Bioreducible Polypeptide Containing Cell-Penetrating Sequence for Efficient Gene Delivery

To design excellent polypeptide-based gene vectors and determine the gene delivery efficiency. Polypeptides (designated as xPolyK(6), xPolyK(6)-R(8)1 and xPolyK(6)-R(8)2), comprising the DNA condensing and buffering peptide HK6H as well as cell penetrating peptide (CPP) R-8 were obtained by the oxidative polymerization of CHK6HC and CR8C at different molar ratios in 4 mL phosphate-buffered saline (PBS) containing 30% (v/v) DMSO at room temperature for 96 h. The cytotoxicity of vectors was studied by MTT assay. Moreover, particle size, zeta potential and morphology along with the in vitro transfection efficiency and cellular uptake of vector/plasmid DNA (pDNA) complexes were characterized at various w/w ratios to determine their potential in gene therapy. All the vectors presented excellent ability of binding and condensing pDNA, additionally with low cytotoxicity. Simultaneously, transfection efficiency of the vectors appeared apparent dependence on the vector composition. The distinct correlation between the content of CR8C with the transfection efficiency demonstrated the effective improvement in transfection efficacy by the oxidative polymerization. Particularly, xPolyK(6)-R(8)2 possessed the highest transfection efficiency at a w/w ratio of 50. Furthermore, xPolyK(6)-R(8)2 also presented the best cellular uptake capability demonstrated by confocal microscopy and flow cytometry. Bioreducible polypeptides incorporating with proper amount of CPP are promising as effective non-viral gene vectors in gene therapy.