*2.2.2. SDS-PAGE*

One of the most used methods in proteomics, electrophoresis, is a procedure that separates proteins in a sample according to their charge. Using a gel of polyacrylamide, a protein solution is applied, and in relation to their charge, proteins will migrate across the gel [53]. The proteins get sorted by molecular weight [53], and staining is needed with an appropriate pigment, for instance, Coomassie blue [54]. The molecular weight is then compared with the one shown by the markers, and a densitometry analysis is performed [33]. This procedure is suitable to characterize the proteins that form the corona, comparing the proteins obtained from the plasma with the ones that are found in the corona. It is thus possible to verify exactly which ones get adsorbed to its surface and, therefore, the ones that have greater affinity to the nanoparticle [43].

spectroscopy is not normally used independently, and it's usually meant to complement other methods, such as FTIR [62]. Together, they provide information about protein's sec-

Protein Interactions and Nanomaterials: A Key Role of the Protein Corona in Nanobiocompatibility

http://dx.doi.org/10.5772/intechopen.75501

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NMR, which stands for nuclear magnetic resonance, is a form of evaluation of proteins, widely used to describe dendrimers, polymers, and fullerenes derivatives, characterizing structure, purity, and functionality [64] and their possible effects in membrane disruption [65]. Usually, it is used to analyze lipids, as they show high affinity for the nanoparticles [66], after a size exclusion chromatography [33]. It still has disadvantages; for instance, it cannot distinguish the distribution of targeting agent density on a population of nanopar-

Considered one of the primary sources of structural information about the protein-ligand complex [67], and it is based on the positions and intensities of the reflections as measured in the diffraction pattern of the crystal [67]. This method has big challenges associated to it, as the quantity of radiation needed may be excessive and cause damages to the proteins before a signal is obtained [68], and there are many uncertainties when applying it, such as identity

After the nanoparticle's entry in the biological fluid, it is important to ensure its internalization into the cells and intracellular transport, as the formation of the protein corona influences directly the cellular uptake and may also have a significant role on the success of the nanoparticle or lack thereof [69]. Most of mammalian cells internalize the nanoparticles through pinocytosis, although big, specialized cells (such as macrophages) are able to do it by phagocytosis [70, 71], which is the uptake of large particles [70]. Some nanoparticles can also do it by passive penetration of the cellular membrane; however, if the nanoparticle is not small enough, it may deform the membrane [72] by forming holes or thinning it [73], increasing the cytotoxicity [71, 74]. Still, this mechanism is useful in drug delivery, as the nanoparticle travels directly to the cytosol, without making use of endocytic vesicles [74], promoting the reach of the intracellular targets [75]. Hence, it is necessary to take that into account, when designing the nanoparticle, as it may be possible to optimally design the surface of the selected nanoparticle for drug delivery and avoid the membrane's deformation [74]. As for the pinocytosis internalization, which is the cellular uptake of small particles (fluids and solutes), it has four

The physical properties of the nanoparticle such as size, net surface charge, and chemical composition determine which endocytosis process is chosen for the internalization [76],

ondary structure [63], plus vibrational and rotational parameters.

*2.2.7. Nuclear magnetic resonance (NMR)*

or location of the proteins to be evaluated [67].

different types of mechanisms [70] (**Figure 2**).

**3. Nanoparticle's intracellular trafficking**

ticles [64].

*2.2.8. X-ray crystallography*

### *2.2.3. Fluorescence spectroscopy*

Fluorescence spectroscopy is a method that allows the measurement of the fluorescence of a compound, when excited at a given wavelength [33]. The fluorescence may come from the protein (intrinsic probes), the nanoparticle, or even a fluorophore added to the complex (extrinsic probes) [33, 55], which will be picked up by the amino groups and then detected in the fluorescence spectrometer [56].
