Tailoring of the internal surface of PEGylated NP to modulate the biological identity of the protein corona towards macrophages in the Atheroma plaque – NanoBioID

It is clearly established that the protein corona formed by interaction with plasma proteins on injected nanoparticles (NPs) determines their biological identity dictating the NP cell uptake properties and their fate in vivo. The NanoBioID project proposes to modulate the protein corona composition related to the synthetic identity of PEGylated magnetic NP inner surface in order to evaluate their ability to target vulnerable atheroma plaques in vivo in atherosclerosis.
To achieve this objective, the chemical functionalities of the internal surface of the PEGylated NPs will be used for the covalent grafting of molecular grafts of different physicochemical natures selected by their proven binding capacity once exposed on NP surface, with an apolipoprotein (Apo) of interest present in the bloodstream. Indeed, HDL containing this Apo is involved in cholesterol efflux occurring in foamy macrophages within the atherosclerotic plaque (FMs) that expose high affinity receptors for this protein. To increase the apparent affinity constant of this protein but also to manage the adsorption of opsonins and dysopsonins, which can undermine the binding with the macrophage receptor, the steric hindrance of the polymer corona will be used as a kinetic parameter limiting their adsorption. The chemical surface modification process will allow applying the same synthetic profiles characterized by a set of analytical tools to two types of metal oxide nanoparticles of the same size (20 nm): (i) fluorescent silica nanoparticles that incorporates two fluorophores in visible and near infrared (NIR) range for in vitro test, cytometry and fluorescence imaging experiments; (ii) maghemite nanoflowers used for their remarkable magnetic relaxivities useful for MRI. The affinity of the Apo with the functionalized internal surface of the PEGylated NPs will be evaluated by physicochemical methods of fluorescence and their conformational properties measured by circular dichroism.
Comparative studies of interactions with FMs of the plaque will be carried out using the different synthetic identities generated by incubation of NPs in sera from healthy mice enriched with Apo or not, and in serum from an APOE-/- mice used as a model in atherosclerosis, decomplemented or not. The bioidentities thus generated will be analyzed by LC-MS/MS and their relative protein abundance quantified by machine learning processing in order to verify the overabundance of Apo and to select the best stoichiometric opsonin/dysopsonin profile. The binding stability of the Apo present on the PEGylated NPs will be evaluated in competition with plasma proteins by mass spectrometry but also by fluorescence correlation spectroscopy.
A set of in vitro tests involving blood cells in order to predict the elimination of NPs towards the liver, experimental models mimicking the FMs of the plaque as well as FMs present in situ (ex vivo interaction tests on biopsies of murine and human aortas) will be implemented to assess the interactions with the generated bioidentities. Finally, in vivo experiments will be carried out in a mouse model of atherosclerosis with PEGylated NPs offering the most favorable proteomic profiles to bind on the receptors exposed on the FMs, which one wishes to target for diagnostic purposes. These biological identities will thus be applied to fluorescent nanoparticles to verify their targeting towards FMs present in atherosclerotic plaques by NIR fluorescence tomography and to magnetic nanoflowers for validation by MRI.
If this project is successful, it will be possible to target vulnerable atheroma plaques of mouse from a given synthetic identity, without resorting to the conjugation of targeting agents.