Project Summary Non-tuberculous mycobacterial (NTM) lung disease represents a major public health challenge. The long treatment period and high-dose systemic administration of antibiotics often leads to adverse effects, resistance development, and suboptimal therapeutic accumulation at the infection site. Identifying new drugs and routes of administration as well as shorter and better-tolerated treatment regimes are highly desired for treating NTM lung infections, including those caused by MAC and M. abscessus, among other species. Here, we propose a novel antibiotic delivery system, cyclodextrin-based metal-organic frameworks (CD-MOFs), to form antibiotic/CD-MOF cocrystals administered through an inhalation route via a dry powder inhaler (DPI) to tackle the challenges in NTM infection treatments. Highlighted innovations include (1) High loading ratios and deep lung delivery with minimized systemic exposures and adverse effects; (2) Cyclodextrin-improved bioavailability and permeability for the boosted antimicrobial activity; (3) Synergistic anti-mycobacterial efficacy via multiple antibiotic integration; (4) Controlled release of antibiotics towards optimized PK/PD performance; (5) MOF-induced intracellular internalization by alveolar macrophages favorable in treating NTM and other intracellular infections; and (6) Significantly lowered production cost with complete avoidance of toxic solvents. Because of the unique value of CD-MOF cocrystals provided, our proposal will offer multiple benefits to the field of pulmonary antibiotic delivery for the improved efficacy and safety in treating NTM lung infections. In Aim 1, we plan to demonstrate the pulmonary anti-mycobacterial feasibility of antibiotics/CD-MOF cocrystals. The investigation starts by synthesizing cocrystals with high loading ratio (>10wt%) and appropriate aerodynamic performance (MMAD: 1-5µm) suitable for deep lung pulmonary delivery. Then, we will evaluate the in vitro anti- mycobacterial efficacy using standard broth microdilution method (M. abscessus, 30±2°C; CAMHB) to determine MIC, providing quantitative insights into the therapeutic potential of the proposed antibiotics/CD-MOF cocrystals. In Aim 2, we will incorporate mycobacterial intracellular infection by introducing Mycobacterium avium (MAC, ATCC 700898) in monocyte-derived macrophages (MDMs), co-cultured with a rationally designed 3D alveolar ALI microtissue (MOI=10), to model the NTM infection in lung. The infected microtissue will be treated in apical surface with (1) air (negative control), (2) standalone antibiotics (positive control), and (3) antibiotic/CD-MOF cocrystals (treatment). Multiple endpoints will be evaluated including anti-mycobacterial killing efficacy (CFU enumeration and log reduction), toxicity (cell viability, pro-inflammatory cytokine expressions, ALI barrier integrity, gene expression of type I/II pneumocytes), cellular internalization, and antibiotic release and absorption profiles. In future SBIR p...