The conversion of colloidal drug carriers/polymeric nanoparticles into dry microparticulate powders (e.g., by spray-drying) is a prominent approach to overcome the aerodynamic limitations of these formulations for delivery via inhalation. However, to what extent such nano-embedded microparticles disintegrate into individual/intact nanoparticles after contacting relevant physiological media has so far not been addressed. Polymeric nanoparticles were spray-dried into nano-embedded microparticles (NEMs) using different amounts of trehalose as embedding matrix excipient. Formulations were characterized and then evaluated for their disintegration behavior after aerosolization onto model mucus. Although a rapid and complete aqueous redispersion was observed for specific excipient/nanoparticle weight ratios (i.e., greater than 1/1), the same formulations revealed no disintegration after deposition onto a static mucus layer. Double-labeled NEMs powders (i.e., dual color staining of polymeric nanoparticles and trehalose) demonstrated rapid matrix dissolution, while the nanoparticle aggregates persisted. When deposited onto agitated mucus, however, sufficient disintegration of NEMs into individual polymeric nanoparticles was observed. These findings indicate that mechanical forces are necessary to overcome the attraction between individual nanoparticles found within the NEMs. Thus, it remains questionable whether the lung mechanics (e.g., breathing, mucociliary clearance) acting on these formulations will contribute to the overall disintegration process.