Cationic liposomal sodium stibogluconate (SSG), a potent therapeutic tool for treatment of infection by SSG-sensitive and -resistant Leishmania donovani

Abstract

Pentavalent antimonials have been the first-line treatment for leishmaniasis for decades. However, the development of resistance to sodium stibogluconate (SSG) has limited its use, especially for treating visceral leishmaniasis (VL). The present work aims to optimize a cationic liposomal formulation of SSG for the treatment of both SSG-sensitive (AG83) and SSG-resistant (GE1F8R and CK1R) Leishmania donovani infections. Parasite killing was determined by the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic counting of Giemsa-stained macrophages. Macrophage uptake studies were carried out by confocal microscopic imaging. Parasite-liposome interactions were visualized through transmission electron microscopy. Toxicity tests were performed using assay kits. Organ parasite burdens were determined by microscopic counting and limiting dilution assays. Cytokines were measured by enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Although all cationic liposomes studied demonstrated leishmanicidal activity, phosphatidylcholine (PC)-dimethyldioctadecylammonium bromide (DDAB) vesicles were most effective, followed by PC-stearylamine (SA) liposomes. Since entrapment of SSG in PC-DDAB liposomes demonstrated enhanced ultrastructural alterations in promastigotes, PC-DDAB-SSG vesicles were further investigated in vitro and in vivo. PC-DDAB-SSG could effectively alleviate SSG-sensitive and SSG-resistant L. donovani infections in the liver, spleen, and bone marrow of BALB/c mice at a dose of SSG (3 mg/kg body weight) not reported previously. The parasiticidal activity of these vesicles was attributed to better interactions with the parasite membranes, resulting in direct killing, and generation of a strong host-protective environment, necessitating a very low dose of SSG for effective cures.

FIG 1

Leishmanicidal activity of graded concentrations of various cationic liposomes against promastigotes of the SSG-responsive strain AG83 (A) and the SSG-resistant strain GE1F8R (B), at 22°C. Values are means ± standard errors of three independent experiments performed in duplicate.

FIG 2

(A and B) Effects of graded concentrations of various cationic liposomes on L. donovani amastigote proliferation within murine peritoneal macrophages, using the AG83 (A) and GE1F8R (B) strains. Values are means ± standard errors of three independent experiments. (C) Uptake of PC-SA (left) and PC-DDAB (right) liposomes inside infected macrophages. Peritoneal macrophages isolated from BALB/c mice were infected with L. donovani promastigotes for 3 h and subsequently treated with fluorescent PC-SA or PC-DDAB liposomes. Macrophage nuclei are stained with DAPI (large blue areas). Intact liposomes are visible close to the amastigote nuclei, colocalizing green and red fluorescence and appearing yellow. Arrows, L. donovani amastigotes (small blue areas) inside macrophages. Magnification, ×40. Data are representative of two independent experiments.

FIG 3

Electron microscopic assessment of L. donovani promastigotes treated with free and SSG-encapsulated PC-DDAB. (A) Control promastigotes demonstrate an elongated body and a normal aspect of the intracellular organelles. (B) The 75% effective dose (ED₇₅) of PC-DDAB induced vacuolization and alterations at the plasma membrane. (C) Parasites treated with the ED₇₅ of PC-DDAB incorporating SSG were spherical, with a complete absence of electron-dense materials. Bars, 2 μm (magnification, ×30,000) (A and B) or 1 μm (magnification, ×60,000) (C). Nu, nucleus; Mt, mitochondria; K, kinetoplast; FP, flagellar pocket; DMt, dilated mitochondria; FN, fragmented nucleus; V, vacuoles; dM, dilated matrix of mitochondria; G, granules; MB, membrane blebbing.

FIG 4

In vivo antileishmanial activity of free and SSG-encapsulated PC-DDAB liposomes. (A, B, D, and E) Therapeutic effects of drugs were analyzed as reductions in weight and Leishman-Donovan units (LDU) in liver (A and D) and spleen (B and E) of AG83-infected (A and B) and GE1F8R-infected (D and E) mice. (C and F) Bone marrow parasite loads of AG83 (C) and GE1F8R (F) were expressed as amastigotes per 1,000 bone marrow nuclei. Values are means ± standard errors (n = 5) of two independent experiments. *, P < 0.05 versus control for organ weight and versus PC-DDAB for LDU, by ANOVA.

FIG 5

Comparative analysis of therapeutic efficacies of PC-SA-SSG versus PC-DDAB-SSG formulations against L. donovani AG83 infections. (A) In vitro leishmanicidal activity was documented as the number of amastigotes in 200 macrophages after 72 h of treatment. Data represent means of three independent experiments performed in triplicate. (B) Suppression of liver and spleen parasite burdens in 3-month-infected and treated BALB/c mice. Data represent means ± standard errors (n = 5) of total parasites per organ. ***, P < 0.001 by Student's t test (A) or P < 0.0001 by ANOVA (B).

FIG 6

ELISA (A and B) and fluorescence-activated cell sorting analysis (C to G) of splenocytes from normal BALB/c mice treated with PC-SA-SSG or PC-DDAB-SSG. (A and B) Splenocytes were isolated from mice treated with PC-SA-SSG or PC-DDAB-SSG liposomes and were cultured for determination of IFN-γ (A) and IL-10 (B) levels. (C to G) Splenocytes treated with PC-SA-SSG or PC-DDAB-SSG were gated for CD4⁺ (C and E), CD8⁺ (D and F), and CD19⁺ (G) cells and permeabilized, and intracellular IFN-γ (C and D) and IL-10 (E, F, and G) levels were determined. Data represent means ± standard errors (n = 4). ***, P < 0.0001; *, P < 0.05.