The use of biodegradable nanoparticles loaded with 5-fluorouracil was investigated as a potential means to sustain the release of this drug. Nanoparticles prepared from four biodegradable polymers were loaded with 5-fluorouracil using three loading concentrations of drug and three different concentrations of added polymer. Washing particles using a centrifugation/re-suspension with ultrasound protocol was found to dislodge the majority of drug, resulting in an over-estimation of incorporation efficiency and low levels of strongly entrapped drug. Increasing the initial 5-fluorouracil concentration before polymer/monomer addition increased the drug loading in both washed and unwashed particles. Increasing the amount of polymer used to make nanoparticles did not increase loadings, but did produce increased amounts of unusable polymer waste. Drug release from nanoparticles was evaluated using a Franz cell diffusion apparatus, which showed an initial burst effect followed by a slower release phase over 24 h. Indeed, nanoparticles prepared from poly(lactide-co-glycolide) released 66% of their 5-fluorouracil payload over this period. It was concluded that 5-fluorouracil-loaded nanoparticles could be readily included into a hydrogel-based delivery system to provide sustained drug release for trans-epithelial drug-delivery applications.