Micro-carriers mediated bacteriophage delivery for targeting intracellular Mycobacterium tuberculosis infection and TB prevention

Abstract

Tuberculosis (TB) is a debilitating infectious disease that afflicts 10 million people every year. Treatment is particularly challenging due to prolonged treatment duration (4-6 months) consisting of an oral regimen of 4 antibiotics. Discontinuation of treatment due to patient non-compliance often results in a relapse of infection and increased antibiotic resistance. Multidrug resistant TB (MDR-TB) is an alarming global health issue, with a prevailing incidence of 450 000 cases and an estimated 200,000 deaths in 2021. There is a clinical need to find effective treatments against drug-resistant strains and develop a patient-compliant method of drug delivery. In this work, we have used polymeric microparticles to establish an inhalation-based platform for delivering TB drugs. We also focus on bacteriophages, which are bactericidal even against antibiotic-resistant strains. We work towards developing microparticle-based approaches to improve their access to intracellular niches and deposition in lungs.

Mycobacterium tuberculosis (Mtb) infects host macrophages and continues to survive and grow intracellularly. To target this intracellular reservoir in macrophages, first, we engineered polylactic-co-glycolic acid (PLGA) microparticles. Positively charged poly-l-lysine-conjugated micron-sized particles demonstrated remarkable internalization by Mtb-infected THP-1 macrophages and primary bone marrow-derived macrophages. Cationic microparticles also exhibited higher uptake in all immune cells and alveolar macrophages upon intra-tracheal delivery in vivo. We then proceeded to extend the application of this platform to deliver bacteriophages within infected macrophages.

Bacteriophages have limited penetration within mammalian cells and cannot efficiently interact with intracellular bacteria. Cationic microparticles served as excellent phage-carriers as they delivered two log-fold higher phages intracellularly compared to non-modified particles. Intracellular Mtb can reside within several intracellular compartments, such as endosomes, lysosomes, or cytosol. To improve the colocalization of microparticles with Mtb, we chemically conjugated Transferrin (Tf), which is known to be recruited by intracellular bacteria. Tf-coated cationic microparticles exhibited enhanced interaction with the mycobacterial phagosome.

Due to the high prevalence of the disease, especially in high-risk populations such as HIV patients, healthcare workers, and family members of TB patients, we envisioned an inhalable formulation that can be administered regularly to prevent the development of infection. We tested if bacteriophages can be used as prophylactic agents for TB and observed significant protective effect. We also synthesized 5-7 µm PLGA porous particles to improve phage delivery.

Bacteriophages are subjected to several biological barriers during in vivo administration which limits their application especially for lung-associated intracellular infections. Biomaterial-based approaches can be applied to improve phage pharmacokinetics and efficiency. Local delivery to infection sites is most preferred as phages require direct contact with bacteria for their action. In this work, we have developed PLGA microparticles to enable bacteriophage internalization within infected cells and bacteriophage deposition in lungs upon intra-tracheal delivery. This work opens doors for further development of inhalable carriers for bacteriophages.