Chronic lung infections caused by Pseudomonas aeruginosa are a major contributor to morbidity and mortality in patients with cystic fibrosis. Biofilm formation and the emergence of antibiotic resistance limit the effectiveness of current inhaled therapies, highlighting the need for innovative formulation strategies that enhance local antibacterial efficacy while preserving epithelial integrity. This study explores a nano-in-micro inhalable formulation design that combines azithromycin (AZM) with the antibiotic adjuvant menadione (MEN) to enhance antibiofilm activity under biorelevant pulmonary exposure conditions. Nano-embedded microparticles (NEMs) were developed as an inhalable “Trojan particle” approach, combining AZM in a microparticle matrix with MEN-loaded polymeric nanoparticles. The formulations were produced by spray drying and characterized in terms of morphology, aerodynamic performance, solid-state properties and redispersibility. Antibiofilm activity against P. aeruginosa biofilms and epithelial safety in Calu-3 cells were evaluated using biorelevant aerosol exposure models, including nebulization and dry powder atomization. NEMs exhibited favorable aerodynamic properties suitable for bronchial delivery. Incorporation of MEN into the NEM system enhanced the antibiofilm efficacy of AZM compared with the microparticle matrix-only formulation, indicating that the adjuvant effect of MEN was retained upon translation into an inhalable formulation. Cytotoxicity studies demonstrated that neither nebulized nor dry powder–delivered formulations compromised the membrane integrity of Calu-3 cells. These results indicate that the combination of azithromycin and menadione within a NEM design preserves drug activity and adjuvant efficacy while enabling effective aerosol delivery. Future studies in more complex and disease-relevant models will further strengthen the understanding of the translational potential of this approach.
International Journal of Pharmaceutics , 2026, 698 126965.