School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin 300350, China.
Department of Hepatopancreatobiliary and Splenic Medicine, Affiliated Hospital, Logistics University of People's Armed Police Force, Chenglin Road 220, Tianjin 300162, China.
J Mater Chem B. 2020 Mar 25;8(12):2418-2430. doi: 10.1039/c9tb02641j.
Bioreducible cationic polymers have gained considerable attention in gene delivery due to their low cytotoxicity and high efficiency. In the present work, we reported a cationic polymer, poly(disulfide-l-lysine)-g-agmatine (denoted as SSL-AG), and evaluated its ability to transfer pEGFP-ZNF580 plasmid (pZNF580) into human umbilical vein endothelial cells (HUVECs). This SSL-AG polymeric carrier efficiently condensed pZNF580 into positively charged particles (<200 nm) through electrostatic interaction. This carrier also exhibited excellent buffering capacity in the physiological environment, good pDNA protection against enzymatic degradation and rapid pDNA release in a highly reducing environment mainly because of the responsive cleavage of disulfide bonds in the polymer backbone. The hemolysis assay and in vitro cytotoxicity assay suggested that the SSL-AG carrier and corresponding gene complexes possessed both good hemocompatibility and great cell viability in HUVECs. The cellular uptake of the SSL-AG/Cy5-oligonucleotide group was 3.6 times that of the poly(l-lysine)/Cy5-oligonucleotide group, and its mean fluorescence intensity value was even higher than that of the PEI 25 kDa/Cy5-oligonucleotide group. Further, the intracellular trafficking results demonstrated that the SSL-AG/Cy5-oligonucleotide complexes exhibited a high nucleus co-localization rate (CLR) value (36.0 ± 2.8%, 3.4 times that of the poly (l-lysine)/Cy5-oligonucleotide group, 1.6 times that of the poly(disulfide-l-lysine)-g-butylenediamine/Cy5-oligonucleotide group) at 24 h, while the endo/lysosomal CLR value was relatively low. This suggested that SSL-AG successfully delivered plasmid into HUVECs with high cellular uptake, rapid endosomal escape and efficient nuclear accumulation owing to the structural advantages of the bioreducible and agmatine groups. In vitro transfection assay also verified the enhanced transfection efficiency in the SSL-AG/pZNF580 group. Furthermore, the results of CCK-8, cell migration and in vitro/vivo angiogenesis assays revealed that pZNF580 delivered by SSL-AG could effectively enhance the proliferation, migration and vascularization of HUVECs. In a word, the SSL-AG polymer has great potential as a safe and efficient gene carrier for gene therapy.
具有生物还原性能的阳离子聚合物由于其低细胞毒性和高效率而在基因传递中受到了广泛关注。在本工作中，我们报道了一种阳离子聚合物，聚（二硫键-赖氨酸）-g-胍丁胺（记为 SSL-AG），并评估了其将 pEGFP-ZNF580 质粒（pZNF580）转染入人脐静脉内皮细胞（HUVECs）的能力。这种 SSL-AG 聚合物载体通过静电相互作用有效地将 pZNF580 凝聚成带正电荷的颗粒（<200nm）。该载体在生理环境中还表现出优异的缓冲能力，对酶降解的良好 pDNA 保护作用，以及在高度还原环境中快速释放 pDNA，主要是因为聚合物主链中二硫键的响应性断裂。溶血试验和体外细胞毒性试验表明，SSL-AG 载体及其相应的基因复合物在 HUVECs 中既具有良好的血液相容性，又具有很高的细胞活力。SSL-AG/Cy5-寡核苷酸组的细胞摄取量是聚（L-赖氨酸）/Cy5-寡核苷酸组的 3.6 倍，其平均荧光强度值甚至高于聚乙烯亚胺 25kDa/Cy5-寡核苷酸组。此外，细胞内转运结果表明，SSL-AG/Cy5-寡核苷酸复合物在 24 小时时表现出高核共定位率（CLR）值（36.0±2.8%，是聚（L-赖氨酸）/Cy5-寡核苷酸组的 3.4 倍，是聚（二硫键-赖氨酸）-g-丁二胺/Cy5-寡核苷酸组的 1.6 倍），而内体/溶酶体 CLR 值相对较低。这表明 SSL-AG 成功地将质粒递送入 HUVECs，具有高细胞摄取率、快速的内体逃逸和高效的核积累，这归因于其生物还原和胍基基团的结构优势。体外转染试验也验证了 SSL-AG/pZNF580 组的转染效率得到了增强。此外，CCK-8、细胞迁移和体外/体内血管生成试验的结果表明，由 SSL-AG 递送入的 pZNF580 能够有效增强 HUVECs 的增殖、迁移和血管生成。总之，SSL-AG 聚合物作为一种安全有效的基因载体，具有很大的基因治疗潜力。
