Article pubs.acs.org/Langmuir
pH-Dependent Interaction and Resultant Structures of Silica Nanoparticles and Lysozyme Protein Sugam Kumar,† Vinod K. Aswal,*,† and P. Callow‡ †
Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085, India Institut Laue Langevin, 6 rue Jules Horowitz, B.P. 156, 38042 Grenoble Cedex 9, France
‡
S Supporting Information *
ABSTRACT: Small-angle neutron scattering (SANS) and UV−visible spectroscopy studies have been carried out to examine pH-dependent interactions and resultant structures of oppositely charged silica nanoparticles and lysozyme protein in aqueous solution. The measurements were carried out at fixed concentration (1 wt %) of three differently sized silica nanoparticles (8, 16, and 26 nm) over a wide concentration range of protein (0−10 wt %) at three different pH values (5, 7, and 9). The adsorption curve as obtained by UV−visible spectroscopy shows exponential behavior of protein adsorption on nanoparticles. The electrostatic interaction enhanced by the decrease in the pH between the nanoparticle and protein (isoelectric point ∼11.4) increases the adsorption coefficient on nanoparticles but decreases the overall amount protein adsorbed whereas the opposite behavior is observed with increasing nanoparticle size. The adsorption of protein leads to the protein-mediated aggregation of nanoparticles. These aggregates are found to be surface fractals at pH 5 and change to mass fractals with increasing pH and/or decreasing nanoparticle size. Two different concentration regimes of interaction of nanoparticles with protein have been observed: (i) unaggregated nanoparticles coexisting with aggregated nanoparticles at low protein concentrations and (ii) free protein coexisting with aggregated nanoparticles at higher protein concentrations. These concentration regimes are found to be strongly dependent on both the pH and nanoparticle size.
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INTRODUCTION Nanoparticles, because of their small size and large surface-tovolume ratio, possess unique and distinct properties that are useful for their numerous applications.1−3 Many of these applications require the interaction of nanoparticles with macromolecules such as surfactants, polymers, and proteins.4−6 In particular, in the field of nanobiotechnology, nanoparticle− protein complexes have attracted sufficient interest because they can gain access to and operate within the cell as a result of their size, which is comparable to that of a cell.7−9 This conjugation of nanoparticles with biomolecules is governed by various interactions such as covalent bonding, hydrogen bonding, electrostatic forces, and so forth, depending on the system properties. The degree of these interactions depends on the characteristics of both nanoparticles and proteins, which can easily be tuned through solution conditions.10−12 The silica nanoparticles and lysozyme proteins are one of the most studied model systems for understanding the behavior of the nanoparticle−biomolecule system.13−15 Proteins are charged molecules, and their function depends on the native folded structure. In the physiological environment, proteins are known to cover nanoparticles immediately; therefore, their structure and functionality may be disturbed. A number of studies have been carried out to examine the adsorption of proteins on nanoparticles and its effect on the protein structure © XXXX American Chemical Society
and activity using different techniques such as ellipsometry, reflectometry, circular dichroism (CD), nuclear magnetic resonance (NMR), Raman spectroscopy, infrared spectroscopy, and so forth.13,15−20 The presence of protein is also known to cause interactional changes in the nanoparticle systems.21−23 The tuning of interaction between nanoparticles, in particular, through the adsorption of different macromolecules, has been of recent great interest because the nanoparticle−protein system can lead from an enhancement in stabilization to the aggregation of particles. The dependence of particle-mediated adhesion energies on their adsorption curves have also been reported.24 The interaction between lysozyme protein and silica nanoparticles provides a system where both components are individual charge-stabilized colloids interacting via a short-range attractive potential combined with long-range repulsion. The interaction in their complexes between nanoparticles and protein is predominantly governed by the resultant electrostatic interactions that are known to lead to many nonspecific associations that are especially relevant to biological systems and have several important applications.25−29 We have recently Received: October 9, 2013 Revised: December 11, 2013
A
dx.doi.org/10.1021/la403896h | Langmuir XXXX, XXX, XXX−XXX
Langmuir
Article
having thickness 2 mm and temperature kept constant at 30 °C during the measurements. The data were corrected and normalized to absolute scale using standard procedure.
studied the structure formed as a result of electrostatic complexion between silica nanoparticles and lysozyme protein at physicochemical pH 7 when nanoparticles and protein both are oppositely charged.22 The protein adsorption was found to lead to the protein-mediated aggregation of nanoparticles that are characterized by the mass fractal structure. These aggregates are found to coexist with unaggregated particles at low protein concentrations and free proteins at higher protein concentrations. The occurrence of these two regimes is believed to be decided by the charge neutralization of nanoparticles by protein molecules.22 The competition of attraction between two components and repulsion between individual components in oppositely charged nanoparticles and protein systems plays an important role in determining the structure of the aggregates and the mechanism governing the morphology of the aggregates.23,29 For this purpose, the nanoparticle curvature and solution properties such as pH become important in controlling their interaction and resultant structure in these systems.15,23 The formation of different nanostructures from randomly branched complexes to single-stranded nanorods has been reported in oppositely charged nanoparticle and polyelectrolyte systems by tuning the electrostatic interactions whereas the growth of 3D networks of silica nanoparticles with different geometries is demonstrated in the nanoparticle−DNA system.30−33 It is interesting to know such an evolution of structures in nanoparticle−protein systems with the systematic variation in their charge states. In this article, we have utilized small-angle neutron scattering (SANS) to study the pH dependence of interactions of differently sized silica nanoparticles with lysozyme protein. SANS is an ideal technique for studying such multicomponent systems because of the unique advantage of easy contrast variation.22,34,35 UV− visible spectroscopy has been carried out to measure the adsorption curves of protein on nanoparticles. SANS measurements have been carried out at different protein concentrations on the adsorption curve with differently sized silica nanoparticles and pH values.
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■
SANS ANALYSIS In SANS experiments, one measures the coherent differential scattering cross section per unit volume (dΣ/dΩ) as a function of Q. For identical particles dispersed in a medium, it can be written as37−40 dΣ (Q ) = nP(Q ) S(Q ) + B dΩ
(1)
where n is the number density. P(Q) is the intraparticle structure factor, and S(Q) is the interparticle structure factor. B is a constant term representing the incoherent background. P(Q) accounts for the scattering from a single particle and hence depends on the shape and size of the particle whereas S(Q) is Fourier transform of the radial distribution function g(r) and hence correlates particles present in the system. For a core−shell structure having inner and outer radii R and R + ΔR, respectively, P(Q) may be given by following equation P(Q ) =
16π 2 [(R + ΔR )3 (ρshell − ρsol )F(Q , R + ΔR ) 9 2 − R3(ρshell − ρcore ) F(Q , R )] (2)
where F (Q , R ) =
3{sin(QR ) − (QR )cos(QR )} (QR )3
(3)
ρcore, ρshell, and ρsol are the scattering length densities of the core, shell, and solvent, respectively. In the case of particle aggregation as characterized by the mass fractal structure, S(Q) is accounted for by the following equation41 Smf (Q ) = 1 +
EXPERIMENTAL SECTION
Dm Γ(Dm − 1) 1 sin{(Dm Dm (QR b) [1 + (Qξ)−2 ](Dm − 1)/2
− 1) tan−1(Qξ)}
An electrostatically stabilized colloidal suspension of three differently sized silica nanoparticles (Ludox SM30, LS30, and TM50) and hen egg protein lysozyme were purchased from Sigma-Aldrich and Fluka, respectively. The adsorption curves of protein interaction with silica nanoparticles in aqueous solution were studied using an ND 1000 nanodrop spectrophotometer. The instrument is based on surface retention technology utilizing the surface tension to hold the sample. A pulsed xenon flash lamp is used as a source to cover the spectrum range from 220 to 750 nm, and the light coming through the sample is analyzed by CCD arrays. Absorbance spectra of protein solutions of different concentrations (wt %) prepared in H2O at three pH values (5, 7, and 9) were recorded as a function of wavelength. Three buffer solutions (20 mM) from acetate buffer for pH 5, phosphate buffer for pH 7, and Borax buffer for pH 9 were used to maintain the pH. D2O was used as the solvent in samples for SANS experiments because it provides better contrast for hydrogenous samples and a low incoherent background. Small-angle neutron scattering experiments were performed at the D22 facility at the Institut Laue Langevin, France.36 The mean wavelength (λ) of the neutron beam used was 6 Å with Δλ/λ ≈ 10%. Neutrons scattered from samples were detected using a 2D 1 m × 1 m 3He detector. Data were collected at three sample-to-detector distances of 2, 8, and 17.6 m to cover a wave vector transfer (Q = 4π sin(θ/2)/λ, where θ is the scattering angle) range of 0.003 to 0.35 Å−1. All of the measurements were carried out for a fixed concentration (1 wt %) of silica nanoparticles and a varying concentration of protein in the range of 0 to 10 wt %. Samples were held in HELMMA quartz cells
(4)
where ξ signifies the maximum length up to which the fractal microstructure exists, Rb is the size of the building block, and Dm is the mass fractal dimension. Therefore, the scattering cross section from mass fractals can simply be obtained by putting eq 4 in eq 1 The expression of S(Q) for the surface fractal structure is given by42,43 Ssf (Q ) = Q−1Γ(5 − Ds)ξ 5 − Ds 5
[1 + (Qξ)2 ](Ds − 2 ) sin[(Ds − 1) tan−1(Qξ)] (5)
In may be mentioned that the scattering intensity from both kinds of fractal structures is governed by power law behavior in a definite Q range.22,41,42 d∑ (Q ) ≈ Q−Dm dΩ
1 1
pH-Dependent Interaction and Resultant Structures of Silica Nanoparticles and Lysozyme Protein Sugam Kumar,† Vinod K. Aswal,*,† and P. Callow‡ †
Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085, India Institut Laue Langevin, 6 rue Jules Horowitz, B.P. 156, 38042 Grenoble Cedex 9, France
‡
S Supporting Information *
ABSTRACT: Small-angle neutron scattering (SANS) and UV−visible spectroscopy studies have been carried out to examine pH-dependent interactions and resultant structures of oppositely charged silica nanoparticles and lysozyme protein in aqueous solution. The measurements were carried out at fixed concentration (1 wt %) of three differently sized silica nanoparticles (8, 16, and 26 nm) over a wide concentration range of protein (0−10 wt %) at three different pH values (5, 7, and 9). The adsorption curve as obtained by UV−visible spectroscopy shows exponential behavior of protein adsorption on nanoparticles. The electrostatic interaction enhanced by the decrease in the pH between the nanoparticle and protein (isoelectric point ∼11.4) increases the adsorption coefficient on nanoparticles but decreases the overall amount protein adsorbed whereas the opposite behavior is observed with increasing nanoparticle size. The adsorption of protein leads to the protein-mediated aggregation of nanoparticles. These aggregates are found to be surface fractals at pH 5 and change to mass fractals with increasing pH and/or decreasing nanoparticle size. Two different concentration regimes of interaction of nanoparticles with protein have been observed: (i) unaggregated nanoparticles coexisting with aggregated nanoparticles at low protein concentrations and (ii) free protein coexisting with aggregated nanoparticles at higher protein concentrations. These concentration regimes are found to be strongly dependent on both the pH and nanoparticle size.
■
INTRODUCTION Nanoparticles, because of their small size and large surface-tovolume ratio, possess unique and distinct properties that are useful for their numerous applications.1−3 Many of these applications require the interaction of nanoparticles with macromolecules such as surfactants, polymers, and proteins.4−6 In particular, in the field of nanobiotechnology, nanoparticle− protein complexes have attracted sufficient interest because they can gain access to and operate within the cell as a result of their size, which is comparable to that of a cell.7−9 This conjugation of nanoparticles with biomolecules is governed by various interactions such as covalent bonding, hydrogen bonding, electrostatic forces, and so forth, depending on the system properties. The degree of these interactions depends on the characteristics of both nanoparticles and proteins, which can easily be tuned through solution conditions.10−12 The silica nanoparticles and lysozyme proteins are one of the most studied model systems for understanding the behavior of the nanoparticle−biomolecule system.13−15 Proteins are charged molecules, and their function depends on the native folded structure. In the physiological environment, proteins are known to cover nanoparticles immediately; therefore, their structure and functionality may be disturbed. A number of studies have been carried out to examine the adsorption of proteins on nanoparticles and its effect on the protein structure © XXXX American Chemical Society
and activity using different techniques such as ellipsometry, reflectometry, circular dichroism (CD), nuclear magnetic resonance (NMR), Raman spectroscopy, infrared spectroscopy, and so forth.13,15−20 The presence of protein is also known to cause interactional changes in the nanoparticle systems.21−23 The tuning of interaction between nanoparticles, in particular, through the adsorption of different macromolecules, has been of recent great interest because the nanoparticle−protein system can lead from an enhancement in stabilization to the aggregation of particles. The dependence of particle-mediated adhesion energies on their adsorption curves have also been reported.24 The interaction between lysozyme protein and silica nanoparticles provides a system where both components are individual charge-stabilized colloids interacting via a short-range attractive potential combined with long-range repulsion. The interaction in their complexes between nanoparticles and protein is predominantly governed by the resultant electrostatic interactions that are known to lead to many nonspecific associations that are especially relevant to biological systems and have several important applications.25−29 We have recently Received: October 9, 2013 Revised: December 11, 2013
A
dx.doi.org/10.1021/la403896h | Langmuir XXXX, XXX, XXX−XXX
Langmuir
Article
having thickness 2 mm and temperature kept constant at 30 °C during the measurements. The data were corrected and normalized to absolute scale using standard procedure.
studied the structure formed as a result of electrostatic complexion between silica nanoparticles and lysozyme protein at physicochemical pH 7 when nanoparticles and protein both are oppositely charged.22 The protein adsorption was found to lead to the protein-mediated aggregation of nanoparticles that are characterized by the mass fractal structure. These aggregates are found to coexist with unaggregated particles at low protein concentrations and free proteins at higher protein concentrations. The occurrence of these two regimes is believed to be decided by the charge neutralization of nanoparticles by protein molecules.22 The competition of attraction between two components and repulsion between individual components in oppositely charged nanoparticles and protein systems plays an important role in determining the structure of the aggregates and the mechanism governing the morphology of the aggregates.23,29 For this purpose, the nanoparticle curvature and solution properties such as pH become important in controlling their interaction and resultant structure in these systems.15,23 The formation of different nanostructures from randomly branched complexes to single-stranded nanorods has been reported in oppositely charged nanoparticle and polyelectrolyte systems by tuning the electrostatic interactions whereas the growth of 3D networks of silica nanoparticles with different geometries is demonstrated in the nanoparticle−DNA system.30−33 It is interesting to know such an evolution of structures in nanoparticle−protein systems with the systematic variation in their charge states. In this article, we have utilized small-angle neutron scattering (SANS) to study the pH dependence of interactions of differently sized silica nanoparticles with lysozyme protein. SANS is an ideal technique for studying such multicomponent systems because of the unique advantage of easy contrast variation.22,34,35 UV− visible spectroscopy has been carried out to measure the adsorption curves of protein on nanoparticles. SANS measurements have been carried out at different protein concentrations on the adsorption curve with differently sized silica nanoparticles and pH values.
■
■
SANS ANALYSIS In SANS experiments, one measures the coherent differential scattering cross section per unit volume (dΣ/dΩ) as a function of Q. For identical particles dispersed in a medium, it can be written as37−40 dΣ (Q ) = nP(Q ) S(Q ) + B dΩ
(1)
where n is the number density. P(Q) is the intraparticle structure factor, and S(Q) is the interparticle structure factor. B is a constant term representing the incoherent background. P(Q) accounts for the scattering from a single particle and hence depends on the shape and size of the particle whereas S(Q) is Fourier transform of the radial distribution function g(r) and hence correlates particles present in the system. For a core−shell structure having inner and outer radii R and R + ΔR, respectively, P(Q) may be given by following equation P(Q ) =
16π 2 [(R + ΔR )3 (ρshell − ρsol )F(Q , R + ΔR ) 9 2 − R3(ρshell − ρcore ) F(Q , R )] (2)
where F (Q , R ) =
3{sin(QR ) − (QR )cos(QR )} (QR )3
(3)
ρcore, ρshell, and ρsol are the scattering length densities of the core, shell, and solvent, respectively. In the case of particle aggregation as characterized by the mass fractal structure, S(Q) is accounted for by the following equation41 Smf (Q ) = 1 +
EXPERIMENTAL SECTION
Dm Γ(Dm − 1) 1 sin{(Dm Dm (QR b) [1 + (Qξ)−2 ](Dm − 1)/2
− 1) tan−1(Qξ)}
An electrostatically stabilized colloidal suspension of three differently sized silica nanoparticles (Ludox SM30, LS30, and TM50) and hen egg protein lysozyme were purchased from Sigma-Aldrich and Fluka, respectively. The adsorption curves of protein interaction with silica nanoparticles in aqueous solution were studied using an ND 1000 nanodrop spectrophotometer. The instrument is based on surface retention technology utilizing the surface tension to hold the sample. A pulsed xenon flash lamp is used as a source to cover the spectrum range from 220 to 750 nm, and the light coming through the sample is analyzed by CCD arrays. Absorbance spectra of protein solutions of different concentrations (wt %) prepared in H2O at three pH values (5, 7, and 9) were recorded as a function of wavelength. Three buffer solutions (20 mM) from acetate buffer for pH 5, phosphate buffer for pH 7, and Borax buffer for pH 9 were used to maintain the pH. D2O was used as the solvent in samples for SANS experiments because it provides better contrast for hydrogenous samples and a low incoherent background. Small-angle neutron scattering experiments were performed at the D22 facility at the Institut Laue Langevin, France.36 The mean wavelength (λ) of the neutron beam used was 6 Å with Δλ/λ ≈ 10%. Neutrons scattered from samples were detected using a 2D 1 m × 1 m 3He detector. Data were collected at three sample-to-detector distances of 2, 8, and 17.6 m to cover a wave vector transfer (Q = 4π sin(θ/2)/λ, where θ is the scattering angle) range of 0.003 to 0.35 Å−1. All of the measurements were carried out for a fixed concentration (1 wt %) of silica nanoparticles and a varying concentration of protein in the range of 0 to 10 wt %. Samples were held in HELMMA quartz cells
(4)
where ξ signifies the maximum length up to which the fractal microstructure exists, Rb is the size of the building block, and Dm is the mass fractal dimension. Therefore, the scattering cross section from mass fractals can simply be obtained by putting eq 4 in eq 1 The expression of S(Q) for the surface fractal structure is given by42,43 Ssf (Q ) = Q−1Γ(5 − Ds)ξ 5 − Ds 5
[1 + (Qξ)2 ](Ds − 2 ) sin[(Ds − 1) tan−1(Qξ)] (5)
In may be mentioned that the scattering intensity from both kinds of fractal structures is governed by power law behavior in a definite Q range.22,41,42 d∑ (Q ) ≈ Q−Dm dΩ
1 1
