Ethanol-Injection Liposomal Formulations

Several liposomal formulations were fabricated using ethanol injection method as previously performed (Nikoofal-Sahlabadi et al. 2018 (link)). The lipid phase consisted of variable molar ratios of the cationic lipid DOTAP, and/or other neutral lipids including DOPE, HSPC, DSPE-mPEG2000, the MMP-2 cleavable mPEG2000-peptide-DOPE, cholesterol and the antioxidant α-tocopherol (Table 1). The total lipid concentration of each formulation was set to 50 mM. Briefly, lipids were dissolved in chloroform and mixed at different molar ratios in a round-bottom flask, as shown in Table 1. The organic solvent was then evaporated by a rotary evaporator (Heidolph, Germany), followed by a 2 h freeze-drying process using the VD-800F lyophilizer (Taitech, Koshigaya, Japan) to remove any residue of the organic solvent in the prepared thin layer lipid film. Next, the lipid film was dissolved in warm absolute ethyl alcohol (65 °C) and injected with pre-heated ammonium sulfate solution (250 mM, 65 °C) at 1:9 v/v ratio. The liposomal formulations were incubated at 65 °C for 1 h while shaking on a vortex shaker. This was followed by a 5 min sonication using a bath sonicator (Bandelin electronic GmbH & Co. KG, Berlin, Germany). The resultant large multilamellar vesicles (MLVs) were then extruded (11×) using the thermobarrel extruder (Avestin, Inc., Ottava, Canada) through 200 nm, 100 nm, and 50 nm polycarbonate filters (Whatman, Maidstone, UK). The final products were dialyzed (cellulose membrane, 12–14 kDa MWCO, MilliporeSigma, Burlington, MA, USA) against dextrose histidine buffer (10 mM, pH 6.5) to remove free ammonium sulfate. To Dox encapsulation, the amount of 1 mg Dox per 12 µmol total lipid from Dox solution was loaded into liposomes after incubation at 65 °C for 1 h, and cooled to room temperature. Free Dox were finally removed by dialysis (cellulose membrane, 12–14 kDa MWCO, MilliporeSigma, Burlington, MA, USA) against dextrose histidine buffer (10 mM, pH 6.5).
The ultimate prepared liposomal formulations were sterilized using 0.22 µm syringe filters and stored at 2–8 °C. Liposomes characterization is explained in detail in the SI.