Decationized polyplexes for targeted delivery of nucleic acids: from carrier design to in vivo evaluation

Abstract

Gene therapy is considered a promising treatment for current intractable diseases. However, the clinical applicability of gene therapy is highly dependent on the development of safe and efficient gene delivery vectors. So far, viral vectors have been used for clinical applications, but due to severe risks associated with viruses, cationic polymers have been evaluated as alternatives to viral vectors. Cationic polymers can easily form nanosized particles with plasmid DNA (pDNA), named polyplexes, via electrostatic interactions and possess an enormous chemical and structural flexibility. Polycation-based vectors have demonstrated high efficiency in vitro, however, they induce severe toxicity and possess suboptimal efficiencies in vivo, mainly due to their cationic nature, which significantly hampers their clinical applicability. With our work we have developed an alternative to conventional polycation-based polyplexes: decationized polyplexes. Unlike the cationic polymer based systems, decationized polyplexes are formed by hydrophilic and neutral polymers and can be obtained by an innovative 3-step process: polyplex formation by electrostatic interaction between pDNA and a polycationic precursor, structure stabilization by disulfide crosslinking, and finally removal of cationic charge - decationization. Structurally, decationized polyplexes consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). Retention of pDNA in the nanoparticles is exclusively based on physical entrapment given by the disulfide crosslinks, which provides an intracellularly triggered release profile, since disulfides are cleaved under the higher reducing environment present inside the cells. Through our study decationized polyplexes have demonstrated important advantages when compared to their cationic counterparts, such as much lower degree of nonspecific uptake and high degree cell specific uptake when decorated with targeting moieties as demonstrated by several cell uptake studies. Furthermore, in vitro studies showed lower cellular toxicity and in vivo nanotoxicity studies using a zebrafish model showed remarkable lower teratogenicity and mortality profile from decationized polyplexes. Stability evaluation in biological fluids a high stability for prolonged periods was found. Finally, in an in vivo biodistribution study, using tumor bearing mice, decationized polyplexes have shown greater retention in blood circulation and higher target tissue (tumor) accumulation. Given their important advantages, decationized polyplexes were also investigated and optimized for small interfering RNA (siRNA) delivery purposes, showing that decationized polyplexes can be used as a platform for different gene delivery modalities. In conclusion, decationzed polyplexes have demonstrated to be an important contribution for the development of safer polymeric gene delivery systems especially for targeted therapies. Importantly, the requirements for decationized polyplexes optimization have been identified, that will be the focus of future studies to further improve transfection efficiency and in vivo performance.