Chitosan nanoparticles (NPs) are biocompatible drug carriers able to cross the blood-brain barrier and represent a promising drug delivery system to the central nervous system. We used chitosan NPs to deliver the D-Ala2-D-Leu5-enkephalin (DADLE) to neuronal cells in vitro. DADLE is a hypometabolising synthetic opioid potentially useful for biomedical applications, but its short plasmatic half-life makes its in vivo administration ineffective. Here, we demonstrate by immunoelectron microscopy that (1) chitosan NPs are capable to deliver the opioid to neuronal cells; (2) DADLE is released from the internalised, opioid-loaded NPs up to 48 h; (3) in the nucleus, DADLE binds the transcription/splicing sites; (4) cells treated with DADLE-loaded NPs undergo a decrease in transcription factor amounts and proliferation rate without damage to cell organelles. In this model, chitosan NPs protected the loaded opioid from degradation, thereby prolonging its intracellular effects. These findings suggest that these NPs are efficient for the systemic and tissue administration of opioids in vivo.