In this work, a magnetic bio-metal-organic framework (MBMOF) nanocomposite with porous-layer open morphology is synthesized through a simple sonochemical approach and its effects on Leishmania major (MRHO/IR/75/ER) under both in vitro and in vivo conditions are investigated. The effects of sonication time, initial concentration of reagents and sonication power on size and morphology of MBMOF nanocomposites have been investigated and optimized. A comparison was then made between the structural information of the nanostructures and that of the bio-metal-organic framework crystals. Using the powder X-ray diffraction (PXRD), field emission scanning electron microscope (FE-SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), energy dispersive analysis of X-ray (EDAX), vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), and Brunauer-Emmet-Teller (BET) techniques, the prepared MBMOF nanocomposites were characterized. The mean numbers of promastigotes (cell/ml) in different MBMOF concentrations (3.12, 6.25, 12.5, 25, 50, 100, 200 and 400 µg mL) were determined by direct counting after 24, 48 and 72 h. Using MTT assays, the cytotoxic impacts of the MBMOF nanocomposites on promastigotes, intracellular amastigotes, and J774 macrophages were estimated. In order to investigate their therapeutic effects, the prepared MBMOF nanocomposites (25 and 12.5 µg mL) were used as ointment three times a week to treat Leishmania major in BALB/c mice. The lesion size and weight of mice were assessed before and during the treatment. The parasitic loads were measured in spleen and liver through the culture. After 72 h, the INF-γ and IL-4 cytokines levels in the supernatant of the spleen culture were measured. To the best of the authors' knowledge, this study is the first to attempt to synthesize the bio-MOFs through an in-situ sonosynthesis route under ultrasound irradiation and examine their cytotoxicity effects on Leishmania major under in vitro and in vivo conditions.

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