Polycationic β-cyclodextrin "Click Clusters":: Monodisperse and versatile scaffolds for nucleic acid delivery
Polycationic β-cyclodextrin "Click Clusters":: Monodisperse and versatile scaffolds for nucleic acid delivery
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DOI：
10.1021/ja074597v
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发表时间：
2008-04-09
影响因子：
15
通讯作者：
Reineke, Theresa M.
中科院分区：
文献类型：
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作者：
Srinivasachari, Sathya;Fichter, Katye M.;Reineke, Theresa M.
在本论文中，我们合成了一系列具有离散分子量的多价多阳离子β-环糊精“点击簇”，对其进行了表征，并作为治疗性的PDNA载体进行了研究。这些材料是基于β-环糊精核心创造性地设计的，以提供生物相容的多价结构和寡乙胺臂，以促进PDNA的结合、包裹和细胞摄取。通过铜催化的1，3-偶极环加成反应，将乙酰化的全叠氮-β-环糊精(4)与一系列含1~5个亚乙胺单元的炔树枝(7a-e)反应，形成一系列含有1，2，3-三唑键的点击簇合物(9a-e)。凝胶电泳法、动态光散射法和透射电子显微镜实验表明，这些大分子结合并致密地形成了大小为80-130 nm的球形纳米粒子。聚阳离子保护PDNA免受核酸酶降解，其中9c、9d和9e结构在有血清存在的情况下最长48h不允许PDNA降解。Opti-MEM对细胞摄取图谱的评估表明，所有的点击簇都有效地将Cy5标记的PDNA运送到HeLa和H9c2(2-1)细胞中，化合物9d和9e的效果与阳性对照Jet-PEI和SuPerfect相似。此外，荧光素酶基因传递实验表明，报告基因的表达水平随着簇臂内寡乙胺数量的增加而增加。通过蛋白质、四甲基偶氮唑蓝和乳酸脱氢酶检测这些材料的细胞毒性，结果表明所有的点击簇在预期的剂量范围内都保持无毒，而阳性对照Jet PEI和SuPerfect具有高度的细胞毒性。特别是，9D和9E是最有效和最有前景的聚阳离子载体，有待于进一步优化，以进行未来的全身给药实验。
Herein, a novel series of multivalent polycationic beta-cycloclextrin "click clusters" with discrete molecular weight have been synthesized, characterized, and examined as therapeutic pDNA carriers. The materials were creatively designed based on a beta-cyclodextrin core to impart a biocompatible multivalent architecture and oligoethyleneamine arms to facilitate pDNA binding, encapsulation, and cellular uptake. An acetylated-per-azido-beta-cyclodextrin (4) was reacted with series of alkyne dendrons (7a-e) (containing one to five ethyleneamine units) using copper-catalyzed 1,3-dipolar cycloaddition, to form a series of click clusters (9a-e) bearing 1,2,3-triazole linkers. Gel electrophoresis experiments, dynamic light scattering, and transmission electron microscopy revealed that the macromolecules bind and compact pDNA into spherical nanoparticles in the size range of 80-130 nm. The polycations protect pDNA against nuclease degradation, where structures 9c, 9d, and 9e did not allow pDNA degradation in the presence of serum for up to 48 h. The cellular uptake profiles were evaluated in Opti-MEM and demonstrate that all the click clusters efficiently deliver Cy5-labeled pDNA into HeLa and H9c2 (2-1) cells, and compounds 9d and 9e yielded efficacy similar to that of the positive controls, Jet-PEI and Superfect. Furthermore, the luciferase gene delivery experiments revealed that the level of reporter gene expression increased with an increase in oligoethyleneamine number within the cluster arms. The cytotoxicity profiles of these materials were evaluated by protein, MTT, and LDH assays, which demonstrate that all the click clusters remain nontoxic within the expected dosage range while the positive controls, Jet PEI and Superfect, were highly cytotoxic. In particular, 9d and 9e were the most effective and promising polycationic vehicles to be further optimized for future systemic delivery experiments.