聚酰基硫脲基因输送体系的构建与评价

基于新型树枝状大分子聚酰基硫脲(PATU)，通过巯基-烯Michael加成反应得到表面N, N-二甲基半胱胺修饰的聚酰基硫脲(PATU-DMCA). 核磁结果表明：PATU-DMCA树枝状结构正确且完整. 琼脂糖凝胶电泳及动态光散射粒径测定结果表明：PATU-DMCA在低代数、低氮磷摩尔比条件下可以有效包载DNA，形成粒径为60~100 nm、Zeta电位为10~20 mV的纳米复合物. 在人宫颈癌细胞HeLa和人肺癌细胞A549上，低氮磷摩尔比的PATU-DMCA的无血清转染效率优于经典的树枝状大分子基因载体聚酰胺胺，而两者细胞毒性相当. HeLa上的亚细胞分布结果表明：纳米复合物在细胞水平的转运过程基本包括黏附细胞膜、内吞入胞、进入溶酶体，通过“质子海绵”效应从溶酶体逃逸后进入细胞质及细胞核进行转染. PATU-DMCA以及PATU本身在基因输送中均具有很大的潜力.

Construction and evaluation of gene delivery system based on polyacylthiourea

N, N-dimethylcysteamine modified PATU (PATU-DMCA) was synthesized through thiol-ene Michael addition reaction based on a novel dendrimer of polyacylthiourea (PATU). NMR results show that the dendrimer structure of PATU-DMCA is correct and integrated. Agarose gel electrophoresis assay and dynamic light scattering results demonstrate that PATU-DMCA of low generations with low molar ratios of N and P can package DNA to form nanoparticles with size of 60~100 nm and Zeta potential of 10~20 mV. In human cervical carcinoma cell HeLa and human lung cancer cell A549, the formed polyplexes with low molar ratios of N and P show higher transfection efficiency in serum-free medium than the classical dendrimer poly (amidoamine), while the cytotoxicity is equal to each other. The subcellular distribution study in HeLa indicate that the intracellular trafficking of the polyplexes include cell membrane adhesion, endocytosis into cell, entering into lysosome, and lysosomal escape via ‘proton sponge effect’ for transfection in cytoplasm and nucleus. Both PATU-DMCA and PATU itself have enormous potential for gene delivery.