Abstract / Summary
Abstract Background: Lower respiratory tract infections caused by multidrug-resistant bacterial pathogens remain a major global health challenge and are frequently accompanied by airway obstruction and exaggerated pulmonary inflammation. Although antimicrobial peptides exhibit potent bactericidal activity against resistant pathogens, their clinical translation is limited by poor stability, rapid enzymatic degradation, and inefficient pulmonary delivery. Simultaneously, low-dose theophylline has attracted renewed interest because of its bronchodilatory and histone deacetylase-2 (HDAC2)-restoring activities. The present study aimed to develop and optimize a multifunctional co-spray-dried dry powder inhaler (DPI) capable of simultaneously delivering the antimicrobial peptide KAMP-18 and theophylline directly to the deep lung to provide combined antimicrobial, bronchodilatory, and anti-inflammatory therapeutic effects. Methods: A three-factor, three-level Box–Behnken experimental design was employed to optimize inlet temperature, feed flow rate, and L-leucine concentration during spray drying. The optimized formulation was comprehensively characterized for production yield, encapsulation efficiency, particle size distribution, powder flow, moisture content, morphology, solid-state properties, thermal behavior, surface charge, and chemical compatibility using laser diffraction, SEM, FTIR, DSC, PXRD, and zeta potential analysis. Molecular docking and 200 ns molecular dynamics simulations investigated interactions between KAMP-18 and bacterial lipopolysaccharide (LPS) as well as theophylline and HDAC2. Aerosol performance was assessed using a Next Generation Impactor (NGI). Biological performance was evaluated through ex vivo tracheal relaxation, antimicrobial and antibiofilm assays, cytocompatibility studies, mechanistic investigations of HDAC2 activation and inflammatory cytokine suppression, pharmacokinetic analysis, stability testing, histopathology, hemocompatibility, and mucus penetration studies. Results: The optimized formulation demonstrated a production yield of 72.4 ± 3.1%, encapsulation efficiencies exceeding 99% for both active agents, and a median particle diameter of 2.45 ± 0.15 μm. SEM revealed spherical corrugated microparticles, while FTIR, DSC, and PXRD confirmed excellent physicochemical compatibility without chemical degradation. Molecular dynamics simulations demonstrated highly stable KAMP-18/LPS and theophylline/HDAC2 complexes with average backbone RMSD values of 0.24 ± 0.03 nm and 0.18 ± 0.02 nm, respectively. NGI analysis showed excellent aerosolization characteristics with emitted doses above 91%, fine particle fractions approaching 79%, and MMAD values around 2.8 μm, indicating efficient deep lung delivery. The formulation preserved potent antibacterial activity against Pseudomonas aeruginosa, Klebsiella pneumoniae, and methicillin-resistant Staphylococcus aureus while reducing mature biofilm biomass by up to 88.4%. Ex vivo studies demonstrated significantly enhanced bronchodilatory activity with an EC50 of 0.66 ± 0.07 μM and a strong synergistic interaction (combination index = 0.34). Mechanistic studies revealed restoration of HDAC2 activity, marked suppression of intracellular reactive oxygen species, and substantial reductions in TNF-α, IL-6, and IL-8 secretion. The formulation exhibited excellent cytocompatibility, prolonged pulmonary peptide retention above the minimum inhibitory concentration for up to 12 h, minimal hemolytic activity, favorable histopathological safety, enhanced enzymatic stability, and efficient mucus penetration. Conclusions: The optimized co-spray-dried KAMP-18/theophylline dry powder inhaler successfully integrates antimicrobial, bronchodilatory, and anti-inflammatory activities into a single inhalable platform. The formulation demonstrated excellent aerosol performance, preserved biological activity after spray drying, prolonged pulmonary retention, remarkable safety, and strong multifunctional therapeutic efficacy. These findings support the considerable translational potential of this dual-drug inhalable system as a promising next-generation therapeutic strategy for the treatment of bacterial pulmonary infections associated with airway inflammation and multidrug resistance. Clinical trial number: Not applicable