In an attempt to circumvent toxic effects of branched polyethylenimine (bPEI, 25 kDa), it was crosslinked with varying proportions of a novel linker, PEG 600 -bis(aminoethylphosphate) (PaP), which resulted in the formation of nanoparticles (PPaP) in the range of 61-99 nm. These nanoparticles were found to have significantly lower toxicity in vitro than the native PEI. GFP expression in cells mediated by PPaP (8.1%)/DNA complex was found to be ∼1.1-4.8 folds higher compared to GenePORTER 2™, Lipofectamine™, Superfect™ and native PEI in HeLa, HEK293 and CHO cells. FACS analysis on HeLa cells revealed ∼62% transfected cells, whereas, in the case of the GenePORTER 2™ transfection reagent, transfected cells were found to be ∼36%. Intracellular trafficking in HeLa cells showed a significant population of PPaP (8.1%) nanoparticles and their DNA complex in nucleus after 1 h of treatment. Also, efficient delivery of GFP specific siRNA resulted in ∼71% suppression of the target gene. DNase protection assay revealed that ∼78% of complexed DNA was protected by PPaP(8.1%) nanoparticles even after 2 h of treatment. In vivo transgene expression studies in Balb/c mice showed significantly higher expression in the spleen. The results advocate the potential of PPaP nanoparticles as efficient carriers of nucleic acids in vivo. © The Royal Society of Chemistry 2011.