Phosphatidylcholine

Docetaxel (DTX, Mw = 807.9 g/mol, 99.4% purity) was obtained from Xingcheng Chempharm (Zhejiang, China) and bicalutamide (BIC, Mw = 430.3 g/mol, 99.7% purity) was obtained from Taizhou Creating Chemical (Taizhou, China). Bovine bile extract, Tween 80, dimethyl sulfoxide and Tris maleate salt were purchased from Sigma-Aldrich (St. Louis, MO, USA), phosphatidylcholine (99%) was purchased from Lipoid (Ludwigshafen, Germany), and sodium chloride (99.5%) was purchased from Fluka Chemie AG (Buchs, Switzerland). Phosphate buffered saline (PBS) and fasted state simulated intestinal fluid (FaSSIF) were purchased from Corning (Midland, MI, USA) and Biorelevant (London, UK), respectively. Ultrapure water (SG Water Purification System, Barsbuttel, Germany) was used for all experiments. All other chemicals and solvents were of analytical grade and were used without further purification.

Four different virosomal preparations have been specifically designed for this study, three of them containing each of the individual antigens, and one containing all the three antigens together. Briefly, a solution containing 1 mg of inactivated Influenza virus A/H1N1/California was pelleted at 286 000g for 1 hour, dissolved in presence of 0.5 ml of PBS containing 0.1 M of Octaethyleneglycol mono (n-dodecyl) ether (OEG; Sigma Aldrich, MO, USA), and then mixed with 32 mg of phosphatidylcholine (Lipoid Ag, Steinhausen, Switzerland) dissolved in 1.5 ml of PBS-OEG 0.1 M. The mixture was centrifuged at 100 000g for 30 min and the supernatant containing Haemagglutinin and Neuraminidase was recovered. For the individual virosomal formulations, the obtained supernatant was then mixed with 2 mg of Leish-F3 (or Leish-F3+), or KMP11 or LJL143 in presence of detergent. Virosomes were then formed by detergent removal and sterile-filtered. The virosome particles containing the mixture of the three antigens were produced similarly, from 1 mg of starting influenza protein mixed with 1 mg of each antigen (Leish F3+, KMP11 and LJL143). Size determination and distribution of the particle population was performed using a Zetasizer Nano instrument (Malvern Instruments, Malvern, UK). Parasite derived and/or VD proteins content in virosome particle was determined by SDS-PAGE Coomassie Stained.

Virosomes containing each of the antigens individually were prepared, as previously described [26 (link)]. Briefly, a mixture of 1 mg of inactivated influenza virus A/H1N1/California and 32 mg of phosphatidylcholine (Lipoid Ag, Steinhausen, Switzerland) was centrifuged at 100,000× g for 30 min, and the supernatant, containing haemagglutinin and neuraminidase, was recovered. These influenza antigens were then used to produce VS that included the vaccine antigens (individually) by detergent removal. The VS were then sterile-filtered and their size distribution was determined using a Zetasizer Nano instrument (Malvern Instruments, Malvern, UK). The parasite-derived and/or virus-derived protein contents of the VS were checked by SDS-PAGE Coomassie staining.