Protein corona

This chapter highlights the specific challenges related to in vitro toxicity testing of nanomaterials. The difficulties presented are related to the very complex behavior of nanomaterials during the in vitro tests, namely, dissolution, aggregation, sedimentation, and formation of a protein corona. All these aspects modify the physicochemical characteristics of nanomaterials and their transport to the cell layer and cellular uptake and affect the effective cellular dose and response. The article underlines the necessity, for the toxicologist, to characterize and control all these features to be able to provide a reliable toxicity result. 

Interaction with proteins from the cell culture media and formation of the protein corona... 

Fig-1 The final NM-induced toxic effect observed in vitro is the result of multiple processes (1) interaction with proteins (formation of the protein corona, activation/inactivation of enzymes) (2) dissolution and release of toxic ions (3) production of ROS at the NMs surface (4) aggregation/agglomeration (5) diffusion and sedimentation that influence NM transport to the cell layer and the final effective concentration (6) interaction with the cell membrane and membrane receptors (activation/inhibition) (7) cell uptake (including receptor-mediated endocytosis and other uptake mechanisms) (8) interaction with intracellular enzymes (activation/inhibition) (9) production of intracellular ROS (10) activation of transcription factors and (11) binding to nucleic acids and genotoxicity, among others. Processes (1)—(5) are closely interconnected. The resulting effect observed is the result of the composite rate of all these different reactions... 

NM Surface Chemistry, Formation of Protein Corona, and Its Role in Mediating the Biological Activity of NMs... 

Apart from the presence of proteins, several other factors can influence NM behavior in culture media, including the salt composition, the pH, or the buffer capacity. Using gold nanoparticles (AuNP) of three sizes Maiorano et al. [24] demonstrated that the nanoparticle-protein interactions are differendy mediated when AuNP are suspended in two common cell culture media (DMEM and RPMI) supplemented with fetal bovine serum. An increased protein coating and different size distribution were observed in AuNP suspended in DMEM in comparison to RPMI. Most impor-tantiy, differences were also found in the biological responses of two cell lines (HeLa and U937), as the intracellular internalization and cytotoxicity were higher in cells exposed to AuNP in RPMI, where the protein corona was less abundant. 

Cellular uptake is initiated by the adhesion of the NMs to the cell membrane, which depends strongly on the NM size, shape, state of agglomeration, surface properties (composition and charge), surface functionalization [45], and the presence of protein corona [41, 46], All these parameters have an influence on the pathways of NM uptake and the uptake efficiency. 

Lesniak A, Fenaroli F, Monopoli MP, Oberg C, Dawson KA, Salvati A (2012) Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells. ACS Nano 6(7) 5845-5857... 

The AuNPs placed in DMEM F-12 cell culture media containing 2.5 mM L-gluta-mine, and 15 mM HEPES without phenol red were well-dispersed and stabilized (Rahman et ak, 2013) however, plasma proteins adsorb on the NP surface, making a protein shell called a protein corona (Cedervall et al., 2007 Lynch et al., 2007 Lynch and Dawson, 2008) within 5 min after mixing (AUdlany and Murphy, 2010) (Figure 5.10). This corona is known to influence the size, shape, and surface chemistry of the AuNPs affecting its interaction with biomolecules and cells (Miclaug et al., 2014). 

Schematic diagram showing the formation of the protein corona on the surface of an AuNP.

Charge and size changes in AuNPs assemblies in cell culture media with and without serum proteins, (a) Zeta potential changes. The numbers over each bar indicate the mean zeta potential of the AuNPs in the various environments, (b) Size distribution of the AuNPs, after formation of the protein corona when the AuNPs are added to cell culture media containing serum protein. The numbers over each bar indicate the mean NP diameter in the various environments. Abscissa labels Au = AuNPs alone DF = DMEMF-12 + r/o FBS alone and DFA = DMEMF-12 + 1% FBS plus AuNPs. 

Fourier transform infrared (FTIR) spectra were carried out using an OPUS/IR spectrometer (Vector 22, Bruker Corporation, MA, USA) in the interval 4000-600 cm at a resolution of 4cm . Control AuNPs and those exposed to culture media were removed from their suspensions by centrifugation at 14,000 rpm and dried at 80 °C into a powder. Figme 5.12b displays the IR spectra of sodium citrate-capped AuNPs and AuNPs in DMEMF-12 culture media with 1% FBS. The major stretching frequencies in the presence of the protein corona on AuNPs surface (Une (ii) of Figure 5.12b) were observed at 3272, 2921, 1734, 1626, 1530, 1244, and 1052 cm, which represents the O—H stretching modes. 

Comparison of AuNPs with and without a protein corona, (a) UV-VIS absorption spectra of AuNPs (black-soiid iine), DIVlEMF-12 media (black-dot line) and DMEMF-12 media with AuNPs (black-dash line) (b) FTIR spectra of AuNPs in the absence (i)and presence (ii)of protein corona (c) photographs above the spectra in (a) show the color appearance of each solution. In these figures, AuNPs, DMEMF-12-F 1% FBS, and DMEMF-12 containing 1% FBS plus AuNPs are represented by Au, DF, and DFA. 

GSH mediation of AuNPs agglomeration in the presence of a protein corona, (a) UV-VIS absorption spectra of AuNPs in DMEMF-12 media (DFA, black-dashed line), AuNPs in DMFMF-12 -H FeCb (DFA-F, gray-solid line), and AuNPs in DMFMF-12-F FeCb + GSH (DFA-FG, black-dotted line). The photograph above (a) shows the color appearance of each sample, (b) FTIR spectra of protein corona-capped AuNPs in the absence (i) and presence (ii) of GSFI. The designation, DMEMF in these experiments indicates that the DMEM culture medium contained 1% FBS. 

In order to confirm the GSH mediation of protein corona-capped AuNPs, the IR spectrum of the protein corona-capped AuNPs in the presence of GSH showed a new weak absorption signal at 2356 cm (line (ii) of Figure 5.20b), which was assigned to S-H vibration (Ding et al., 2009) indicating the thiol conjugation with the AuNPs surface. This peak was not present in protein corona-capped AuNPs not reacted with GSH (line (i) of Figure 5.20b). 

Hamad-Schifferh, K., 2013. How can we exploit the protein corona Nanomedicine 8, 1-3. 

Miclau , T., Bochenkov, V.E., Ogaki, R., Howard, K.A., Sutherland, D.S., 2014. Spatial mapping and quantification of soft and hard protein coronas at silver nanocubes. Nano Lett. 14, 2086-2093. 

Lundqvist, M. Stigler, J. Elia, G. Lynch, I. Cedervall, T. Dawson, K. A. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc. Nat Acad. Sci. U.S.A., 2008, i05(38), 14265-14270. 

CedervaU T, Lynch I, Lindman S, Berggard T, Thubn E, Nilsson H, et al. Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc Natl Acad Sci USA 2007 104 2050-5. 

The surface chemistry of mesoporous silica particles can influence the reactivity, solubility, interaction and agglomeration of particles in different environments, as well as their accumulation in organs and tissues. Surface chemistry will also strongly influence the nature of adsorbed protein corona and the interaction strength with it (Lynch et al. 2009). 

S. Tenzer, D. Docter, ]. Kuharev, A. Musyanovych, V. Fetz, R. Hecht, E Schlenk, D. Fischer, K. Kiouptsi, C. Reinhardt, K. Landfester, H. Schild, M. Maskos, S.K. Knauer, and R.H. Stauber, Rapid formation of plasma protein corona critically alfects nanoparticle pathophysiology, Nat Nanotechnol, 8 (10), 772-81,2013. 

J.L. Betker, J. Gomez, and T.J. Anchordoquy, The effects of fipoplex formulation veuiables on the protein corona and comparisons with in vitro transfection efficiency, / Control Release, 171 (3), 261-8, 2013. 

Elia, G and Dawson, K. (2011) The evolution of the protein corona around nanoparticles a test study. ACS Nano,... 

SakuUchu, U., Mahmoudi, M., Maurizi, L., Salaklang, J., Hofmann, H., 2014. Protein corona composition of superparamagnetic iron oxide nanoparticles with various physico-chemical properties and coatings. Sci. Rep. 4, 5020. 

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