Intracellular Trafficking of DNA/Copolymer Complexes

Gene therapy treats a genetic deficiency by delivering genetic information in the form of nucleic acids (DNA) to the targeted cells. The main bottleneck in the advancement of gene therapy is the development an efficient, non-toxic gene delivery system. One of the main goals of such an efficient delivery system is to deliver the exogenous plasmid containing therapeutic gene to the nucleus of the cells while preventing it from degradation in the low pH, nucleases rich environment of endosomes/ and lysosomes. The positively charged groups of these cationic polymers condense the negatively charged DNA into nanoparticles called polyplexes.

Novel pentablock copolymers of poly(diethylaminoethylmethacrylate) (PDEAEM), poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO), have been synthesized in our laboratories and are being investigated for their efficiency to deliver genes to the cancer cells. The tertiary amines of PDEAEM group condense the DNA in polyplexes. These polyplexes are hypothesized to be up-taken by the cells via endocytosis, after which they get trapped into the endosomes. However, the tertiary amines of PDEAEM groups get protonated in the low-pH environment of the endosomes, resulting in an influx of water and chloride ions into the endosomes, making they ultimately get ruptured, thus enabling the release of the trapped polyplexes. This work demonstrates intracellular paths taken by polyplexes using confocal fluorescence microscopy.