Gene expression with nonviral vectors is usually transient and lasts for only a few days. Therefore, repeated injection of the expression vector is required to maintain a therapeutic protein concentration in the target tissue. Biodegradable nanoparticles (approximately 200 nm diameter) formulated using a biocompatible polymer, poly(D,L-lactide-co-glycolide) (PLGA), have the potential for sustained gene delivery. Our hypothesis is that nanoparticle-mediated gene delivery would result in sustained gene expression, and hence better efficacy with a therapeutic gene. In this study, we have determined the antiproliferative activity of wild-type (wt) p53 gene-loaded nanoparticles in a breast cancer cell line. Nanoparticles containing plasmid DNA were formulated using a multiple-emulsion-solvent evaporation technique. To understand the mechanism of sustained gene expression with nanoparticles, we monitored the intracellular trafficking of both the nanoparticles and the nanoparticle-entrapped DNA, and also determined p53 mRNA levels over a period of time. Cells transfected with wt-p53 DNA-loaded nanoparticles demonstrated a sustained and significantly greater antiproliferative effect than those with naked wt-p53 DNA or wt-p53 DNA complexed with a commercially available transfecting agent (Lipofectamine). Cells transfected with wt-p53 DNA-loaded nanoparticles demonstrated sustained p53 mRNA levels compared to cells which were transfected with naked wt-p53 DNA or the wt-p53 DNA-Lipofectamine complex, thus explaining the sustained antiproliferative activity of nanoparticles. Studies with fluorescently labeled DNA using confocal microscopy and quantitative analyses using a microplate reader demonstrated sustained intracellular localization of DNA with nanoparticles, suggesting the slow release of DNA from nanoparticles localized inside the cells. Cells which were transfected with naked DNA demonstrated transient intracellular DNA retention. In conclusion, nanoparticle-mediated wt-p53 gene delivery results in sustained antiproliferative activity, which could be therapeutically beneficial in cancer treatment.