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.