**1. Introduction**

Nanotechnology is becoming everyday a more valuable resource in developing strategies of diagnostics and therapeutics; in fact, a new area is arising which is named nanomedicine [1]. From the use of nanoparticles [2] to nanorobots [3] or nanosensors [4], there is no shortage of ways to apply it to nanomedicine's benefit. Nanoparticles are particularly useful as theranostic agents, as a multifunctional platform which combine both therapeutic and diagnostic applications simultaneously [5]. However, nanoparticles must gather a number

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of characteristics in order to be considered as good theranostic agents, such as suitable size [6] and shape [7] for cell penetration, biocompatibility, surface charge, efficient targeting [8], and fluorescence, among others [9]. Despite of these advantages and promising applications, there are still many problems associated to the entrance of the nanoparticle in a physiological environment, which may be justified with different intrinsic characteristics of the nanoparticles [10]. In general, there are two different nanoparticle identities, such as "synthetic identity," which refers to their intentional physicochemical properties [11], and a "biological identity," which is related to the physicochemical properties shown by the nanoparticle after its application in a physiological environment and interaction with the presented biomolecules [10]. This "biological identity" is profoundly related to the formation of the protein corona, as it significantly alters the size, shape, and surface charge of the nanoparticle [12, 13]. The protein corona is formed after the entrance of the nanoparticle in a physiological environment, such as the bloodstream and/or peripheral blood, where the presence of thousands of proteins [14] (among other biomolecules) causes their adsorption onto the nanoparticle surface [15], in a corona shape [16]. The formation of this corona is energetically favorable, with a decrease of enthalpy and increase of entropy [15]. It can be divided into two categories: a "hard" corona, and a "soft" corona. The "soft" corona is based on abundant proteins that firstly bind to the nanoparticle through low-affinity bonds, and the "hard" corona is more dense [17], based on sparse proteins that replace the "soft" proteins over time, due to their higher affinity bonds [11], which is known as the Vroman effect [18]. The composition of the corona is directly dependent on the biomolecular composition of the physiological environment that surrounds it [19, 20], the time of exposure [11, 17, 21], and also incubation conditions (such as temperature or mild stirring), among others. Moreover, it is clear that the protein corona is not static and varies in the course of time; in other words, it's dynamic [17, 21]. Eventually, it will reach a state of equilibrium, steady stochastic state, where the association and dissociation rates for each protein occur equally [21], unless it is further incubated in a different biological media or proximal biological fluid, with a formation of a new corona [22]. The great majority of the proteins that form the corona are independent of the size and surface charge of the nanoparticle but are very influenced by the chemical properties of the material that constitutes the nanoparticle [23]. However, there are still sensitive proteins to size and surface charge, whose nature can change and consequently alter the interactions between nanoparticles and cells with the consequent alteration of the biological outcome and biological impacts [23]. For instance, if opsonins bind to the nanoparticle in the protein corona, they will be recognized as a "threat," and consequently are phagocytosed by macrophages [24]. It is crucial to prevent opsonization, "camouflage" the nanoparticle to avoid the phagocytosis, and keep the nanoparticles in circulation, which can be achieved by the application of a polymer coating, such as poly(ethylene glycol) (PEG) [25]. This polymer coating also prevents the formation of the protein corona, which can later compromise the nanoparticle internalization by the cells [25]. It is thus important to study the influence of the protein corona in the internalization of the nanoparticle, as the interactions of the nanoparticles with cells in in vivo studies are much different from the in vitro ones [26–28], which can prove to be an obstacle in the generalized application of this theranostic approach based on nanomaterials.

**2. Characterization of protein corona by proteomic strategies**

Since the formation of the protein corona has a great impact on the nanoparticle's performance when applied to a biological system, it is important to assess its structure and composition, in order to minimize the adverse effects it may have on the nanoparticle's use. Any alteration in shape, size, electron transfer, or others may come from the binding of the protein corona to the nanoparticle and may be used as parameters of comparison to be tested between nanoparticles, before and after administration to a biological fluid [29]. However, it is necessary to separate the nanoparticle-protein complex from the excess of plasma proteins [30] before assessing the composition of the protein corona. This is frequently made by centrifugation [30, 31], but it can have many adverse effects in the corona, due to the alterations caused by washing steps as well as gradient and volume variations [30, 32, 33]. In order to avoid loss of proteins from the corona, or even tainting the protein corona sample with the proteins in excess from the plasma, centrifugation can be accompanied by other procedures, such as size exclusion chromatography [32] or microfiltration [33, 34]. In the case of magnetic nanoparticles, a one-step centrifugation does not work, as it agglomerates the nanoparticles, making it necessary to perform a magnetic separation [35, 36]. As said by Megido et al. [33], the main methods of

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

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evaluation can be held as qualitative or quantitative, being summarized in **Figure 1**.

Quantitative proteomics is the collection of techniques that allow the determination of the number of proteins in a sample, which may be its absolute amount or just the relative change

**Figure 1.** Summary of methodological approaches useful for the characterization of the protein corona (figure based on

**2.1. Quantitative proteomics**

Megido et al. [33]).
