Histidylated cationic polyorganophosphazene/DNA self-assembled nanoparticles for gene delivery

Abstract

Cationic polyorganophosphazene has shown the ability to deliver gene. To obtain more efficient transfection, His(Boc)-OMe bearing histidine moiety was introduced to synthesize a new derivative of cationic polyphosphazenes with another side group of 2-dimethylaminoethylamine (DMAEA). The poly(DMAEA/His(Boc)-OMe)phosphazene (PDHP) and DNA could self-assemble into nanoparticles with a size around 110 nm and zeta potential of +15 mV at the PDHP/DNA ratio of 10:1 (w/w). The maximum transfection efficiency of PDHP/DNA self-assembled nanoparticles (PHSNs) against 293 T cells was much higher than that of poly(di-2-dimethylaminoethylamine) phosphazenes (PDAP)/DNA self-assembled nanoparticles (PASNs) and PEI 25/DNA self-assembled nanoparticles (PESNs) at the polymer/DNA ratio of 10:1, but the cytotoxicity of PDHP assayed by MTT was much lower than that of PDAP and PEI 25. These results suggested that PDHP could be a good candidate with high transfection efficiency and low cytotoxicity for gene delivery.

Introduction

The cationic polymers are one of the most widely investigated non-viral vectors for gene delivery owing to their obvious advantages such as stability in storage, production in large-scale easily, minimal immunogenicity, molecular diversity of chemical or cell specific targeting moieties modification, etc. (Spack and Sorgi, 2001, Merdan et al., 2002). Unfortunately, a major problem associated with cationic polymers is their lower transfection efficiency compared with viral vectors (El-Aneed, 2004). Thus, many efforts have been made to increase their transfection efficiency with low toxicity. Because endosomal escape is thought to be one of the major bottlenecks in non-viral nucleic acid delivery (Singh et al., 2004), a pervasive approach to improve transfection efficiency of cationic carriers is the inclusion or co-application of endosomolytic agents to protect the plasmid from hydrolytic digestion within endosome and/or enable it escape from endosome. The classic endosomolytic agents such as chloroquine (Erbacher et al., 1996, Ciftci and Levy, 2001) or fusogenic peptide (Wagner et al., 1992, Lee et al., 2001) are impractical for in vivo gene therapy because of their cellular toxicity, immunogenicity and side effects. Recently, the polymers that were conjugated with histidine or other moieties containing imidazole group showed a significant enhancement of gene expression without increasing toxicity compared with non-modified polymers (Roufaï and Midoux, 2001, Kima et al., 2003, Mishra et al., 2006, Park et al., 2006, Swami et al., 2007). In this case, histidine or other moieties containing imidazole group could favor polymers/DNA complexes escaping from endosome by a “proton sponge” mechanism, which the imidazole heterocycles display a pK_a around 6, thus possessing a buffering capacity in the endolysosomal pH range, inducing membrane destabilization after their protonation and facilitating complexes releasing to cytosol.

Polyphosphazenes have been investigated for many different biomedical and pharmaceutical applications (Aldini et al., 2001, Zhang et al., 2005, Andrianov et al., 2005, Greish et al., 2005) due to their biodegradability, versatile physicochemical properties and nontoxic degradation products. Polyphosphazenes have not been explored for gene delivery until Hennink's group synthesized and characterized a biodegradable cationic polyphosphazene substituted with side group of 2-dimethylaminoethylamine (DMAEA) (Luten et al., 2003). This poly(di-DMAEA)phosphazene (PDAP) could condense DNA to form positively charged complex nanoparticles, which showed transfection activity in vitro and gene expression at a distant tumor site after intravenous administration (de Wolf et al., 2005). However, few studies have been done for gene delivery using other derivates of polyphosphazenes.

The purpose of this work is to develop a novel derivate of poly(organo)phosphazene with more efficient transfection activity and low cytotoxicity for gene delivery. In this work, a new cationic derivative of poly(organo)phosphazene by introducing His(Boc)-OMe and DMAEA as side groups was synthesized. The synthesis, characteristics, cytotoxicity and transfection activity of poly(DMAEA/His(Boc)-OMe)phosphazene (PDHP) were investigated. The influence of histidine moiety on transfection activity of PDHP was discussed as well.