Polylactide-co-glycolide-rifampicin-nanoparticles efficiently clearMycobacterium bovisBCG infection in macrophages and remain membrane-bound in phago-lysosomes

• DOI: 10.1242/jcs.121814
• Publication Date: 2013-05-18T05:32:11Z
• Authors: Raja Kalluru; Federico Fenaroli; David Westmoreland; Lilia Ulanova; Atoosa Maleki; Norbert Roos; Marie Paulsen Madsen; Gerbrand Koster; Wolfgang Eggerli ...; Steven Wilson; Hanna Roberg-Larsen; G. K. Khuller; Amandeep Singh; Bo Nyström; Gareth Griffiths
• Keywords: Male; Drug Carriers; 0303 health sciences; Macrophages; Cell Membrane; Colony Count, Microbial; Mycobacterium bovis; Cell Line; 3. Good health; Mice; 03 medical and health sciences; Polylactic Acid-Polyglycolic Acid Copolymer; Phagosomes; Animals; Nanoparticles; Female; Lactic Acid; Rifampin; Antibiotics, Antitubercular; Polyglycolic Acid

Nanoparticles (NPs) are increasingly used as biodegradable vehicles to selectively deliver therapeutic agents such as drugs or antigens to cells. The most widely used vehicle for this purpose is based on co-polymers of lactic acid and glycolic acid (PLGA) - that has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we fabricated PLGA NPs containing either high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was two-fold: first to resolve the controversial issue of whether, after phagocytic uptake, PLGA NPs remain membrane-bound or whether they escape into the cytoplasm, as has been widely claimed. Second, we sought to make NPs enclosing sufficient rifampicin to efficiently clear macrophages of infection with Mycobacterium bovis-BCG. Using fluorescence microscopy and immuno-electron microscopy, in combination with markers for lysosomes, we show that BCG bacteria, as expected, localized to early phagosomes, but at least 90 percent of PLGA particles were targeted to, and remained in low pH, hydrolase-rich phago-lysosomes. Our data collectively argue that PLGA NPs remain membrane-enclosed in macrophages for at least 13 days and degrade slowly. Importantly, provided that the NP's are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9–12 days treatment, as shown by estimates of the number of bacterial colonies in vitro.