PEC structure influence on protein corona formation

FBS with a concentration of 30% (v/v) was added to the 500 µl of prepared PECs containing 100 µg of nanoparticles, and the samples were incubated at 37 ^oC for 1 h. To remove the soft corona and evaluating the hard corona, the incubated PECs were separated by centrifuging at 13000 rpm and washed two times by adding 1 ml of phosphate buffer solution (PBS, pH 7.4) and centrifuging at 15000 rpm for 30 min³⁹. The nanoparticles were dispersed in PBS (pH 7.4) for further analysis.

Size distribution and zeta potential of the PECs were determined subsequent to protein corona formation in FBS before and after removing soft corona.

The hard corona was investigated using acrylamide SDS-page electrophoresis. Following formation of protein corona, washing and dispersing the PECs in PBS, the proteins interacted with nanoparticles was eluted using SDS-sample buffer and boiled for 5 min at 100 ^oC. The samples were then investigated by 1D gel 12% Acrylamide SDS- page electrophoresis⁴⁰.

LC-Ms/Ms was performed to determine protein corona construction, spectral count of peptides (SPC) and relative quantity of each protein emerged on PEC corona as described in Supporting Information (^S7)^41,42.

It was explained in Supporting Information ^S8.