Squalene

Squalene (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) exists as an oily liquid at room temperature [17 (link)]; therefore, the concentration of Squalene is expressed by the volume fraction. Dimethyl sulfoxide (DMSO) (Sangon Biotech Co., Ltd., Shanghai, China) was used as the solvent. The Squalene samples were dissolved in 6% DMSO and 90% E3 to form a 4% stock solution. The stock solution was diluted to different concentrations with E3 before use, such that the concentrations of the test sample were 0, 0.05%, 0.2%, 0.5%, 0.7%, and 1%. H₂DCFDA (Merck Co., Ltd., Shanghai, China) was prepared as a stock of 20 µg/mL in DMSO and frozen at −20 °C. Its working solution was prepared on the day of the experiment.

DHA-loaded NLCs were fabricated through ultrasonication using 800 mg of monostearin (Yuanye Biotechnology Co., Shanghai, China), 200 mg of squalene (Yuanye Biotechnology Co., Shanghai, China), 500 mg of DHA (Maclean Biochemical Technology Co., Shanghai, China), 270 mg of Tween 80 (Sinopharm Group Chemical Reagent Co., Ningbo, China), 30 mg of glycerol (Xintong Fine Chemical Co., Tianjin, China), and 7200 mg of ultrapure water. Briefly, ultra-pure water was added to an oil phase composed of two lipids (monostearic acid and squalene), DHA, and two surfactants (Tween 80 and glycerol). Then, the reagents were heated together at 70 °C to promote binding. Subsequently, the liquid was agitated for 30 min at 500 rpm (IKA Instruments & Equipment Co., Guangzhou, China) to create a milky pre-emulsion. The DHA-loaded NLCs (DHA-NLC) were then obtained by using a sonicator (Scientz Biotechnology Co., Ningbo, China) at 60% amplitude for 5 min.

Squalene (B50732), lanosterol (B50780), cholesterol (B20272), and stigmasterol (B20314) were purchased from Yuanye Biotechnology; cycloartenol (C923402) was purchased from Macklin; and mevinolin (PHR1285), fosmidomycin (F8307), terbinafine (T8826), and ketoconazole (PHR1385) were purchased from Merck.