Construction of cell penetrating peptide vectors with N-terminal stearylated nuclear localization signal for targeted delivery of DNA into the cell nuclei.

Cellular uptake and nuclear localization are two barriers to gene delivery. Here, we designed new gene delivery carriers with an N-terminal stearylated (STR) nuclear localization signal (NLS), PKKKRKV, present in the Simian Virus 40 large T antigen with the aim to overcome limitations, such as cell membrane and nuclear pores, offering attractive possibilities to enhance gene delivery. Four vectors with different structures of N-stearylated nuclear localization signal-octaarginine peptide (STR-PKKKRKV-R(8) or STR-NLS-R(8), STR-VKRKKKP-R(8) or STR-reverse NLS-R(8), PKKKRKV-R(8) or NLS-R(8), and VKRKKKP-R(8) or reverse NLS-R(8)) were compared. The gene expression mediated by these vectors in dividing and non-dividing cells (both in 293T and HeLa cell lines) was investigated. The most efficient N/P ratio was 4 for STR-PKKKRKV-R(8,) STR-VKRKKKP-R(8,) and 0.25 for PKKKRKV-R(8), VKRKKKP-R(8.) The maximum transfection activity of these vehicles (VKRKKKP-R(8)) was up to 80% as effective as jetPEI™ and the vehicles did not exhibit cytotoxicity. Interestingly, N-stearylated peptides presented lower transfection activity compared to peptides without N-stearylation at lower N/P ratios (0.25 to 1). Confocal study showed that the vectors could effectively promote the nuclear translocation.