3.2.5. Protein Corona

Since NPs are injected into the bloodstream, they are exposed to a large amount of biomolecules that form a corona around them [76] (Figure 5). Protein corona is mainly composed of proteins with di fferent affinity interactions: immunoglobulin G, serum albumin, fibrinogen, clusterin, and apolipoproteins [77]. Therefore, NPs experiment changes in their physicochemical properties and their biological identity once the protein corona is formed. Therefore, in order to know the possible adverse e ffects of the physicochemical, kinetic, dynamic, and thermodynamic interactions of NPs, the characterization of these NP-protein interactions has become one of the main challenges of nanomedicine.

**Figure 5.** Schematic protein corona formation. First, the introduction of a nanoparticle to fluid/medium enriched in protein content takes place (I). Then, the nanoparticle is coated with proteins, which are abundant and highly mobile (II). Lastly, the protein species are exchanged over time, which results in hard corona of strongly bound proteins (III).

When NPs are incubated in a biological medium, a competitive dynamic process (between soluble biomolecules and surface) take place to form the protein corona. This process is based on the a ffinity adsorption of proteins on NP surfaces and on protein-protein interactions. According to the Vroman effect [78], the first are bound to NP surface proteins with a high concentration and low a ffinity and then are gradually replaced by higher a ffinity proteins present in low concentrations. The protein corona is classified into hard and soft depending on the duration of protein exchanges. Hard corona is a bound layer of proteins with high a ffinity and long exchange time. Proteins of the hard corona form the closest layer to the NP surface, so they are susceptible to thermodynamically favorable conformational changes (irreversible) depending on the chemistry functionalization, the hydrophobicity or hydrophily, the nature of proximal biological fluid, and the temperature [79]. Soft corona is a low a ffinity layer of proteins with a fast exchange over time. A recent model [80] suggests that hard corona is bound in a hard way to the NP surface and the soft corona is not directly bound to the NP but with a certain (low) degree of biomolecule interactions. As a result, the protein concentration, particle size, type of nanomaterial, and the surface properties are factors determining the layers of biomolecules and the protein corona density [81].

Depending on the type of administration routes, NPs are subjected to interactions with di fferent kind of biomolecules [82]. The biological environment is another key factor that plays a determinant role in the protein corona formation: the media components, temperature, pH, and the physiological state of the medium. The "in vivo" protein corona formation of biomedical liposomes seems to be more complex than "in vitro" [83]. In consequence, the "in vivo" protein corona characterization is fundamental for biomedical applications.

Di fferent methods and techniques are needed to determine proteins interactions in di fferent biological media because of the large number of proteins at di fferent concentrations that compete to functionalize with the NP surface [84]. Techniques usually described for protein corona evaluation are based on proteomic analysis [80], centrifugation, isothermal calorimetry titration, Ultraviolet and Visible (UV-Visible) spectrometry, Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) quantification, and sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis [85].

Therefore, it is essential to understand the relationship between the di fferent properties of nanomaterials and a concrete biological environment in order to understand their stability, viability, behavior, and the results obtained in the di fferent areas of research.
