Adsorption of Myoglobin and Corona Formation on Silica Nanoparticles

Abstract

The adsorption of proteins from aqueous medium leads to the formation of protein corona on nanoparticles. The formation of protein corona is governed by a complex interplay of protein-particle and protein-protein interactions, such as electrostatics, van der Waals, hydrophobic, hydrogen bonding, and solvation. The experimental parameters influencing these interactions, and thus governing the protein corona formation on nanoparticles, are currently poorly understood. This lack of understanding is due to the complexity in the surface charge distribution and anisotropic shape of the protein molecules. Here, we investigate the effect of pH and salinity on the characteristics of corona formed by myoglobin on silica nanoparticles. We experimentally measure and theoretically model the adsorption isotherms of myoglobin binding to silica nanoparticles. By combining adsorption studies with surface electrostatic mapping of myoglobin, we demonstrate that a monolayered hard corona is formed in low salinity dispersions, which transforms into a multilayered hard + soft corona upon the addition of salt. We attribute the observed changes in protein adsorption behavior with increasing pH and salinity to the change in electrostatic interactions and surface charge regulation effects. This study provides insights into the mechanism of protein adsorption and corona formation on nanoparticles, which would guide future studies on optimizing nanoparticle design for maximum functional benefits and minimum toxicity.

Figure 1

(a) TEM image of silica NPs showing the diameter (∼30 nm) with a spherical shape. (b) ζ-Potential of silica NPs as a function of pH. The silica NPs are negatively charged in the range 4 < pH < 11. (c) Net charge on myoglobin as a function of pH. Silica NPs and myoglobin are oppositely charged below IEP_MGB, i.e., 4 < pH < 7. (d) X-ray crystal structure of horse myoglobin (PDB # 5ZZE). The yellow line structures indicate histidine residues in the protein. The His-93 and His-64 residues play a key role in charge regulation effects due to their physiologically relevant pK_a values.

Figure 2

(a) Schematic of the experimental procedure to determine the adsorption isotherms of myoglobin on silica NPs. Myoglobin and silica NPs are mixed at a desired pH and equilibrated for 24 h at room temperature. After separating silica NPs from the aqueous phase, the amount of unadsorbed protein was determined by measuring the absorbance of the supernatant at 280 nm. (b) Schematic representation of hard corona (solid-line circle) and soft corona (dashed circle) around a silica NP. (c) Absorption spectrum of myoglobin (conc. = 2.0 mg mL^–1) before adding silica NPs (red line) and after adding silica NPs and centrifugation at equilibrium (dashed line). (d, e) Sample cell of myoglobin in water showing the change of color before adding silica NPs (d) and after adding silica NPs and separating the NPs with adsorbed myoglobin using centrifugation (e).

Figure 3

Amount of protein adsorbed (Γ) as a function of the equilibrium concentration of protein (c_eq) at different pH and salinity. (a, b) Adsorption isotherms for myoglobin binding to silica NPs with no added NaCl at (a) pH < IEP_MGB and (b) pH > IEP_MGB. The scattered points are the experimental data, and the lines represent the best fit using the GAB model given in eq 2. For pH < IEP_MGB, the amount of myoglobin adsorbed rapidly reaches the maximum value due to the strong electrostatic attraction between the protein and NP. For pH > IEP_MGB, the amount of myoglobin adsorbed on NPs shows a gradual increase. (c, d) Experimental isotherms showing the adsorption of myoglobin on silica NPs with 100 mM NaCl added to solution at (c) pH < IEP_MGB and (d) pH > IEP_MGB.

Figure 4

(a) Schematic representation of adsorption constants K_hard for hard corona formation driven by protein–NP interaction and K_soft for soft corona formation driven by protein–protein interaction. The values of K_hard and K_soft are estimated by fitting the experimental adsorption isotherms using the GAB model, as given in eq 2. (b) Change in the maximum amount of protein adsorbed in hard corona (Γ_m) as a function of dispersion pH with and without adding NaCl. In the absence of added NaCl, the Γ_m is maximum at pH 8 due to the preferred orientation of the adsorbed protein on silica surface. (c, d) Adsorption constants for hard corona and soft corona, respectively, as a function of pH and salinity.

Figure 5

(a–c) Electrostatic surface maps for myoglobin (PDB # 5ZZE) at pH 4 (a), 8 (b), and 10 (c). The negative and positive residues on the protein are represented in red and blue, respectively. The inset in (a) is the color scale in the unit of elementary charge, e. The electric dipole moment vector of the protein is indicated by the arrow. Here, the view of the protein is fixed and the changes in the dipole moment with pH are represented by the change in the length and orientation of the vector shown by the arrow. (d) Magnitude of the dipole moment of myoglobin as a function of pH. The value of the total dipole moment shows a maximum near IEP_MGB due to asymmetric charge distribution on the protein surface.

Figure 6

(a) Fraction of myoglobin in the soft corona as a function of pH and concentration of NaCl. (b, c) Schematic representation of the structural change of the protein corona around a silica NP upon adding NaCl to a myoglobin–silica NP dispersion at pH < IEP_MGB (b) and pH > IEP_MGB (c).