The purpose of this work was to develop a new type of nanoparticles made of PEG-grafted chi-tosans (CS-g-PEG) using tripolyphosphate (TPP) as a polyanionic crosslinker and to investigate the potential of these nanostructures as gene carriers. The formation of these nanoparticles was optimised by the evaluation of the combined effects of pH, PEGylation degree and chitosan/crosslinker ratio on the particle formation. The selected CS-g-PEG/TPP nanoparticles were studied with regard to their physico-chemical properties, DNA association efficiency as well as to their toxicity and gene expression in vitro. Furthermore, the best performing nanoparticle prototypes were also evaluated for their potential for in vivo gene delivery. CS-g-PEG/TPP nanoparticles displayed a high DNA association efficiency combined with high stability and low cellular toxicity. The results of the in vitro transfection assays showed positive effects of the PEGylation in the case of particles prepared from high molecular weight chitosan, while the presence of PEG slightly decreased the efficiency of the nanoparticles based on low molecular weight chitosan. Overall, high and long lasting gene expression levels could be observed for all types of nanoparticles. Moreover, CS-g-PEG/TPP nanoparticles also mediated high gene expression levels in vivo, following nasal administration. These results indicate the potential of ionically crosslinked CS-g-PEG/TPP nanoparticles as transmucosal gene delivery systems.