Dispersion protocols have minimal impact on the biomolecular corona of advanced nanomaterials in cell culture assays

Industrial sectors have largely invested in the use of advanced nanomaterials (NMs), which are currently being implemented in a wide range of applications. However, the potential exposure to living beings and the environment still remains a concern. While some of these materials were not designed to be dispersible in aqueous media, the development of dispersion protocols to ensure compatibility with the in vitro and in vivo assays has become crucial for the correct assessment of the studies. NMs' identity in biological media is significantly influenced by the formation of a biomolecular corona on its surface. However, this corona might be affected by the dispersion method, altering their physicochemical characteristics and complicating the understanding of their interactions with biological systems. Therefore, understanding the efficiency of dispersion protocols and their influence on the biological identity of NMs is fundamental. However, systematic studies on the effects of dispersion protocols are still lacking, making this a crucial yet overlooked aspect in the field. This study aims to compare two standard dispersion protocols, commonly known as Harvard and Nanogenotox, and evaluate their impact on the biomolecular corona formation across a selection of advanced industrial NMs. To this aim, different techniques were used to assess particle size, colloidal stability and ion release, as well as protein and sialic acid content and abundance in the corona. Results show that the dispersion protocol modestly alters nanoparticle size and agglomeration state, and proteomics analysis revealed that each nanoparticle type forms a distinct corona, influenced by the distinct surface modifications. The presence of bovine serum albumin (BSA) in the Nanogenotox protocol minimally affected the overall trends in protein composition between the two protocols. These findings emphasize the significance of the dispersion protocol in nanotoxicology assays and demonstrate that variations between these methods do not play a decisive role in shaping the bio-identity and potential biological effects of advanced and multicomponent NMs.