Mycobacterial lipid-derived immunomodulatory drug- liposome conjugate eradicates endosome-localized mycobacteria

Abstract

Tuberculosis is a challenging disease due to the intracellular residence of its pathogen, Mycobacterium tuberculosis, and modulation of the host bactericidal responses. Lipids from Mycobacterium tuberculosis regulate macrophage immune responses dependent on the infection stage and intracellular location. We show that liposomes constituted with immunostimulatory lipids from mycobacteria modulate the cellular immune response and synergize with sustained drug delivery for effective pathogen eradication. We evaluate the pH-dependent release of Rifampicin from the mycobacterial-lipid-derived liposomes intracellularly and in vitro, their cell viability, long-term stability, and antimicrobial efficacy. Intracellular drug levels were higher following liposome treatment compared with the free drug in a temporal fashion underlying a sustained release. The drug-encapsulated liposomes were taken up by clathrin-mediated endocytosis and elicited a robust pro-inflammatory immune response while localizing in the recycling and late endosomes. Notably, these were the same cellular compartments that contained the pathogen underlying localized intracellular targeting. Our results also imply a lipid-centric and species-specific selectivity of the liposomal drug formulations. This work provides a proof-of-concept for the dual-action of liposomes derived from the pathogen itself for their effective eradication, in conjunction with the attuned host immunomodulation.

Keywords: Drug delivery; Immunomodulation; Infectious diseases; Intracellular antimicrobial activity; Liposomes; Mycobacterial lipids.

Fig. 1

(A) Representative SEM image of (left) Rif-loaded BL-d liposomes representing the uniformity in size, (middle) image of untreated THP-1 macrophages, and (left) represent the SEM image of THP-1 cells treated with BL-d liposomes for 2 h. Scale Bar = 200 nm. (B) In-vitro cytotoxicity of BL-d liposomes (–Rif, +Rif) on THP-1 cells at various indicated concentrations. Physicochemical characterization of bacterial lipid-derived Rif-loaded liposomes (C) Schematic representation of physicochemical characteristics (size, charge, lamellarity, fluidity, packing, and immunogenic) of an anionic liposome (D) (UPPER PANEL) Schematic representation of destabilization of the liposomal membrane before and after fusion with the endosomal membrane, showing a maximum release at endosomal pH. (LOWER PANEL) In vitro cumulative drug release of Rifampicin from BL-d liposomes at various pH. Effect of pH on the fluidity and anisotropy of drug-loaded and empty BL-d liposomes (E) Generalized polarization (F) Laurdan anisotropy (G) DPH anisotropy. BL-d liposome induces pro-inflammatory interleukins in THP-1 macrophages (H) Quantification of various interleukins levels per macrophage. ND: not detected. Concentrations were calculated by fitting the data with four parametric regression methods. Data represent mean ± SD of three independent experiments. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparison tests, N = 3.

Fig. 2. Cellular uptake and drug retention profile of BL-d liposomes in THP-1 macrophages.

(A) Representative confocal images of free N—Rh DHPE dye and BL-d liposomes loaded with N-Rh-DHPE (25 μg/mL) in a time-dependent manner. Scale bar: 10 μm, 63× oil objective. (B) Mean fluorescence intensity of free N—Rh DHPE BL-d liposomes loaded with N—Rh DHPE (25 μg/mL) using FACS. (C) Area abundance of Rifampicin extracted from THP-1 macrophages treated with free rifampicin (25 μg/mL), and BL-d Rifampicin liposomes (25 μg/mL). The area is calculated using the standard curve of Rifampicin, prepared in methanol with a serial dilution and fitted with a linear regression curve. Data represents the mean ± SD of three independent experiments (A-B), while in (C) data is from two independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison test.

Fig. 3. Concentration-dependent and time-dependent intracellular antimicrobial efficacy of free RIF and Rif-loaded BL-d liposomes in THP-1 macrophages.

(A) The concentration-dependent anti-mycobacterial activity of free drug and Rif-loaded BL-d liposomes (5, 25, and 50 μg/mL) was determined using CFU counting by serial dilution and plate counting method for 24 h. (B—D) Time-dependent representative confocal images of Msm-infected THP-1 macrophages treated with Rifampicin (25 μg/mL) and Rif-loaded BL-d liposomes (25 μg/mL). Data represent the means ± SD of three independent experiments. Statistical Significance was determined using one-way ANOVA with Tukey’s multiple comparison test. Scale bar: 10 μm, 63× oil objective.

Fig. 4. Uptake mechanism of N-Rh-DHPE-labelled BL-d liposomes (BL-d Rh Lipo).

(A-C) Representative confocal images of THP-1 cells showing inhibition of the uptake of BL-d Rh liposomes after treatment with indicated endocytosis inhibitors for 1 h. (D) Quantitative estimation of the inhibition of liposomal uptake after additional 2 h incubation with BL-d liposomes. (E-J) Subcellular localization of BL-d Rh liposomes. Representative confocal images of THP-1 macrophages treated with BL-d Rh Lipo (25 μg/mL) and later stained with various endosomal antibodies in a time-dependent manner © EEA-1; 30 min (F) EEA-1; 24 h (G) RAB-7; 30 min (H) RAB-7; 24 h (I) LAMP-1; 30 min and (J) LAMP-1; 24 h. The relative intensity of each condition with Pearson correlation coefficients given at the corner of the RI graph and zoomed inset of each image are highlighted with white borderline and the arrow. (K) Manders correlation M2 coefficients depicting the extent of colocalization between the BL-d Rh Lipo and probed the various endosomal markers at various time points. We have used the weighted colocalization coefficients for analysis. (L) Pearson correlation coefficients of each time point and condition treated with BL-d Rh liposomes (25 μg/mL). Confocal images represent BL-d Rh liposomes in red, endosomal markers in green, nuclei in blue, overlay of the compartment, and colocalized particles in yellow. Data represent the means ± SD of three independent experiments. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparison test. Scale bar: 10 μm, 63× oil objective. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Intracellular trafficking and subcellular distribution of M. smegmatis (Msm), BL-d liposomes, and endosomal markers. Representative confocal images of BL-d liposomes showing subcellular distribution in the cytoplasm and other intracellular or endosomal regions within the THP-1 macrophages such as (A) early endosome, EEA-1 (B) late endosomes, RAB-7 (C) lysosomes, LAMP-1. Liposomes are shown in red, endosomal compartments in magenta, bacteria (Msm) in green, and nuclei in blue, and overlay of the compartment and particle in whitish the compartment and particle in whitish-blue containing relative intensities with Pearson correlation coefficients at the corner side of the graph. (D) No. of colocalized bacteria with liposomes containing endosomes/macrophages. (E) The bar graph represents the colocalization profile through Manders correlation coefficients in between the M. smegmatis and BL-d liposomes vs endosomes, BL-d liposomes vs endosomes, and endosomes vs M. smegmatis. Data represent the means ± SD of three independent experiments. Two-way ANOVA (Sidak multiple comparison test) was performed, with a statistical analysis of p > 0.05, p > 0.005, and p > 0.0005. Scale bar: 10 μm, 63× oil objective. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)