Intracellular plasmid DNA delivery by self-assembled nanoparticles of amphiphilic PHML- b -PLLA- b -PHML copolymers and the endocytosis pathway analysis

This work presents a new series of polycationic nanoparticles of ( l-)-lysine conjugated amphiphilic triblock copolymer poly(hydroxyletheyl methacrylate-L-lysine)- b-poly(L-lactide)- b-poly(hydroxyletheyl methacrylate-L-lysine)s (PHML- b-PLLA- b-PHML) as potent low cytotoxic vectors for intracellular plasmid DNA delivery. First, the triblock PHML- b-PLLA- b-PHML copolymers were prepared via a combination of metal-free controlled ring opening polymerization and successive atom transfer radical polymerization. Then the cationic PHML- b-PLLA- b-PHML nanoparticles were further prepared by solution self-assembly. The particle size, zeta potential and morphology of as-prepared PHML- b-PLLA- b-PHML nanoparticles were characterized by dynamic light scattering and atomic force microscopy, respectively. The plasmid DNA binding affinities and polyplex stabilities were separately explored by agarose gel retardation and DNase I degradation assays. Then in vitro cytotoxicity and gene transfection efficiency of the PHML- b-PLLA- b-PHML nanoparticles vectors as well as relevant polyplex endocytosis pathway were investigated with H1299 cells. It was revealed that the PHML- b-PLLA- b-PHML nanoparticles exhibited low cytotoxicity, strong plasmid DNA binding affinity, high polyplex stability and efficient plasmid DNA transfection even under serum conditions (10% FBS). Moreover, the endocytosis analysis results disclosed that the PHML ₃₀ - b-PLLA- b-PHML ₃₀ nanoparticle/plasmid DNA polyplexes were predominantly involved in lipid-raft-mediated endocytosis pathway, similar to that of SV40 virus-based vectors.