Functionalized Immunomodulatory Biopolymeric Nanosystem For The Targated Deliveryof Anti-Tuberculosis Bioactives

Abstract

Tuberculosis (TB) continues to be the leading cause of death from a single infectious disease globally, with over 6.2 million new cases and nearly 2 million deaths annually. Pulmonary TB, which affects the lungs, accounts for approximately 83% of cases and is particularly concerning due to its high contagiousness. Despite the availability of treatment options, including first-line drugs requiring a six-month regimen and second and third-line drugs for drug-resistant TB, current therapies face significant limitations. These include prolonged treatment duration, high pill burden, poor patient compliance, and the emergence of multidrug- resistant (MDR) TB strains. The cost of treating MDR-TB is prohibitively high, and the side effects are more severe, especially in patients with co-infections, exacerbating drug-drug interactions. TB's ability to hijack the host immune system during infection necessitates novel therapeutic strategies to target TB bacteria and modulate the host immune response. This study aimed to develop a biocompatible, multifunctional drug delivery system using 1,3-β- glucan-functionalized chitosan-polycaprolactone nanoparticles (1,3-β-Glu-CS-PCL- VITD3/RIF) for co-delivering Rifampicin (RIF) and Vitamin D3 (VITD3) to alveolar macrophages, where TB bacteria reside. The nanoparticles exhibited spherical shapes with average particle sizes of 182.90 ± 1.15 nm (Glu-CS-PCL-VITD3) and 122.63 ± 5.11 nm (Glu- CS-PCL-RIF) and zeta potentials of +24.8 ± 0.73 mV and +19.5 ± 0.21 mV, respectively. They demonstrated sufficient entrapment efficiency (54% for VITD3 and 62% for RIF) and pH- responsive, sustained drug release over 48 hours (86% for VITD3 and 96% for RIF at pH 6.4). Cytocompatibility studies with RAW 264.7 cells confirmed 100% cell viability. ROS generation was significantly 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 significantly stimulated TNF-α secretion (1.8-fold increase), while CS-PCL nanoparticles showed increased IL-4 levels (48.58 ± 10.16 pg/mL), indicating a balanced M1/M2 response. Minimum Inhibitory Concentration (MIC) studies revealed that Glu-CS-PCL-RIF exhibited superior bactericidal activity against drug-susceptible laboratory (H37Rv) strain and clinically resistant isolates (MDR11) of M. tuberculosis (Mtb), with a remarkably low MIC of 0.00149 μg/mL on day 7, two times lower than standard RIF. The combination of Glu-CS-PCL-RIF and Glu-CS- PCL-VITD3 demonstrated significant potency against MDR11, with the nanoparticles containing 0.745 μg/mL of RIF, four times lower than standard treatment. Intracellular killing of H37Rv TB in THP-1 macrophages demonstrated that CS-PCL-RIF achieved higher bactericidal activity with significantly lower Mtb CFU counts (4.05E+02 and 3.27E+03 CFU/mL) despite containing approximately 15 times less RIF than the standard 50 μg/mL RIF treatment. CS-PCL-VITD3 exhibited a highly significant reduction in CFUs compared to the untreated group and CS-PCL, indicating that VITD3 potentiates bactericidal mechanisms within macrophages. The Glu-CS-PCL-VITD3 exhibited higher bactericidal activity than non- functionalized nanoparticles, demonstrated by Mtb CFU counts of 1.08E+04 compared to 7.85E+04 for CS-PCL-VITD3. This enhanced activity is attributed to the successful functionalisation with 1,3-β-glucan. In conclusion, this study developed multifunctional 1,3-β- glucan-functionalized nanoparticles with immunomodulatory capabilities (host-directed therapy) and potent antibacterial activity. This nanosystem offers a promising approach for targeted anti-TB treatment, with significant potential for further development and clinical application.