Dual-Loaded Multilamellar Vesicles Preparation

A double-emulsion procedure (Li et al., 2019 (link)) was used to prepare MVLs containing ioversol and doxorubicin hydrochloride. Briefly, 1 mL of chloroform containing the lipids (41 mg HSPC:40.5 mg cholesterol:5 mg DPPG: 11.25 mg triolein) in 1 mL aqueous solution (the first aqueous solution) was emulsified by Scientz-IID Ultrasonic Homogenizer (Ningbo Scientz Biotechnology Co., Ltd. Ningbo, China) for 30 sec (30% power output, 25 °C) to produce a w/o emulsion. The first aqueous solution contains 1 mg of doxorubicin hydrochloride in 1 mL of 320 mg ioversol injection. This w/o emulsion (2 mL) was subsequently emulsified with 6 mL of the second aqueous solution containing 4% glucose (wt/vol) and 20 mM lysine at 2800 r/min by XHF-D mixer (Ningbo Scientz Biotechnology Co., Ltd. Ningbo, China) at 40 °C to prepare w/o/w emulsion. Then the w/o/w emulsion was diluted with 4 mL of second aqueous solution poured into 100-mL egg type flask. Chloroform was removed by flushing nitrogen over the surface of the double emulsion at 35–37 °C. The resultant MVLs were collected at 100g for 10 min, and resuspended in sterile saline solution after discarding the supernatant. BD Falcon™ cell strainers (BD Biosciences, USA) were used to isolate different size MVLs, improve the uniformity of MVLs. The ioversol and doxorubicin hydrochloride concentration in MVLs were determined by HPLC.