The protein corona of nanoparticles

In this thesis, the interaction of proteins with nanoparticles and the resulting protein corona will be analyzed. The protein corona can be defined as the adsorbed layers of proteins on the surface of nanoparticles. While the proteins in the innermost layer interact preferentially with the surface of the nanoparticle, the rest of the proteins interact with each other. Based on the Vroman effect discussed in the previous section, in blood serum and other complex protein solutions, the surface of the nanoparticles is first covered with proteins that show higher diffusion and are present at higher concentrations, which gradually exchange to proteins that have a higher affinity to the surface [16, 18, 50, 52].

The different layers of the protein corona are referred to as “hard corona” and “soft corona”, but their definition in the literature is still ambiguous. In this dissertation, the innermost layer formed by proteins that strongly interact with the surface of the nanoparticle will be referred to as the “hard corona”, while the rest of the multi-layer protein coverage will be discussed under the name of “soft corona”. The qualitative

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analysis of the hard corona proteome is presented in Chapters 7 and 9, which sheds light on the adsorption and exchange of the different corona proteins in the culture medium and inside the cell.

The emerging use of nanotechnology in pharmaceutical and industrial applications demands the detailed study of nanoparticle-biosystem interactions. Due to their high concentration and great variety of functions, the investigation of protein-nanoparticle interactions is of high interest. While most effects responsible for protein adsorption and competition on solid surfaces are applicable for the surface of nanoparticles as well, major differences can arise from their small size and surface curvature. The small size of nanoparticles results in an asymmetrical force field rendering higher energy levels for the surface atoms [56-58]. Such high surface energy is thermodynamically unfavorable and the processes that lower the surface energy are prioritized. The adsorption of proteins on the surface of the nanoparticles reduces the surface energy by the net change in the Gibbs free energy originating from the protein binding [56-58], which makes the interaction energetically favorable. Therefore, it is expected that in a complex protein solution, the surface of nanoparticles will always be covered with proteins, unless it is functionalized to repel them [59].

The conformational changes observed in the case of adsorption on macroscopic solid surfaces can take place during the nanoparticle-protein interactions as well, although, they strongly depend on the surface characteristics of the nanoparticles. Individual particles with a larger diameter and, therefore, smaller surface curvature were shown to cause more severe conformational changes due to the larger surface provided for interactions [60]. It has been shown that an oligopeptide on thiolated gold nanoparticles could change its primarily α-helical structure into β-sheets [61], while in another study, β-sheets were found to change into α-helices upon adsorption [62]. The conformational changes of proteins often result in changes in their function, thus, they must be addressed in every experiment aiming at revealing nanoparticle-protein interactions.

The literature focusing on nanoparticle-protein interactions usually describes experiments either in pure protein solutions [22, 23, 25, 63] or highly complex solutions with hundreds to thousands of protein species, such as blood sera or cell lysates [34, 35, 64-66]. The former systems are adequate to determine the surface interactions, but they inform less

9 about binding kinetics, contrary to the latter, which describe the adsorption and competition in a complex solution but often lose the information about exact surface interactions.

There have been debates in the literature about the interaction of citrate-stabilized gold nanoparticles and proteins. Brewer and colleagues showed that bovine serum albumin adsorbs on the surface of nanoparticles by interacting purely with the citrate layer and they proposed the indirect protein-nanoparticle interaction based on electrostatic effects while rejecting the possibility of the exchange of surface-adsorbed citrate ions [25]. Some studies have proposed the same electrostatic interactions as well [24, 63], while others proposed the direct protein-nanoparticle interactions due to the exchanged citrate ions on the surface [22, 63]. Further studies are needed to identify the nature of surface interactions since it has implications for the corona formation, stability, and the extent of protein denaturation in the corona.

The studies of the composition and evolution of the protein corona have also yielded controversial results. Some of them propose that the composition of the corona changes dynamically over time [12, 67-69], while a nowadays often considered concept is that the protein corona forms rapidly and only the quantitative ratio of the proteins changes [32].

However, this latter, widely accepted theory can only stand its ground in a homogeneous protein solution, because the intracellular processing of nanoparticles and their direction to digestive cellular compartments are expected to significantly alter the corona composition. In this thesis, the interactions of proteins with citrate-stabilized gold nanospheres of uniform size will be studied by surface-enhanced Raman scattering (SERS) in the solutions of pure proteins and in protein mixtures (Chapters 5, 6, and 8).

Even though nowadays nanoparticles can be engineered with different shapes, sizes, and surface functionalization, in a complex protein solution, their behavior can strongly differ from the expected due to the adsorption of the protein corona [12, 18]. This new surface functionalization by the adsorbed proteins, also known as the “biological identity” [12], determines the path of the nanoparticles in the biomolecular system, the residence time, and the site and extent of accumulation [70-73]. In other words, this new layer is what the cell “sees” [19]. Previous studies have shown that the presence of the protein corona can reduce the cellular stress caused by the nanoparticles and their inherent cytotoxicity [73],

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while others suggest an increment in the adverse effects of the nanoparticles [74] based on the interaction of the protein corona with the biomolecular environment. However, knowing only the composition of the in vitro protein corona does not guarantee the suitable prediction of the in situ effects of the nanoparticles [75], as in living cells, each cellular compartment where the nanoparticles can reside has their own chemical environment, which can strongly influence the corona composition. Therefore, to characterize the protein corona formed in situ, new analytical methods have to be developed.