Department of Organic Chemistry, ‡Department of Physics, and §Department of Microbiology and Cell Biology, Indian Institute of Science , Bangalore 560 012, India.
Bioconjug Chem. 2013 Sep 18;24(9):1612-23. doi: 10.1021/bc400247w. Epub 2013 Aug 22.
Dendrimers as vectors for gene delivery were established, primarily by utilizing few prominent dendrimer types so far. We report herein studies of DNA complexation efficacies and gene delivery vector properties of a nitrogen-core poly(propyl ether imine) (PETIM) dendrimer, constituted with 22 tertiary amine internal branches and 24 primary amines at the periphery. The interaction of the dendrimer with pEGFPDNA was evaluated through UV-vis, circular dichroism (CD) spectral studies, ethidium bromide fluorescence emission quenching, thermal melting, and gel retardation assays, from which most changes to DNA structure during complexation was found to occur at a weight ratio of dendrimer:DNA ∼ 2:1. The zeta potential measurements further confirmed this stoichiometry at electroneutrality. The structure of a DNA oligomer upon dendrimer complexation was simulated through molecular modeling and the simulation showed that the dendrimer enfolded DNA oligomer along both major and minor grooves, without causing DNA deformation, in 1:1 and 2:1 dendrimer-to-DNA complexes. Atomic force microscopy (AFM) studies on dendrimer-pEGFP DNA complex showed an increase in the average z-height as a result of dendrimers decorating the DNA, without causing a distortion of the DNA structure. Cytotoxicity studies involving five different mammalian cell lines, using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) assay, reveal the dendrimer toxicity profile (IC50) values of ∼400-1000 μg mL(-1), depending on the cell line tested. Quantitative estimation, using luciferase assay, showed that the gene transfection was at least 100 times higher when compared to poly(ethylene imine) branched polymer, having similar number of cationic sites as the dendrimer. The present study establishes the physicochemical behavior of new nitrogen-core PETIM dendrimer-DNA complexes, their lower toxicities, and efficient gene delivery vector properties.
树状聚合物作为基因传递载体已得到确立，主要是利用到目前为止少数几种突出的树状聚合物类型。我们在此报告一种氮核聚丙基醚亚胺（PETIM）树状聚合物的 DNA 复合效率和基因传递载体特性研究，该树状聚合物具有 22 个三级胺内部支链和 24 个位于外围的伯胺。通过紫外-可见光谱、圆二色性（CD）光谱研究、溴化乙锭荧光发射猝灭、热融和凝胶阻滞实验评估了该树状聚合物与 pEGFPDNA 的相互作用，在复合物中发现大多数 DNA 结构的变化发生在树状聚合物与 DNA 的重量比约为 2:1 时。表面等电位测量进一步证实了这种电中性的化学计量比。通过分子建模模拟了 DNA 低聚物与树状聚合物复合物的结构，模拟结果表明，在 1:1 和 2:1 的树状聚合物与 DNA 复合物中，树状聚合物沿主要和次要沟折叠 DNA 低聚物，而不会引起 DNA 变形。原子力显微镜（AFM）研究表明，由于树状聚合物修饰 DNA，DNA 上的平均 z 高度增加，而不会导致 DNA 结构的扭曲。涉及五种不同哺乳动物细胞系的细胞毒性研究，使用[3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴化物]（MTT）测定法，揭示了树状聚合物毒性谱（IC50）值约为 400-1000μg/mL，取决于所测试的细胞系。使用荧光素酶测定法进行定量估计表明，与具有与树状聚合物相似数量的阳离子位点的支化聚亚乙基亚胺相比，基因转染至少高 100 倍。本研究确立了新型氮核 PETIM 树状聚合物-DNA 复合物的物理化学行为、较低的毒性和有效的基因传递载体特性。
