Assessing the effect of protein corona formation in the process of EV surface engineering

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as Extracellular Vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, up to date, works dealing with the functionalization of EV surface with proteins have never considered the biomolecular corona (BC) “variable”, namely the fact that extrinsic biomolecules, mainly proteins, may spontaneously adsorb on the EV surface in biofluids, contributing to determine the biological identity of the EV. In this work, we explore and compare EVs modified with the antibody Cetuximab (CTX) by chemisorption (covalent binding of CTX via biorthogonal click-chemistry) and by physisorption (formation of a CTX corona). Results (surprisingly) indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significative differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells in Fetal Bovine Serum (FBS)-supplied culture medium, very likely due to the higher robustness of the CTX anchorage that resists to the formation of a BC due to interaction with the FBS proteins.