LDE-MTX was prepared from a lipid mixture composed of 100 mg cholesteryl oleate, 200 mg egg phosphatidylcholine (Lipoid, Germany), 10 mg triglycerides, 12 mg cholesterol and 60 mg of MTX (5 (link),19 (link)). The aqueous phase (100 mg of polysorbate 80 and 10 mL of Tris-HCl buffer, pH 8.05) was kept at room temperature. The pre-emulsion was obtained adding the hydrophilic phase to the oil phase by ultrasonic radiation until complete dissolution of the drug. Emulsification of the compounds was obtained by high-pressure homogenization using an Emulsiflex C5 homogenizer (Avestin, Canada). After homogenization at constant temperature, the nanoemulsion was centrifuged at 1800 g for 15 min at 4°C to separate the emulsified from unbound MTX. The nanoemulsion was sterilized by passage through 0.22 μm pore filter (Millipore, USA) and kept at 4°C until it was used. The association of MTX to LDE was measured by HPLC before the treatment (22 (link)).
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