Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan, P. R. China.
Bioconjug Chem. 2012 Jan 18;23(1):125-34. doi: 10.1021/bc2005472. Epub 2011 Dec 21.
Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs to TAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TAT-based peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.
细胞摄取和核定位是基因传递的两个主要障碍。为了评估额外的核定位信号 (NLS) 是否可以提高基因转染效率，我们将不同类型的 NLS 引入 TAT 基基因传递系统中，形成三种复合物，包括 TAT-PV/DNA、TAT/DNA/PV 和 TAT/DNA/HMGB1。通过琼脂糖凝胶电泳评估不同载体的 DNA 结合能力。在不同条件下，由不同复合物介导的体外转染。用不同的复合物处理的细胞通过共聚焦显微镜观察。MTT 分析表明，所有复合物在 HeLa 和 Cos7 细胞系中即使在高 N/P 比下也没有明显的细胞毒性。与 TAT/DNA 复合物相比，TAT-PV/DNA 复合物介导的荧光素酶报告基因表达增强了约 200 倍。共聚焦研究表明，除了 TAT/DNA/PV，所有其他复合物在 HeLa 和 Cos7 细胞系中均表现出增强的核积累和细胞摄取。这些结果表明，核定位信号的引入可以增强基于 TAT 的肽的转染效率，这表明这里展示的基于 TAT 肽的载体在基因传递中具有很大的潜力。
