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Structural and Kinetic Visualization of the Protein Corona on Bioceramic Nanoparticles Ramon Rial, Brandon Tichnell, Brendan Latimer, Zhen Liu, Paula V. Messina, and Juan M. Ruso Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03573 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018
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Structural and Kinetic Visualization of the Protein Corona on Bioceramic Nanoparticles Ramón Riala, Brandon Tichnellb, Brendan Latimerb, Zhen Liub, Paula V. Messinac, Juan M. Rusoa*
(a) Soft Matter and Molecular Biophysics Group, Department of Applied Physics, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain. (b) Department of Physics and Engineering, Frostburg State University, Frostburg, MD, USA 21532. (c) Department of Chemistry, Universidad Nacional del Sur, INQUISURCONICET, B8000CP, Bahía Blanca, Argentina *Author to whom correspondence should be addressed: email:
[email protected] Abstract
Bioceramic nanoparticles exhibit excellent features that enable them to function as ideal material for hard tissue engineering. However, to fully understand its behavior, it is of crucial importance to understand their behavior within the fluids of the human body. To achieve this goal, we have studied the interaction between hydroxyapatite nanorods (HA) and bovine serum albumin (BSA). First we describe the surface morphology of the nanoparticle. Then, the main characteristics of the physiological interplay of BSA and hydroxyapatite nanoparticle are presented by using a battery of techniques: ITC, zeta potential, UV-vis, fluorescence and CD. Experimental data was analyzed by developing specific approaches to determine important parameters such as rates, affinities, and stochiometries of protein associated with the nanoparticles. ITC has been confirmed as a powerful technique to determine the affinity, binding, and thermodynamics of BSA-nanoparticle interactions. Careful quantitative assessment of the kinetics properties of the adsorption was revealed by UV-vis and fluorescence measurement. Finally, CD measurements highlight the important role of the protein flexibility in these kind of systems.
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Introduction
It is beyond expectation on what the bionanotechnology has accomplished and reached in just a matter of time. It has the potential to provide benefits to people and societies and improve health and environment. Bionanotechnology led to increasing the interest in the study of the interplay of low dimensional structures with biological entities. In parallel, important research in the area of biomedical research have demonstrated the usefulness of nanoparticles as the centerpiece of drug/gene delivery, visualization, or tissue engineering1. However, the biological effect of the nanoparticles depends on their ability to reach the target organs or cells inside the body. Achieving these targets involves routes of administration such as intravenous, where the nanoparticles interact with
blood
components,
namely
plasma
proteins,
thus
determining
their
hemocompatibility. Hence, the evaluation of hemocompatibility requires several approaches, including, in particular, the study of these interactions. One of the concepts that best characterizes and defines these types of relationships is the “protein corona”, that is, the protein layer absorbed on the nanoparticle. Although this term was first used to describe the adsorption of blood proteins onto polymeric nanoparticles2, it is usually considered as the starting point of regular studies on this topic in the work published by Cedervall et al3. The concepts “hard” and “soft” protein corona have been proposed to designate tightly and loosely adsorbed protein coatings over the nanoparticle surface, respectively. The first soft corona can be detected already at early stages and will derive over time to become the hard corona4. However, the organization and structure of these protein layers depends on both the intrinsic properties of the surface of adsorption and the proteins (size, charge, shape) as well as various extrinsic factors (time, concentration, velocity)5. For example, the evolution of bovine serum albumin on Au nanoparticles from weakly connected to an irreversible strong bonded layer with time was monitored by using different experimental techniques6. The effect of nanoparticle size is a key factor, especially when nanoparticles are smaller than most proteins, with a consequent lack of information of their biological repercussions. It was recently demonstrated that the thickness and density of this protein coating depended strongly on particle size7. Variations of ionic strength or pH can alter nanoparticle collision rates leading to
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modifications in their stability and promoting dissolution and change the molecularscale interaction with cells8. The effect of the surface charge has also been evaluated. It has been found that the adsorption of HSA is independent of the sign of surface charge. In addition, both the adsorption model (slightly anti-cooperative) and the number of adsorbed protein molecules are similar regardless of the sign of the net electric charge of the nanoparticles9. The role of temperature on the formation of the corona has been approached much less frequently, despite its enormous relevance. Mahmoudi et al10 demonstrated that it is determinant in the coverage rate and in the configuration of the corona, magnifying its influence in the range of physiological temperatures. Computational approaches have also contributed to characterize the protein corona. Specifically, they have concluded that the surface adsorption energy is a key driver when nanoparticles are introduced into biological systems11. From the proteins spatial location perspective, functional motifs and binding sites among others determine the adsorption process: stochastic, reversible, irreversible12. However, nanoparticles can alter both the biological activity and the three-dimensional structure of proteins, simply through soft interactions that are size function13. Finally, it is clear that the combination of both entities offers many opportunities: BSA corona contribute to decrease the high level of virulence of gold nanorods by cancelling the interactions that can cause damage to the lipid membrane of the cells14; functionalization of nanoparticles surfaces can change steric barriers, which causes an important reduction in both the amount of protein adsorbed and the recognition by macrophages15. While protein corona onto metallic nanoparticles has been deeply studied, the formation onto nanobioceramics is still largely unexplored. Only a few papers have systematically focused on this topic16,17. This fact draws much attention, especially considering that synthesis of nanobioceramics are cheaper than the metallic ones and avoid the manipulation of harsh, harmful products used in the latter. In light of this reality it is obvious that a more systematic research is required to ensure a consistent knowledge of this topic. For this reason, in this paper we have used a multiple experimental approach to understand and parameterize the interactions of bovine serum albumin (BSA) with hydroxyapatite (HA) nanoparticles. BSA was chosen because is a well-known model protein and HA because of its excellent biocompatiblilty, bioactivity and osteoconductivity properties18. The combination of
these elements offers a promising avenue for applications in health and biomedical areas. ACS Paragon Plus Environment
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Materials and methods
Reagents Hexadecyl-trimethyl ammonium bromide (CTAB, MW =364.48 g mol−1, 99% Sigma), polypropylene glycol (PPG, molecular weight: 425 g mol−1, density: 1.004 g cm−3 at 25 °C), sodium phosphate, calcium chloride and sodium nitrite were purchased from Sigma-Aldrich and they were used directly without purification. For solution preparation, only triple-distilled water was used.
Preparation of HA and BSA samples Hydroxyapatite nanorods were prepared using a modification of the method proposed by Liu et al.19. First, 35 mL of a 3.13 mM CTAB aqueous solution was mixed with 2 mL of polypropylene glycol and mechanically agitated for 10 min. Second, 20 mL of 2 M NaNO2 solution and 0.22 g of CaCl2 were subsequently incorporated. At the end, 20 mL of 140 mM of Na3PO4 aqueous solution was added to the above mixed solution, in a constant drip, at room temperature under mechanical shaking. Once all the components were added, the solution was stirred constantly for one hour. The next step consisted in introducing the mixture for 24 hours in an autoclave at 100 ° C. At the end of this step, we proceeded to filtering and washing with distilled water of the materials in order to minimize the presence of impurities and then dried at 50ºC for 24 h. Finally, the resulting material was ignited in the muffle furnace for 3 h at about 400 ºC. Hydroxyapatite nanoparticles of 8 ± 1 nm diameters and 28 ± 3 nm length were made by a previously described methodology.20 To prepare the stock solutions of HA, 0.0301 g and 0.0030 g of HA powder were respectively added to vials with 0.015 L of distilled water. These solutions were vigorously sonicated for 10 minutes to obtain homogeneous dispersions. Protein solutions were freshly prepared by dissolving a known amount of the protein in buffer (PBS, pH 7.4) and used the day of each experiment. Therefore, 2.0 x 10-4 mol L-1 of BSA was prepared by dissolving 0.1992 g of solid BSA in vials with 0.015 L of buffer and stored at 273-277 K. Before the beginning of each experiment, BSA solutions were exposed to room temperature (RT) for a maximum of 1 h.
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Structural characterization of HA nanorods. X-ray powder diffraction. The samples were analysed on Philips type powder diffractometer fitted with Philips “PW1710” control unit, Vertical Philips “PW1820/00” goniometer and FR590 Enraf Nonius generator. The equipment consisted of a graphite diffracted beam monochromator and copper radiation source (λ(Kα1)=1.5406Å), operating at 40 kV and 30mA. The X-Ray powder diffraction pattern (XRPD) has been collected by measuring the scintillation response to Cu Kα radiation versus the 2Θ value over a 2Θ range of 10-70, with a step size of 0.02° and counting time of 2 s per step.
Field emission scanning electron microscopy (FE-SEM). Surface morphology was evaluated using a field emission scanning electron microscope (ZEISS FE-SEM ULTRA PLUS). To acquire all the SEM images a secondary electron detector was used. The accelerating voltage (EHT) applied was 3.00 kV with a resolution (WD) of 2.1 nm. The associated energy-dispersive spectrophotometer provided qualitative information about surface elemental composition.
TEM. Particle size distributions were characterized by transmission electron microscopy (TEM) with a 200 kV ultrahigh resolution analytical electron microscope JEOL JEM-2010.
Isothermal titration calorimetry. Experiments were carried out in a VP-ITC microcalorimeter (MicroCal Inc., Northampton, U.S.).21 In order to determine the binding isotherms, the hydroxyapatite solutions (0.2 mM and 0.5 mM) were introduced into the syringe (296 µL), while the BSA solutions (0.02 mM and 0.05 mM) were introduced into the sample cell (1.4166 mL). To avoid HA precipitation, the stirring was kept constant at 416 rpm. An important factor to take into account is the equilibrium time necessary before starting each experiment, in our case it was one hour, more than enough for the power base line to remain stable. Injections of 10 µL at a constant rate of 0.5 µL s-1 were performed every 300 s. To eliminate negative signals, a reference power of 25 µJ s-1 was applied. Thus, it is guaranteed that the signal is not altered by any overcompensation mechanism. Dilution experiments of pure BSA were also conducted. The values obtained were systematically subtracted from those measured for the HABSA systems. In this way it is guaranteed that all the heat produced in the cell is due
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solely to the binding process. Following the described procedure, experiments were performed at a temperature of 298.15 K.
Fluorescence measurements. Fluorescence measurements were performed on a Cary Eclipse spectrofluorimeter. The excitation and emission splits were 5 nm. The synchronous fluorescence spectra were obtained by setting the data interval at 1 nm and the averaging time at 0,5 seconds. The range used was 290-600 nm upon excitation at 280 nm. The fluorescence spectra of BSA-HA were recorded at 288.15, 298.15 and 309.15 K maintaining the BSA concentration constant at 0.05 mM, and increasing concentrations of HA from 0 mM to 1 mM.
UV-Vis-IR Spectral Software from
FluorTools was used for data processing22,23.
UV-vis absorption spectra. The UV-vis absorption measurements were recorded with a Cary 100 Bio UV-Vis Spectrophotometer. The investigated spectral range was 250-600 nm. Two different tests were performed: first a series of BSA solutions with increasing concentrations of HA, 0-0.5 mM, were studied; secondly two dynamic tests were carried out, in which the BSA concentration was maintained at 0.05 mM, and the HA concentrations were 0.05 and 0.5 mM, respectively. The absorption spectra were measured 1, 3, 6, and 24 hours after the samples preparation and they were constantly stirred at 250 rpm during the whole experiment.
Zeta potential. A Malvern Zeta Sizer Nano (ZS90) with a He-Ne laser ( = 633 nm) was used for zeta potential measurements. Malvern`s software provides the zeta potential from electrophoretic mobilities using the Henry equation.
CD spectroscopy. Far-UV circular dichroism (CD) spectra were obtained using a JASCO-715 automatic recording spectropolarimeter (Japan) with a JASCO PTC-343 Peltier-type thermostated cell holder. Quartz cuvettes with 0.2-cm pathlength were used. CD spectra of pure BSA and nanoparticle_BSA dilute solutions were recorded from 190 to 270 nm. The following setting was used: resolution, 1 nm; bandwidth, 1 nm; sensitivity, 50 mdeg; response time, 8 s; accumulation, 3; and scan rate, 50 nm/min. Corresponding absorbance contributions from buffer solution were subtracted with the same instrumental parameters. Data was reported as molar ellipticity.
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Results and discussion
The obtained XRD patterns of our materials are shown in Figure 1. It can be observed that they perfectly match with the bibliographical patterns; JCPDS file no. 9-432. All peaks were indexed as hexagonal Ca5(PO4)3(OH) and no peaks associated with pollutants have been found, e. g. NaNO2 or Na3PO4. The XRD peaks had markedly broadening effect. This fact suggests that HA structures were nanosized19. The rate of crystalline phase was obtained by using the formula 1 ⁄ ⁄ where I300 correspond to the (300) peaks and V112/300 is the ratio between (112) and (300) diffraction signals of HA. The value obtained was 0.76. This value indicates poor crystallinity, which is in very good agreement with biogenic hydroxyapatite. is a very relevant magnitude, it has been demonstrated that as the crystallinity decreases the material is more resorbable and consequently more beneficial for early bone growth24.
Figure 1. X-ray diffraction pattern of hydroxyapatite
The general morphology of the samples was revealed by SEM images, top images of Figure 2. The samples have a uniform and homogeneous structure. At higher magnification it can be observed that the surface has a desultory network pattern composed by highly agglomerated nanoparticles. The clumped nanoparticles seem to have a very similar size. This fact reveals that Ostwald ripening does not play an
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important role in this process and making this morphology thermodynamically stable. Quantitative examination of SEM images shown that the size distribution of the nanoparticles is unimodal with an average length of 96 nm. As it was expected from the bibliographical source, resulting in a very large aspect ratio19. Although several studies have emphasized the importance of oriented assembly in the development of several nanomaterials, there are very few works that include quantitative information on this subject. Chen et al24 revealed that the aligned selfassembly of HA nanorods through certain directions, contrasting stochastic arrangement, could be achieved by hydrothermal treatment. This fact would add another option by opening new routes in the architecture and synthesis of innovative biomaterials. For this purpose, we analyzed the SEM images, intending to determine if the caked particles that make up our material had a random orientation or not. The orientation was calculated as the angle between the line through the long axis of the nanoparticle and the horizontal X-axis. For the surfaces analyzed the histogram obtained is plotted in Figure 3. The distribution is observed to be bimodal with peaks centered on 43º and 139º suggesting that the nanoparticle aggregation process is not isotropic but has favored arrangements probably related with the size and shape of the nanoparticles.
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Figure 2. Top: SEM images of HA samples at different scales, 1 µm (left) and 200 nm (right). Bottom: TEM images of the same HA samples.
Figure 3. Particle size distribution of HA nanoparticles (Left). Histogram of HA angle (Right).
For the complete characterization of the nanoparticles that make up our materials, these were diluted in butanol and sonicated. After that, samples obtained were observed by TEM (Figure 2 bottom). These images clearly show that the material is composed by uniform rod-like particles with similar aspect ratios (length/diameter). The size
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distribution histogram displays mean values of 9 ± 2 nm in diameter and 50 ± 7 nm in length. In order to explore the interaction of BSA with the nanoparticles, the isothermal titration calorimetry (ITC) technique was used. The amount of BSA in the calorimetric cell and the HA in the syringe were 0.05 and 0.5 mM, respectively. Figure 4 shows the dependence between the amount of heat released per injection and the molar ratio of the HA to the BSA. Reported values were calculated after subtracting the blank from the experiments to neglect heats from protein and HA dilution. The results clearly show that the binding interaction between BSA and HA nanoparticles is exothermic and gradually decreased with increasing HA/BSA ratio. This result falls within the standard, most of the nanoparticle-protein interactions are exothermic in a monotonic way, just a small number of them are endothermic24.
Figure 4. Heat of interaction for titration of HA nanoparticles into BSA solution at 298.15 K. The solid line corresponds to a fit of the experimental points to a one site binding equation. The values of the thermodynamic functions of the adsorption process can be obtained by fitting the experimental values of heat of interaction to isothermal functions. After trying different models to determine which of them best fits the data, we picked a unique set of indistinguishable sites in the binding analysis which means the protein adsorbed in the same way onto the nanoparticle surface. The binding constant (K),
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enthalpy change (∆H), and binding stoichiometry (n) were obtained from the results of the fit of the curves. The entropy changes (∆S) were obtained from the standard relationships of thermodynamics. The values obtained were: n= 1.1 ± 0.3; K = (4.87± 1.23)×104 M-1; ∆H = -1093 ± 280 kJ·mol-1 and ∆S = -9.23 ± 3.67 kJ·mol-1 K-1. The obtained quantity of enthalpy is directly related to the electrostatic and hydrophobic interactions, formation of hydrogen bonds and π-π interaction. On the other hand, hydration and conformational restriction of the amino acid residues on protein`s surface after adsorption commit to adverse entropy loss. Thus, according to our values, in the interaction of BSA with HA nanoparticles the enthalpy plays a key role in revealing that the impelling forces behind the adsorption process were non-covalent bondings25. Paralleling ITC, the z-potential measurements also showed an evolution of the protein corona. Figure 5 demonstrates the zeta potential measurements of the complex HA-BSA against BSA/HA ratio. The initial negative value is very similar to those of pure HA nanoparticles (-16 mV). This value is consistent with previous understanding where it was found that the zeta potential of HA nanoparticles increases when their size decrease26. As the protein concentration increases an expeditious decrease in the zeta potential was found out, clearly suggesting the binding of BSA. After this minimum a slightly increase and a final plateau is observed. In this media, both the nanoparticles and the protein have negative charge, however, this is not surprising, it was previously reported that protein adsorption onto nanoparticles generates a shift in zeta potential regardless of original surface charge27. Regarding the changes in zeta potential profile while considering the isotropic surface of the nanoparticles, intuitively the obtained results stemmed from the architectural peculiarities of BSA.
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Figure 5. Zeta potential of HA nanoparticles as a function of BSA/HA ratio.
The presence of chromophores in most proteins makes them viable for fluorescence spectroscopy analysis. BSA has two tryptophan placed at locations 134 and 212 of the chain. Trp-212 is placed in a region of the BSA where clusters of residues with hydrophobic moieties coexist and Trp-134 is placed on the exterior of the protein27. Figure 6 pictures the fluorescence emission spectra of pure BSA and BSA with different concentrations of HA nanoparticles. During observation, BSA demonstrates a great fluorescence emission band at 345 nm, while first being excited with a wavelength of 280 nm. The fluorescent emission intensity of BSA decreased regularly when it is titrated with various concentrations of HA. The great quenching of the BSA fluorescence suggests that the microenvironment around the Trp 134 residue was in an intensive hydrophobic surrounding when HA is added. In addition, no changes in shape or red/blue shifts have been observed in the peak. Considering independent and non-interactive binding sites, the experimental data were evaluated by the well-known Stern-Volmer equation:
1
(1)
F0 and F are the steady-state fluorescence intensities with and without quencher. Ksv represents Stern-Volmer quenching constant and [Q] corresponds to the concentration of the quencher, in this case, is hydroxyapatite.
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Figure 6. Fluorescence emission spectra of BSA in the absence and presence of different concentrations of HA nanoparticles (left). Overlap of the fluorescence spectrum of BSA and de absorbance spectrum of HA. [BSA]/[HA] = 1 (right)
The shape of the F0/F against [Q], plots informs about the quenching type. These are either static or dynamic. When the quenching process is both static and dynamic, the Stern-Volmer plot follows a continuous ascending curve28. In this case, linear correlations were observed between the intensities and the HA concentration (R2=0.924, 0.973, 0.945) which suggests that all Trp(s) in the protein differ slightly in accessibility29. Ksv, obtained from the slopes, were (8.90 ± 0.01) × 103, (8.69 ± 0.01) × 103 and (6.68 ± 0.01) × 103 L mol-1 at 288.15, 298.15 and 309.15 K. As the binding constant increases with a decrease in temperature, we can infer that a static quenching procedures is the most likely mechanism of fluorescence in BSA and HA. The assumption of the static quenching process reveals the generation of a system bounded by HA and BSA. This system was verified from the results obtained for the quenching rate constant. This constant represents the highest scatter collision quenching constant of different quenchers with the BSA, ⁄ , here τ0 represents the mean half-life of the molecule in the absence of quencher. The value of τ0 in the present case is 10-8 s-1 30
, therefore, Kq has a value of: 8.69×1010 L M-1 s-1. This value is greater than the
highest scatter collision quenching constant of different quenchers with the BSA which is 2 x 1010 L·mol-1·s-1 31. Kq values of BSA quenching process originated by HA are superior to the Kq of the scattered procedure indicates that the probable quenching mechanism involves complex formation rather than dynamic collisions32.
Fluorescent measurements allow us to get quantitative information about the binding process. Assuming proteins are attached one by one to a set of equivalents sites on nanoparticle surface, the following equation can be applied33
( )
! " ∙
(2)
Ka is the binding constant of HA with BSA and n the number of binding sites per BSA molecule. Linear fitting of experimental data indicates values of (3.80 ± 0.83) × 103 M-1 and 1.24 ± 0.22 for Ka and n, respectively. We conclude there is only one independent
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class of binding sites between HA and BSA, demonstrating astounding agreement with ITC measurements. The length between the protein and the attached HA can be calculated from fluorescent resonance energy transfer (FRET). Energy transfer take places when the fluorescent emission band of the donor overlaps with an excitation band of the acceptor molecule that is within 2-8 nm.34 The overlay of both plots: fluorescence of BSA and absorption of HA at 298.15 K is pictured in Figure 6. The transfer rate for a donor and acceptor located between each other at a length r is given by: 35 $ % (&)
'( ) * +( , -
.
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/ (01 2 34 567
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