene activities and the gene delivery vector could be used not only in vitro but also in vivo.