Rifampicin-Liposomes for Mycobacterium abscessus Infection Treatment: Intracellular Uptake and Antibacterial Activity Evaluation

Abstract

Treatment of pulmonary infections caused by Mycobacterium abscessus are extremely difficult to treat, as this species is naturally resistant to many common antibiotics. Liposomes are vesicular nanocarriers suitable for hydrophilic and lipophilic drug loading, able to deliver drugs to the target site, and successfully used in different pharmaceutical applications. Moreover, liposomes are biocompatible, biodegradable and nontoxic vesicles and nebulized liposomes are efficient in targeting antibacterial agents to macrophages. The present aim was to formulate rifampicin-loaded liposomes (RIF-Lipo) for lung delivery, in order to increase the local concentration of the antibiotic. Unilamellar liposomal vesicles composed of anionic DPPG mixed with HSPC for rifampicin delivery were designed, prepared, and characterized. Samples were prepared by using the thin-film hydration method. RIF-Lipo and unloaded liposomes were characterized in terms of size, ζ-potential, bilayer features, stability and in different biological media. Rifampicin's entrapment efficiency and release were also evaluated. Finally, biological activity of RIF-loaded liposomes in Mycobacterium abscessus-infected macrophages was investigated. The results show that RIF-lipo induce a significantly better reduction of intracellular Mycobacterium abscessus viability than the treatment with free drug. Liposome formulation of rifampicin may represent a valuable strategy to enhance the biological activity of the drug against intracellular mycobacteria.

Keywords: Mycobacterium abscessus; antibiotic resistance; liposomes; rifampicin.

Figure 1

Results of investigation on physicochemical stability over time. Effect of storage temperature (room temperature (RT) and 4 °C) on hydrodynamic diameter and ζ-potential of (A) empty liposomes and (B) RIF-loaded liposomes. (C) Effect of THP culture medium at different incubation times on hydrodynamic diameter and ζ-potential of empty liposomes (Lipo) and RIF-loaded liposomes (RIF–Lipo). Pre-exp. values refer to liposomes before incubation with THP culture medium. (D) Stability studies over time at two different storage temperatures for free RIF and RIF loaded into liposomes.

Figure 2

TEM images obtained by PTA staining of RIF-loaded HSPC–DPPG liposomes dissolved in Hepes buffer (A) and in THP culture medium (B). Bar represents 100 nm.

Figure 3

AFM images (top panels) and corresponding height profiles evaluated on the marked section (bottom panels) of RIF-loaded liposomes in different media: (A,B) Hepes and (C,D) THP culture medium. Bars represent 200 nm.

Figure 4

Profile of release of rifampicin over 24 h. Data were obtained as the mean of three independent experiments.

Figure 5

Intracellular mycobacterial killing is improved by rifampicin-loaded liposomes with no cytotoxicity effect. dTHP-1 (5 × 10⁵ cells/mL) were infected with M. abscessus (MOI 10) for 3 h and the extracellular bacilli were killed with 250 µg/mL Amikacin for 1 h. (A) Cells were treated for 18 h with RIF-loaded liposomes (RIF–Lipo) and free drug (RIF) at different concentrations and bacterial growth was quantified by CFU assay. Results are shown as mean ± standard deviation (SD) of CFU values obtained from triplicate cultures and are representative of three independent experiments. (B) Cells were treated with unloaded liposomes (Lipo Empty), 96 µM rifampicin-loaded liposomes (RIF–Lipo) and 96 µM free drug (RIF) for 18 h, and bacterial growth was assessed by CFU assay. Results are shown as mean ± SD of CFU values obtained from triplicate cultures and are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.0001 by two sided Student’s t-test. (C) Liposome formulations and rifampicin do not exert cytotoxicity on macrophages. dTHP-1 (2 × 10⁵ cells/200 µL) were stimulated with unloaded liposomes (Lipo Empty), 96 µM rifampicin-loaded liposome (RIF–Lipo) and 96 µM free rifampicin (RIF) for 18 h and then were subjected to MTT Assay. Cells were treated with 0.1% saponin at 37 °C for 30 min as negative control (Sap). Results are shown as mean ± standard deviation (SD) of OD_540nm values performed in triplicate and are representative of three independent experiments.

Figure 6

Liposome internalization analysis within macrophages. dTHP-1 (5 × 10⁵ cells/mL) were stimulated for 18 h with empty liposomes (Lipo Empty) or with RIF-loaded liposomes (RIF–Lipo), containing or not calcein (Cal). Cells were collected and liposome uptake was analyzed by flow cytometry. Results are shown as mean ± SD of mean fluorescence intensity (MFI) values obtained from three independent experiments (A) of which representative flow cytometry histograms are reported (B).