Amphiphilic Phosphoprotein-Controlled Formation of Amorphous

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Amphiphilic Phosphoprotein-Controlled Formation of Amorphous Calcium Carbonate with Hierarchical Superstructure Yan Liu, YongJian Cui, and Rong Guo* College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, P. R. China S Supporting Information *

ABSTRACT: Amorphous calcium carbonate (ACC) plays important roles in biomineralization, and the phosphoproteins extracted from biogenic stable ACC can induce and stabilize synthetic ACC in vitro. Here, mineralization of square-shaped ACC plates with micrometer-sized channels has been reported in the presence of the amphiphilic phosphoprotein casein. Casein can be assumed to take a key role during ACC plate formation, where it serves as an effective stabilization agent for ACC and assembles spherical ACC particles into ACC plates. The stabilizing effect of casein arises from the electrostatic attraction between phosphate groups as well as carbonate groups (especially the former) and the calcium ions, preventing the transformation from unstable ACC to the more stable crystalline phase of CaCO3. The assembling effect of casein mainly comes from the hydrophobic interaction between casein molecules bound on CaCO3 particle surface. The inclusion of casein in ACC plates revealed by the thermogavimetric analysis confirms the proposed stabilizing and assembling mechanism. The ability to fabricate such novel hierarchical structured ACC holds the promise for creating more complex micro- and nanostructured materials by use of biological proteins with special structure.

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combination with magnesium ions.11,19−21 Aizenberg et al. found that such macromolecules from ACC produced by a sponge are proteins rich in glutamic acid and glutamine; however, proteins extracted from calcite phase are rich in aspartic acid.3 It is demonstrated that phosphoproteins from bivalves can effectively inhibit calcium carbonate crystallization; however, dephosphorylation of these proteins almost negates their inhibitory activity.22,23 Inoue and Hecker also found that phosphorylated proteins are associated with stable crustacean ACC.24,25 Inspired by such findings, the design and synthesis of relatively stable ACC with some additives under mild conditions in vitro have aroused interest. It is demonstrated that the transformation of ACC can be inhibited by addition of some additives, such as biomolecules,26 magnesium ions,27,28 triphosphate,29 or polyphosphonate species.30 Donners et al. reported the formation of stable micrometer-sized ACC particles in the presence of certain dendrimer−surfactant mixtures.31 Stable ACC microparticles with hollow spherical superstructures have been obtained by the addition of phytic acid.32 ACC films can be formed in the presence of a novel acid polysaccharide template.33 Understanding the modes of formation and stabilization of amorphous calcium carbonate is of much importance in many

INTRODUCTION Biomineralization, a very common and important phenomenon in nature, has become an active area of research recently.1−3 Calcium carbonate is of enormous interest because it is widely used as a model system for studying the biomimetic process due to its abundance in nature.4−8 Calcium carbonate exists as three anhydrous crystalline polymorphs (calcite, aragonite, vaterite), two hydrated metastable forms (monohydrocalcite and calcium carbonate hexahydrate), and an unstable amorphous phase. Among these forms, amorphous calcium carbonate (ACC) is the least stable phase and transforms rapidly into one of the crystalline polymorphs unless it is stabilized by specific additives. ACC has several potential biological functions. Due to the high solubility of ACC relative to the crystalline phases, ACC can be used as temporary storage deposits when needed.9 Also, it is demonstrated that ACC can act as a nanoparticle precursor material for the generation of the crystalline polymorphs, since ACC has no long-range crystalline order and preferred symmetry.10−13 Although ACC is difficult to produce in vitro due to its lower stability,14,15 the biogenic ACC has been found to exist widely in living organisms, such as in spicules from sponge and mollusk shells. 3,16 A key principle in the nature of biomineralization is the involvement of biological macromolecules that have high binding affinity to the surface of CaCO3 crystallites, which leads to the control and directing of the crystallization of minerals.9,17,18 Biogenic ACC stabilization has been attributed to specific specialized proteins, often in © 2012 American Chemical Society

Received: January 21, 2012 Revised: March 14, 2012 Published: March 14, 2012 6097

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Figure 1. XRD pattern (a) and FTIR spectra (b) of CaCO3 obtained in the presence of 2.0 g L−1 casein at 10 °C. [CaCl2] = 10 mM.

Figure 2. SEM images of CaCO3 obtained in the presence of (a−c) and in the absence of (e) 2.0 g L−1 casein at 10 °C. [CaCl2] = 10 mM. Crossedpolarized optical micrographs of the prepared sample (d) and the control sample (f). 6098

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Figure 3. TEM and SEM images of CaCO3 samples produced at the early reaction stages in the presence of 2.0 g L−1 casein at 10 °C. The inset in part b is the SAED pattern of the precursor particle. The reaction time is: (a) 5 min, (b) 10 min, (c) 20 min, and (d) 30 min.

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fields of research. Furthermore, the relatively high solubility of ACC may be advantageous in the pharmaceutical industry, water treatment, filtration, and catalysis. We are very interested in the understanding of the fundamental biomineralization mechanisms in phosphoprotein systems as well as its potential importance for developing novel materials and new applications. Important sources of phosphoproteins are the casein proteins, consisting of a highly repetitive sequence of glutamic acid (Glu) (23% of all residues in casein). Also, the main two casein constituents, αs1- and β-casein have eight and five phosphoserine residues, respectively. Thus, casein has the ability to bind calcium ions via both phosphate and carboxylate groups of phosphoserine and glutamic acid residues. Furthermore, caseins can be thought of as amphiphilic block copolymers consisting of blocks with high levels of hydrophobic or hydrophilic amino acid residues. Thus, caseins exhibit a strong tendency to self-assemble into casein micelles because of its amphiphilic property in aqueous solution.34,35 All these can influence the crystallization process of CaCO3. In this work, mineralization of ACC with novel hierarchical structures has been obtained in the presence of casein micelles. The influence of reaction time, temperature, pH, and reagent concentration on the mediation of the phase transformation and morphology change of CaCO3 crystals in the presence of casein has been investigated systematically. The results demonstrate that kinetic and thermodynamic control can prominently regulate the polymorphs and morphologies of CaCO3 mineral in casein solution. Our studies may contribute to the understanding of biological mechanisms in ACC stabilization and hold the promise for creating more hierarchical structured materials by use of proteins with special structure.

EXPERIMENTAL SECTION

Materials. Casein [isoelectric point (pI) = 4.5 and average molecular weight (MW) = 2.1 kDa], casein hydrolysate, and dephosphorylated casein were all purchased from Sigma. Analytical grade sodium carbonate and calcium chloride were purchased from Shanghai Chemical Reagent Co. and used as received without further purification. Distilled water was used as the solvent. Experimental Procedures. In a typical procedure, 1 mL of 1 M CaCl2 aqueous solution was added to 50 mL of 2.0 g/L soluble casein aqueous solution, and then the pH was adjusted to a fixed value using 1 M HCl and NaOH solution after stirring for 30 min. Then, 50 mL of sodium carbonate solution (10 mM) was added into the above solution. After stirring for 3 h, the obtained precipitate was centrifuged and rinsed several times with ethanol and distilled water and dried at a temperature below 10 °C for at least 48 h. Characterization. X-ray powder diffraction (XRD) patterns were recorded using a Bruker D8 Advanced XRD diffractometer with Cu KR radiation at a scanning rate of 0.04° s−1. Scanning electron microscope (SEM) images were taken with a JEOL JSM-6700FXIB, fitted with a field emission source and working at 20 kV. All samples were mounted on copper stubs and sputter-coated with gold prior to examination. Infrared spectroscopic analysis was performed in transmission mode (FT-IR) using a Nicolet Aexus 470, with scanning from 4000 to 500 cm−1 by using KBr pellets. Transmission electron microscopy (TEM) images were obtained on a 150 kV H-800 microscope. High-resolution transmission electron microscopy (HRTEM) and selected-area electron diffraction (SAED) images were recorded by using a JEM-2010 UHR high-resolution transmission electron microscope (Japan Electron Co.). Optical micrographs were taken with a Nikon ECLIPSE E600 fluorescence microscope equipped with polarizers. 6099

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1−2 μm size (Figure 3b). The amorphous feature of the precursor particles was confirmed by the appearance of diffuse rings in the SEAD pattern (Figure 3b inset). Indeed, magnified TEM images, shown in Figure S3 (Supporting Information), reveal that these spherical particles are constructed from smaller subunits. After a reaction time of 20 min, there is a gradual buildup of these spherical-like particles (1−2 μm), with aggregation and coalescence occurring synchronously (Figure 3c). SEM image of higher magnification also confirms that these spherical particles are constructed from smaller subunits (inset in Figure 3c). With time, the degree of aggregation and coalescence increases (Figure 3d), and finally, square-shaped ACC plate superstructures are formed after a prolonged reaction time (Figure 2). Figure 4 shows the FTIR spectra of

RESULTS AND DISCUSSION ACC Superstructure Formation in the Presence of Casein. The X-ray diffraction (XRD) pattern of the precipitate obtained in casein solution at 10 °C is shown in Figure 1a, clearly indicating that the formed product is composed of pure amorphous calcium carbonate.28,32,36,37 The phase of the product was also confirmed by the FTIR spectrum (Figure 1b). The two splitting bands at 1412 and 1483 cm−1 and the carbonate out-of-plane bending absorption at around 866 cm−1 are characteristic of the ACC phase. The very broad and weak absorption at around 710 cm−1 also confirms the formation of the ACC phase.9,38−40 Furthermore, the spectrum exhibits a broad water absorption band around 3400 cm−1, which indicates that the formed ACC contains water molecules in its lattice structure.38−40 Earlier studies have shown that the stable forms of biogenic ACC contain water molecules in their lattice structure, whereas the known examples of transient forms of ACC do not.9,15 The infrared data display clearly the CONH band at 1654 cm−1, confirming that casein molecules remain attached in the separated samples. Figure 2a−c shows representative scanning electron microscopy (SEM) images of ACC. As shown, square-shaped ACC plate with about micrometer-sized channels on the surface are obtained. The size and the thickness of the ACC plate are about 30 and 3 μm, respectively. Indeed, SEM images of higher magnification, shown in Figure 2c, reveal that the superstructures with rough surfaces are actually constructed from many smaller subunits. Notably, a few particles with the distinctive appearance of calcite were present in the inset of the square-shaped plate, as indicated by white arrows in Figure 2c. Unfortunately, it is indeed difficult to unambiguously prove whether calcium carbonate is truly amorphous or not in this case. However, if the rhombohedral structured particle is truly calcite, the fraction of the calcite must be too small to be detected by XRD and FTIR measurements. Figure 2d shows the polarized optical micrograph of the sample obtained in casein solution at 10 °C. Its amorphous nature was confirmed by the darkness under polarized light. In a control experiment with the absence of any additives, well-defined rhombohedral calcite crystals can be formed (Figure 2e). Figure 2f shows optical micrographs, taken between crossed polarizers, of the control samples, confirming the rhombohedral crystals. For the formed superstructure there was no change of morphology and phase within the investigated time (36 h), that is, the reaction time has little or no influence on the morphology of ACC (Figure S1a,b, Supporting Information). The air-dried synthetic ACC obtained was stable and showed no sign of crystallization after more than 2 months under ambient conditions. However, the ACC started transforming to vaterite and calcite about 3 months later (Figure S2, Supporting Information). To the best of our knowledge, this is the first synthesis of novel ACC superstructure by protein molecules in vitro, which indicates that the added protein plays an important role in preventing the ACC transformation into crystalline forms. To explore further the growth and assembly mechanism of the ACC superstructure, we examined the intermediate products at different stages with TEM, SEM, and FTIR measurements. At the very early stage of mineralization (after reaction for 5 min), around 5−12 nm sized ACC nanoparticles were observed (Figure 3a). Later, casein-capped nanoparticles grow and aggregate into larger spherical-like particles of about

Figure 4. FTIR spectra of CaCO3 samples at 20 min (down) and 30 min (up). Ccasein = 2.0 g L−1.

CaCO 3 precipitates collected at 20 and 30 min of mineralization in the presence of casein. It can be seen that both spectra taken at the two times exhibit the two characteristic splitting bands at 1415 and 1481 cm−1 (ν3) and the carbonate out-of-plane bending absorption at around 870 cm−1, indicating that the intermediate precipitate is also ACC. Morphology and Phase Transformation Control of CaCO3. Traditional strategies for selection of polymorphs in calcium carbonate often involve altering reaction conditions to influence kinetics over thermodynamics or vice versa. To further demonstrate the details of the growing process, we conducted an experimental series of different environmental conditions, varying temperature, casein concentration, and different starting pH values. When the reaction temperature was increased to 25 °C, the CaCO3 crystals formed mainly are spheres, and some of them are interconnected as twins with pricks (Figure 5a). The XRD pattern of this sample is shown in Figure 5b, which shows weak and broad diffraction peaks from vaterite, in addition to the sharp diffraction peaks from calcite. The corresponding FT-IR spectrum (Figure S4, Supporting Information) also shows the presence of a band at 744 cm−1, which is characteristic of vaterite, in addition to the bands corresponding to calcite.41,42 This indicates that the obtained particles consist mainly of calcite with only a small fraction of the vaterite. The formation and further characterization of the novel prickly structure is under progress. Notably, ACC can only be obtained at temperatures below 10 °C. When the reaction temperature increases, the rate of the dissolution−recrystallization process is accelerated, and it contributes to the dissolution of ACC 6100

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Figure 5. SEM image (a) and XRD pattern (b) of CaCO3 samples obtained in the presence of 2.0 g L−1 casein at 25 °C. [CaCl2] = 10 mM.

Figure 6. SEM images of CaCO3 samples obtained in the presence of casein with different concentrations: Ccasein = 1.0 g L−1 (a) and 4.0 g L−1 (b). (c) A typical TEM image of the dendritic CaCO3 in part b. The inset in part c is the SAED pattern taken from the selected area marked in c.

Figure 7. XRD pattern (a) and FTIR spectra (b) of CaCO3 obtained in the presence of casein with different concentrations. [CaCl2] = 10 mM, Ccasein = 1.0 g L−1 (up) and 4.0 g L−1 (down).

concentration of 1.0 g L−1 is calcite (Figure 7). Here, the effective concentration of casein required for ACC formation is above 2.0 g L−1 (for CaCO3 concentration of 10 mM). At a concentration lower than 2.0 g L−1, casein loses its ability to control the formation of ACC. The variation of pH also changed the morphology and phase of the produced CaCO3 crystal. SEM and FTER experiments were conducted under the standard conditions at three typical pHs, 3.0, 5.0, and 10.0. When the pH was above the pI value of casein (pI = 4.5), square-shaped ACC plates are still formed (Figure S5, Supporting Information). However, spherical-like CaCO3 is obtained at pH 3.0, where the pH is below the pI of casein (Figure 8a). FTIR results show that CaCO3 sample prepared at pH 3.0 is calcite (Figure 8c). Furthermore, the presence of excess CO32‑ ions in the solution also had a

formed at an early reaction stage,43 which is helpful for the formation of the more stable crystalline phase of CaCO3. The variation of casein concentration can also effectively modulate the morphologies and phase modifications of CaCO3. At the casein concentration of 1.0 g L−1, CaCO3 crystals formed mainly exhibited ellipsoid-like structures with a rather rough surface and have a size of about 1.6 μm in length and a maximum diameter of about 0.8 μm (Figure 6a). With the concentration of casein increased further up to 4.0 g L−1, dendritic CaCO3 with a rather rough surface is mainly formed (Figure 6b). The selected-area electron diffraction (SAED) pattern (inset in Figure 6c) suggests that the dendritic CaCO3 is amorphous in nature, which is further confirmed by the FTIR and XRD results shown in parts a and b of Figure 7, respectively. The CaCO3 sample prepared at a casein 6101

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Figure 8. SEM images and XRD patterns of CaCO3 samples obtained in the presence of 2.0 g L−1 casein. (a, c) [CO32‑] = [Ca2+] = 10 mM, pH 3.0. (b, d) [CO32‑] = 30 mM, [Ca2+] = 10 mM, pH 7.0.

Figure 9. SEM images of CaCO3 samples obtained in the presence of 0.2 g L−1 dephosphorylated casein (a) and 2.0 g L−1 casein hydrolysate (b). [CaCl2] = 10 mM.

significant effect on the formation of CaCO3 crystal. For example, when the molar ratio CO32‑/Ca2+ was increased from 1:1 to 3:1, SEM results show no evidence of square-shaped ACC plates, but pure elliptical-like aggregate around 1.5 μm in length and 0.7 μm in width consisting of rhombohedra (Figure 8b) is observed. FTIR results show that the CaCO3 sample prepared is calcite (Figure 8d). It can be seen that lower pH and excess CO32‑ are not favorable for the formation of ACC. Formation Mechanism of Square-Shaped ACC Plates. Casein with an isoelectric point of about 4.6 is negatively charged and has several phosphoserine residues in addition to a high content of glutamic acid residues. Thus, casein has the ability to bind calcium ions via both phosphate and carboxylate

groups of phosphoserine and glutamic acid residues, which contributes to the formation of ACC.44,45 The presence of strong electrostatic attraction between casein and Ca2+ can be proved by the effect of pH and excess CO32‑ on the polymorph of CaCO3. As discussed above, lower pH and excess CO32‑ are not favorable for the formation of ACC. At a pH below the pI of casein, the electrostatic attraction between Ca2+ and the negatively charged groups is absent due to the almost protonated acid groups. Also, an excess of CO32‑ induces a net negative surface charge at the interface with the growing nanoparticles, so electrostatic interactions with the negatively charged group of casein are also significantly reduced. 6102

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Information), it can be seen that the formed CaCO3 is calcite. Obviously, ACC induced by the casein molecules also disappears once the protein is hydrolyzated. The results indicate that the amphiphilic property also matters much in the stabilization of ACC. In addition to the role of stabilizing ACC, the amphiphilic phosphoprotein also plays a crucial role in fabricating hierarchical microstructures of ACC. First, casein micelle stabilized nanosized ACC is formed at an early reaction stage (Figure 3a). Subsequently, casein-capped nanoparticles grow and aggregate into spherical-like particles in size of about 1.0 μm, as shown in Figure 3b. During the process, the structure of casein micelles is destroyed, and casein molecules bind to the CaCO3 surface via the electrostatic attraction. Then, these larger spherical microparticles further self-assemble into superstructured ACC by coagulation and coalescence (Figure 3c). They further grow and aggregate by an Ostwald ripening process and produce the square-shaped ACC plate superstructures with microchannels eventually (Figure 2). Generally, van der Waals force is believed to drive the aggregate growth of nanostructures in biomimetic mineralization. Here, in addition to van der Waals force, the hydrophobic interaction between casein molecules may be one of the main driving forces for the nanoparticle assembly because of the amphiphilic casein-capped CaCO3 surface.

Therefore, the strong binding of casein on CaCO3 surface is inhibited, and ACC cannot be obtained in both cases. We further demonstrated the inclusion of casein in ACC plate using TG measurement. The TG curve of the ACC sample shows a three-step weight loss (Figure S6, Supporting Information). The weight loss below 250 °C can be attributed to the dehydration process. The weight loss of 8.5% from 250 to 500 °C was due to the thermal decomposition of the protein casein, which means that an amount of casein was included during the ACC formation process. CaCO3 started to decompose at 700 °C, corresponding to the third weight loss in the TG curve. As is well-known, ACC is a highly thermodynamically unstable polymorph of CaCO3 that readily transforms to the more stable crystalline phase of CaCO3.46 Biogenic ACC stabilization has been attributed to specific biomacromolecules. Earlier studies found that biomacromolecules from ACC are proteins rich in glutamic acid and glutamine3 or phosphorylated proteins.22−25 In vitro, the former protein molecules alone cannot induce ACC in the absence of magnesium ions, but the latter protein can. It has been found that phosphonates adsorb to ionic crystals much more strongly than other additives. They preferentially interact with kinks on the crystal nucleus surface and thus efficiently inhibit crystal growth. 3 Similarly, phosphoserine residues of casein preferentially interact with kinks on the crystal nucleus surface and thus efficiently inhibit crystal growth, leading to the formation and stabilization of ACC. To confirm the crucial role of the phosphate group in casein molecules, dephosphorylated casein was used as a control. In the case of dephosphorylated casein, the product is mainly an elliptical-like aggregate around 5.5 μm in length and 3.0 μm in width with clearly calcite crystal faces (Figure 9a). Obviously, ACC induced by casein molecules disappears once the protein is dephosphorylated. Notably, compared with the case of Glu (23%), the content of the phosphate group is very low (2.5%); however, the phosphate group in casein still plays a crucial role in the formation and stabilization of ACC. Of course, the electrostatic attraction between negative carboxylate group and Ca2+ also exists, and this is confirmed by the formation of elliptical-like aggregate consisting of rhombohedra shown in Figure 9a.47 More importantly, caseins can be thought of as amphiphilic block copolymers consisting of blocks with high levels of hydrophobic or hydrophilic amino acid residues. Thus, casein exhibits a strong tendency to self-assemble into casein micelles because of its amphiphilic property in aqueous solution.34,35 The hydrated mineral is confined in a hydrophobic coat, so the presence of water in the coordination sphere around the calcium ions must also prevent reorganization into one of the stable crystalline anhydrous phase.3 Our controlled experiments on the effect of casein concentration on the polymorphs of the product support the effect of casein micelles. In a word, ACC could not be formed in the solution with a casein concentration lower than the critical micellar concentration of 1.0 g L−1.48 To confirm the role of the amphiphilic property of casein molecules, casein hydrolysate was also used as a control. The casein hydrolysate is a mixture of small peptides, and some of them bear phosphate groups; however, casein micelles are absent in the system. In the presence of casein hydrolysate, the product is mainly ricelike CaCO3, and some irregular aggregates also exist (Figure 9b). According to the FTIR spectrum of the sample shown in Figure S7 (Supporting

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CONCLUSIONS In the present work, mineralization of square-shaped ACC plates, possibly with a very small fraction of calcite, has been reported in the presence of the amphiphilic phosphoprotein casein. By regulating the synthesis conditions such as temperature, the pH of the solution, the reaction time, and the concentration of casein, we can control the phase transformation and morphology change of CaCO3 crystals. The results demonstrate that kinetic and thermodynamic control can prominently regulate the polymorphs and morphologies of CaCO3 mineral in the presence of casein. The phosphate group as well as carboxylate groups in casein works cooperatively with the amphiphilic property of casein molecules, in the stabilization and formation of ACC hierarchical structure. The obtained ACC plates with micrometer-channels may find wide applications in drug delivery, filters, catalysis, and biomedical engineering, because of their effective permeability and nontoxicity. These observations may well provide novel ideas for improved materials synthesis.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S7. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-514-87971858. Fax: +86-514-87311374. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundations of China (20803061 and 21073156) and PAPD. 6103

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