Targeted intracellular delivery of antituberculosis drugs to Mycobacterium tuberculosis-infected macrophages via functionalized mesoporous silica nanoparticles

Abstract

Delivery of antituberculosis drugs by nanoparticles offers potential advantages over free drug, including the potential to target specifically the tissues and cells that are infected by Mycobacterium tuberculosis, thereby simultaneously increasing therapeutic efficacy and decreasing systemic toxicity, and the capacity for prolonged release of drug, thereby allowing less-frequent dosing. We have employed mesoporous silica nanoparticle (MSNP) drug delivery systems either equipped with a polyethyleneimine (PEI) coating to release rifampin or equipped with cyclodextrin-based pH-operated valves that open only at acidic pH to release isoniazid (INH) into M. tuberculosis-infected macrophages. The MSNP are internalized efficiently by human macrophages, traffic to acidified endosomes, and release high concentrations of antituberculosis drugs intracellularly. PEI-coated MSNP show much greater loading of rifampin than uncoated MSNP and much greater efficacy against M. tuberculosis-infected macrophages. MSNP were devoid of cytotoxicity at the particle doses employed for drug delivery. Similarly, we have demonstrated that the isoniazid delivered by MSNP equipped with pH-operated nanovalves kill M. tuberculosis within macrophages significantly more effectively than an equivalent amount of free drug. These data demonstrate that MSNP provide a versatile platform that can be functionalized to optimize the loading and intracellular release of specific drugs for the treatment of tuberculosis.

Fig 1

Upper panel: TEM images of MSNP (A), PEI-coated MSNP (B), and MSNP equipped with pH-operated nanovalves (C). The inserts show higher magnification of the particles delineated by red boxes, revealing their pore structure. Lower panel: the surface functionality of each MSNP.

Fig 2

Antituberculosis drug-loaded MSNP are internalized efficiently by human macrophages infected with M. tuberculosis (A to D) and by uninfected macrophages (E). PMA-differentiated THP-1 cells were infected with M. tuberculosis for 90 min and then incubated with 125 μg/ml NP-RIF. The infected monolayer was fixed 3 days later and analyzed by confocal microscopy. Abundant red fluorescent NP-RIF (A) is seen in the immediate vicinity of the green fluorescent protein-expressing M. tuberculosis (arrow) (B) in the infected macrophage, whose nucleus is stained blue (C). The merged image is shown in panel D. We have obtained similar results with human MDM. (E) The macrophage-like THP-1 cells were left untreated (dashed line) or were incubated with 20 μg/ml of fluorescent NP-RIF (RIF-loaded NP) for 6 h (solid line) and then fixed and evaluated by fluorescence-activated cell sorting (FACS). The majority of the macrophages that were incubated with RITC-labeled NP-RIF internalized the fluorescent nanoparticles and exhibited a 10-fold-greater mean fluorescence intensity than the untreated control macrophages.

Fig 3

MSNP traffic to lysosomal compartments in human macrophages. THP-1 cells were incubated for 3 h with 15 μg/ml of green fluorescent nanoparticles (uncoated FITC-MSNP [top row, A to D] or PEI-coated FITC-MSNP [bottom row, E to H]). Macrophage plasma membranes were stained with Alexa Fluor 633-WGA (shown as pseudocolor blue in merged images, D and H). After fixation and permeabilization, the late endosomal-lysosomal marker, CD63, was stained with a red fluorescent antibody (B and F), and the macrophage nuclei were stained blue with DAPI (shown in merged images in C and G). Both the uncoated and PEI-coated MSNP (several of which are indicated by arrows) colocalized extensively with CD63 (96% and 89% colocalization, respectively). Several uncoated MSNP located outside the macrophages, indicated by arrowheads (A to D), did not colocalize with CD63. Approximately 11% of intracellular PEI-coated MSNP did not colocalize with CD63 (three noncolocalizing MSNP are indicated by arrowheads; E to H). We have observed a similar high level of colocalization of MSNP with lysosomal compartments in human MDM.

Fig 4

Time course release of RIF from uncoated MSNP (A) or PEI-coated (B) MSNP.

Fig 5

PEI coating on MSNP enhances the delivery of RIF to M. tuberculosis-infected human macrophages. PMA-differentiated THP-1 cells were infected with M. tuberculosis for 90 min and subsequently treated with free RIF (A), NP-RIF (B), or PEI-NP-RIF (C). CFU of M. tuberculosis from the infected macrophage monolayer were enumerated 3 days postinfection. Log differences in CFU/monolayer between the no-treatment condition and selected treatments are indicated within brackets. All differences indicated by brackets were statistically significant at P < 0.01 (analysis by one-factor ANOVA).

Fig 6

MSNP equipped with pH-sensitive valves as a nanodelivery platform for antituberculosis drugs. (A) Illustration of the principle by which the pH-sensitive valve is operated. (B) Release profiles of Hoechst 33342 dye. The pH-dependent release mechanism can be monitored using this model cargo. Acid was added at 30 min to adjust the solution pH. (C) Time course release of the antituberculosis drug INH from the particles after change from neutral pH to pH 1.8. Acid was added at 15 min to adjust the solution pH.

Fig 7

pH-gated NP loaded with INH kill M. tuberculosis in human macrophages. PMA-differentiated THP-1 cells were incubated with M. tuberculosis for 90 min and then treated with free INH (A and B) or pH-gated NP-INH at concentrations of 31 to 250 μg/ml (C). CFU of M. tuberculosis in treated or untreated infected macrophages were enumerated 3 days postinfection. Neutral and acid eluates from 250 μg/ml pH-gated NP-INH served as controls for the amount of INH leakage at neutral pH and INH loading on the particles, respectively. Acid treatment of INH had no effect on its antimycobacterial activity (A). Log differences in CFU/monolayer between the no-treatment condition and selected treatment conditions are indicated within brackets (C). The differences between results for the pH-gated NP-INH-treated monolayer and no treatment and between pH-gated NP-INH and neutral eluate were significant at the P < 0.001 level (one-factor ANOVA). The difference between the 250 μg/ml pH-gated NP-INH-treated monolayer and the acid eluate treated monolayer was significant at the P < 0.01 level (one-factor ANOVA).