Enhancing TB Therapy with Precision: Immunomodulatory Nanoparticles Co-Delivering Rifampicin and Vitamin D3 for Macrophage-Targeted Treatment

Abstract Tuberculosis (TB) remains a leading cause of death worldwide, with over 6.2 million cases and nearly 2 million deaths annually. Despite available chemotherapies, significant challenges persist, including prolonged treatment, poor compliance, and the rise of multidrug-resistant (MDR) strains. This study developed a biocompatible, multifunctional drug-delivery system using 1,3-β-glucan-functionalized chitosan-polycaprolactone nanoparticles (1,3-β-Glu-CS-PCL) for co-delivery of Rifampicin (RIF) and Vitamin D3 (VITD3) to alveolar macrophages. Reactive oxygen species (ROS) generation was enhanced by Glu-CS-PCL-RIF (1.5- to 1.8-fold) and Glu-CS-PCL-VITD3 (1.7-fold higher than non-functionalized nanoparticles). Glu-CS-PCL-VITD3 also stimulated TNF-α secretion (1.8-fold increase), indicating a shift in the immune response towards macrophage activation. Minimum inhibitory concentration (MIC) studies showed superior bactericidal activity of Glu-CS-PCL-RIF against a drug-susceptible laboratory strain and clinically resistant isolates of Mycobacterium tuberculosis (Mtb), with a MIC of 0.00149 μg/mL, 2-fold lower than that of standard RIF. The combination of RIF and VITD3 demonstrated significant potency against drug-resistant TB with an effective dose 4x lower than standard treatment. Intracellular killing studies in THP-1 macrophages revealed that CS-PCL-RIF significantly reduced Mtb CFU counts (while containing approximately 15 times less RIF than the standard 50 μg/mL treatment). Glu-CS-PCL-VITD3 showed a notable reduction in CFUs compared to non-functionalized counterparts, illustrating the impact of 1,3-β-glucan functionalization. In conclusion, these multifunctional nanoparticles demonstrate strong potential for targeted anti-TB therapy by combining immunomodulation with enhanced antibacterial efficacy for future clinical applications.