Gallium nanoparticles facilitate phagosome maturation and inhibit growth of virulent Mycobacterium tuberculosis in macrophages

Abstract

New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs.

Fig 1. SEM images of nanoparticles containing Ga(III) or rifampin.

Nanoparticle morphologies are different depending upon the drug encapsulated. GaD is a rough rectangular shape, while RifD is a long rod shape. GaM shows aggregation of particles. GaF, GaM, GaNP and RifNP are rectangular, with different sizes. GaTP: Gallium(III) meso tetraphenylporphyrine chloride; GaNP: nanoparticle encapsulating GaTP; GaF: folate-conjugated nanoparticles encapsulating GaTP; GaM: mannose-conjugated nanoparticles encapsulating GaTP; RifD: dendrimer nanoparticle encapsulating rifampin; and RifNP: P118 nanoparticle encapsulating rifampin.

Fig 2. Ga(III) uptake (A), retention (B), and cytotoxicity (C) following incubation of THP-1 macrophages with Ga(III) nanoparticles.

(A) Gallium associated with THP-1 cells levels was measured 2, 4, 8 and 24 h following treatment of THP-1 macrophages with Ga(III) nanoparticles. (B) Ga retained in macrophages over time was determined at day 1, 5, 10, and 15 following treatment of THP-1 macrophages with the nanoparticles. THP-1 cells were administered 300 μM nanoparticles containing Ga(III). (C) Cytotoxicity of Ga(III) nanoparticles on THP-1 macrophages as measured by reduction of resazurin. GaTP: gallium(III) meso tetraphenylporphyrine chloride GaF: folate-conjugated nanoparticles encapsulating GaTP. GaM: mannose-conjugated nanoparticles encapsulating GaTP.

Fig 3. Anti-tuberculous activities of nanoparticles encapsulating Ga(III) or rifampin in M.tb infected macrophages.

(A) Pretreatment: MDMs were infected with M.tb (H37Rv, MOI = 0.2, 24 h) at days 5, 10, and 15 following MDM treatment with nanoparticles (300 μM Ga) and then cultured for 2 days, following which the MDM were lysed and M.tb CFU determined. (B) Treatment post-infection: THP-1 macrophages were infected with M.tb (H37Ra, MOI = 1, 1 h) followed by 24 h treatment with specific nanoparticles. They were then incubated for an additional 3 or 6 days. Following that the MDM were lysed and viable intracellular M.tb determined by CFU. Data represent the mean ± SEM of triplicate samples (n = 3). Statistical differences were determined using Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with non-drug treated control.

Fig 4. Co-localization of M.tb (H37Rv) and nanoparticles within MDMs.

MDMs were treated with the fluorescein-conjugated nanoparticles (PLGA-GaNP and PLGA-RifNP, Red) for 24 h before infection with M.tb H37Rv expressing green fluorescent protein (GFP). The relative locations of the nanoparticles and M.tb were determined by confocal microscopy.

Fig 5. Confocal images of macrophages treated with GaNP.

a) Fluorescein-conjugated GaNP (Red), b) macrophages and GaNP, c) galectin 3 (A) or cathepsin D (B), d) Nuclei (Blue). Nanoparticle-treated MDMs were incubated with primary rabbit galectin 3 or cathepsin D. These proteins were visualized by incubation with Alexa Fluor^®488 goat-anti-rabbit antibody. The scale bar represents 25 μm.

Fig 6. Expression of cathepsin D and galectin 3 in MDMs.

MDMs treated with drugs were infected with H37Ra for 4 h (MOI = 1), following which the cells were lysed and levels of cathepsin D and galectin 3 were determined by immunoblot and quantitated by densitometry. Data represent the mean ± SEM of triplicate samples (n = 3). Statistical differences were determined using Student’s t test: *p < 0.05, **p<0.01 compared with non-drug treated (+) control.

Fig 7. Confocal microscopy assessment of the effect of Ga nanoparticles on phagolysosome fusion in THP-1 macrophages infected with M.tb H37Ra.

A) Co-localization of LysoTracker Red DND-99 and FITC-labeled H37Ra. B) Percentage of FITC-labeled bacteria co-localized with acidic compartments labeled with LysoTracker in control and Ga nanoparticle treated MDM. Data are the mean ± SEM determined by analyzing 40 ~ 70 cells. Statistically significant differences were determined using Student’s t test: **p<0.01.

Fig 8. Inhibition of phagosome maturation by M.tb and restoration by Ga(III) nanoparticles.

Early endosome containing Ga nanoparticles fuse with early mycobacterial phagosomes to form a phagosome vacuole containing M.tb and Ga nanoparticles. EEA1: early endosome antigen 1.