NLS (PKKKRKV)

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.

A spatial effect of targeted signal distribution is found in catiomer-gene polyplexes which strongly influences the gene transfection profiles. The nuclear localization sequence PKKKRKV (NLS) is chosen as the target ligand. For a given gene delivery system of constant composition, size, and charge, it is engineered such that the NLS is the only structural variable in polyplexes. This unique architecture of polyplexes relies on successive electrostatic interaction. The structural features of the PKKKRKV sequence in polyplexes are investigated by NMR. The gene transfection profiles of the polyplexes are found to be well correlated with the spatial distribution of the nuclear localization sequence. Upon modification of nuclear localization, the gene transfection efficiency is found to increase remarkably from 40% to 60%. The drastic improvement of gene transfection is explained by a NLS spatial distribution mechanism. High gene transfection based on spatial NLS distribution provides a new base for the design and development of non-viral gene delivery vectors.

Porphyrin-peptide conjugates bearing multiple nuclear localization sequences (NLS) could show increased tumor cell uptake and affinity for nuclear receptors, and consequently increased photodynamic activity. Previous studies suggest that an increase number of NLS might enhance the nuclear uptake of proteins and other macromolecules. We report the syntheses and investigation of a series of multimeric porphyrin-NLS conjugates bearing two, three or four peptides with the minimum sequence PKKKRKV, linked via PEG or 5-carbon linkers, and with different distributions at the porphyrin periphery. Our results show that the tumor cell uptake and phototoxicity of these conjugates is mainly determined by their amphiphilic character, and not the number of NLS residues per molecule, contrary to previous studies. The mono- and di-substituted photosensitizers bearing one or two PEG linkers and up to three peptide sequences were found to be the most phototoxic toward human carcinoma HEp2 cells, while the tetra-NLS conjugates symmetrically substituted around the porphyrin ring accumulated the least within cells and were non-phototoxic. All conjugates localized intracellularly within endosomal vesicles and lysosomes, probably as a result of an endocytic mechanism of uptake; as a consequence no nuclear uptake was detected by fluorescence microscopy.