Enhanced nuclear import and transfection efficiency of TAT peptide-based gene delivery systems modified by additional nuclear localization signals.

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.

Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate.

The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.

PEGylated peptide based reductive polycations as efficient nonviral gene vectors.

To overcome the critical barriers in gene delivery, a series of reducible polycations (RPCs) based on low molecular weight (LMW) peptides, i.e. PolyHK6 H, PolyHK6 H-mPEG1 , PolyHK6 H-mPEG2 , and PolyHK6 H-mPEG3 , with different poly(ethylene glycol) (PEG) contents, are synthesized and evaluated as nonviral gene vectors. All the RPCs exhibit lower cytotoxicity compared with 25 kDa polyethyleneimine (PEI) and PEGylated PEI (PEI-mPEG: PEI-mPEG1 , PEI-mPEG2 , and PEI-mPEG3 ). PolyHK6 H-mPEG1 and PolyHK6 H-mPEG2 can bind and condense plasmid deoxyribonucleic acid (pDNA) efficiently with a particle size of about 200 nm. Moreover, they display much higher transfection efficiency than that of 25 kDa PEI especially in serum-supplemented medium. Moreover, PolyHK6 H-mPEG1 has equal transfection efficiency with PEI-mPEG1 which is optimal in the PEI-mPEG, but PolyHK6 H-mPEG1 exhibits significantly lower cytotoxicity than PEI-mPEG1 . This is attributed to the fact that inter-peptide disulfide bonds can increase the stability of RPCs/pDNA complexes in extracellular environment and thereafter cleave in cytoplasm to facilitate the release of pDNA in intracellular environment. The PEGylated RPCs demonstrate here improved intracellular gene transfer performance and will have great potential applications in vivo.

Tyroserleutide-based gene vector for suppressing VEGF expression in cancer therapy.

A small interfering RNA (siRNA) plasmid DNA (pYr-1.1-hU6-EGFP-siVEGF) was constructed and used for suppressing vascular endothelial growth factor (VEGF) expression and inhibiting tumor growth. Then, a (tyrosyl-seryl-leucine)-polyethyleneimine-poly(ethylene glycol) (YSL-PEI-PEG) conjugate was designed and synthesized as a gene carrier for the delivery of pYr-1.1-hU6-EGFP-siVEGF plasmid. The therapeutic peptide YSL was conjugated to PEI to improve the anti-cancer efficiency, and the PEG chain was introduced to reduce the serum protein adsorption and improve the stability of the complex in the systemic circulation. It was found that YSL-PEI-PEG could efficiently condense plasmid DNA when the vector/DNA weight ratio was higher than 2. Compared with PEI 25 kDa, YSL-PEI-PEG exhibited higher transfection efficiency and lower cytotoxicity. More importantly, the results showed that the gene delivery system owned strong ability to inhibit cancer cell proliferation in vitro and tumor growth in vivo. YSL-PEI-PEG has great potential as a gene vector for clinical applications.

A linear-dendritic cationic vector for efficient DNA grasp and delivery.

This paper presents an attempt to design an efficient and biocompatible cationic gene vector via structural optimization that favors the efficient utilization of amine groups for DNA condensation. To this end, a linear-dendritic block copolymer of methoxyl-poly(ethylene glycol)-dendritic polyglycerol-graft-tris(2-aminoethyl)amine (mPEG-DPG-g-TAEA) was prepared with specially designed multiple functions including strong DNA affinity, endosomal buffering and expected serum-tolerance. Based on the transfection in serum-free and serum-conditioned media, the influences of the polymer structures including the degree of polymerization of DPG and TAEA substitution degree were explored. As compared to polyethylenimine (M(w)=5 kDa) (PEI5k) with similar molecular weight and higher amine density, mPEG-DPG-g-TAEA displayed comparably high DNA affinity due to the special linear-dendritic architecture. Consequently, at very low N/P ratio, mPEG-DPG-g-TAEA vectors could mediate efficient in vitro luciferase expression at levels that are comparable with or even superior to the commercially available Lipofectamine™ 2000, while being apparently higher than PEI5k. The designed vectors exhibit considerably higher cell biocompatibility and better resistance against bovine serum albumin adsorption than PEI5k. The stability of the complexes on coincubation with heparin was found to be largely dependent on the polymer structure. As concluded from the comparative transfection study in the absence/presence of chloroquine, it is likely that the polycation itself could produce endosomal buffering. This linear-dendritic vector shows promising potential for the application of gene delivery.