This study was aimed to develop poly(DL-lactide-co-glycolide) (PLGA) nanoparticle of highly water soluble antibiotic drug, netilmicin sulfate (NS) with improved entrapment efficiency (EE) and antibacterial activity. Dextran sulfate was introduced as helper polymer to form electrostatic complex with NS. Nanoparticles were prepared by double emulsification method and optimized using 2(5-1) fractional factorial design. EE was mainly influenced by dextran sulfate: NS charge ratio and PLGA concentration, whereas particle size (PS) was affected by all factors examined. The optimized NS-loaded-NPs had EE and PS of 93.23 +/- 2.7% and 140.83 +/- 2.4 nm respectively. NS-loaded-NPs effectively inhibited bacterial growth compared to free NS. Sustained release protected its inactivation and reduced the decline in its killing activity over time even in presence of bronchial cells. A MIC value of 18 mu g/mL was observed for NPs on P. aeruginosa. Therefore, NPs with sustained bactericidal efficiency against P. aeruginosa may provide therapeutic benefit in chronic pulmonary infection, like cystic fibrosis.