title: Poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear Mycobacterium bovis BCG infection in macrophages and remain membrane-bound in phago-lysosomes.
abstract: 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 copolymers of lactic acid and glycolic acid (PLGA) and has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we describe fabrication of PLGA NPs containing either a high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was twofold: 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 that enclosed 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 that at least 90% 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 NPs are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9-12 days of treatment, as shown by estimates of the number of bacterial colonies in vitro.
authors.affiliation: Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: Raja
authors.initials: R
authors.lastname: Kalluru
authors.forename: Federico
authors.initials: F
authors.lastname: Fenaroli
authors.forename: David
authors.initials: D
authors.lastname: Westmoreland
authors.forename: Lilia
authors.initials: L
authors.lastname: Ulanova
authors.forename: Atoosa
authors.initials: A
authors.lastname: Maleki
authors.forename: Norbert
authors.initials: N
authors.lastname: Roos
authors.forename: Marie
authors.initials: M
authors.lastname: Paulsen Madsen
authors.forename: Gerbrand
authors.initials: G
authors.lastname: Koster
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Egge-Jacobsen
authors.forename: Steven
authors.initials: S
authors.lastname: Wilson
authors.forename: Hanna
authors.initials: H
authors.lastname: Roberg-Larsen
authors.forename: Gopal K
authors.initials: GK
authors.lastname: Khuller
authors.forename: Amandeep
authors.initials: A
authors.lastname: Singh
authors.forename: Bo
authors.initials: B
authors.lastname: Nyström
authors.forename: Gareth
authors.initials: G
authors.lastname: Griffiths
mesh_terms: D000818:Animals; D000904:Antibiotics, Antitubercular / Q000008:administration & dosage*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015169:Colony Count, Microbial; D004337:Drug Carriers / Q000737:chemistry*; D005260:Female; D019344:Lactic Acid; D008264:Macrophages / Q000382:microbiology*; D008297:Male; D051379:Mice; D009163:Mycobacterium bovis / Q000187:drug effects*; D053758:Nanoparticles / Q000737:chemistry*; D010588:Phagosomes; D011100:Polyglycolic Acid; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D012293:Rifampin / Q000008:administration & dosage*
chemical_list: D000904:Antibiotics, Antitubercular; D004337:Drug Carriers; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D012293:Rifampin
keywords: Macrophages; Mycobacteria; Nanoparticles