Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity
Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity
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DOI：
10.1021/bc700397x
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发表时间：
2008-03-01
影响因子：
4.7
通讯作者：
Kim, Sung Wan
中科院分区：
文献类型：
--
作者：
Ou, Mei;Wang, Xu-Li;Kim, Sung Wan
设计合成了结构明确、转染效率高、细胞毒性低的新型可生物降解聚二硫胺作为非病毒基因传递载体。在N，N'-半胺双丙烯酰胺（CBA）和三种受N- boc保护的二胺（N- boc -1,2-二氨基乙烷、N- boc -1,4-二氨基丁烷和N- boc -1,6-二氨基己烷）之间进行Michael加成，然后在酸性条件下进行N- boc脱保护，最终得到具有二硫键的阳离子聚合物，主链上有叔胺基团，侧链上有伯胺基团。高分子结构经H-1 NMR确证，分子量在3.3 ~ 4.7 kDa之间，聚分散性较窄（1.12 ~ 1.17）。酸碱滴定试验表明，在pH值7.4 ~ 5范围内，聚二硫胺对支链PEI 25 kDa具有较好的缓冲能力。1，这可能有助于DNA从内体室逃逸。凝胶阻滞实验表明，在5.0 MM DTT存在的情况下，1 h内观察到明显的多聚体解离，表明DNA在还原条件下如细胞质中由于二硫键的断裂而快速释放。这些聚二氟胺介导的遗传转染是侧链间隔长度依赖的。在293T细胞、Hela细胞和NIH3T3细胞中，带有六乙烯间隔的聚（CBA-DAH）的转染效率与bPEI 25 kDa相当。在C2C12细胞（小鼠成肌细胞系）中，这种聚二氟胺介导的荧光素酶表达比bPEI 25 kDa高7倍，C2C12细胞是一种难以用许多阳离子聚合物转染的细胞系。此外，MTT试验表明，这三种聚二氟胺s/pDNA复合物的毒性明显低于bPEI/pDNA复合物。
Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N,N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines (N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by H-1 NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5. 1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 MM DTT within I h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disuffide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disuffide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disuffide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.