Iwanaga Marie, Kodama Yukinobu, Muro Takahiro, Nakagawa Hiroo, Kurosaki Tomoaki, Sato Kayoko, Nakamura Tadahiro, Kitahara Takashi, Sasaki Hitoshi
a Department of Hospital Pharmacy , Nagasaki University Hospital , Nagasaki , Japan.
b Department of Clinical Pharmacokinetics, Graduate School of Biomedical Sciences , Nagasaki University , Nagasaki , Japan.
J Drug Target. 2017 Apr;25(4):370-378. doi: 10.1080/1061186X.2016.1274996. Epub 2017 Jan 12.
The purpose of this study was to develop a ternary complex of plasmid DNA (pDNA) electrostatically assembled with dendrigraft poly-l-lysine (DGL) and biodegradable glycosaminoglycan for effective and secure gene delivery. High gene expression of pDNA/DGL complex was confirmed with slight cytotoxicity and erythrocyte agglutination. Anionic ternary complexes of 55.4-223.8 nm were formed by the addition of a glycosaminoglycan such as chondroitin sulfate A (CS-A), chondroitin sulfate B (CS-B), chondroitin sulfate C (CS-C) or hyaluronic acid (HA). Using the cell line B16-F10, most of the ternary complexes showed only weak gene expression and little cytotoxicity, although the pDNA/DGL/CS-A complexes maintained a certain level of gene expression. In particular, the pDNA/DGL/CS-A8 complexes showed significantly higher gene expression than pDNA/DGL complexes in the presence of fetal bovine serum. Gene expression from the pDNA/DGL/CS-A8 complex was inhibited by a high concentration of CS-A and endocytosis inhibitors. After intravenous administration of the pDNA/DGL/CS-A8 complex and the pDNA/DGL complex into ddY mice, high gene expression was observed in the reticuloendothelial systems, the pDNA/DGL/CS-A complex is expected to be useful for gene therapy.
本研究的目的是开发一种由质粒DNA（pDNA）与树枝状聚-L-赖氨酸（DGL）静电组装而成，并结合可生物降解糖胺聚糖的三元复合物，用于有效且安全的基因递送。pDNA/DGL复合物具有高基因表达，且细胞毒性和红细胞凝集作用轻微。通过添加硫酸软骨素A（CS-A）、硫酸软骨素B（CS-B）、硫酸软骨素C（CS-C）或透明质酸（HA）等糖胺聚糖，形成了粒径为55.4 - 223.8nm的阴离子三元复合物。使用B16-F10细胞系，大多数三元复合物仅表现出较弱的基因表达和较小的细胞毒性，尽管pDNA/DGL/CS-A复合物保持了一定水平的基因表达。特别是，在存在胎牛血清的情况下，pDNA/DGL/CS-A8复合物的基因表达明显高于pDNA/DGL复合物。高浓度的CS-A和内吞作用抑制剂会抑制pDNA/DGL/CS-A8复合物的基因表达。将pDNA/DGL/CS-A8复合物和pDNA/DGL复合物静脉注射到ddY小鼠体内后，在网状内皮系统中观察到高基因表达，pDNA/DGL/CS-A复合物有望用于基因治疗。
