Biocorona: The Shadowed Side of Nanoparticles

Particles with dimensions ranging from few to several hundred nanometers are defined as nanomaterials. Humankind has been always fascinated by nanosize materials as shown by the creation of special handcrafted objects during the Roman Empire with distinct features to change color depending of the light incidence such as the Lycurgus cup [1]. In the last decade impressive progress has been made in colloid synthesis [2] throughout the establishment of sol-gel techniques [3], mechanical alloying of metal [4] and molecular self-assembly [5], thereby significantly contributing to the flourishing field of nanomaterials. Indeed, the research focusing on potential fields of application of nanomaterials has not only reached industrial manufacturing of paints [6] and solar cells [7] but also various medical disciplines ranging from oncology [8] to cardiology [9]. Current applications of nanomaterials in medicine range from bioimaging [10] to nanoformulations [11]. Nanoformulations are nanosized pharmaceutical preparation of drug in the nanosized range [12]. Especially for nanomaterials used as nanocontainers of bioactive compounds [13-17], it is of fundamental importance to understand not only the intrinsic properties of the nanomaterial, but also the properties acquired once administered in vivo. The main goal of nanoformulations for oncological application is the prevention of severe side effects from the toxic compounds used in therapy [18]. However, the systemic administration of such nanocontainers has to be carefully investigated. One factor assumed to contribute to a change of material properties is the direct interaction with patient derived molecules. Accordingly, nanomaterials with potential application in life sciences need to be investigated considering these possible interactions with biological fluids which are characterized by a high content of proteins and salts [19]; molecular entities which are often not in the focus during synthesis and evaluation of nanomaterials. An excellent review of Del Pino and coworkers has described the protein adsorption onto nanoparticles using the Hill coefficient n, together with a detailed mathematical description of protein adsorption phenomena [20].


Introduction
Particles with dimensions ranging from few to several hundred nanometers are defined as nanomaterials. Humankind has been always fascinated by nanosize materials as shown by the creation of special handcrafted objects during the Roman Empire with distinct features to change color depending of the light incidence such as the Lycurgus cup [1]. In the last decade impressive progress has been made in colloid synthesis [2] throughout the establishment of sol-gel techniques [3], mechanical alloying of metal [4] and molecular self-assembly [5], thereby significantly contributing to the flourishing field of nanomaterials. Indeed, the research focusing on potential fields of application of nanomaterials has not only reached industrial manufacturing of paints [6] and solar cells [7] but also various medical disciplines ranging from oncology [8] to cardiology [9]. Current applications of nanomaterials in medicine range from bioimaging [10] to nanoformulations [11]. Nanoformulations are nanosized pharmaceutical preparation of drug in the nanosized range [12]. Especially for nanomaterials used as nanocontainers of bioactive compounds [13][14][15][16][17], it is of fundamental importance to understand not only the intrinsic properties of the nanomaterial, but also the properties acquired once administered in vivo. The main goal of nanoformulations for oncological application is the prevention of severe side effects from the toxic compounds used in therapy [18]. However, the systemic administration of such nanocontainers has to be carefully investigated. One factor assumed to contribute to a change of material properties is the direct interaction with patient derived molecules. Accordingly, nanomaterials with potential application in life sciences need to be investigated considering these possible interactions with biological fluids which are characterized by a high content of proteins and salts [19]; molecular entities which are often not in the focus during synthesis and evaluation of nanomaterials. An excellent review of Del Pino and coworkers has described the protein adsorption onto nanoparticles using the Hill coefficient n, together with a detailed mathematical description of protein adsorption phenomena [20].
The scope of this review is to describe a general method to tackle the identification and measurement of the protein-particle interactions and their physiological effects in case a therapeutic treatment is established with nanomaterials.
Due to the large surface area, nanosize biomaterials have high free energy which has the tendency to dynamically interact with molecular entities present in the surrounding [21]. Proteins are the major entity strongly interacts with the surface of nanoparticles. As soon as a nanomaterials is in contact with biological fluid, proteins are adsorbing on the surface forming a surrounding layer. The binding of this layer is assumed to be tight, however reversible, resulting in a dynamic exchange of proteins with the microenvironment. The protein layer, is also called biocorona, and is composed of a hard corona and a soft corona, characterized by a slow exchange and a fast exchange, respectively, of proteins. Interestingly it has been observed that among the 3,400 proteins of human plasma only a minor part of them is directly interacting with nanoparticles. However, these

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proteins lead to striking effects in terms of cellular uptake [22][23][24][25]. For instance the adsorption of serum albumin on the surface of nanoparticles can lead to the perturbation of the protein structure depending on the surface charge of the nanomaterials [26]. This modification can drive the complex nanoparticlesprotein towards albumin receptors or scavenger receptor as demonstrated by Fleischer and collaborator [27]. Therefore, the "physiological coating" is the main part of the complex coming in contact with the cell and determining the consequent cellular uptake [28].
Protein adsorption has widely been studied in the field of biomedical nanoparticles [29]. However, Domke and collaborators were the first investigating protein adsorption on the surface of biomedical materials via atomic force microscopy (AFM) [30]. Interestingly, adsorbed proteins can be "scratched" off using the sharp point of the microscope cantilever, thereby providing evidence that proteins interact with the material surface via weak interactions. This finding initiated the development of novel protein adsorption-resistant biomaterials [31,32]. In contrast to static techniques such as AFM, hydrodynamic investigations of the nanoparticle diameter can be conducted. Incubation with biological media are more realistic and describe the biocoronaprocesses better and in a more complex environment. This is due to the fact that the nanoparticles are still in contact with the biological fluid during measurement. For this reason, dynamic light scattering (DLS) [21], differential centrifugal sedimentation (DCS) [33] and fluorescence correlation spectroscopy (FCS) [34] are very well established techniques to describe the dynamic binding of proteins to nanoparticle surfaces. Throughout the DLS technique peaks referring to the diameter of the nanoformulations are reported during the analysis. Therefore, the interaction between nanoparticles and proteins is clearly visible as shift in diameter compared to the initial recorded dimensions [29,35]. Furthermore, DCS is the optimal technique to allow a real time evolution of the biocorona [33]; and using FCS investigation of the interaction protein-particle is feasible [34].
However, identification of the biocorona protein components is very challenging. As diverse as the material is, so are the affinities to various plasma proteins. Therefore, valuable identification method for the determination of the adsorbed protein is the analysis via LC-MS [25]. In addition, these biomolecules govern the fate of the nanocarriers upon administration in vivo. Biodistribution and cellular uptake and trafficking can be influenced by the biocorona [36]. In this case, opsonins, ie fibrinogen and complement factor, can promote macrophage uptake with consequent clearance of the nanocarriers from the circulation [37,38]. In contrast, di opsonins, i. e. human serum albumin and apolipoproteins, can increase blood circulation time [39].
Dynamicity of the biocorona is one peculiarity of this system. Monopoli and collaborators have investigated in detail how the adsorbed protein to the surface of nanomaterials change in time showing a progressive displacement of protein with lower affinity in favor of more affine proteins which are reaching the nanoparticle surface in a second time [21]. Moreover, the binding of more affine protein is significantly more stable allowing the formation of a compact protein coating with a stabilizing role for the formulation while lowering the free surface energy. It seems note worthy that the formation of a rapid coating of exogenous materials in the human organism significantly influences unwanted pathological effects such as coagulation, thrombosis or even cell death [29,40].

Conclusion
The evaluation of the protein adsorption on the surface of nanoparticles is a phenomenon with significant importance. Several techniques have to be considered for a better understanding of the biocorona. In addition, as underlined by our review, nanoparticles for therapeutic application cannot be considered as isolated entities but as a continuum with the surrounding physiological medium. For this reason, the identification of the adsorbed proteins can be beneficial to allow a fine tuning of nanoparticles properties in order to achieve the desired therapeutic effects.