title: Enhanced nuclear import and transfection efficiency of TAT peptide-based gene delivery systems modified by additional nuclear localization signals.
abstract: 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.
authors.affiliation: Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan, P. R. China.
authors.forename: Wen-Jie
authors.initials: WJ
authors.lastname: Yi
authors.forename: Juan
authors.initials: J
authors.lastname: Yang
authors.forename: Cao
authors.initials: C
authors.lastname: Li
authors.forename: Hui-Yuan
authors.initials: HY
authors.lastname: Wang
authors.forename: Chen-Wei
authors.initials: CW
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Tao
authors.forename: Si-Xue
authors.initials: SX
authors.lastname: Cheng
authors.forename: Ren-Xi
authors.initials: RX
authors.lastname: Zhuo
authors.forename: Xian-Zheng
authors.initials: XZ
authors.lastname: Zhang
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D024243:HMGB1 Protein / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019913:Nuclear Localization Signals / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods*
chemical_list: D015696:Gene Products, tat; D024243:HMGB1 Protein; D019913:Nuclear Localization Signals; D004247:DNA