The development of new vectors to deliver DNA into cells for therapy of cancers or genetic diseases has been a major area of research for many years. However, the clinical application of this technology requires the development of efficient, reliable and sterile vectors enabling the transfer of genes in vivo. Non viral, polymer or lipid-based vectors offer a new impetus to gene therapy because they are less toxic than viral vectors (no endogenous recombination, fewer immunological reactions, easy production and delivery of large-sized plasmid). The aim of this study is to develop a new tool for DNA delivery composed of methacrylic polymeric (Eudragit® RS and RL) nanoparticles. These nanoparticles were prepared by two methods: nanoprecipitation and double emulsion. The nanoparticles were characterized by their size, zeta potential and amount of DNA adsorption. Cytotoxicity tests based on mitochondrial activity (MTT test) revealed that the nanoparticles had limited cytotoxicity and that this depended on both the cell type and the nanoparticle concentration. Transgene expression was observed using the Green Fluorescence Protein gene as reporter gene, and was evaluated by flow cytometry in FaDu, MDA-MB 231 and MCF-7 cell lines. The results showed that transfection rates ranging between 4 and 7% were achieved in FaDu and MDA-MB 231 cells with nanoparticles prepared by the nanoprecipitation method. In MCF-7 cells transfected with nanoparticles prepared by either the double emulsion or the nanoprecipitation method, the transfection efficiency was between 2 and 4%. Nanoparticles prepared by nanoprecipitation were slightly more efficient than nanoparticles prepared from a double emulsion. Particle size was not an important factor for transfection, since no significant difference was observed with size between 50 and 350 nm. We showed that Eudragit® RS and RL nanoparticles could introduce the transgene into different types of cells, but were generally less effective than the lipofectamine control.