ProteinâNanoparticle Interactions: What Are the ProteinâCorona

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Protein-Nanoparticle interactions: What are the protein-corona thickness and organization? Laurent Marichal, Gael Giraudon--Colas, Fabrice Cousin, Antoine Thill, Yves Boulard, Jean-Christophe Aude, Jean Labarre, Serge Pin, and Jean Philippe Renault Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01373 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Protein-Nanoparticle interactions: What are the protein-corona thickness and organization? Laurent Marichal1,2, Gaël Giraudon--Colas1, Fabrice Cousin3, Antoine Thill1, Jean Labarre2, Yves Boulard2, JeanChristophe Aude2, Serge Pin1, Jean Philippe Renault1 1NIMBE, 2I2BC,

IRAMIS, DRF, CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France

JOLIOT, DRF, CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France

3Laboratoire

Léon-Brillouin, UMR 12 CEA-CNRS, Université Paris-Saclay, CEA-Saclay, Gif-sur-Yvette, France

Abstract Proteins adsorption on surfaces is generally evaluated in terms of evolution of the proteins’ structures and functions. However, when the surface is those of a nanoparticle, the protein corona formed at its surface possesses a particular supramolecular structure that gives a "biological identity" to the new object. Little is known about the actual shape of the protein corona. Here, the protein corona formed by the adsorption of model proteins (myoglobin and hemoglobin) on silica nanoparticles was studied. Small-angle neutron scattering and oxygenation studies were combined in order to assess both the structural and functional impact of the adsorption on proteins. Large differences in terms of oxygenation properties could be found while no significant global shape changes were seen after adsorption. Moreover, the structural study showed that the adsorbed proteins form an organized yet discontinuous monolayer around the nanoparticles.

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Introduction Adsorption of proteins on surfaces is a ubiquitous phenomenon and occurs readily on almost every nanoparticle, leading to the apparition of the so-called protein corona 1. Usually, proteins interacting with a surface can be divided into two categories 2: proteins whose adsorption has an impact on their structure ("soft proteins") and proteins that are not impacted by the adsorption ("hard proteins"). Soft proteins are susceptible to loose part of their structure and to spread on the surface in order to maximize their interactions 2, which of course induce important functional and dynamic impacts. On the contrary, hard proteins are supposed to keep the majority of their structure and their function. However, this paradigm has been questioned as many globular proteins thought to be "hard" have their structure and/or function substantially modified by the adsorption (3-5). Although much work was done characterizing protein/nanoparticle systems, very few studies were aimed at determining the precise shape and internal structure of the protein corona 6. Yet this matter should be of upmost importance considering the difficulties developing nanosystems for biomedical purposes 7. Studying proteins adsorbed on flat surfaces (e. g. by atomic force microscopy 8) does not allow us to assess the complexity of the protein corona structure. In this respect, only ensemble-averaging techniques such as smallangle scattering can give us access to this kind of information. In this study, we used small angle neutron scattering to analyze the shape of adsorbed proteins. Neutron scattering allowed us to tune the contrast between the solvent (aqueous buffer) and the solute of interest (the proteins). We studied pure myoglobin (Mb, monomer of 17 kDa) and hemoglobin (Hb, tetramer of 64.5 kDa 9) adsorbed on monodisperse silica nanoparticles (Ludox NP, nanospheres of 13.0 nm of radius). Working on hemoproteins gave us the possibility to probe both the structural and functional impact of the protein adsorption without artefacts due to the NP presence 5. Hb is more susceptible to deformation on the surface than Mb, its four subunits being able to move with respect to one another 10. Prior to these measurements, we documented the adsorption behavior of these two proteins in deuterated water in order to know whether the thermodynamic and functional properties of the studied system were retained in these new conditions of solvent.

Experimental Chemicals Pure Milli-Q water (MilliPore, 18 MΩ.cm) was used for samples preparation and experiments. Phosphate buffers were prepared by dissolving disodium phosphate (28029 VWR Chemicals, purity ≥ 99.5%) and monosodium phosphate (28015 VWR Chemicals, purity ≥ 99%) salts in either pure water or in deuterated water (Eurisotop D214H ≥ 99.9% D). Silica nanoparticles (NP) were LUDOX® TM-50 (Merck) that are nanospheres with a physical radius of 13.0 nm (see Fig. S1) and a specific surface area of 110 m2.g-1. Before the experiments, NP were dialyzed twice against 100 volumes of pure water or D2O (using 3.5 kDa dialysis membranes Spectrum™ 132724) in order to eliminate


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excess salts. The suspension was then filtered by a 0.45 µm syringe filter (17598, Sartorius) and the NP mass concentration was measured by desiccation and dry mass weighing. Metmyoglobin from equine heart (Mb) was purchased in lyophilized powder (M1882, Merck), solubilized, dialyzed twice against 100 volumes of D2O and centrifuged (20 000 g) for 10 min before use. The Mb concentration was measured by spectrophotometry at 623 nm with ε = 3500 L.mol-1.cm−1 9. Porcine hemoglobin (Hb) was purified in its oxygenated form from fresh blood following modified standard preparation

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using erythrocyte membrane precipitation. Hb solution was extensively dialyzed against pure

water at 4°C, stripped by passing hemoglobin desalted solution through a mixed-bed ion-exchange resin (AG® 501-X8, Bio-Rad) 12 and centrifuged at 20 000 g for 10 min. Two more dialysis were done against 100 volumes of D2O at 8°C. The Hb concentration was measured by spectrophotometry at 576 nm with ε = 15 150 L.mol1.cm−1 9.

Adsorption isotherms of Mb and Hb on NP were measured by the depletion method 13. For one isotherm, a set of samples containing a constant concentration of NP (1 g.L-1) and varying concentrations of proteins (ranging from 0.01 to 2 g.L-1) was prepared. The samples were mixed gently at room temperature for 3h and were then centrifuged at 20 000 g for 10 min. Protein concentration in the supernatant was finally measured by spectrophotometry. The amount of adsorbed protein m∞ is expressed in mg of adsorbed protein per square meter of NP surface and in number of adsorbed proteins per NP. Each solution was diluted in deuterated phosphate buffer with a final concentration of 0.1 mol.L-1 and pD 7.4 in order to control the pD over the entire experimental time. pD was obtained by measuring the apparent pH (pH*) using the following equation: 𝑝𝐷 = 𝑝𝐻 ∗ +0.4 14

Oxygenation study Oxygen binding was measured in a tonometer by spectrophotometry at 25°C. An integrated sphere module was associated to the spectrophotometer in order to decrease the scattering contribution of NP. HbO2 was gently mixed for 2 hours with or without NP in D2O phosphate buffer 0.1 mol.L-1 (pD 7.4) at 25°C. We used Hb at a concentration of 65 mM and for experiments with Hb adsorbed on NP, the concentration of NP was 38.4 g/L for studies in H2O and 40.5 g/L for D2O (calculated from the adsorption isotherms in order to have 95% of adsorbed proteins). HbO2 was deoxygenated under an argon flow and oxygen binding was recorded by following the absorption at several wavelengths. No desorption of hemoglobin from NP was observed during deoxygenation. All the measurements were done in triplicate. The experimental oxygen binding curves were fitted with the Hill equation: 𝑌

log 1 ― 𝑌 = 𝑛 ∙ log 𝑃𝑂2 ― log 𝑃1/2 where Y is the fraction of HbO2 and PO2 the oxygen partial pressure used to determine the Hill coefficient n and the oxygen partial pressure at half saturation P1/2.


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Small Angle Neutron Scattering (SANS) experiments were performed at the instrument PAXY (Laboratoire Léon-Brillouin). Two approaches were carried out. First, proteins and NP were studied in 100% D2O buffer in order to optimize their scattering. Secondly, samples were placed in a mixture of 60% D2O/40% H2O buffer in order to match the silica’s scattering length density (SLD) with the solvent’s one (Fig. S7). In this case only the protein scattering signal is measured. Four instrumental setups have been used in order to cover a broad scattering vector (Q) range over more than 2 decades (0.002−0.4 Å−1) (𝑄 =

4𝜋 𝜆 sin 𝜃)

where 2θ is the scattering angle) with different incident wavelength

and sample/detector distances (6Å at 1m, 6 Å at 3 m, 8.5 Å at 5 m, and 15 Å at 6.7 m) . All samples were loaded in quartz cells (Hellma) of small path length (1mm for 60% D2O and 2mm for 100% D2O) and placed in a temperature controlled (20°C) sample changer. The azimuthally averaged spectra were corrected for solvent, cell, and incoherent scattering as well as for background noise 15. The silica matched scattering curve was fitted by a vesicle model 16 using the Sasview software. 17

Results and discussion The adsorption of Mb and Hb on silica NP in D2O buffer was quantified by the depletion method 13 (Fig. 1 and Tab. 1). Mb and Hb have different adsorption behaviors, both in terms of maximum amount of adsorbed proteins (m∞) and affinity (Kads). Hb is five times more affine than Mb. Such a difference had previously been observed in H2O solutions (13, 5). This can be explained by the presence of arginine moieties in Hb (3 or 5 per globin) and not in Mb. Indeed, arginine is one of the main contributors to the protein’s affinity for silica surfaces 18

Figure 1: Adsorption isotherms of myoglobin (red dots) and hemoglobin (blue squares) adsorbed on silica nanoparticles in D2O phosphate buffer 0.1 mol.L-1 (pD 7.4). The experimental points are from two separate isotherms and the continuous lines are fits by the Langmuir model.


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Table1: Fits of the adsorption isotherms (Fig. 1) by the Langmuir adsorption model give the adsorption constants (Kads) and maximum amount of adsorbed protein (m∞). The maximum number of adsorbed proteins per NP (N) is calculated from m∞. The last column Ntheory is the theoretical number of proteins, considered as hard spheres, necessary to completely cover the NP surface 19.

Mb Hb

Kads (L.mol-1) 1.02.105 4.92.105

m∞ (mg.m-²) 0.88 2.20

Corresponding N (protein/NP) 246 161

Ntheory (protein/NP) 340 150

Mb and Hb have also significantly different adsorption behaviors in terms of m∞. In order to visualize the kind of coverage that m∞ represents we can apply a geometrical model that estimates the maximum number of spheres that can be adsorbed on a nanosphere in order to cover it (19, last column of Tab. 1, and Fig. S3). A complete coverage with folded Mb would theoretically correspond to 340 molecules, whereas only 240 sites are observed here. This would mean that either Mb spread on the surface, 2, 20, 21, or that they cannot occupy certain site on the surface. On the contrary, the surface coverage seems complete with Hb. The Kads measured here is globally representative of the measurement conducted in H2O on different NP (Tab. S1), considering that deuteration can impact both the electrostatic properties of the silica surface and of the protein. In terms of m∞, the values measured in D2O are also within the (quite wide) boundaries of what is measured either with the same NP in H2O buffer, or in other systems found in the literature. Therefore, the adsorption behavior in heavy water can be considered similar to the one in light water. Since oxygenation behavior of hemoglobin is known to be impacted both by adsorption and deuteration, we also measured the oxygenation curves of free and adsorbed Hb in D2O phosphate buffers (Fig. 2 and Tab. 2).

Figure 2: Effect of adsorption on hemoglobin activity. Oxygen binding curves of Hb with (red dots) and without NP (blue squares) in D2O phosphate buffer 0.1 mol.L-1 (pD 7.4) at 25°C. The red curve was obtained with more than 95% of the Hb adsorbed, according to the isotherms measured in figure 1. The continuous lines show the best fits obtained by the Hill equation.


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Table 2: Oxygen partial pressure at half saturation (P1/2) and Hill Coefficient (n) of Hb oxygen binding with and without NP in D2O and H2O phosphate buffers 0.1 mol.L-1 (pD 7.4 and pH 7.0 respectively) at 25°C.

P1/2 (mmHg) n

D2O Without NP 15.5 2.8

D2O With NP 8.5 2.5

H2O Without NP 9.4 2.9

H2O With NP 6 2.6

Regarding the effect of deuteration, the negative impact of D2O on the Hb affinity for O2 observed here (increase of P1/2 in table 2) has already been studied for human hemoglobin 22. However, in that case, not only was the oxygen affinity decreased, but also the Hill number describing the extent of the cooperativity was decreased down to 2. These modifications were attributed to changes in the protein dynamic upon deuteration 23,

but a direct viscosity effect on O2 diffusion cannot be excluded 24.

Regarding the effect of adsorption, the interaction of Hb with polydisperse silica NP leads to a decrease of the P1/2 in H2O buffer 5, probably through stiffening of the protein structure 25. The interaction of Hb with Ludox NP in H2O (Tab. 2) also shows an increase in the O2 affinity of the adsorbed Hb. Shifting to D2O does not change this trend, with a decrease of P1/2 from 15.5 to 8.5 mmHg upon adsorption. The second main result from these oxygenation curves is the conservation of the Hill coefficient n (Tab. 2) that proves that the typical cooperative behavior of hemoglobin is preserved upon adsorption both in H2O and in D2O. It therefore suggests that the Hb quaternary structure remains intact after the adsorption. Having assessed that deuteration does not drastically modify the behavior of the proteins under scrutiny once adsorbed on nanoparticles, small-angle neutron scattering (SANS) measurements were performed to determine their shape. The scattering properties of isolated systems, respectively Ludox NP, Mb and Hb were measured first (Fig. S1 and S2). Ludox NP are monodisperse nanospheres with a radius of 13.0 ± 0.2 nm (Fig. S1), in perfect agreement with previous measurements 26. SANS scattering of Mb and Hb are similar to the ones in the literature 27 28.The gyration radius were obtained from a Guinier fitting of the curves in the low q region and gave 1.4 ± 0.1 nm for Mb (very close to the value of 1.5 nm obtained for Mb also from horse heart but complexed with azide in reference

28)

and of 2.3 ± 0.2 nm for Hb (consistent with the value of 2.37 nm for

human hemoglobin in 27). The SANS curve of Mb presents a single inflexion point at q = 0.15 Å -1, while the curve of Hb presents two shoulders at q = 0.12 and q = 0.2 Å -1 corresponding roughly to intra and inter subunit distances. For measurements on adsorbed proteins, we chose experimental conditions maximizing the amount of adsorbed protein and minimizing the amount of non-adsorbed protein (Tab. S2). The drawback of this approach is that the surface coverage is only partial in these conditions. The SANS signal of Mb or Hb adsorbed on nanoparticles (Mb/NP and Hb/NP assemblies) are presented respectively in figures 3A and 3B, after subtraction from the contribution of free proteins estimated from the adsorption isotherms (the total scattering is shown in figure S4 in supporting information and the comparison


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between the scattering prior and after subtraction is shown in Figure S5) . For both assemblies, the signature of the form factor of monodisperse silica nanoparticle (first oscillation at around 4.47/R ~ 0.034 Å -1) can be observed. This oscillation appears to be slightly shifted towards the lower Q values in presence of proteins, suggesting that the covered NP is bigger than the naked NP. This shift is more visible in the Fig. 3B (see also in Fig. S4) than 3A due to the fact that a Hb corona is larger than a Mb one. However, the shift is limited and its Qvalue cannot be linked directly to the size of a corona. Indeed, for the Hb/NP assembly, a fit of the oscillation by a sphere model would give a radius of roughly 14.5 nm for the covered NP, while the addition of the size of the Hb to the size of the NP would provide an overall radius of approximately at 17 nm in the case of all the NP surface is covered by proteins. However, we will see later that the scattering curve is strongly affected by an intra protein-protein structure factor in this Q-region thus sizes cannot be assessed by simple form factors. Moreover, the contrast in a corona layer is probably complex, as even compact assemblies like protein crystals may contain very large amount of water. Another salient feature is the increase of the scattering at small q with a power law is the signature of the formation of fractal aggregates. The fractal dimension D of such aggregates can be deduced from the scattering law: 𝐼(𝑄) ∝ 𝑃(𝑄) × 𝑆(𝑄) Where P(Q) is the form factor and S(Q) is the structure factor. At small Q (QRg < 1), P(Q) is constant for our objects. So, the equation can be written as follow: 𝐼(𝑄) ∝ 𝑆(𝑄) ∝ 𝑄 ―𝐷

29

Here, D equals 2.1, which is classically found in case of reaction-limited aggregation (RLA) process

30, 31

. Such

process occurs in systems for which attractions are dominating at short range but that are weakly repulsive at longer range. Such small repulsive energy barrier between the particles has thus to be overcome for the aggregation to happen.


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Figure 3: SANS spectra in 100% D2O phosphate buffer (pD 7.4). (A) Scattering of free Mb (red dashed line), free NP (blue circles) and a Mb/NP assembly (green squares). (B) Scattering of free Hb (red dashed line), free NP (blue circles), and a Hb/NP assembly (green squares). The NP concentration is constant at 50 g.L-1. The free protein spectra were measured at 10 g.L-1 and then normalized in order to correspond to the same concentration as the adsorbed protein concentration in the mixture (2.2 g.L-1 and 6.0 g.L-1 for Mb and Hb


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respectively). The contribution to the scattering from the non-adsorbed protein fraction was subtracted from the overall scattering, assuming that there is a linear combination of decorated and free proteins (see Fig. S5). (C) Structure factor of the Hb/NP assembly. In yellow, scattering spectrum of the Hb/NP assembly (from (B)) divided by the signal of Hb, in equal amount of the adsorbed Hb fraction. In black, fit of the correlation peak by the hard sphere structure factor with Percus-Yevick closing (effective radius of 2.1 nm). We must notice that, if NP aggregation is currently used in biological tests through protein ligand interaction 32, such a spontaneous assembly triggered by the introduction of a pure protein has seldom been observed in the literature 33. On the contrary, folded proteins are usually thought to stabilize colloidal assemblies,

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whereas

unfolded ones can destabilize them 35. We cannot deduce from our data whether the aggregation occurs through a NP-protein-NP or NP-proteinprotein-NP interaction, or even by a depletion mechanism

33.

However, one must keep in mind that such

assembly requires only one bridging point between NP to occur, and any vacant adsorption sites may allow the adsorption of a protein already interacting with another NP. We can also notice that this aggregation behavior is probably very general, as it can explain the use of centrifugation to separate NP/protein assemblies from their non-bound components

36.

Considering the

potential role of NP aggregation on their toxicology, 37 it should probably be more thoroughly studied. The second salient point in figures 3A and 3B is the fact that the form factor of the protein is preserved. Indeed, in the high Q region (Q > 0.1 Å -1), the scattering signal is completely dominated by the scattering of proteins as the scattering of NP strongly decays like Q-4 (Porod law, Fig S1) and is lower than 0.01 cm-1 for Q > 0.1 Å -1. Moreover, the protein/NP assembly curves show a strong decay with a Q-4 power law. At the very large Q, (Q > 0.2 Å -1), such scattering essentially comes from the protein form factor. The Q-4 decay shows that the proteins maintains its globular structure. Indeed, a fully unfolded protein would have a behavior of a wormlike chain, i.e. a polymer in good solvent, and thus a decay like Q-1.7

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A partial unfolding would lead to an overall decay

between these two limit behaviors 39 40 Indeed, for both Mb and Hb, the scattering of the decorated NP superimposes to those of pure proteins to a constant factor, demonstrating that their shape is preserved. Such a preservation was also recently observed for Mb absorbed inside mesoporous silica 41. We attempted to fit the whole experimental data by simple models, first a core-shell model with a homogeneous core of silica and a homogeneous corona of protein, and secondly a “Raspberry” model, i.e. a homogeneous core of silica decorated by spheres with the SLD of proteins (see Fig. S6). The fits are impossible to make, especially in the intermediate Q region (around 0.05 Å -1), as there is a strong decrease in intensity in the experimental data of the decorated particle compared to the curve of the naked NP. It is likely that simple models cannot properly model the partial structure factor Sprot_NP that accounts for proteins/NPs interactions. However, for the Hb/NP assembly (Fig. 3B), it appears that there a marked increase of intensity in intensity at Q = 0.16 A-1., which is concentration dependent as it is greatly reduced in mixtures with fewer adsorbed proteins (Fig. S4). Such intensity may be related to the appearance of an intra protein-protein structure factor onto the


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NP related to the main first neighbor distance between the proteins. Indeed, such correlation also appears in highly concentrated protein solutions 42 43 44 and is the signature of a short distance order. In order to obtain a better insight on such intra structure factor, we calculated it crudely dividing the signal of the Hb/NP assembly by the signal of Hb (Fig. 3C). A correlation peak is now clearly visible at Q = 0.15 Å -1 and a correlation hole at lower Q. This corresponds in direct space to a distance of ~2/0.15 = 42 Å, which almost corresponds to the distance where two Hb proteins are in close contact (Hb diameter = 4.6 nm). Such structure factor recalls thus those of a system of hard spheres, suggesting that proteins can be regarded as solutions of hard spheres when adsorbed on the NP surface45 46 . To test such an assumption, we modeled the structure factor by the PercusYevick structure factor that described 3-D systems of had spheres. The fit gives an effective diameter of the system of around 4.2 nm., close to those of the Hb. , even though this latter is not perfectly spherical. From our current understanding of such a colloidal media this suggests some mobility on the surface and a repulsive potential between the adsorbed proteins. We must notice that such a signal cannot be seen for Hb solutions of comparable concentration in the absence of NP, but are observed in the literature for free hemoglobin at concentration higher than 300 g.L-1 47. This intra structure factor is not visible for the Mb/NP assembly on the Q-range that is actually probed by the experiment. Indeed, such correlation peak would occur at ~ 2/dMb = 0.42 Å-1, where dMb is the myoglobin diameter (~15 Å), and thus at a very large Q value for which the signal-to-noise ratio is very weak. It would be obviously tempting to try to extract more information from these data, and more specifically, the layer thickness and density in protein. However, it would require, as stated earlier, to postulate both the protein/nanoparticle and nanoparticle/nanoparticle partial structure factors. Such assumption would then lead probably to meaningless results. Therefore, we tried to simplify the systems and suppress these contributions, by varying the neutron contrast of the solutions in order to match the scattering length density of the nanoparticles by using 60% (v/v) of D2O (see Fig. S7). In absence of proteins, the scattering of the pure solution of NP must thus completely vanish, which has been checked. (Fig. 4, blue circles). In such condition, the contrast between the solvent and the proteins (prot – solvent)2 decreases also and the coherent scattering intensity of the of the proteins (Fig. 4) decreases by a factor 11 since prot corresponds to 43% (v/v) of D2O. In the meantime, the incoherent scattering is increased by at last 1 order of magnitude. The overall signal/noise ratio is lowered by two orders of magnitude compared to the scattering in 100% D2O (Fig. 3). As a consequence, the scattering of the protein/NP assembly is also much weaker than in 100% D2O. The information that can be obtained by the Mb/NP scattering curve are limited (Fig. 3A, green squares): at Q > 0.05 Å -1, the signal to noise ratio is too weak to capture the features of the scattering with confidence. However, we can still see at Q < 0.05 Å -1, a strong decay with a power law Q-2.1 characteristic of the reactionlimited aggregation similar to what was observed in 100% D2O.


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Figure 4: SANS spectra in 60% D2O phosphate buffer. (A) Scattering of free Mb (red dashed line), free NP (blue circles) and a Mb/NP assembly (green squares). (B) Scattering of free Hb (red dashed line), free NP (blue circles), and a Hb/NP assembly (green squares). The NP concentration is constant at 50 g.L-1. The free protein concentrations correspond to the same concentration as the adsorbed protein concentration in the mixture (2.2 g.L-1 and 6.0 g.L-1 for Mb and Hb respectively). The contribution to the scattering from the non-adsorbed protein fraction was subtracted from the overall scattering, assuming that it is a linear combination of decorated proteins and free proteins (See Fig. S8). The continuous curve is the fitting of the Hb/NP assembly with a vesicle model (vesicle wall thickness fixed to 40 Å, black line). For the Hb/NP assembly, the overall scattering is larger as the mass of the Hb is 4 times larger than of the mass of Mb (Fig. 3B, green squares). The characteristic features of the scattering can thus be obtained: i) a strong upturn of the intensity at low Q, ii) two minima at intermediate Q around 0.02 A-1 and 0.04 A-1 and iii) a decay at high Q which superimposes with Hb form factor (for more details look at Fig. S8). We first attempted to model such scattering by the raspberry model (small spherical particles decorating a larger spherical core 48) that appeared to be well suited at first sight (for illustration, see Fig. S3). The model was however not satisfactory and the data were better fitted by a vesicle model (a continuous layer surrounding a spherical hollow core 49). Please note that the modeling by a vesicle model was not possible in the 100%D2O contrast, pointing out the influence of the protein-NP partial structure factor on the overall scattering curve. In these conditions, the adsorbed proteins layer appears as a corona surrounding the nanoparticles. In order to fit the data with the fewest possible number of parameters, we used, for the diameter of the core of the vesicle, the value determined above for the Ludox NP (26 nm, Fig. S1). It was then possible to fit the data by adjusting the corona thickness to 4.0 ± 0.5 nm. Within experimental errors, this value corresponds to the effective diameter of the hard sphere (4.2 nm determined above) and to the Hb tetramer gyration diameter (4.6 nm, Fig. S2). We therefore confirm that the tetrameric structure of Hb remains intact onto the surface and we can propose that the hemoglobin is sitting flat on the surface (interaction by two to three out of the four subunits) in a close-packed structure.


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Conclusions The overall picture that can be drawn from these experiments on mixtures of hemoproteins and silica nanoparticles is the following: i) The celebrated expression "protein corona" is particularly well chosen, as a layer of nanometric thickness appears when the protein adsorb on the surface, ii) Proteins conserve their shape and their quaternary structure in this layer and iii) there is some degree of organization in the protein monolayer, as shown by the appearance of a short distance repulsive liquid-like order. Therefore, the current picture drawn from studies on flat surfaces of protein deforming and spreading on the surface is, by no means, generalizable to nanoparticles. Therefore, the change in protein functionality upon adsorption (here an increase of oxygen binding activity) and the nanoparticle aggregation cannot be explained by a dramatic change in protein conformation. We also demonstrated that aggregation of NP can be induced by protein adsorption, a phenomenon probably neglected in recent descriptions of the impact of protein corona on nanoparticle bioactivity.

Author information Corresponding author [email protected] Author contribution The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This research was supported by a grant from the « Programme de Toxicologie » (NaToM grant) of the CEA. L.M. was supported by a CFR grant from the CEA. Supporting information 3D representation of protein-like objects adsorbed on a sphere, dcattering spectrum of Ludox NP, SANS spectra of Mb and Hb and NP, Graphic representation of the contrast matching principle, Adsorption data of Mb and Hb on various silica surfaces in H2O solutions, Experimental conditions used for the SANS experiments, Density (ρ) and Neutron Scattering Length Density (SLD) of the molecules involved in this study

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Figure 1: Adsorption isotherms of myoglobin (red dots) and hemoglobin (blue squares) adsorbed on silica nanoparticles in D2O phosphate buffer 0.1 mol.L-1 (pD 7.4). The experimental points are from two separate isotherms and the continuous lines are fits by the Langmuir model 89x69mm (150 x 150 DPI)


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Figure 2: Effect of adsorption on hemoglobin activity. Oxygen binding curves of Hb with (red dots) and without NP (blue squares) in D2O phosphate buffer 0.1 mol.L-1 (pD 7.4) at 25°C. The red curve was obtained with more than 95% of the Hb adsorbed, according to the isotherms measured in figure 1. The continuous lines show the best fits obtained by the Hill equation. 89x69mm (150 x 150 DPI)


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Figure 3: SANS spectra in 100% D2O phosphate buffer (pD 7.4). (A) Scattering of free Mb (red dashed line), free NP (blue circles) and a Mb/NP assembly (green squares). (B) Scattering of free Hb (red dashed line), free NP (blue circles), and a Hb/NP assembly (green squares). The NP concentration is constant at 50 g.L-1. The free protein spectra were measured at 10 g.L-1 and then normalized in order to correspond to the same concentration as the adsorbed protein concentration in the mixture (2.2 g.L-1 and 6.0 g.L-1 for Mb and Hb respectively). The contribution to the scattering from the non-adsorbed protein fraction was subtracted from the overall scattering, assuming that there is a linear combination of decorated and free proteins (see Fig. S5). (C) Structure factor of the Hb/NP assembly. In yellow, scattering spectrum of the Hb/NP assembly (from (B)) divided by the signal of Hb, in equal amount of the adsorbed Hb fraction. In black, fit of the correlation peak by the hard sphere structure factor with Percus-Yevick closing (effective radius of 2.1 nm). 84x209mm (150 x 150 DPI)


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Figure 4: SANS spectra in 60% D2O phosphate buffer. (A) Scattering of free Mb (red dashed line), free NP (blue circles) and a Mb/NP assembly (green squares). (B) Scattering of free Hb (red dashed line), free NP (blue circles), and a Hb/NP assembly (green squares). The NP concentration is constant at 50 g.L-1. The free protein concentrations correspond to the same concentration as the adsorbed protein concentration in the mixture (2.2 g.L-1 and 6.0 g.L-1 for Mb and Hb respectively). The contribution to the scattering from the non-adsorbed protein fraction was subtracted from the overall scattering, assuming that it is a linear combination of decorated proteins and free proteins (See Fig. S8). The continuous curve is the fitting of the Hb/NP assembly with a vesicle model (vesicle wall thickness fixed to 40 Å, black line). 363x138mm (96 x 96 DPI)


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ProteinâNanoparticle Interactions: What Are the ProteinâCorona

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