Liang Xuan, Ren Xianyue, Liu Zhenzhen, Liu Yingliang, Wang Jue, Wang Jingnan, Zhang Li-Ming, Deng David Yb, Quan Daping, Yang Liqun
Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-Sen University, Guangzhou, People's Republic of China.
Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China.
Int J Nanomedicine. 2014 Jan 31;9:419-35. doi: 10.2147/IJN.S51919. eCollection 2014.
The purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector.
A series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino)-1-propylamine (DMAPA-Glyp) and 1-(2-aminoethyl) piperazine (AEPZ-Glyp) residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid-base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA) condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney) and the CNE2 (human nasopharyngeal carcinoma) cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley rats) was evaluated to identify the safety and transfection efficiency.
The hyperbranched cationic glycogen derivatives conjugated with DMAPA and AEPZ residues were synthesized. They exhibited better blood compatibility and lower cytotoxicity when compared to branched polyethyleneimine (bPEI). They were able to bind and condense pDNA to form the complexes of 100-250 nm in size. The transfection efficiency of the DMAPA-Glyp/pDNA complexes was higher than those of the AEPZ-Glyp/pDNA complexes in both the 293T and CNE2 cells, and almost equal to those of bPEI. Furthermore, pDNA could be more safely delivered to the blood vessels in brain tissue of Sprague Dawley rats by the DMAPA-Glyp derivatives, and then expressed as green fluorescence protein, compared with the control group.
The hyperbranched cationic glycogen derivatives, especially the DMAPA-Glyp derivatives, showed high gene-transfection efficiency, good blood compatibility, and low cyto toxicity when transfected in vitro and in vivo, which are novel potential nonviral gene vectors.
本研究旨在合成并评估超支化阳离子糖原衍生物作为一种高效的非病毒基因递送载体。
合成了一系列与3-(二甲氨基)-1-丙胺（DMAPA-Glyp）和1-(2-氨基乙基)哌嗪（AEPZ-Glyp）残基共轭的超支化阳离子糖原衍生物，并通过傅里叶变换红外光谱和氢-1核磁共振光谱对其进行表征。通过在NaCl水溶液中的酸碱滴定评估其缓冲能力。使用琼脂糖凝胶电泳评估糖原衍生物对质粒脱氧核糖核酸（pDNA）的凝聚能力以及对DNase I降解的保护作用。测量糖原衍生物/pDNA复合物的zeta电位和粒径，并使用原子力显微镜观察复合物的图像。分别通过溶血试验和MTT（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐）试验评估血液相容性和细胞毒性。通过流式细胞术和荧光显微镜在293T（人胚肾）和CNE2（人鼻咽癌）细胞系中评估阳离子糖原衍生物介导的pDNA转染效率。评估pDNA在模型动物（Sprague Dawley大鼠）体内的递送情况，以确定安全性和转染效率。
合成了与DMAPA和AEPZ残基共轭的超支化阳离子糖原衍生物。与支链聚乙烯亚胺（bPEI）相比，它们表现出更好的血液相容性和更低的细胞毒性。它们能够结合并凝聚pDNA，形成尺寸为100-250 nm的复合物。在293T和CNE2细胞中，DMAPA-Glyp/pDNA复合物的转染效率高于AEPZ-Glyp/pDNA复合物，且几乎与bPEI相当。此外，与对照组相比，DMAPA-Glyp衍生物能够更安全地将pDNA递送至Sprague Dawley大鼠脑组织的血管中，并表达为绿色荧光蛋白。
超支化阳离子糖原衍生物，尤其是DMAPA-Glyp衍生物，在体外和体内转染时均表现出高基因转染效率、良好的血液相容性和低细胞毒性，是新型潜在的非病毒基因载体。
