rifampin and coumarin-6

Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.

Topics: Animals; Anti-Bacterial Agents; Biological Transport; Coumarins; Drug Carriers; Embryo, Nonmammalian; Granuloma; Lactic Acid; Macrophages; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Nanoparticles; Optical Imaging; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rhodamines; Rifampin; Thiazoles; Tuberculosis; Zebrafish

Although treatment of pulmonary tuberculosis with respirable microspheres (MS) with an incorporated antituberculosis drug is expected to be highly effective, this treatment seems to achieve a much lesser effect than expected in the case of killing Mycobacterium tuberculosis residing in the lungs. To elucidate the reason for this weaker effect, we examined the distribution and accumulation of respirable MS consisting of poly(lactic-co-glycolic) acid (PLGA) in rat lungs. For this, we delivered the PLGA MS containing fluorescent coumarin 6 or an antituberculosis agent, rifampicin (RFP), by insufflation via the trachea and then determined the pulmonary distribution by counting the number of the MS in lung cryosections observed under a microscope. In addition, the uptake of MS by alveolar macrophage (AMφ) was determined by immunostaining for Mφ cell marker CD68 and RFP content in the cells. Approximately half of the fluorescent PLGA MS reached the alveoli without entrapment by trachea and primary bronchi and were then ingested by the AMφ cells up to 24h after insufflation. RFP in a form of PLGA MS was markedly transported into AMφ at an amount 10 times greater than that for the free RFP powder. However, a large proportion of RFP was eliminated from the lungs by 6h after insufflation.

Topics: Administration, Inhalation; Animals; Antibiotics, Antitubercular; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Coumarins; Fluorescence; Immunoenzyme Techniques; Lactic Acid; Lung; Macrophages, Alveolar; Male; Microspheres; Phagocytosis; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Wistar; Rifampin; Thiazoles; Tissue Distribution; Trachea