Development of a reduction-sensitive diselenide-conjugated oligoethylenimine nanoparticulate system as a gene carrier.
Development of a reduction-sensitive diselenide-conjugated oligoethylenimine nanoparticulate system as a gene carrier.
复制标题
开发还原敏感的二硒化物共轭寡乙烯亚胺纳米颗粒系统作为基因载体
DOI：
10.2147/ijn.s32961
复制
发表时间：
2012
影响因子：
8
通讯作者：
Gu Z
中科院分区：
文献类型：
--
作者：
Cheng G;He Y;Xie L;Nie Y;He B;Zhang Z;Gu Z
还原敏感阳离子聚合物是一种很有前途的非病毒基因载体。到目前为止，二硫键一直是其设计的唯一黄金标准。本研究的目的是开发一种新型的还原反应性阳离子聚合物作为基因载体。方法采用支化低聚亚胺800 Da (OEI800)，通过含二硒键的活性酯加成合成多阳离子载体。合成了与OEI800-SSx交联的二硫键和与OEI800-Sex交联的单硒键，并进行了比较。用凝胶渗透色谱法测定了它们的分子量和降解性能。在还原环境下，通过动态光散射、透射电子显微镜和电泳分析研究了颗粒大小、形态和DNA结合的变化。在小鼠黑色素瘤细胞系（B16F10）和人宫颈上皮细胞系（HeLa）中评估细胞毒性和体外转染，并用fitc标记的OEI800衍生物和cy5标记的DNA通过共聚焦激光扫描显微镜研究细胞内降解和与DNA的解离。结果成功合成了二硒共轭OEI800 （OEI800- sesex）高分子量聚合物载体。与DNA压实后，在适当的C/P比下，OEI800-SeSex聚合物形成平均尺寸为140 nm的纳米颗粒。通过凝胶渗透色谱、动态光散射和透射电镜分析，OEI800-SeSex表现出与OEI800-SSx相似的还原响应降解特性。在B16F10和HeLa细胞中，OEI800- sesex的细胞毒性明显低于PEI25k，转染效率明显高于OEI800。转染荧光素酶的OEI800-SeSex组与标准PEI25k和传统还原敏感聚合物OEI800-SSx组相当。此外，共聚焦激光扫描显微镜也证实了OEI800-SeSex的细胞内降解和与DNA的解离。结论所获得的OEI800-SeSex能有效结合质粒DNA生成纳米级颗粒，且具有与OEI800-SSx相同的还原敏感性。体外实验证实其细胞毒性低，转染能力强。二硒烯键可以作为一种有效的新型还原敏感键用于基因传递。
Background The reduction-sensitive cationic polymer is a promising nonviral carrier for gene delivery. Until now, disulfide bonds have been the only golden standard for its design. The aim of this research was to develop a novel reduction-responsive cationic polymer as a gene carrier. Methods Polycationic carriers were synthesized by addition of branched oligoethylenimine 800 Da (OEI800) via an active ester containing diselenide bonds. Disulfide bonds cross-linked with OEI800-SSx and monoselenide bonds linked with OEI800-Sex were synthesized and compared. Their molecular weights and degradation properties were determined using gel permeation chromatography. Changes in particle size, morphology, and DNA binding were investigated by dynamic light scattering, transmission electron microscopy, and electrophoresis assay in a reduction environment. Cytotoxicity and transfection in vitro were evaluated in a murine melanoma cell line (B16F10) and a human cervical epithelial carcinoma cell line (HeLa), while intracellular degradation and dissociation with DNA were studied by confocal laser scanning microscopy with FITC-labeled OEI800 derivatives and Cy5-labeled DNA. Results Diselenide-conjugated OEI800 (OEI800-SeSex) polymer carriers of high molecular weight were successfully synthesized. After compacting with DNA, the OEI800-SeSex polymers formed nanoparticles with an average size of 140 nm at an adequate C/P ratio. OEI800-SeSex showed reduction-responsive degradation properties similar to those of the OEI800-SSx via gel permeation chromatography, dynamic light scattering, and transmission electron microscopy. OEI800-SeSex showed much lower cytotoxicity than PEI25k, and significantly higher transfection efficiency than OEI800 in both B16F10 and HeLa cells. Transfection of luciferase in the OEI800-SeSex group was comparable with that of standard PEI25k and traditional reduction-sensitive polymer OEI800-SSx groups. Furthermore, intracellular degradation of OEI800-SeSex and dissociation with DNA were also confirmed by confocal laser scanning microscopy. Conclusion The OEI800-SeSex obtained was able to bind plasmid DNA efficiently to yield nanosized particles and had reduction sensitivity which is as efficient as that for OEI800-SSx. In vitro experiments confirmed its low cytotoxicity and high transfection ability. Diselenide bonds can be used as effective and novel reduction-sensitive linkages for gene delivery.