The main objective of WP5 is to characterise the “biological identities” of different nanomaterials after exposure in different biological media, i.e. blood plasma, serum, cell culture media, etc. These complexes will be characterised by diverse physiochemical approaches and proteomics in order to identify the identity of the biomolecules. It is expected that the main outcome of this study will be relevant for the correct understanding of nanoparticles related toxicity which will impact future safe implementation of nanomaterials.

Outcomes of the Workpackage 

Nanotechnology has recently acquired new dimensions since advances in synthetic chemistry have made it possible to produce ENM of an extraordinary variety. For instance, it is now possible to synthesize them in a large range of sizes and shapes, and precisely control their physico-chemical aspects such as surface charge, hydrophobicity and surface modification. Regardless of the numerous applications that nanotechnology offers, their large scale production inevitably implies increased exposure to humans during the manufacture process but also throughout the material life cycle.

Nanomaterials behave significantly differently than other chemicals. Size and the material surface energy are considered to be key distinct properties conferring identity to particles at the nanoscale. Nanomaterial size is comparable to several biomolecules and it confers their ability to penetrate membranes, to be recognized by specific receptors and trigger potential adverse responses to cells or in an organism, and to accumulate in cellular compartments with unknown consequences. Therefore, an accurate study of the size and colloidal stability of ENM dispersions is a crucial step to ensure high quality dispersions and to identify the absence of different subpopulations in size or agglomeration/aggregation that will produce unreliable data.

The interactions between ENM and proteins are highly affected by the material physico-chemical properties, such as the chemical composition, the surface chemistry, the shape, the porosity, and the surface morphology which determine the nature of the protein corona identities; both qualitatively (types of biomolecule forming the corona) and quantitatively (absolute amount). This means that for each ENM type that will be exposed in biological fluid, a distinct biomolecular corona is formed that will affect the biological outcome.

The protein corona is derived from proteins present in biological fluids, many of which are glycosylated. To date, the glycans on the proteins have been largely overlooked mainly because of the lack of dedicated infrastructure and workflow that could tackle the multidisciplinary aspects of the study. Within NANOSOLUTIONS, UCD has developed a running platform that has shown for the first time that the biomolecular corona is highly glycosylated and that changes in the glycan expression on the corona have a dramatic effect on the ENM bionano recognition. Quite remarkably, biomolecular corona are formed by proteins with high affinity with the ENM surface, regardless of their abundance in the media of origin as previously reported. Further studies have shown that glycosylation of the corona affects the cellular recognition and cellular response.

ENM physico-chemical properties govern the biomolecular corona formation and have a strong impact in the biological fate of ENM as approaching living organisms. Along with the protein component, the carbohydrate biomolecules should be taken into account as they are capable of controlling the ENM colloidal stability and the cellular response. The ENM dispersion complexity did not allow a straightforward correlation with the ENM properties and proteins, as by changing one parameter, such as charge or shape, the ENM distribution is also altered and the material surface area might be strongly affected. Lastly the initial corona composition study has revealed that the majority of the corona proteins are mostly dysopsonin proteins and thus these materials are likely not to be seen by the immunological system, however fluctuation in their abundance is specific to material and functional group.

A new platform has been set up for epitope mapping by UCD, where the protein orientation around ENM can be predicted by means of immuno-labels. This platform allows determining the statistical distribution of exposed protein epitopes presented across the nanoparticle corona surface on a particle-by-particle basis, identifying the epitopes expressed as well as their organization in relation to one another. The study has been published in Nature Nanotechnology and it has been shown to be a robust platform to map the detailed and exposed motifs on the ENM surface.

Physicochemical characterization of different ENMs in the presence of biological fluids, in particular human plasma, and quantitative composition analysis of the “hard corona” by a combination of proteomics and mass spectrometry with new methodologies based on immunolabelling with QDs have been carried out. The importance of profiling not only the protein corona composition as a whole, but also specific protein sequences that can potentially engage in nanoparticle-cell receptor interactions, has led to the development of a new platform for the molecular characterization of the protein corona. The platform has a focus on the organization and exposure of motifs or epitopes relevant for cell receptor interactions. A screening platform has been set up which enables the multi-detection of relevant specific protein sequences presented on the nanoparticle surface by monitoring qualitatively and quantitatively the interaction of QD immunoprobes with biomolecular corona protein domains on the NP surface. These interactions can be measured by using steady-state fluorescence spectroscopy or flow cytometry.

Mapping relevant epitopes on the biomolecular corona of nanoparticles (and their specific distribution both in relation to epitopes of the same class and epitopes of different classes) allows us to connect the real nature of the bionanointerface to cellular interactions, therefore obtaining a closer connection between a particle and its biological impact. Transfected cells exhibited increased uptake of particles compared to non-transfected cells in various concentrations of IgG-depleted serum, suggesting specific receptor-protein interactions play a role in particle recognition. Results show that a higher number of particles are endocytosed into the transfected cells compared to the controls for all studied conditions.

A set of platforms to characterize the ENM biomolecular corona complexes in realistic environments and to study the interactions with specific cellular receptors has been developed in order to provide capacity for profiling the ENM corona to predict early interaction with selected biological targets (receptors). Highly monodisperse (in biological media), LPS free, particles have been used to established the methodology and other ENMs have been tested, showing the connection between specific motifs of the protein-corona composition and the ENM-corona binding partners.