Synthesis of Lipid Nanoparticles by Thin-Film Hydration

Synthesis of lipid nanoparticles (LNPs) is done by a thin layer evaporation method [21 (link)]. In brief, DSPC, cholesterol, and polyethylenimine (PEI) (4:1:2) were dissolved in Chloroform and Methanol (1:1) and kept on magnetic stirrer for 4 hr at 25 °C and 200 rpm. Next, the solvent was evaporated using a rotary evaporator (DLAB RE-100 Pro) at 50 °C, 40 RPM, to obtain a thin layer. The obtained thin layer of lipids was dispersed in doubled distilled water by sonication (Sonics, Vibra cell VCX 500) at 50 amplitude (10 s on-off cycle) for 10 min and extruded using a 200 nm then 100 nm polycarbonate membrane at 50 °C to control the LNPs size.