The purpose of this study was to produce mucoadhesive nanocarriers made from chitosan (CS) and hyaluronic acid (HA), and containing the macromolecular drug heparin, suitable for pulmonary delivery. For the first time, this drug was tested in ex vivo experiments performed in mast cells, in order to investigate the potential of the heparin-loaded nanocarriers in antiasthmatic therapy. CS and mixtures of HA with unfractionated or low-molecular-weight heparin (UFH and LMWH, respectively) were combined to form nanoparticles by the ionotropic gelation technique. The resulting nanoparticles loaded with UFH were between 162 and 217 nm in size, and those prepared with LMWH were 152 nm. The zeta potential of the nanoparticle formulations ranged from +28.1 to +34.6 mV, and in selected nanosystems both types of heparin were associated with a high degree of efficiency, which was approximately 70%. The nanosystems were stable in phosphate buffered saline (PBS), pH 7.4, for at least 24h, and released 10.8% of UFH and 79.7% of LMWH within 12h of incubation. Confocal microscopy experiments showed that fluorescent heparin-loaded CS-HA nanoparticles were effectively internalized by rat mast cells. Ex vivo experiments aimed at evaluating the capacity of heparin to prevent histamine release in rat mast cells indicated that the free or encapsulated drug exhibited the same dose-response behaviour.