P53 mediated apoptosis by reduction sensitive shielding ternary complexes based on disulfide linked PEI ternary complexes

摘要

Reduction-sensitive hyaluronic acid derivatives (HA-SS-COOH) were shielded on the DNA/polyethylenimine (PEI) to construct ternary complexes (DNA/. PEI/HA-SS-COOH, DPS ternary complexes) with efficient gene transfection. Details studied were conducted to investigation of factors influencing transfection efficiency, including the gene compression by fluorescence resonance energy transfer (FRET) spectrum and the intracellular fate of fluorescent labeled complexes by the confocal laser scanning microscope (CLSM). In the FRET study, DPS complexes were found to enhance condensation of DNA in preparation, while timely loosen gene under exposure to reductive reagent. Similar cellular uptake levels were observed for the designed reduction sensitive complexes and the stable one (DNA/. PEI/. HA, DPH ternary complexes), but the intracellular process was strikingly different for the two types of complexes. Only DPS showed obvious desired intracellular deshielding and endosomal escape, which contributed to highly efficient gene delivery. After loading with p53 plasmid, DPS complexes achieved significantly up-regulated p53 tumor suppressor gene expression at both mRNA and protein levels, as revealed by quantitative polymerase chain reaction (qPCR) and western blot investigations. Transgene induced apoptosis was evaluated by propidium iodide staining and flow cytometry analysis of cell cycle. Tumor cells transfected by DPS complexes containing p53 gene displayed almost 50% higher suppression in proliferation compared to those untreated cells, accompanied with a 46% elevation in the number of cells at sub-G1 phase and remarkable p53 dependent cell cycle perturbations prior to apoptosis. These results demonstrated that targeted delivery of p53 gene via reduction-sensitive DPS ternary complexes enabled up-regulated cellular p53 mRNA level through the exogenous p53 gene, inducing a significant p53-dependent anti-proliferative effect on tumor cells, which could be effective means of cancer treatment.