Functionalization of CoCr alloy with Electrochemical Reduced Graphene Oxide. Proteomic analysis of the Macrophages-ErGOCoCr surface interaction

Electrochemically reduced graphene oxide (ErGO) films on a biomedical grade CoCr alloy have been generated and the macrophage cellular response was here analyzed through a proteomic approach for its possible biomedical application. CoCr discs of 38 mm diameter and 2 mm thickness were polished on successive abrasive papers in silicon carbide grade from 600 to 2000 grain size. Immediately after, polished CoCr disks were immersed in an aqueous suspension of graphene oxide (GO) at 4g/L in order to perform direct electrochemical reduction of graphene oxide on the CoCr surface. An Autolab potentiostat/galvanostat was used for the electrochemical reduction of GO, which was performed in a three-electrode electrochemical cell consisting of a graphite bar used as counter electrode, Ag/AgCl as reference electrode and CoCr discs as working electrode. The electrochemical reduction method used was cyclic voltammetry (CV), carried out from -2.1V to -0.5V (vs. Ag/AgCl) at a scan rate of 10 mV/min. Study of cell-material interactions was focused on the innate immune system response by analyzing the macrophage proteome, since it may reveal new insights in molecular mechanisms and signaling pathways being trigger upon biomaterial interactions. Mouse macrophages (J774A.1) were seeded on tissue culture dishes on CoCr (Control group), and on CoCrErGO surfaces (CoCrErGO group). After 72h and 96h of cell culture with the biomaterial, cells were lysed in RIPA buffer containing protease inhibitors. Samples of same protein concentration were taken for SDS/PAGE, excised and trypsin digested, and peptides were analyzed in Q-Exactive. Mass spectra were searched against the Mus musculus database using Mascot search engine through Proteome Discoverer. Peptide identification was performed at a FDR of 1% Annotated MS data were filtered with Matlab 2020b based on maintained patterns of expression observed both at 72h and 96h. Protein abundance was normalized following data flow in (1). Differentially