Publication informationNature nanotechnology, 7 (12): 779-786Publisher Nature Publishing Group Item record/more information

It is now clearly emerging that besides size and shape, the other primary defining element of nanoscale objects in biological media is their long-lived protein ("hard") corona. This corona may be expressed as a durable, stabilizing coating of the bare surface of nanoparticle (NP) monomers, or it may be reflected in different subpopulations of particle assemblies, each presenting a durable protein coating. Using the approach and concepts of physical chemistry, we relate studies on the composition of the protein corona at different plasma concentrations with structural data on the complexes both in situ and free from excess plasma. This enables a high degree of confidence in the meaning of the hard protein corona in a biological context. Here, we present the protein adsorption for two compositionally different NPs, namely sulfonated polystyrene and silica NPs. NP-protein complexes are characterized by differential centrifugal sedimentation, dynamic light scattering, and zeta-potential both in situ and once isolated from plasma as a function of the protein/NP surface area ratio. We then introduce a semiquantitative determination of their hard corona composition using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray liquid chromatography mass spectrometry, which allows us to follow the total binding isotherms for the particles, identifying simultaneously the nature and amount of the most relevant proteins as a function of the plasma concentration. We find that the hard corona can evolve quite significantly as one passes from protein concentrations appropriate to in vitro cell studies to those present in in vivo studies, which has deep implications for in vitro-in vivo extrapolations and will require some consideration in the future.

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What the biological cell, organ, or barrier actually "sees" when interacting with a nanoparticle dispersed in a biological medium likely matters more than the bare material properties of the particle itself. Typically the bare surface of the particle is covered by several biomolecules, including a select group of proteins drawn from the biological medium. Here, we apply several different methodologies, in a time-resolved manner, to follow the lifetime of such biomolecular "coronas" both in situ and isolated from the excess plasma. We find that such particle-biomolecule complexes can be physically isolated from the surrounding medium and studied in some detail, without altering their structure. For several nanomaterial types, we find that blood plasma-derived coronas are sufficiently long-lived that they, rather than the nanomaterial surface, are likely to be what the cell sees. From fundamental science to regulatory safety, current efforts to classify the biological impacts of nanomaterials (currently according to bare material type and bare surface properties) may be assisted by the methodology and understanding reported here.