中国组织工程研究Issue(47)：8162-8168,7.DOI:10.3969/j.issn.2095-4344.2013.47.004
聚乙烯亚胺-壳聚糖/DNA纳米粒的制备及介导体外转染关节软骨细胞
Preparation of polyethylenimine-chitosan/DNA nanoparticles for transfecting articular chondrocytes in vitro
摘要
Abstract
BACKGROUND:Chitosan is wel known as good biocompatibility and biodegradability;however, its extensive use in biomedical applications is restricted due to its poor transfection efficiency. OBJECTIVE:To prepare the polyethyleneimine-chitosan/DNA nanoparticles loading enhanced green fluorescent protein gene, and to detect their physicochemical properties and gene transfection efficiency towards chondrocytes in vitro. <br> METHODS:Low molecular weight polyethyleneimine was covalently linked to chitosan backbone to construct chitosan-graft-polyethyleneimine;then the chitosan-graft-polyethyleneimine was mixed with DNA nanoparticles, which loaded enhanced green fluorescent protein gene, by a complex coacervation method. The nanoparticle morphology was observed under a scanning electron microscopy. The sizes and zeta-potentials of the <br> nanoparticles were measured by a Marven-nano laser diffractometer. The binding capacity of plasmid DNA was evaluated by agarose gel electrophoresis analysis. The gene transfection experiments in vitro were performed towards rabbit’s chondrocytes. The gene transfection efficiency was measured with flow cytometry and under fluorescence microscope. How marked DNA entered into the nucleus of chondrocytes mediated by the nanoparticles was detected by laser scanning confocal microscopy. <br> RESULTS AND CONCLUSION:The prepared nanoparticles were mainly spherical, with an average size of (154.6±18.6) nm, and zeta-potential of (24.68±6.82) mV. The agarose gel electrophoresis analysis confirmed that the nanoparticles could effectively protect plasmid DNA from DNase Ⅰ-induced degradation. Gene transfection in vitro proved that the nanoparticles were efficient in transfecting rabbit’s chondrocytes and the expression of green fluorescent proteins was observed under fluorescent microscope, with a transfection efficiency of (23.80±1.74)%that was significantly higher than that of the naked plasmid DNA and chitosan/DNA nanoparticles (P<0.05). But no significant differences were observed between polyethyleneimine-chitosan/DNA nanoparticles and LipofectamineTM 2000. These findings indicate that the polyethyleneimine-chitosan/DNA nanoparticles can effectively protect plasmid DNA from nuclease degradation, and exhibit the favorable transfection ability towards articular chondrocytes.关键词
生物材料/组织工程软骨材料/壳聚糖/聚乙烯亚胺/非病毒基因载体/软骨细胞/基因转染/DNA纳米粒/国家自然科学基金分类
医药卫生引用本文复制引用
卢华定,戴驭虎,连礼熠,吕璐璐,赵慧清..聚乙烯亚胺-壳聚糖/DNA纳米粒的制备及介导体外转染关节软骨细胞[J].中国组织工程研究,2013,(47):8162-8168,7.基金项目
国家自然科学基金资助项目(82172040，30600632)，广东省科技计划基金资助项目(2012B031800451)，广东省自然科学基金资助项目(S2011010004808)@@@@the National Natural Science Foundation of China, No.82172040,30600632 （82172040，30600632）
the Science and Technology Project of Guangdong Province, No.2012B031800451 （）
the Natural Science Foundation of Guangdong Province, No. S2011010004808 （）
