Surface functionalization of MPs using liver adhesive ligand
The protocol for covalent bonding of proteins on PFC- MPs was similar to that reported in previous studies.[17] The activation of proteins was achieved by mixing 10 μg.ml-1 of each respective ECM protein solution for 30 minutes at room temperature in a solution containing 2mMN -ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) (Sigma Aldrich, cat# E7750) in ultrapure water (18.2 MΩ) and 5mM N-hydroxysuccinimide esters (NHS) (Sigma Aldrich, cat#130672) in 0.1M 2-(N-morpholino) ethanesulfonic acid hemisodium salt (MES) buffer (Sigma Aldrich, cat#M3671. PFC- MPs were also dispersed in 0.1M MES and their pH was adjusted to four different values including 5.90, 6.20, 6.50, and 6.80 by addition of 1M NaOH. Afterward, activated proteins were added to the particles to react with the free amino groups available on MP surfaces. The mixture was kept under mild shaking for 5 h at room temperature, dialyzed for 48h using SnakeSkin™ Dialysis Tubing (3.5K MWCO, Fisher scientific, cat#PI68035) against distilled water included 0.05% triton 100X (Sigma Aldrich, cat#X100-100ML) and then freeze-dried. The amount of protein conjugated to MPs was measured by Bicinchoninic acid (BCA) assay (Thermofisher, cat# PI23235) using manufacturer’s directions, as well as via XPS surface analysis, as described above. For simplicity, we picked MPs decorated with laminin-111 as a model to study efficiency of protein grafting.