M199 medium

N-butyl-cyanoacrylate monomer (10 mg/mL, BBraun, Germany). Dextran sulphate (70,000 Da, Sigma-Aldrich, Denmark). Albumin, bicinchoninic kit (QuantiPro BCA Assay Kit), Dimethyl sulfoxide, ethanol, hydrochloric acid, isopropanol, methanol, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and sodium hydroxide were from Sigma-Aldrich, MO, USA. Cellulose membrane (Spectra/por 2, 12 to 14 kDa, Spectrapore, USA). M199 medium, RPMI 1640 medium and Müller-Hinton broth were from Vitrocell, Brazil. Rapid panoptic dye (Laborclin, Brazil). Amphotericin B was donated by Cristália, Brazil. Polymyxin B sulphate (500,0000 UI) was donated by Química Haller, Brazil. Distilled water was used for chemical assays and ultrapure water (Milli Q, 18.2 MΩ.cm at 25°C and a TOC value below 5 ppb) for biological experiments and formulations.

Leishmania (Leishmania) amazonensis (IFLA/BR/67/PH8) was isolated from lesions on C57BL/6 mice and then propagated as promastigotes at 26 °C in M199 medium (Vitrocell) supplemented with 40 mM of HEPES, 2.5 µg/mL of hemin, 10 mM of adenine, 2 mM of L-glutamine, 2 µg/mL of D-biotin, 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 20% inactivated fetal bovine serum (FBS, from Vitrocell), at pH 7.2. Subcultures were prepared weekly at initial density of 5 × 10⁵ promastigotes/mL up to 6 passages. To generate axenic amastigotes, stationary-phase promastigote cultures were incubated at 2.5 × 10⁷/mL in M199 media containing 0.25% glucose, 0.5% trypticase, 40 mM sodium succinate (at pH 4.5), 20% FBS, and 5% penicillin/streptomycin at 32 °C for 7 days. Parasites were washed 3 times in phosphate-buffered saline (PBS) before use in experiments.