We have recently reported the formation of a new type of nanoparticles consisting of blends of poly (lactic-co-glycolic acid) (PLGA) and polyethylene oxide (PEO) derivatives, which exhibit the capacity to associate and release plasmid DNA in a controlled manner. In the present work our goal was to investigate the ability of these nanoparticles to overcome cellular and mucosal barriers (i.e. nasal mucosa) and thus, to work as gene delivery carriers. First, we studied the in vitro cellular uptake (HEK 293 cell line) of FITC-labelled plasmid DNA nanoencapsulated in PLGA: Pluronic F68 and PLGA: Tetronic T904 particles by confocal microscopy. Second, we investigated the uptake of rhodamine-labelled nanoparticles by the nasal mucosa following intranasal administration to mice. Third, we monitored the immune response generated by the nanoparticles containing a beta-galactosidase encoding gene, following nasal administration to mice, using the ELISA technique. The results of the in vitro cell culture studies showed the ability of these new nanoparticles to enter the cells and transport the associated DNA molecule across the cell membrane. Moreover, the results obtained following in vivo administration of the fluorescent nanoparticles evidenced their capability to overcome the nasal mucosal barrier. Finally, the results of the immunisation studies showed that DNA-loaded nanoparticles elicit a fast and strong response, significantly more pronounced than that corresponding to the naked plasmid DNA for up to 6 weeks. Overall, these results suggest that these new nanoparticles have a potential as carriers for the delivery of DNA across the nasal mucosa.