To prepare the liposome, the detergent remove method was used. Cholesterol (Sigma Aldrich, USA), L-alpha phosphatidylcholine (PC) (Sigma Aldrich, USA), biotinyl phosphoethanolamine (biotin-PE) (Avanti Polar Lipids, USA), and Ni-NTA-DGS (Avanti Polar Lipids, USA) were first dissolved in 60 ˚C N-Octylglucoside (Sigma Aldrich, USA) solution to make lipid stock solutions. The liposome for the experiment was generated by mixing the lipids with a ratio of PC: Cholesterol: Ni-NTA-DGS: biotin-PE = 16:2:2:1, to achieve a total lipid concentration of 800 mM. After mixing, 540 ul Tris-buffered saline (0.01 M, pH 7.4, 150 mM NaCl) was added to the mix and followed by eliminating N-Octylglucoside with the Pierson detergent removal column (Thermo Fisher, USA). Then, the detergent-free lipid mix was vortex and extruded through 0.2 μm syringe filter at 60 ˚C for 15 times to construct the liposome with unified size.
To functionalize the liposomes, the His-tagged human recombinant E-selectin (10335-H03H, Sino Biological, USA) was used. 45 μl of 0.58 μM E-selectin solution was added to the liposome solution. To collect the E-selectin conjugated liposome, the solution was centrifuged at 100,000xg for 3 hours at 4 °C. After the centrifuge, the supernatant was removed, and the pellet was resuspended in 100 μl Tris-buffered saline. The liposome samples were stored at 4 °C and used within 24 hours.
To functionalize the liposomes, the His-tagged human recombinant E-selectin (10335-H03H, Sino Biological, USA) was used. 45 μl of 0.58 μM E-selectin solution was added to the liposome solution. To collect the E-selectin conjugated liposome, the solution was centrifuged at 100,000xg for 3 hours at 4 °C. After the centrifuge, the supernatant was removed, and the pellet was resuspended in 100 μl Tris-buffered saline. The liposome samples were stored at 4 °C and used within 24 hours.