Efficient gene and siRNA delivery with cationic polyphosphoramide with amino moieties in the main chain

Abstract

Although a series of cationic polymers has been designed and synthesized by various kinds of strategies, the lower transfection efficiency and higher cytotoxicity are still a major problem for successful gene therapy. In this study, a novel cation polyphosphoramide (denoted as PPA) with amino moieties in the main chain was synthesized by polycondensation of ethyl dichlorophosphate with N¹-(2-aminoethyl)-N¹-(1-methyl)-1, 2-ethanediamine and investigated as non-viral vectors for gene transfection in target cells. Gel retardation analysis showed that PPA could efficiently retard the mobility of DNA at a lower N/P ratio. The size and zeta potential measurements found that these PPA/pDNA polycomplexes exhibited a decreased size and increased zeta potential. The cytotoxicity assay further revealed that PPA was non-toxic to different cells even at higher concentrations. It was also observed that polyionic complexes at a lower ratio of PPA/DNA (3 : 1) exhibited higher transfection efficiency. Interestingly, protein phosphorylation and a luciferase reporter assay showed that overexpression of the target gene (GFP-PKD2) transfected with PPA could enhance phosphorylation of PKD2, downstream IκB degradation and luciferase reporter activity of NF-κB signalling pathway in response to PMA, the agonist for PKD2 and NF-κB activation, indicating that the synthesized PPA could effectively deliver more than one exogenous genes into target cells and their proteins showed a functional role in target cells. More importantly, Western blotting and immunofluoescence staining of NF-κB p65 nuclear translocation showed that siRNA could be also delivered by PPA effectively, and exhibited silencing of the target gene as well as the signaling pathway in A549 cancer cells and HeLa cells. These results suggested that this novel cation polyphosphoramide could be used as an efficient carrier for plasmid and siRNA in future gene therapy applications.