Annatto (Bixa orellana L.) is extensively used as food pigment worldwide. Recently, several studies have found it to have healing and antioxidant properties, as well as effective action against leishmaniasis. Therefore, the purpose of this study was to incorporate the oil obtained from annatto seeds into a nanostructured lipid carrier (NLC) and evaluate its physicochemical properties and biological activity against Leishmania major. Nanoparticles were prepared by the fusion-emulsification and ultrasonication method, with the components Synperonic™ PE (PL) as the surfactant, cetyl palmitate (CP) or myristyl myristate (MM) as solid lipids, annatto oil (AO) (2% and 4%, w/w) as liquid lipid and active ingredient, and ultra-pure water. Physicochemical and biological characterizations were carried out to describe the NLCs, including particle size, polydispersity index (PDI), and zeta potential (ZP) by dynamic light scattering (DLS), encapsulation efficiency (EE%), thermal behavior, X-ray diffraction (XRD), transmission electron microscopy (TEM), Electron Paramagnetic Resonance (EPR), cytotoxicity on BALB/c 3T3 fibroblasts and immortalized human keratinocyte cells, and anti-leishmaniasis activity in vitro. Nanoparticles presented an average diameter of ~200 nm (confirmed by TEM results), a PDI of less than 0.30, ZP between −12.6 and −31.2 mV, and more than 50% of AO encapsulated in NLCs. Thermal analyses demonstrated that the systems were stable at high temperatures with a decrease in crystalline structure due to the presence of AOs (confirmed by XRD). In vitro, the anti-leishmania test displayed good activity in encapsulating AO against L. major. The results indicate that the oily fraction of Bixa orellana L. in NLC systems should be evaluated as a potential therapeutic agent against leishmaniasis.

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