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Functional Nanostructured Materials (including low-D carbon)
Nanoparticle Concentration vs. Surface Area in the Interaction of Thiol Containing Molecules: Towards a Rational Nano-Architectural Design of Hybrid-Materials Keshav Goel, Matias Zuñiga-Bustos, Caitlin Lazurko, Erik Jacques, Constanza Galaz-Araya, Francisco Velenzuela-Henriquez, Natalia L. Pacioni, Jean-François Couture, Horacio Poblete, and Emilio I. Alarcon ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03942 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019
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ACS Applied Materials & Interfaces
Nanoparticle Concentration vs. Surface Area in the Interaction of Thiol Containing Molecules: Towards a Rational Nano-Architectural Design of Hybrid-Materials Keshav Goel, †,¥, ‡ Matias Zuñiga-Bustos, §, ‡ Caitlin Lazurko, †,¥ Erik Jacques, † Constanza Galaz-Araya,§ Francisco Valenzuela-Henriquez,£ Natalia L. Pacioni, ∂,ø Jean-François Couture,¥ Horacio Poblete, §,,* and Emilio I. Alarcon†,¥* †Division
of Cardiac Surgery, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, Canada, K1Y 4W7. of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada, K1H 8M5. §Center for Bioinformatics and Molecular Simulations, Facultad de Ingeniería, Universidad de Talca, Campus Lircay S/N, Talca, Chile, 3460000. £Instituto de Matemática, Pontificia Universidad Católica de Valparaíso, Blanco Viel 596, Cerro Barón, Valparaíso, Chile, 2350026. ∂Universidad Nacional de Córdoba, Facultad de Ciencias Químicas, Departamento de Química Orgánica. Haya de la Torre y Medina Allende s/n, Ciudad Universitaria, Córdoba, Argentina, X5000HUA. øConsejo Nacional de Investigaciones Científicas y Técnicas (CONICET), INFIQC, Buenos Aires 1418, Córdoba, Argentina, X5000IND. Núcleo Científico Multidisciplinario, Dirección de Investigación. Universidad de Talca, Talca, Chile, 3460000. Millennium Nucleus of Ion Channels-Associated Diseases (MiNICAD), Talca, Chile, 3460000. KEYWORDS: Nanosurface engineering, isothermal titration calorimetry, molecular dynamics, association models, cystein containing peptides, silver nanoparticles, polydispersity. ¥Department
ABSTRACT: The effect of accounting for total surface in the association of thiol-containing molecules to nanosilver was assessed using isothermal titration calorimetry, along with a new open access algorithm which calculates the total surface area for samples of different polydispersity. Further, we used advanced molecular dynamic calculations to explore the underlying mechanisms for the interaction of the studied molecules in the presence of a nanosilver surface in the form of flat surfaces or as 3D pseudo-spherical nanostructures. Our data indicate that not only the total surface area available for binding but also the supramolecular arrangements of the molecules in the near proximity of the nanosilver surface strongly affects the affinity of thiol-containing molecules to nanosilver surfaces.
Introduction The increasing demand for functional materials with superior biological activities has stimulated the development of composites with improved biocompatibility.1-3 Although synthetic materials present attractive venues for improving the functionalities of biotemplates; our team and others have systematically demonstrated that incorporating sub-nanomolar concentrations of nanoparticles within biomimetic matrices are enough to modulate properties such as elasticity, resistance to enzymatic degradation, antimicrobial effects, and electroconductivity.4-17 However, when using nanoparticles as structural components in regenerative medicine; difficulties in calculating the nanoparticle concentration along with the still uncertain impact of the nanoparticle diameter on the interaction with the biomatrix, have hampered the rational progress in nanoscale engineering. Difficulties in calculating nanoparticle concentration are paradoxically a direct consequence of the nanoparticle formation’s mechanism. Thus, although molecules of the same kind are
created equally, this does not apply for nanoparticles, where even those originating from the same metal precursor under comparable experimental conditions are found in different diameters and sometimes shapes (a.k.a. polydispersity and polymorphism).9 Consequently, the very first step in advancing the rational design of nanoparticle-based functional materials for designing novel functional materials is to identify molecular motifs with high affinities for nanometric surfaces. However, measuring thermodynamic parameters for the interaction of molecules with nanoparticles that incorporates nanoparticle polydispersity, which is key for identifying these motifs that could be used downstream as novel nano-tools for biomaterial, remains elusive. Very recently, our research team has developed the first openaccess algorithm able to link nanoparticle size distribution, independent of the sample dispersion, with its concentration in solution: Nanoparticle Polydispersity Corrector (NANoPoLC).9 In the present work, we have further expanded the usability of NANoPoLC to provide
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total surface area available and use the concentration and surface area obtained for nanosilver structures, of different polydispersity, to determine thermodynamic parameters in solution using isothermal titration calorimetry for the association of three thiol containing molecules: lipoic acid (LA) and two peptide sequences: CLK-GP-Hyp-GP-HypGP (peptide 1) and CLK-K(-KLC)-GP-Hyp-GP-Hyp-GP (peptide 2). Experimental thermodynamic data was also compared with energy calculations carried out using advanced molecular dynamic simulations for the structural quantifications, as well as, free energy calculations of the adsorption of the peptides to nanosilver surfaces.18 Results Numerical calculation of total surface area We have previously described a mathematical model that incorporates a distribution function to describe the size distribution of nanoparticle systems (NP) using a normalized Gaussian distribution.9 This model can be applied to any size distribution of colloidal nanoparticles. In our equation, the probability of finding a NP of diameter “d” is expressed as: 𝑑 + ℎ―
∫𝑑 ―
(1)
𝑔(𝑡)𝑑𝑡 ≈ 𝑔(𝑑)ℎ
In eq. (1), (𝑎 ― ≤ 𝑑 < 𝑑 + ℎ ― ≤ 𝑏) and ℎ ≈ 0. We have previously shown that the concentration of nanoparticles can be expressed according to equation (2):6
use this equation and the fillable excel file has been also made available. Association of lipoic acid onto nanosilver surfaces Nanosilver particles were prepared following a protocol our research team has optimized over the years for the synthesis of biocompatible materials.4-5 This protocol uses sodium citrate as capping agent for the newly formed nanoparticles, whose size distribution can be tuned depending on the initial citrate concentration in solution.19 As our ultimate goal was to demonstrate the importance of considering total surface area in the calculations for the binding; once the nanoparticles were prepared the final citrate concentration were adjusted to 1.0 mM in all cases, see materials and methods. Figure 1 contains the data measured when lipoic acid (LA) was used to modify the citrate protected nanosilver surface. Fig. 1A schematically depicts how the replacement of citrate by LA takes place. In this study, we have used nanosilver samples with different polydispersities as shown in Fig. 1B (9±5 nm and 19±5 nm, p peptide 1 > lipoic acid. Interestingly, for the 19 nm nanosilver particles, there was larger associativity to the nanosilver surface when compared to the 9 nm samples. This could be explained in terms of the total surface area available alongside with intrinsic differences in the molecular packing in the near proximity of the nanoparticle surface. Our experimental data suggest that, for example, engineering of biomimetic structures with polymers bearing 2 CLK moieties as structural arms would allow for better anchoring of nanosilver to the 3D matrices, preventing metal oxidation and minimizing organ infiltration. Further studies using our algorithm and binding model will allow for screening of more complex structures including larger peptides and oligomers, which will pinpoint moieties with high affinity for other metal nanosurfaces, for example. Materials and Methods Chemicals and Reagents Silver nitrate (AgNO3), trisodium citrate (Na3C6H5O7), and 2Hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (I-
2959) were purchased from Sigma-Aldrich and used without further purification. Custom synthesized peptides (purity > 95%): Peptide 1: (CLK-GP-Hyp-GP-Hyp-GP, M-W = 1,051 g/mol), and peptide 2: (CLK-K(-KLC)-GP-Hyp-GP-Hyp-GP, M-W = 1,523 g/mol) from CanPep were used as received. All solutions were prepared using MilliQ water. Synthesis of citrate capped nanosilver Citrate capped nanosilver (AgNPs) were prepared as described with minor modifications.4, 25-26 Briefly, a deoxygenated (45 min N2) aqueous solution containing 0.2 mM AgNO3, 0.2 mM I-2959, and 1.0 mM, 0.2 mM, or 0.1 mM9, 19 sodium citrate was irradiated with UVA light (8 lamps, Luzchem LZC-4 photoreactor at 25.0±0.5°C) for 45 minutes. Following irradiation, sodium citrate was added to the 0.2 mM and 0.1 mM citrate solutions to obtain a final citrate concentration of 1.0 mM. Yellow translucent solutions were obtained for the 1.0 mM and 0.2 mM citrate solutions and a green translucent solution was obtained for the 0.1 mM citrate solution.19 The solutions were stored at room temperature and protected from light. Surface plasmon band spectra The surface plasmon absorption band (SPB) was followed throughout the absorbance spectra in a Libra S50 UV–Vis spectrophotometer (Biochrom) at room temperature, using 1.0 cm path length cuvettes. Dynamic light scattering (DLS) measurements Hydrodynamic size and zeta potential of the citrate capped AgNP solutions were measured using a Malvern Zetasizer Nano ZS at 20°C in 1.0 cm path-length disposable plastic cuvettes. Reported values correspond to the average of three independent batches, each measured in triplicate. Transmission electron microscopy (TEM)
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ACS Applied Materials & Interfaces Samples were prepared by applying ~5.0 μL (1/10th diluted) of fresh AgNP solution to carbon-coated copper grids (400 mesh) and dried in a vacuum system for at least three days. High-resolution electron microscopy images were obtained using a FEI-Tecnai G2 F20 TEM operating at 75 kV. Nanosilver stability The stability of the AgNPs following addition of the peptides was determined by monitoring the absorbance of the AgNPs between 360550 nm at room temperature, using 1.0 cm path length cuvettes. Different concentrations of the peptides were tested until a final concentration was determined to be stable. To test the stability, 150 µL of AgNPs were added to wells of a 96 well plate (M2 SpectraMax, 350550 nm). Next, increasing volumes between 1 µL to 10 µL of the peptides were added to ten wells, at increments of 1 µL. The absorbance was measured to determine NP stability. Tests were completed in triplicate to confirm AgNP stability. Nanosilver stability under high ionic strength was assessed in solutions containing 150 mM NaCl, see Fig. S1. Isothermal titration calorimetry (ITC) Stable AgNPs samples were analysed using a VP-ITC MicroCalorimeter from Malvern Isothermal titration calorimeter system. Briefly, 2 mL of either 1 mM, 0.2 mM or 0.1 mM sodium citrate capped AgNPs and 2 mL dH2O were degassed for 15 min and loaded into the ITC sample cell and reference cell, respectively. Temperature was calibrated to 25 °C and the injection syringe was loaded with 300 µL of peptides 1, 2, or lipoic acid at concentrations of 250 µM or 500 µM, based on stability. Parameters were set as: 20 injections, 1 µL per injection, 10 s injection time, and 600 s injection spacing. For some of the peptide 1 samples, parameters were set as 20 injections, 0.5 µL per injection for the first 5 injections then 1 µL for the remaining injections, 10 s injection time, and 600 s injection spacing. Each run was analyzed using Microcal Origin and data extracted for plotting in Kaleida Graph 4.5, see below. Association constant calculations from ITC In our experiments, the ITC cell contained AgNP@citrate, which implies there is a pre-bounded population of citrate on the nanoparticle surface. Thus, when the ITC experiment began, the new ligand e.g. lipoic acid (LA) would replace citrate from the AgNP surface. Thus, the actual binding should be described using a competitive binding model. However, as Ka and H for the interaction between citrate and nanosilver remain unknown; it is not possible to determine the exact binding constant but rather an apparent association constant (KLapp): 𝐿 𝐾𝐿𝑎𝑝𝑝 = 𝐾𝑎 (1 + 𝐾𝑐𝑖𝑡 𝑎 [𝑐𝑖𝑡𝑟𝑎𝑡𝑒])
(5)
Thus, the heat (Q) released during an ITC titration is defined as
𝑄 = 𝑎[𝑐𝑜𝑚𝑝𝑙𝑒𝑥]; 𝑎 is VHo
(6)
Now, we will assume every AgNP has n identical binding sites and, binding is not competitive so this equilibrium can be written as:27
𝑠𝑖𝑡𝑒 + 𝐿⇌𝑐𝑜𝑚𝑝𝑙𝑒𝑥 then
𝐾𝐿𝑎𝑝𝑝 =
[𝑐𝑜𝑚𝑝𝑙𝑒𝑥]
(7)
[𝑠𝑖𝑡𝑒][𝐿]
Taking into account eq. 6, and the mass balance eq. 7 can be written as:
𝐾𝐿𝑎𝑝𝑝 =
𝑄/𝑎 𝑄
𝑄
([𝑠𝑖𝑡𝑒]0 ― 𝑎 )([𝐿]0 ― 𝑎 )
(8)
The binding site surface density for the AgNP and the ligand can be written as:
[𝑠𝑖𝑡𝑒]0 = n
𝑚𝑁𝐴𝑆𝑁𝑃 𝑇𝐴 𝑉
= n[A] (9)
where [A] is the AgNP average total surface area v𝑖𝑏𝑁𝐴𝑆𝐿𝐴
[𝐿]0 =
(10)
𝑉
where b is the number of moles in a liter of ligand stock solution, vi is the injection volume, V is the cell volume, NA is the Avogadro 𝐿 number and 𝑆𝐴 is the surface area per ligand molecule. Then, replacing [site]0 in eq. 10 𝑄/𝑎
𝐾𝐿𝑎𝑝𝑝 =
𝑄
(11)
𝑄
(𝐧[𝐴] ― 𝑎 )([𝐿]0 ― 𝑎 )
and solving for Q that becomes (see further details in the supporting information) 𝑎
𝑄=2
{(
1 𝐾𝐿𝑎𝑝𝑝
) (―
+ 𝐧[A] + [𝐿]0 +
𝟐
1 𝐾𝐿𝑎𝑝𝑝
)
} (12)
2
― 𝐧[A] ― [𝐿]0
― 4𝐧[A][𝐿]0
Now, if we differentiate eq. 12 𝑑𝑄 𝑎 𝑎 =2+2 𝑑[𝐿]0
(―
1 𝐾𝐿 𝑎𝑝𝑝
(―
― 𝐧[A] ― [𝐿]0 ― 2𝐧[A])( ― 1)
(13)
2
1 𝐾𝐿 𝑎𝑝𝑝
) ― 4𝐧[A][𝐿]
― 𝐧[A] ― [𝐿]0
0
Then, rearranging 𝑑𝑄 𝑑[𝐿]0
𝑎
(
(
=2 1+
1 𝐾𝐿 𝑎𝑝𝑝
+ [𝐿]0 ― 𝐧[A]
)
)
2 𝟐𝐧[𝐿]0 1 𝟐𝐧[A] ( 𝐿 ) + (𝐧[A])2 + [𝐿]02 + 𝐿 ― 2𝐧[𝐴][𝐿]0 + 𝐿 𝐾𝑎𝑝𝑝 𝐾𝑎𝑝𝑝 𝐾𝑎𝑝𝑝
(14)
𝐿 and replacing with XS:[𝐿]0/[𝐴] and 1/r:[A] 𝐾𝑎𝑝𝑝
𝑑𝑄 𝑑[𝐿]0
=
𝑉∆𝐻 2
(1 +
𝑟 + 𝑋𝑆 ― 𝐧
(𝑋𝑆)2 ― 2𝐧𝑋𝑆(1 ― 𝑟) + (𝐧 + 𝑟)𝟐
)
(15)
Statistical analyses Otherwise indicated, data was expressed as the mean ± standard error. Statistical analyses were carried out using Kaleida Graph 4.5® (Synergy Software). All values were tested for significance (P
