Alkyne-functionalized cationic polysiloxane polymers conjugated with targeting molecules by click reactions for DNA delivery
Alkyne-functionalized cationic polysiloxane polymers conjugated with targeting molecules by click reactions for DNA delivery
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炔烃官能化阳离子聚硅氧烷聚合物通过点击反应与靶向分子缀合用于 DNA 递送
DOI：
10.1166/jnn.2017.13121
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
and Haruki M.
中科院分区：
文献类型：
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作者：
Kihara Y;Maeda R;Imaizumi A;Ichikawa T;Nemoto N;Ishihara T;Hirano N;and Haruki M.
通过靶向分子偶联到基因载体上的细胞特异性基因传递是一种很有前途的基因治疗策略。点击化学是这种结合的方便工具。我们开发了基于硅氧烷的两亲性聚合物，具有炔功能化和季铵化的咪唑盐（PIm），用于形成能够通过点击化学偶联叠氮功能化靶向分子的纳米乳液。由于带正电荷，这些聚合物有望用于靶向基因传递。本研究采用click化学方法将PIm与肝细胞上被asialalglyprotein receptor （ASGP-Rs）识别的乳糖偶联，并在HepG2和NIH3T3细胞中检测其DNA结合、细胞毒性和体外转染。琼脂糖凝胶阻滞实验使用5.3 kbp的质粒DNA (pDNA)，证实在氮/磷（N/P）比为8时，乳糖偶联聚合物与DNA之间形成复合物。当浓度达到50 μg/mL时，聚合物无明显的溶血活性。聚合物- pdna复合物具有较低的细胞毒性，在转染条件下，在N/P比高达12的情况下，细胞存活率保持在70%以上。荧光素酶分析显示，在HepG2细胞中，低水平乳糖偶联的PIm比未修饰的PIm在N/P比为12时的转染效率高29倍，而在不表达ASGP-Rs的NIH3T3细胞中则没有。这些结果表明，乳糖偶联能显著增强HepG2细胞向PIm的基因转移能力，这很可能是由于该聚合物与HepG2细胞上的ASGP-Rs结合所致。因此，PIm可能是一种很有前途的基因传递载体，可用于偶联细胞特异性靶向分子。
Cell-specific gene delivery through conjugating targeting molecules to gene carriers is a promising strategy for gene therapy. Click chemistry is a convenient tool for such conjugations. We have developed siloxane-based amphiphilic polymers with alkyne-functionalized and quaternized imidazolium salts (PIm) for forming nanoemulsions capable of conjugating azide-functionalized targeting molecules by click chemistry. Being positively charged, these polymers were expected to be applicable to targeted gene delivery. In this study, PIm was conjugated with lactose, which is recognized by asialoglycoprotein receptors (ASGP-Rs) on hepatocytes, using click chemistry and was examined for DNA binding, cytotoxicity, and in vitro transfection in HepG2 and NIH3T3 cells. The agarose gel retardation assay using a 5.3-kbp plasmid DNA (pDNA) confirmed complex formation between the lactose-conjugated polymers and DNA at a nitrogen/phosphorous (N/P) ratio of 8. The polymers exhibited no significant hemolytic activity up to 50 μg/mL. The polymer-pDNA complexes have low cytotoxicity, which maintained a cell survival rate greater than 70% at N/P ratios of up to 12 under transfection conditions. The luciferase assay revealed that PIm with a low level of lactose conjugation showed a 29-fold higher transfection efficiency than unmodified PIm at an N/P ratio of 12 in HepG2 cells, but not in NIH3T3 cells that do not express ASGP-Rs. These results demonstrate that the lactose conjugation confers significantly enhanced gene transfer capability for HepG2 cells to PIm most likely due to binding of the polymer to ASGP-Rs on HepG2 cells. Therefore, PIm could be a promising gene delivery vehicle clickable for conjugating cell-specific targeting molecules.