Repeated pulmonary dosing of β-glucan-chitosan-PLGA nanoparticles controls <i>Mycobacterium tuberculosis</i> in mice

To address limitations in tuberculosis (TB) therapy, we developed an inhalable, immunomodulating, biocompatible nanoparticle system (β-C-P) encapsulating rifampin that targets alveolar macrophage. The nanoparticle consists of a poly(lactic-co-glycolic acid) (PLGA) core, a chitosan coating, and a surface functionalized with 1,3-β-glucan for enhanced macrophage uptake and immunomodulation. We evaluated the safety, immunological effects, and efficacy of rifampin-loaded β-C-P nanoparticles delivered via oropharyngeal aspiration (OPA) in healthy mice and in a low-dose <i>Mycobacterium tuberculosis (Mtb</i>) BALB/c model treated weekly for 4 weeks, as well as in a low-dose <i>Mtb</i> C3HeB/FeJ model treated weekly for 8 weeks. In healthy mice, cell pellets isolated by bronchoalveolar lavage (BAL) showed higher pulmonary exposure (AUC) of rifampin with 20% β-C-P nanoparticles versus 5% β-C-P nanoparticles, while no rifampin was detected in the oral rifampin group. Flow cytometry revealed no significant changes in lung immune cell populations except for a transient neutrophil increase at day 21 in the 5% β-C-P group. In the <i>Mtb</i> BALB/c mouse model, weekly OPA administration of 5%, 10%, and 20% β-C-P nanoparticles significantly reduced lung CFU by 0.5-1.11 log₁₀, comparable to daily oral rifampin. In the <i>Mtb</i> C3HeB/FeJ (Kramnik) mouse model, weekly OPA administration of 10% and 20% β-C-P nanoparticles significantly reduced lung CFU, comparable to daily oral rifampin. Collectively, these findings demonstrate that weekly pulmonary nanoparticle delivery of rifampin-loaded β-C-P nanoparticles achieves sustained rifampin exposure and therapeutic efficacy comparable to daily dosing, without pulmonary toxicity or systemic immune activation. This supports the potential of long-acting inhalable formulations for simplified TB therapy.