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Mechanisms of Modulation of Calcium Phosphate Pathological Mineralization by Mobile and Immobile Small-Molecule Inhibitors Meng Li, Jing Zhang, Lijun Wang, Baoshan Wang, and Christine V Putnis J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10956 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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The Journal of Physical Chemistry

Mechanisims of Modulation of Calcium Phosphate Pathological Mineralization by Mobile and Immobile Small-Molecule Inhibitors

Meng Li,† Jing Zhang,† Lijun Wang,*,† Baoshan Wang,*,‡ and Christine V. Putnis§,¶

†

College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China ‡

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China § Institut für Mineralogie, University of Münster, 48149 Münster, Germany ¶

Department of Chemistry, Curtin University, Perth, Western Australia 6845, Australia

*

To whom correspondence should be addressed.

Lijun Wang College of Resources and Environment Huazhong Agricultural University Wuhan 430070, China Tel/Fax: +86-27-87288382 Email: [email protected]

Baoshan Wang College of Chemistry and Molecular Sciences Wuhan University, Wuhan 430072, China Email: [email protected]

ABSTRACT: Potential pathways for inhibiting crystal growth are either via disrupting local microenvironments surrounding crystal−solution interfaces or physically blocking solute molecule attachment. However, the actual mode of inhibition may be more complicated due to

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the characteristic time scale for the inhibitor adsorption and relaxation to a well-bound state at crystal surfaces. Here we demonstrate the role of citrate (CA) and hydroxycitrate (HCA) in brushite (DCPD, CaHPO4·2H2O) crystallization over a broad range of both inhibitor concentrations and supersaturations by in situ atomic force microscopy (AFM). We observed that both inhibitors exhibit two distinct actions: control of surface crystallization by the decrease of step density at high supersaturations; and the decrease of the [1�00]Cc step velocity at high inhibitor concentration and low supersaturation. The switching of the two distinct modes

depends on the terrace lifetime, and the slow kinetics along the [1�00]Cc step direction provides specific sites for the newly formed dislocations. Molecular modeling shows the strong HCA-

crystal interaction by molecular recognition, explaining the AFM observations for the formation of new steps and surface dissolution along the [101]Cc direction due to the introduction of strong

localized strain in the crystal lattice. These direct observations highlight the importance of the

inhibitor coverage on mineral surfaces, as well as the solution supersaturation in predicting the inhibition efficacy, and reveal an improved understanding of inhibition of calcium phosphate biomineralization, with clinical implications for the full therapeutic potential of small-molecule inhibitors for kidney stone disease.

INTRODUCTION Mineralized tissues normally have a high degree of complexity and significant hierarchical structure.1-3 Metabolic abnormality leads to pathological mineralization, such as kidney stones, that occur as mixed calcium oxalate (CaOx) and calcium phosphate (Ca-P) phases.4, 5 At the initial stages of stone formation, Ca−P crystals are found within the apical surface of renal papillae and the urinary space.6 It has been suggested that brushite (DCPD, CaHPO4·H2O) is present inside


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spherical apatites with a radial distribution of acicular crystallites.5 As the initial nidus,7-9 DCPD may drive the CaOx nucleation in Randall’s plaque.5 The inhibitors known to prevent stone formation include proteins,10 peptides,11, 12 and small organic molecules.10, 13, 14 Inhibitor-crystal recognition and interaction influence both the initial formation of metastable nuclei15, 16 and the stable mineral phase with different crystallites and sizes.16, 17 Moreover, inhibitors modify morphological features of the calcified tissues.5 It is well accepted that the basic mode of action for inhibitors to modulate mineralization is to impede solute molecule attachment18 through the initial site-specific adsorption on crystal surfaces,19, subsequent incorporation21,

22

20

and inclusion inside the crystal.23-25 As the adsorbed inhibitor

exhibits stronger competition with solute ions or molecules, it may permanently block kink sites along the advancing steps,26-28 thereby slowing the step movement velocity and interfering with or preventing crystal layer growth. In contrast to kink blocking, inhibitors also modulate step density, another factor determining mineralization rates.29 For the above two pathways, some problems remain since inhibitor adsorption requires a certain length of time to relax to be in a well-bound state.30, 31 Thus, an appropriate thermodynamic driving force is required to be chosen, allowing the adsorption and relaxation of inhibitor molecules through a slow crystallization kinetic process. Citrate (CA) is a therapeutic agent for inhibiting kidney stone growth,32, 33 and hydroxycitrate (HCA), with nearly an identical structure as CA, could induce localized strain to the crystal lattice and dissolve the calcium oxalate monohydrate (COM) (010) surface at the appropriate supersaturation and coverage.34 This additional alcohol hydroxyl (-OH group) substantially alters inhibitor specificity and efficacy,34 and exerts significant contributions to mineral dissolution even in a supersaturated growth solution through a synergy between carboxyl (-OOH group) and hydroxyl groups.34 This synergistic effect was also observed in dicarboxylic acids with different



numbers of alcohol hydroxyls controlling DCPD dissolution by specific molecular recognition and stereochemical conformity between hydroxyl-carboxyl groups of organic acids and atomic steps.35 The stereochemical relationship and bonding configuration may also account for changes of step kinetics and bulk crystal habits of growing stones.36 In the present study, we present a comprehensive description of two molecular inhibitors of CA and HCA to modulate the DCPD (010) surface growth using a combination of in situ atomic force microscopy (AFM) and density functional theory (DFT)-based molecular modeling. We show two distinct actions of inhibitors, controlling crystallization by step density or step velocity, and their switching depends on the moving terrace lifetime. We found that the strong HCA-crystal interaction originating from molecular recognition and the introduction of strong localized strain to the crystal lattice accounts for the formation of new steps and crystal face dissolution. These direct observations may improve our understanding of inhibition of pathological mineralization and provide clues for designing more effective therapeutic agents of renal stone disease.

EXPERIMENTAL SECTION DCPD Crystal Synthesis in the Absence and Presence of CA and HCA. DCPD crystals were synthesized by slow diffusion in silica gel, as described previously.37, 38 To investigate the effect of HCA and CA (Sigma-Aldrich, St. Louis, Missouri) on the DCPD crystallization, the inhibitors with a molar ratio of Ca2+/inhibitors of 800 (1.25 mM CA or HCA) were incorporated into the gel with 1 M potassium dihydrogen phosphate (Sigma-Aldrich, St. Louis, Missouri). Subsequently, 0.5 M calcium chloride (Fluka, St. Louis, Missouri) was added in the top of a tube. Morphology and phase of these synthesized millimeter-sized DCPD crystals in the absence and presence of CA or HCA were assessed using a light microscope (Olympus BX51) and Bruker D8


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X-ray diffraction (XRD) (Billerica, Massachusetts), respectively. In situ AFM Imaging of the DCPD Surface Growth. All in situ AFM images were collected using Nanoscope V-Multimode 8 AFM (Bruker, Santa Barbara) with an O-ring-sealed fluid cell at room temperature (25 °C). Continuous imaging was performed in contact mode with a scan rate of 2-4 Hz at 256 lines per scan with a Si3N4 tip (Bruker NP-10, spring constants of 0.12-0.35 N/m). A range of supersaturated DCPD solutions (σDCPD = 0.357-0.503, ionic strength IS = 0.15 M, pH = 5.6) (Table S1) in the absence and presence of 0.85-34 μM HCA or CA were passed through the fluid cell containing the anchored DCPD crystals at a constant flow rate of 1 mL/min with a highprecision springe pump (Razel Scientific Instruments model R100-E, Saint Albans, Vermont). All supersaturated solutions were prepared using a method as described previously.20 The inhibitor concentrations of 0.85-10.6 μM used in our experiments had a negligible effect on supersaturation considering the molar ratio Ca2+/inhibitor > 800. For each experimental condition, three independent replications were performed on more than three crystals. Image processing and data analysis were performed by the NanoScope Analysis 1.80 software (Bruker). Contact Angle Measurements for Calculations of Surface Free Energy. The static contact angle (θ) was measured with Drop Shape Analyzer-DSA100 instrument (KRUSS, Hamburg, Germany) at 25 °C. The crystal-air interfacial energy (γS-air, a proxy for the crystal-fluid interfacial energy γS-L ) was calculated using a well-established method,39,40 and the θ values of three different testing liquids (ultrahigh purity water (>18 MΩ·cm), glycerol (99%; Sigma), and ethylene glycol p

(99.9%; Sigma)) with known polar (γL ) and dispersive (γdL ) surface energy components were measured with three repeats for each test liquid on DCPD (010) surfaces pre-adsorbed with different concentrations of CA or HCA at 0, 3.4, 8.5, or 10.6 μM. As γS-air is a composite term, which is the sum of the dispersive (γS ) and polar (γS ) component p


p


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as shown by39 γS-air = γS +γdS p

(1)

The unknown components of γS and γdS are obtained by solving the relationship between θ and the p

known parameters of the testing liquids according to the following expression:39,40 γL (cosθ+1) 2�γdL

p

=

�γdS

+

p �γS (

�γL �γdL

)

(2)

p

Because γL is the sum of

p γL

and γL , consequently, the left side of Eq.2 and d

�γL �γdL

should be linearly

related, where γS and γdS corresponding to the square of the slope and the square of the y-intercept, p

respectively. Molecular Modeling Using Density Functional Theory (DFT) Calculations. The adsorption of CA or HCA on the [1� 00]Cc and [101]Cc steps has been calculated using the slab

models with DFT. Gradient-corrected functional Perdew−Burke−Ernzerhof (PBE)41 was employed with the all-electron double numerical plus polarization (DNP) basis set.42 Detailed calculation procedures can be found in our previously published research.3

RESULTS AND DISCUSSION Effects of HCA and CA on the DCPD (010) Surface Growth. AFM images showed that the growing DCPD (010) surface exhibits typical triangular-shaped hillocks with corresponding step heights of 7.6 Å (Figure 1A), exactly matching theoretical molecular step height. According to the crystallographic model of a DCPD (010) growth surface with step assignment for Cc space group,43 we were able to unambiguously determine the surface orientation and assign the step


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directions as [101� ]Cc , [101]Cc and [1� 00]Cc (Figure 1B). From the atomic structure of the (010) surface, the [1� 00]Cc step direction is quite different from the other two steps in that the calcium

cations and hydrogen phosphate anions are in alignment rather than in corrugated rows.44 The acidic hydrogen atom points into solution in the [1�00]Cc step edge, resulting in an additional

activation barrier for solute molecules adsorbed onto the step.44 Differences of atomic arrangement

result in the three steps having anisotropic spreading velocities, among which the speed along the [1� 00]Cc step direction is the slowest in supersaturated solutions with σ ranging from 0.397 to 0.503 (Figure 1D).

Two inhibitors of CA and HCA (Figure 1C) exhibit nearly identical structure, except for HCA with an extra alcohol group; both caused no significant changes in step velocities at σ > 0.397 and/or ci (concentrations of inhibitor CA or HCA) < 8.5 μM. An appreciable decline of the [1� 00]Cc

step velocities at σ ≤ 0.397 and/or ci ≥ 8.5 μM was observed, and more than about 40% and 19% inhibition for CA and HCA, respectively, was acquired at σ = 0.357 and ci = 10.6 μM (Figure 1E and F). We noted that the presence of up to 23.4% standard error in the measurement of the [1� 00]Cc

step velocity at σ ≤ 0.397 can be ascribed to the unstable thermodynamic adsorption of inhibitors (Figure 1E and F). At pH = 5.6, CA has secondary dissociation and HCA is fully dissociated.34 Nevertheless, the inhibition induced by CA is greater than that by HCA along the [1� 00]Cc step

direction. Moreover, CA and HCA did not markedly change the growth rates of the other two steps (the variations were less than 10%) under the same experimental conditions (Figure S1). The bulk growth rate R is determined by both step velocity and step density,28, 45 suggesting

that the inhibitor efficacy is dependent on a combination of step velocity and step density. While CA shows a stronger inhibitor efficacy on the [1� 00]Cc step movement than HCA at certain conditions (Figure 1E and F), HCA is more effective on the decrease of step density (Figure 2A).



According to the classic terrace−ledge−kink (TLK) model at near equilibrium conditions,46,47 both step velocity and step density exhibit monotonic increase with increasing supersaturation. As shown in Figure 2B, the step widths are anisotropic in the order [101�]Cc ≥ [101]Cc > [1�00]Cc at σ

ranging from 0.357 to 0.503 in the absence of inhibitors. Increasing ci from 0.85 to 10.6 μM

resulted in a remarkable increase in terrace width (λ) with the increment order of [101� ]Cc ≥ [101]Cc > [1� 00]Cc (Figure 2C and 2D, Figure S2). For the [1� 00]Cc steps, the increment in terrace width up to 1.64 times by HCA is slightly larger than that about 1.47 times by CA (Figure 2C and

2D). Concerning the inhibitory effect on step velocity and step density, it is difficult to discriminate which inhibitor is more effective in inhibiting DCPD crystallization. Real-time AFM observations of the DCPD (010) surface growth at σ = 0.357 and ci = 10.6 μM (Figure 3A) showed that new dislocations generated on the [1�00]Cc steps following the addition of CA (Figure 3A). The corresponding step height of newly formed steps exactly matches

the monomolecular step height of 7.6 Å20, 44 (Figure 3B). Reintroduction of pure supersaturated solutions led to the reoccurrence and recovery of original growth hillocks (Figure 3A). It was noteworthy that the [1�00]Cc steps exhibited serrate and rough edges after the introduction of CA, whereas subsequently formed macrosteps (bunches of monolayer steps due to different dislocation

sources) retarded the formation of roughened edges (Figure 3A), suggesting that CA hinders solely the motion of elementary steps while it is unable to block the macrosteps.48 This was also observed in the presence of HCA. In addition to the above step modifications, we observed the formation of etch pits after 10 min of the HCA addition (Figure 3C) at σ = 0.357 and ci = 10.6 μM. Depth of etch pits further increased after 44 min (Figure 3D), and the direction of the newly formed etch pits was not parallel to the original [1� 00]Cc steps (Figure 3C). We also tested other solution conditions and found that ACS Paragon Plus Environment

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etch pits appeared only in ranges of both 0.357 ≤ σ ≤ 0.397 and 8.5 ≤ ci ≤ 10.6 μM. This is consistent with the narrow range of HCA concentrations used for the dissolution on the COM (010) surface.34 As the [1� 00]Cc step growth and dissolution occur simultaneously, the measured dissolution rate of newly formed pits may not reflect the real dissolution process.

To further investigate the differences in the mode and efficacy of action by HCA, we purposely increased the HCA concentration up to 34 μM while maintaining the supersaturation at σ = 0.357 (Figure 4). This led to a low molar ratio of Ca2+ ions to inhibitor (about 270), probably altering the solution supersaturation by HCA complexing with free Ca2+ ions. Nevertheless, no etch pits were observed on the DCPD (010) surface (Figure 4A). It can be rationalised that the high coverage of HCA on DCPD decreases the distance of adsorbed molecules, thereby retarding the release of solute ions as lattice components from the crystal surface.34 Besides, 34 μM HCA serrated the [1�00]Cc steps (Figure 4A) and even induced the formation of new steps parallel to the [101]Cc steps (Figure 4A and B), resulting in the occurrence of quadrilateral spirals. This was also

observed at σ = 0.397 and cHCA = 10.6 μM (Figure S3). After changing supersaturated solutions to

undersaturated solutions at σ = -0.096, the dissolution of spirals occurred preferentially along the [101� ]Cc step (Figure S4). Thus, the morphology change in the growth of hillocks caused by the slight decrease of supersaturation can be ignored. We also noted that the quadrilateral spirals were unstable and the newly formed [101]Cc step could be distorted and finally disappeared (Figure 4B),

most likely due to the interference of adjacent steps. Finally, we need to mention that 34 μM CA did not virtually alter the overall hillock shape (Figure 4C and D). Effects of HCA and CA on the Bulk DCPD Growth. The bulk DCPD crystallites exhibited the macroscopic triangular habit (Figure 5A), consistent with the morphologies of spiral and etch pits on the (010) face.20, 29, 38 When CA and HCA were introduced to solutions with Ca2+/inhibitor



molar ratio of 800, a single bulk crystal best approximates needle-like (Figure 5B) and quadrilateral shapes (Figure 5C), respectively. This is consistent with the inhibition effects of CA on the [1�00]Cc step (Figure 1E) and of HCA on the newly formed [101]Cc step (Figure 4C).

According to the noncentrosymmetric monoclinic structure of brushite,38 the (010) faces dominate the macroscopic habit of brushite, leading to a typical plate-like shape with trianglular etch pits or hillocks.38 This may explain how differences in inhibitor behavior in different step directions on the (010) face may translate into an altered macroscopic morphology as shown in Figure 5. X-ray diffraction pattern (XRD) analyses suggested that the presence of HCA shifted the peaks of the DCPD (020) surface (up to 0.1°) (Figure 5D). According to the Prague equation, 2d sin θ = nλ, θ is negatively related to interplanar spacing d in the given X-ray wavelength λ. Therefore, the increased θ correlated with decreased d, demonstrating that HCA, as potential immobile inhibitors, can strongly adsorb to the interlayer compared to CA with a relatively high mobility on DCPD. XRD shifts and dislocation formations (Figure 3C) could be the result of defects generated during growth by the inhibitory effects of HCA via a dissolution mechanism, i.e., HCA may strongly complex with the surface Ca2+ ions to dissolve the crystal surface. Measurements of Surface Free Energy. To understand the observed differences we carried out a series of contact angle measurements on DCPD substrates before and after adsorption with various concentrations of CA (Figure 6A) and HCA (Figure 6B). The measured surface free energy determines the strength of substrate interaction with liquid and is supposed to manifest in the extent of the hydrophilicity of the substrates.40, 49 The difference of γS-air, a proxy for γSL at the solid (S)−liquid (L) interface40 showed that HCA, at concentrations greater than 8.5 μM, dramatically increased DCPD-liquid interfacial energy compared to CA (Figure 6C). Due to γSL correlating with the mean value of all crystal-plane-step energies,29, 50 a higher γSL is associated with higher step


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edge free energy (γstep) that will delay the formation of critical 1D steps.47 Consequently, the high affinity of HCA bound to DCPD displays stronger potency in decreasing step density than that of CA (Figure 2C and D). Molecular Modeling. The binding energies of deprotonated CA and HCA docking to the [1�00]Cc and [101]Cc steps were calculated by DFT-based simulations with energy minimization (Figure 7). The molecular modeling has not considered ion hydration and steric match for

simplification according to the previous work.35 As shown in Figure 7, in the CA-[1� 00]Cc step, the

–COO- groups in CA is bound to three Ca2+ sites together with a H-bond to the H2O molecule on the outermost surfaces of DCPD. In contrast, two –COO--Ca2+ complexations, one H-bond between –COO- and H2O, and alcohol –OH-Ca2+ complexation in the HCA-[1�00]Cc step were

revealed (Figure 7). The calculated binding energy of 203.9 kJ/mol for CA at the [1� 00]Cc step is slightly higher than 195.4 kJ/mol for HCA (Figure 7A and 7B). Apparently, the binding preference

of CA for the [1� 00]Cc step is responsible for the AFM observations of a slightly stronger inhibition

effect of CA on the [1� 00]Cc step movement speed than HCA (Figure 1E and F). However, the binding energy of HCA to the [101]Cc step, 90.3 kJ/mol, is much greater than that of CA, 64.7

kJ/mol (Figure 7C and 7D), providing the most likely explanation for the occurrence of the newly formed [101]Cc step following the adsorption of high concentrations of HCA (Figure 4A and B) rather than CA (Figure 4C and D). In addition, comparing the binding energies of CA, HCA, and

HPO43- complexations with calcium on the DCPD (010) surface revealed that the HCA-Ca2+ binding was the most energetically favorable. The present study of two structural analogs reveals the mechanism that is different from step density determined crystallization rates,29, 50 and kink blocking.26,27 Commonly, inhibitors adsorb to step edges or terraces ahead of migrating steps and impede the addition of solute molecules.18,



51

Since the molar ratio of calcium to inhibitor (up to 10.6 μM in our experiments) in supersaturated

solutions is larger than 800, the thermodynamic effect, i.e., inhibitor-solute complexation changing solute supersaturation, can be ruled out. For the bi-stable kinetic inhibition observed in the present study, inhibitors will undergo multiple structural conformations before reaching a minimum energy state52 to accommodate Ca2+inhibitor complexation on the step edge.36 Consequently, an inhibitor will be well-bound to DCPD before advancing steps arrive at inhibitor binding sites, blocking solute access to kinks and limiting the step advancement.18, 53-55 If not well-bound, step propagation can remove the weakly bound inhibitors.53, 54 The inhibition effect on step propagation depends on the characteristic time scale τ for inhibitor adsorption and relaxation into the well-bound state.53 In addition, the inhibitor adsorption is related to the terrace exposure time t30 given by λ/ʋ.53 In this study, inhibition of the [1� 00]Cc step advancement gives 110 ± 30 s < τ < 160 ± 50 s. Only at t > τ, the well-bound HCA and CA are sufficient to retard step propagation. This is also related to supersaturation: at relatively low supersaturation (σ < 0.397), the inhibitor (8.5 μM < ci

Mechanisms of Modulation of Calcium Phosphate ... - ACS Publications

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