He Pan, Hagiwara Kyoji, Chong Hui, Yu Hsiao-hua, Ito Yoshihiro
Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Responsive Organic Materials Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Biomed Res Int. 2015;2015:406389. doi: 10.1155/2015/406389. Epub 2015 Oct 11.
Owing to its hydrophilicity, negative charge, small size, and labile degradation by endogenous nucleases, small interfering RNA (siRNA) delivery must be achieved by a carrier system. In this study, cationic copolymers composed of low-molecular-weight polyethylenimine and polythiophenes were synthesized and evaluated as novel self-tracking siRNA delivery vectors. The concept underlying the design of these copolymers is that hydrophobicity and rigidity of polythiophenes should enhance the transport of siRNA across the cell membrane and endosomal membrane. A gel retardation assay showed that the nanosized complexes formed between the copolymers and siRNA were stable even at a molar ratio of 1 : 2. The high cellular uptake (>80%) and localization of the copolymer vectors inside the cells were easily analyzed by tracking the fluorescence of polythiophene using fluorescent microscopy and cytometry. An in vitro luciferase knockdown (KD) assay in A549-luc cells demonstrated that the siRNA complexes with more hydrophobic copolymers achieved a higher KD efficiency of 52.8% without notable cytotoxicity, indicating protein-specific KD activity rather than solely the cytotoxicity of the materials. Our polythiophene copolymers should serve as novel, efficient, low cell toxicity, and label-free siRNA delivery systems.
由于小分子干扰RNA（siRNA）具有亲水性、负电荷、体积小以及易被内源性核酸酶降解的特性，因此必须通过载体系统来实现其递送。在本研究中，合成了由低分子量聚乙烯亚胺和聚噻吩组成的阳离子共聚物，并将其评估为新型的可自我追踪的siRNA递送载体。这些共聚物设计的基本理念是，聚噻吩的疏水性和刚性应能增强siRNA跨细胞膜和内体膜的转运。凝胶阻滞试验表明，即使在1:2的摩尔比下，共聚物与siRNA形成的纳米级复合物也是稳定的。通过荧光显微镜和细胞计数法追踪聚噻吩的荧光，能够轻松分析共聚物载体在细胞内的高摄取率（>80%）和定位情况。在A549-luc细胞中进行的体外荧光素酶敲低（KD）试验表明，与疏水性更强的共聚物形成的siRNA复合物实现了52.8%的更高KD效率，且无明显细胞毒性，这表明具有蛋白质特异性KD活性，而非仅仅是材料的细胞毒性。我们的聚噻吩共聚物应可作为新型、高效、低细胞毒性且无标记的siRNA递送系统。
