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

To be profitably exploited in medicine as drug delivery carriers, nanosized systems should be endowed with essential properties, including high biocompatibility, stealth ability to escape the immune system, prolonged circulation lifetime, and targeting abilities towards the pathological tissues. Currently, the choice of biogenic nanoparticles as Extracellular Vesicles (EVs) represents the most promising strategy to deal with clearance issues. EV targeting properties and pharmacokinetics could be augmented by surface engineering methods by inserting specific target ligands, including antibodies or small peptides. Biorthogonal chemistry allows for the formation of a stable covalent bond among these moieties and EV surface, ensuring the maintenance of biomolecule’s reactivity. As for synthetic nanoparticle counterparts, it has recently been acknowledged proteins might physically adsorb on the EV surface in biofluids, forming a protein corona (PC), thus modifying EV biological identity. This phenomenon cannot be overlooked when investigating the properties and behavior of nanosized systems in biological media. In this work, we explored the possibility to engineer the EV surface with a modified antibody (Cetuximab, CTX) by both chemisorption (via click chemistry reaction) and physisorption (forming a protein corona) mechanisms. We found that both mechanisms are suitable to engineer the EV surface and, at the molecular level, SPR analysis showed that EV formulations functionalized by CTX, through its physisorption or chemisorption presented a comparable affinity for EGFR. However, at the cellular level, the improved stability for CTX-EV prepared by click chemistry strategies confers superior binding and uptake ability toward target cells. These results highlight for the first time the EV-PC formation during surface engineering processes and suggest its functional role, which should be carefully estimated in EV-based drug delivery system development due to the lower stability of the EV-PC compared to the covalent chemical binding between EV and CTX.