AbstractThe present work describes the development, characterization, and in vitro evaluation of novel poly(L‐lysine)‐based polyplexes as nonviral gene delivery systems. Initially, a well‐defined hybrid block copolymer comprising poly(ethylene glycol) methacrylate) (POEGMA) and poly(L‐lysine) (PLL) blocks was successfully synthesized and characterized. The hybrid copolymer shows high ability to condense DNA into stable polyplexes in aqueous media with sizes of approx. 100 nm. The nanoplexes were evaluated for cellular toxicity in A549 alveolar and HepG2 (hepatocarcinoma) cell lines. The nanoparticles cell internalization and transfection ability were assessed in HepG2 cells. The initial experiments showed that DNA was successfully transfected into the nucleus of human liver cancer cells and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. These results revealed that the newly synthesized POEGMA‐b‐PLL diblock copolymer might be very attractive candidate as a nonviral gene delivery vector.