Liposomal resiquimod for the treatment of Leishmania donovani infection

Abstract

Objectives: The imidazoquinoline family of drugs are Toll-like receptor 7/8 agonists that have previously been used in the treatment of cutaneous leishmaniasis. Because of the hydrophobic nature of imidazoquinolines, they are traditionally not administered systemically for the treatment of visceral leishmaniasis. We formulated liposomal resiquimod, an imidazoquinoline, for the systemic treatment of visceral leishmaniasis.

Methods: By using lipid film hydration with extrusion, we encapsulated resiquimod in liposomes. These liposomes were then injected intravenously to treat BALB/c mice infected with Leishmania donovani.

Results: Treatment with liposomal resiquimod significantly decreased the parasite load in the liver, spleen and bone marrow. In addition, resiquimod treatment increased interferon-γ and interleukin-10 production in an antigen recall assay. Resiquimod was shown to be non-toxic in histology and in vitro culture experiments.

Conclusions: FDA-approved resiquimod, in a liposomal formulation, displays promising results in treating visceral leishmaniasis.

Keywords: drug delivery; imidazoquinoline; therapy; visceral.

Figure 1.

Chemical structure of resiquimod.

Figure 2.

LDUs in the liver (a), spleen (b) and bone marrow (c) of BALB/c mice infected with L. donovani. Mice were treated 2 weeks post-infection with tail vein injections of empty liposomes, resiquimod (7.6 µg)-loaded liposomes or 500 mg/kg SSG. LDUs were determined 2 weeks post-injection using the number of amastigotes/500 nuclei. Data are presented as mean with 95% CI. n = 5 for empty or resiquimod-loaded liposomes and n = 4 for SSG.

Figure 3.

Proliferation of T cells isolated from the spleens of BALB/c mice infected with L. donovani and exposed to antigen. Mice were treated 2 weeks post-infection with empty liposomes, resiquimod (7.6 µg)-loaded liposomes or 500 mg/kg sodium SSG. Spleens were removed 2 weeks post-injection and pulsed with L. donovani antigen (20 µg/mL), and T cell proliferation was measured after 72 h. Data are presented as mean with 95% CI. n = 5 for empty or resiquimod-loaded liposomes and n = 4 for SSG.

Figure 4.

Production of IFN-γ (a) or IL-10 (b) from splenic cells isolated from BALB/c mice infected with L. donovani. Mice were treated 2 weeks post-infection with empty liposomes, resiquimod (7.6 µg)-loaded liposomes or 500 mg/kg SSG. Spleens were removed 2 weeks post-injection and pulsed with L. donovani antigen (20 μg/mL), and cytokine production was measured after 72 h. Data are presented as mean with 95% CI. n = 5 for empty or resiquimod-loaded liposomes and n = 4 for SSG.

Figure 5.

Macrophage viability after being cultured with free resiquimod, resiquimod-loaded liposomes or empty liposomes. Cells were seeded at 5 × 10⁴ cells/well and treated for 24 h before viability was measured with respect to the untreated cells. Data are presented as mean ± SD. Statistical significance with respect to free resiquimod is presented as *P < 0.05 and significance with respect to empty liposomes is presented as †P < 0.05.

Figure 6.

Quantification of the liver enzymes ALT and AST in the serum of mice infected with L. donovani. Mice were treated 2 weeks post-infection with empty liposomes, resiquimod (7.6 µg)-loaded liposomes or 500 mg/kg SSG. Blood was collected 2 weeks post-injection, and serum levels are presented as mean with 95% CI. n = 5.