Yang Sen, Meng Zhao, Kang Ziyao, Sun Chao, Wang Taoran, Feng Siliang, Meng Qingbin, Liu Keliang
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology Beijing 100850 China
RSC Adv. 2018 Aug 7;8(50):28356-28366. doi: 10.1039/c8ra04101f.
We designed a series of peptide vectors that contain functional fragments with the goal of enhancing cellular internalization and gene transfection efficiency. The functional fragments included a cell-penetrating peptide (R), a cationic amphiphilic α-helical peptide [(LLKK)-H or (LLHH)], a stearyl moiety, and cysteine residues. Vectors were also synthesized with D-type amino acids to improve their proteolytic stability. The conformations, particle sizes, and zeta potentials for complexes of these peptides with pGL3 plasmid DNA were characterized by circular dichroism and dynamic light scattering. In addition, cellular uptake of the peptide/DNA complexes and gene transfection efficiency were investigated with fluorescence-activated cell sorting and confocal laser-scanning microscopy. Greater transfection efficiency was achieved with the vectors containing the R segment, and the efficiency was greater than Lipo2000. In addition, the D-type C-c(llkk)ch-r had about 7 times and 5.5 times the transfection efficiency of Lipo2000 in 293T cells and NIH-3T3 cells at the N/P ratio of 6, respectively. Overall, the multifunctional peptide gene vectors containing the R segment exhibited enhanced cellular internalization, a high gene transfection efficiency, and low cytotoxicity.
我们设计了一系列含有功能片段的肽载体，目的是提高细胞内化和基因转染效率。这些功能片段包括细胞穿透肽（R）、阳离子两亲性α-螺旋肽[（LLKK）-H或（LLHH）]、硬脂基部分和半胱氨酸残基。还合成了含有D型氨基酸的载体以提高其蛋白水解稳定性。通过圆二色性和动态光散射对这些肽与pGL3质粒DNA形成的复合物的构象、粒径和zeta电位进行了表征。此外，利用荧光激活细胞分选和共聚焦激光扫描显微镜研究了肽/DNA复合物的细胞摄取和基因转染效率。含有R片段的载体实现了更高的转染效率，且该效率高于Lipofectamine 2000。此外，在N/P比为6时，D型C-c(llkk)ch-r在293T细胞和NIH-3T3细胞中的转染效率分别约为Lipofectamine 2000的7倍和5.5倍。总体而言，含有R片段的多功能肽基因载体表现出增强的细胞内化、高基因转染效率和低细胞毒性。
