分枝型聚乙烯亚胺／寡核苷酸组装体的制备及细胞摄取研究

Study on Preparation of Branched Polyethylenimine/Oligonucleotide Assembly and its Role in Enhancing Cellular Uptake

目的制备分枝型聚乙烯亚胺(B-PEI)/反义寡核苷酸(ASON)超分子组装体(assembly)。方法分析(Zetasizer)组装体的粒径分布及Zeta电位,观察组装体的形态,单因素实验优化制备工艺条件。琼脂糖凝胶电泳分析组装体中ASON的包封率及组装体中ASON抗DNaseⅠ降解的能力。激光扫描共聚焦显微镜观察细胞的摄入情况。结果N/P(B-PEI含有氮原子的摩尔数/ASON含有磷原子的摩尔数)为4·0时制得粒径较小,Zeta电位较高的B-PEI/ASON超分子组装体,组装体呈球状,形态规则。N/P高于3·5时ASON的包封率接近100%,B-PEI/ASON超分子组装体荷载的有较强的抗酶解能力。共聚焦显微镜观察结果表明,组装体明显提高细胞对ASON的摄入率。结论B-PEI是一种组装能力强的多聚阳离子,B-PEI/ASON超分子组装体具有较好的物理化学性质,并能够显著增加细胞摄入率。

OBJECTIVE To prepare supramolecule assembly for branched Polyethylenimine/oligonucleotide （B-PEI/ASON）. METHODS The single factor experiment was used to optimize the preparation conditions for supramolecule assembly of B-PEI/ ASON. The size distribution and surface charge of the assembly and the morphology of assembly were measured. The ASON incorporation efficiency of assembly and the ability of protecting ASON against DNasel-mediated degradation were analyzed by agarose gel electrophoresis. Celluar uptake of B-PEI/ASON assembly was determined by eonfocal microscopy. RESULTS The assembly of uniform spheres with small diameter and high positive charge was obtained at a N/P ratio of 4. 0. The ASON was almost assembled by B-PEI at the N/P ratio of above 3.5 and the ASON incorporated by B-PEI/ASON supramolecule assembly was efficiently protected from DNase I-mediated degradation. The result froln confoeal microscopy showed that the enhanced ability of uptake ASON was obtained. CONCLUSION ASON could be assembled strongly and compactly by B-PEI. B-PEI/ASON supramolecule assembly possessed good physieoehemical properties and cellular uptake efficiency of ASON was remarkably improved by B-PEI.