Morphological transformation of calcium phenylphosphonate microspheres induced by micellization of γ-polyglutamic acid

Journal of Colloid and Interface Science 556 (2019) 33–46

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Morphological transformation of calcium phenylphosphonate microspheres induced by micellization of c-polyglutamic acid Xue Wang a,1, Xue Li a,1, Guangli Ou a, Xin Shi a,⇑, Zhi Liu b,⇑ a b

Institute of Chemistry for Functionalized Materials, School of Chemistry and Chemical Engineering, Liaoning Normal University, 850 Huanghe Road, Dalian 116029, China Department of Chemistry, College of Science, Shantou University, Shantou, Guangdong 515063, China

g r a p h i c a l a b s t r a c t It has been found for the first time that c-PGA exhibits the ‘‘schizophrenic” micellization behavior, in that it induces and controls a series of interesting morphology transformations of hierarchical calcium phenylphosphonates. In particular, a rare, reversible morphology transformation between solid microspheres and hollow microspheres is observed with increasing pH.

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Article history: Received 19 April 2019 Revised 8 August 2019 Accepted 9 August 2019 Available online 9 August 2019 Keywords: Calcium phenylphosphonate c-polyglutamic acid ‘‘Schizophrenic” micellization Morphological transformation Lead(II) ions Removal

a b s t r a c t The c-polyglutamic acid (c-PGA), an anionic homo-polyamide similar to naturally occurring polyaspartic acid in mollusk shells, is selected for morphosynthesis of calcium phenylphosphonates. It has been found that they exhibit a series of interesting morphological transformation with changing c-PGA amount and initial pH. A rare, reversible transformation between solid and hollow microspheres, in particular, is observed. Systematic investigation suggests, for the first time, that c-PGA exhibits the ‘‘schizophrenic” micellization behavior in response to structural and morphological changes caused by spontaneous hydrophobic modification with phenylphosphonic acids and calcium ions. The present work introduces a new paradigm for biomimetic synthesis. The results not only bring insight into the template mechanism of biomacromolecules from spontaneous hydrophobic modification, but also provide a universally applicable strategy for morphosynthesis of metal phosphonates containing hydrophobic groups. In addition, solid and hollow calcium phenylphosphonate microspheres with hierarchical structures are excellent adsorbents in the removal of aqueous lead ions. They consistently exhibit 100% Pb2+ removal efficiency when Pb2+ concentration is not more than 600 mg L 1 and even after four recycles, making them promising candidates as efficient and reusable adsorbents. Ó 2019 Elsevier Inc. All rights reserved.

⇑ Corresponding authors. 1

E-mail addresses: [email protected] (X. Shi), [email protected] (Z. Liu). These authors contributed equally.

https://doi.org/10.1016/j.jcis.2019.08.039 0021-9797/Ó 2019 Elsevier Inc. All rights reserved.

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1. Introduction In nature, biomacromolecules play important roles in directing and controlling the formation of biominerals with various morphologies and sophisticated structures. Biomimetic synthesis using natural biomacromolecules not only brings insights into their structure-directing roles and templating mechanism in the biomineralization process, but also inspires the design and synthesis of new functional materials [1–3]. Many natural biomolecules, including amino acids [4], peptides [5], proteins [5–6] and DNA [7], have been consequently used in controlling the morphology and structure of calcium phosphate materials, and the area has seen a lot of encouraging results during the past two decades. It is commonly recognized that the electronrich functional groups in biomolecules, for example, the extensively studied carboxyl groups, are able to interact with divalent calcium ions to provide nucleation sites for the formation of crystallites, and can control the subsequent growth of crystallites through interactions with certain crystal faces of the crystallites. Moreover, the strength of the interactions can be modulated by changing the pH value of the solution so as to control the length and aspect ratio of the crystallites [8]. However, questions remain on how the structural and morphological changes of biomacromolecules take place during biomineralization and their roles in directing and controlling the formation of calcium phosphates. In order to simulate biomacromolecules naturally existed in the biominerals, various biopolymers and synthetic polymers containing carboxyl groups, such as polyamino acids [9–11], polypeptides [12–13] and block copolymers [14–15], have been used in biomimetic synthesis of calcium phosphates. Furthermore, the doublehydrophilic block copolymers (DHBCs) known as ‘‘schizophrenic” polymers were also utilized [15–20]. In general, DHBCs consist of two or multiple water-soluble blocks, of which one or two hydrophilic blocks can be transformed into hydrophobic ones upon changing the external environmental conditions (such as temperature, pH value, ionic strength, light, and the complexation with oppositely charged metal ions, small molecules or polymer chains etc.), while the other hydrophilic blocks promote the dissolution and dispersion of copolymers. Such a transformation occurs in respond to external physical or chemical stimuli, which cause DHBCs to exhibit ‘‘schizophrenic” micellization behavior and form various different self-assembled aggregates in solution so that DHBCs are known as ‘‘schizophrenic” polymers [20–21]. In the biomimetic synthesis of calcium phosphates using DHBCs, DHBCs are also likely to exhibit the ‘‘schizophrenic” micellization behavior in the presence of a large number of calcium ions or/and by changing temperature and pH, which in turn might affect the morphology and structure of calcium phosphates. Despite its importance, the role of this unique behavior of DHBCs on the formation of calcium phosphates has not attracted much attention. Herein, c-polyglutamic acid (c-PGA), a natural anionic homopolyamide similar to naturally occurring polyaspartic acid in the mollusk shells, was selected and used in the morphosynthesis of calcium phenylphosphonates. It has been found for the first time that c-PGA exhibits the ‘‘schizophrenic” micellization behavior to induce and control the morphology transformation of calcium phenylphosphonates. As far as we know, polyglutamic acid generally presents itself as a hydrophilic block in DHBCs that are able to respond to external environmental changes, however, itself alone cannot exhibit the ‘‘schizophrenic” micellization behavior in aqueous solution [21–23]. The ‘‘schizophrenic” micellization of c-PGA occurs in this synthetic system only if c-PGA experiences structural and morphological changes in the co-participation of calcium ions and phenylphosphonic acids, which is somewhat similar to the phosphorylation of proteins accompanied by proteins’

hydrophility/hydrophobicity transformation and conformational change. As a result, calcium phenylphosphonates exhibit a series of interesting morphology transformations with the increase of c-PGA addition amount and initial pH value. It should be noted that mutual morphology transformation between solid microspheres and hollow microspheres has rarely been encountered before. To further understand the role of c-PGA and its ‘‘schizophrenic” micellization behavior in the formation of calcium phenylphosphonates, the effect and mechanism of c-PGA in the morphology transformation and crystal phase transition of calcium phenylphosphonates was investigated and discussed in detail. Considering that phosphonates themselves are effective flocculants for heavy metal ions, calcium ions are environment friendly and biocompatible, and the calcium phenylphosphonate products exhibit solid or hollow microsphere morphology and hierarchical structure, such calcium phenylphosphonate microspheres thus possess the characteristics of an excellent adsorbent for efficient, eco-friendly and reusable removal of very toxic aqueous lead ions. 2. Experimental section 2.1. Materials and characterization All the chemicals were obtained commercially and directly used without further purification. Phenylphosphonic acid (PPA, 98%) was purchased from Acros. Calcium chloride anhydrous (CaCl2), and NaOH were analytical grade and purchased from Tianjin Kemiou Chemical Reagent Co. The c-polyglutamic acid (c-PGA) (MW 700–1000 kDa) was purchased from Xi’an Tongze Biotechnology Co. Ltd. Scanning electron microscopy (SEM) was performed on a SUPRA55 SAPPHIRE electron microscope operating at an accelerating voltage of 1–30 kV. Powder XRD pattern was recorded on Empyrean powder diffractometer using Cu Ka radiation of 0.15406 nm wavelength. X-ray photoelectron spectroscopy (XPS) of calcium phenylphosphonates was analyzed on Thermofisher ESCALAB 250Xi. The N element content of calcium phenylphosphonates was obtained on Elementar Vario EL Cube CHNOS. N2 adsorption-desorption isotherms were obtained on a Quantachrome ASIQM000-4 automated analyzer. Prior to the measurement, the samples were out gassed at 80 °C for at least 24 h. The surface area was estimated according to the BET method [24–25], and the pore size distribution was obtained by using DFT method [26–28]. In the experiments using calcium phenylphosphonates in the removal of Pb2+ ions, the concentration of Pb2+ ions was quantitated by using atomic absorption spectrophotometer on Shanghai Tianmei AA6100. 2.2. Synthesis Calcium phenylphosphonates were synthesized by using solution method with C6H5PO3H (PPA) and CaCl2 as the precursors in the presence of c-polyglutamic acid (c-PGA). In a typical synthesis, 50 mg of c-PGA was added to a solution containing 7.5 mL of 0.08 mol L 1 CaCl2 at pH 8 to obtain a mixture, which was stirred at 50 °C for 30 min. Simultaneously, 7.5 mL of 0.08 mol L 1 PPA solution with a pH value of 8 (adjusted by NaOH) was kept stirred and held at 50 °C. Then, the PPA solution was added into the mixture containing CaCl2 and c-PGA under stirring and held at 50 °C for another 30 min. The resulting precipitate was collected by centrifugation, washed with pure water and dried at room temperature in a vacuum oven. Hereafter, the as-synthesized calcium phenylphosphonate was designated as CaPP-c50/pH8. c50 represents that the amount of c-PGA added was 50 mg, whereas pHn means the initial pH value of the reaction system was 8. The

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different products obtained with different amounts of c-PGA or at different initial pH values were also synthesized with the above synthetic procedure and named as CaPP-cm/pHn. For comparison, the calcium phenylphosphonate samples synthesized without c-PGA were called CaPP-c0/pHn. 2.3. Calcium phenylphosphonate microspheres used as adsorbents in the removal of Pb2+ ions CaPP-c50/pH8 and CaPP-c50/pH9 were pretreated at 80 °C for 24 h in a vacuum oven beforehand. The stock standard solution was used to prepare a series of Pb2+ solutions with the concentration in the range from 50 to 1000 mg L 1 at pH 5.0. Then, 10 mg of the adsorbent was suspended in 10 mL of Pb2+ solution at different concentration, and the resulting mixture was continuously shaken in a shaking bath (120 r min 1) at 293 K for 24 h. The supernatant was separated from the adsorbent by centrifugation at 6000 rpm for 15 min [29] and the Pb2+ concentration of the supernatant was measured by atomic absorption spectrophotomete (AAS) at the wavelength of 283.3 nm [30]. For the adsorption kinetics, 10 mg of adsorbent was suspended in 10 mL of 250 mg L 1 Pb2+ solution with a pH value of 5.0, and the mixture was shaken in a shaking bath (120 r min 1) at 293 K for different intervals. The supernatant was separated from the adsorbent by centrifugation at 6000 rpm for 15 min [29] and the Pb2+ content of supernatant was measured by atomic absorption spectrophotomete (AAS) at the wavelength of 283.3 nm [30].

For in situ multiple-recycle test, 1.3 g of CaPP-c50/pH9 adsorbent was filled in a column with an inner diameter of 1.1 cm [31–32]. First, 10 mL of 1000 mg L 1 Pb2+ solution with a pH value of 5.0 was added to the column and retained for 24 h, then the column was repeatedly washed with pure water. Then, 10 mL of 0.8 mmol L 1 EDTA-2Na solution was added into the column and retained for 2 h to remove Pb2+ ions from CaPP-c50/pH9 adsorbent, and the column was repeatedly washed with pure water for the next cycle. In each cycle, the residual Pb2+ concentration in the solution was measured by atomic absorption spectrophotomete (AAS) at the wavelength of 283.3 nm [30] to obtain the removal efficiency of Pb2+ ions for CaPP-c50/pH9 adsorbent. 3. Results and discussion 3.1. The effect of c-PGA addition amount on the morphology and crystal phase composition of calcium phenylphosphonates The facile synthesis of calcium phenylphosphonates was implemented by a solution method at 50 °C when the initial pH value of reaction system was 8. As shown in Fig. 1A, the calcium phenylphosphonates synthesized with different added amount of c-PGA were characterized by SEM, in comparison with that in the absence of c-PGA. It is clear that the morphology of calcium phenylphosphonates changes from huge thin sheets to microspheres when the amount of c-PGA was increased from 0 to 80 mg. In Fig. 1A(a), calcium phenylphosphonate presents huge

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Fig. 1. (A) The morphology of calcium phenylphosphonates synthesized at an initial pH value of 8 with varying amount of c-PGA: (a) 0 mg; (b) 5 mg; (c) 7 mg; (d) 10 mg; (e) 15 mg; (f) 30 mg; (g) 50 mg; (h) 80 mg; (B) XRD patterns of selected samples: (a) CaPP-c0/pH8; (g) CaPP-c50/pH8; (h) CaPP-c80/pH8 (the diffraction peaks with asterisk refer to Ca(C6H5PO3)
2H2O crystal phase).

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thin sheets of irregular shape when the synthesis was conducted without the addition of c-PGA. When the added amount of cPGA is 5 mg (Fig. 1A(b)), the morphology of calcium phenylphosphonate exhibits nonuniform ellipsoids with several dozens of micrometers in length and twenty to thirty micrometers in diameter. The SEM image at higher magnification (inset of Fig. 1A(b)) further reveals that each ellipsoid consists of a multitude of flakes with a thickness of one to two hundred nanometers and a width of a dozen micrometers. Increasing the amount of c-PGA to 7 mg (Fig. 1A(c)), calcium phenylphosphonate exhibits flower-like microspheres of a dozen micrometers in diameter, which are constituted of criss-cross nanoflakes passing through a central core. Besides flower-like microspheres, half-baked microspheres and many unassembled fan-shaped nanoflakes with the size of several to a dozen micrometers can be observed in Fig. 1A(c). When the dosage of c-PGA reaches 10 mg (Fig. 1A(d)), calcium phenylphophonate appears as spherical cornflower, which is composed of a mass of loosely arranged nanoflakes in the shape of chrysanthemum petal with one to two micrometers in width. At 15 mg of c-PGA (Fig. 1A(e)), it can be observed from the incomplete microspheres in SEM image that calcium phenylphosphonate exhibits hollow flower-like microspheres, which are constituted by bundles of nanoflakes in the shape of narrow strip and packed more closely than that of CaPP-c10/pH8. Further increasing the added amount of c-PGA to 30 mg (Fig. 1A(f)) led to the emergence of two different kinds of microspheres, including large microspheres of a dozen micrometers in diameter and small microspheres with a diameter of several micrometers. The large microspheres are constructed by narrow nanoflakes with less than one micrometer in width, similar to CaPP-c15/pH8 but with narrower nanoflakes in a more compact arrangement, whereas the small microspheres have relatively smooth surfaces and are constituted of a large number of closely packed nanorods, same as cauliflower-like CaPP-c50/pH8. After the added amount of c-PGA exceeds 50 mg, calcium phenylphosphonate always presents uniform cauliflower-like microspheres with a diameter of c.a. 7 lm, which are constructed by many bunches of closely packed nanorods. Estimated from the crack on the microspheres at higher magnification (insets of Fig. 1A(g) and A(h)), the nanorods are tens of nanometers in diameter and tend to gather into bundles. Further increase of c-PGA from 50 mg to 80 mg has little effect on the morphology of microspheres, only that the crack becomes larger on the microspheres. To analyze the crystal phase of calcium phenylphosphonates, the above samples were further characterized by powder XRD. The XRD patterns of the samples synthesized using 50 mg and 80 mg of c-PGA are displayed in Fig. 1B along with that of the sample obtained without c-PGA. The XRD pattern of CaPP-c0/pH8 (Fig. 1B(a)) exhibits the strongest diffraction peak at 2h of 5.81° and the basal spacing d is 15.20 Å, which is consistent with that of Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O reported previously [33–34]. In comparison with CaPP-c0/pH8, CaPP-c50/pH8 and CaPP-c80/pH8 show new diffraction peaks as shown in Fig. 1B(g) and B(h), respectively, confirming that a new crystal phase appears in these two samples. According to earlier reports, these new diffraction peaks can be assigned to the Ca(C6H5PO3)
2H2O crystal phase [33–34]. In other words, the calcium phenylphosphonate samples synthesized in the presence of c-PGA are compose of both Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O and Ca(C6H5PO3)
2H2O crystal phases. 3.2. The effect of initial pH value on the morphology and crystal phase composition of calcium phenylphosphonate microspheres As the above result shows, calcium phenylphosphonates with uniform microsphere morphology can be synthesized at an initial pH value of 8 when the added amount of c-PGA was not less than

50 mg. Then, we fixed the added amount of c-PGA at 50 mg, the effect of pH was investigated systematically. As the pH value constantly changes during the reactions, the final pH values after the reactions are recorded (Fig. S1) and compared with the initial pH values. For convenience, the initial pH value is used throughout the discussion later. The calcium phenylphosphonate samples synthesized at different initial pH values were characterized by SEM as shown in Fig. 2A. Interestingly, calcium phenylphosphonates experience mutual morphology transformation, from solid microspheres to hollow microspheres and further from hollow microspheres to solid microspheres by increasing the initial pH of the reaction systems. From Fig. 2A(a) to A(e), the calcium phenylphosphonate microspheres gradually change from solid microspheres to hollow microspheres when the initial pH value were increased from 8 to 9. Correspondingly, the thickness of the spherical shells decreased and the nanorods constituting of microspheres shorten from several micrometers to several hundred nanometers. When the initial pH value reached 9, the hollow microspheres possess the thinnest spherical shell about five hundred nanometers in thickness. Conversely, when the initial pH value was further increased from 9 to 11, the hollow microspheres of calcium phenylphosphonate evolve into solid microspheres (Fig. 2A(e) to A(i)), during which the shell thickness gradually increased and the nanorods made of microspheres grow longer. As far as we know, the morphology transformation from solid microspheres to hollow microspheres has been reported [35–38], however, mutual morphology transformation between solid microspheres and hollow microspheres is not. The crystal phase composition of representative calcium phenylphosphonates synthesized at different initial pH values was investigated by powder XRD. It can be seen from Fig. 2B that the XRD patterns of CaPP-c50/pH9 and CaPP-c50/pH11 are almost identical to that of CaPP-c50/pH8, indicating that the three samples have the same crystal phase composition. It has already been discussed above that CaPP-c50/pH8 is a mixed crystal phase of Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O and Ca(C6H5PO3)
2H2O. Thus, it may be concluded that all calcium phenylphosphonate microspheres synthesized in the presence of 50 mg c-PGA are composed of mixed crystal phase of Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O and Ca(C6H5PO3)
2H2O even at different initial pH values. For comparison, powder XRD of calcium phenylphosphonates synthesized at initial pH values of 8, 9 and 11 in the absence of c-PGA was also conducted and shown in Fig. 2C. Based on early analyses [33–34], CaPP-c0/pH8 can be attributed to the Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O crystal phase, while CaPP-c0/pH9 and CaPP-c0/pH11 can be assigned to the Ca(C6H5PO3)
2H2O crystal phase, indicating that the pH value determines the crystal phase of calcium phenylphosphonates when no c-PGA is added. By comparison of XRD patterns of the samples synthesized in the presence of c-PGA with that in the absence of c-PGA, it is clear that c-PGA can affect the formation of a mixed crystal phase of Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O and Ca(C6H5PO3)
2H2O. 3.3. The mechanism of c-PGA in the formation of hierarchical calcium phenylphosphonate microspheres 3.3.1. Micellization of c-PGA The role of c-PGA in CaCl2 solution with different initial pH values was investigated by the DLS technique. DLS measurements (Fig. S2) show that the size and the size distribution of 50 mg cPGA in CaCl2 solution at initial pH values of 8, 9 and 11 is 224 ± 25, 284 ± 50, and 181 ± 13 nm, respectively, suggesting that c-PGA can self-assemble into polyion complex (PIC) micelles through a-carboxyl groups in the structure by binding with calcium ions. As shown in Fig. 3, the a-carboxyl groups in c-PGA bind calcium ions to form a Ca-c-PGA complex leading to spontaneous formation of PIC micelles. In these micelles, the chain segments

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Fig. 2. (A) SEM images of hierarchical calcium phenylphosphonate microspheres synthesized in the presence of 50 mg c-PGA at different initial pH values: (a) pH 8; (b) pH 8.4; (c) pH 8.6; (d) pH 8.8; (e) pH 9; (f) pH 10; (g) pH 10.6; (h) pH 10.8; (i) pH 11; (B) XRD patterns of the selected samples of (a) CaPP-c50/pH8; (e) CaPP-c50/pH9; (i) CaPP-c50/pH11 (the diffraction peaks with asterisk refer to Ca(C6H5PO3)
2H2O crystal phase); (C) XRD patterns of the samples synthesized without addition of c-PGA as a control experiment: (a) CaPP-c0/pH8; (e) CaPP-c0/pH9; (i) CaPP-c0/pH11.

of a-carboxyl groups that bind Ca2+ ions serve as the core, and the chain segments of a-carboxylates that interact with Na+ form a water-compatible corona surrounding the core. Moreover, the electrostatic repulsion of anionic hydrophilic corona chains on the surface of the core-shell structure prevents further aggregation of PIC micelles. It is clear from the result of DLS measurements that the size and the size distribution of PIC micelles self-assembled from Ca-c-PGA complex increases at first and then decreases when the initial pH value is increased from 8 to 11. This behavior may arise from the different amount of calcium ions bound to a-carboxyl groups in c-PGA at different final pH values of

5.27, 6.65 and 9.41. It has been reported that the ability of a-carboxyl groups in c-PGA to bind calcium ions becomes stronger at first and reaches a maximum, but weakens with further increase of the pH value [39]. Under acidic conditions, a-carboxyl groups in c-PGA gradually become deprotonated as the pH value is increased, which leads to stronger Ca2+ binging ability for c-PGA. At a pH value of 5.27, the a-carboxyl groups in c-PGA only partially deprotonate to form a-carboxylate ions to bind calcium ions [39], while the a-carboxyl groups without deprotonation in c-PGA cannot bind calcium ions, which results in weaker binding ability of c-PGA. At basic pH, the bond between OH ions and

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Fig. 3. The schematic diagram of the formation of Ca-c-PGA complex and PPA-Ca-c-PGA complex, as well as the formation of PIC micelles from Ca-c-PGA complex, and the formation of different aggregates (micelles or vesicles) self-assembled by PPA-Ca-c-PGA complex with the initial pH value increasing from 8 to 11.

calcium ions inhibits calcium ions binding to a-carboxylates in cPGA, also weakens the binding ability of c-PGA to calcium ions. Hence, when the final pH is close to neutral at 6.65, fully deprotonated a-carboxyl groups in c-PGA bind the maximum amount of calcium ions to form PIC micelles with the largest size. After a solution of phenylphosphonic acid (PPA) with the initial pH value corresponding to the CaCl2 solution is added to the mixture containing the Ca-c-PGA complex, spontaneous hydrophobic modification of the Ca-c-PGA complex occurs through the coordination of its calcium ions with PPA to form PPA-Ca-c-PGA complex as shown in Fig. 3. Similar to double-hydrophilic block copolymers (DHBCs) known as ‘‘schizophrenic” copolymers, the Ca-c-PGA complex has two hydrophilic chain segments, among which one being the Na+-c-PGA chain segment, the other being the Ca2+-c-PGA chain segment which can be further transformed to hydrophobic PPA-Ca-c-PGA chain segment through further coordination of the calcium ions with PPA. Thus, the addition of PPA containing hydrophobic phenyl group into the reaction system causes the Ca-c-PGA complex with DHBC property to evolve into a PPACa-c-PGA complex, which is an amphiphilic block copolymer that can self-assemble into aggregates, such as micelles or vesicles. In other words, c-PGA experiences a two-stage process in the coparticipation of calcium ions and PPA to give rise to the ‘‘schizophrenic” micellization behavior. First, c-PGA binds to calcium ions and form a Ca-c-PGA complex with DHBC properties. After the addition of PPA containing hydrophobic phenyl groups, the coordination of calcium ions from the Ca-c-PGA complex with PPA causes the hydrophobic modification of Ca-c-PGA complex to occur spontaneously, leading to the transformation from a Ca-c-PGA complex to a PPA-Ca-c-PGA complex that belongs to amphiphilic block copolymers. As previously reported, a change in the number of hydrophobic blocks and hydrophilic blocks causes amphiphilic block polymer to exhibit different self-assembled aggregates (micelles or vesicles) [40]. Here, ICP results show that

Ca% in the final product of CaPP-c50/pH8, CaPP-c50/pH9 and CaPP-c50/pH11 is 9.1%, 18.8% and 9.2%, respectively, which suggests that the number of calcium ions bound to a-carboxyl groups in c-PGA increases at first and then decreases with increasing pH. Variation in the number of bound Ca2+ directly affects the number of hydrophobic PPA-Ca-c-PGA blocks in the PPA-Ca-c-PGA complex and, in turn, the morphology of self-assembled aggregates, so that the PPA-Ca-c-PGA complex self-assembles into different aggregates (micelles or vesicles) at different initial pH values. At an initial pH value of 8, the number of PPA-Ca-c-PGA blocks and Na+-c-PGA blocks causes the PPA-Ca-c-PGA complex to form micelles, which consist of a hydrophobic PPA-Ca-c-PGA core and a hydrophilic Na+-c-PGA corona surrounding the core. As the initial pH is increased to 9, the number of hydrophobic PPA-Ca-c-PGA blocks increases because more calcium ions are bound to the carboxylate groups in c-PGA, and the number of the hydrophilic Na+-c-PGA blocks reduces but the total number of blocks remains unchanged, which causes the PPA-Ca-c-PGA complex to selfassemble into vesicles with a cavity full of solution. In the vesicles, the hydrophobic PPA-Ca-c-PGA blocks constitute the hydrophobic bilayer, whose surface is covered by hydrophilic Na+-c-PGA chains. When the initial pH is further increased to 11, the PPA-Ca-c-PGA complex again self-assembles into micelles. At this moment, the number of PPA-Ca-c-PGA blocks decreases as less calcium ions are now bound to a-carboxylate groups in c-PGA at higher pH. But the number of Na+-c-PGA blocks increases to ensure that l (the number of Na+-c-PGA blocks) plus m (the number of PPACa-c-PGA blocks) is equal to n (the total number of blocks). So with the initial pH value increased from 8 to 11, the self-assembled aggregates formed by the PPA-Ca-c-PGA complex experience a morphology transformation from micelles to vesicles and further from vesicles to micelles. Most importantly, the spontaneous hydrophobic modification of c-PGA by PPA containing hydrophobic phenyl group via calcium

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ions causes c-PGA to experience structural and morphological changes, which are a prerequisite for c-PGA to exhibit ‘‘schizophrenic” micellization behavior and form different selfassembled aggregates (micelles or vesicles) at different initial pH values. Control experiments were also conducted to verify that the hydrophobic phenyl group is key to the formation of selfassembled aggregates (micelles or vesicles). Acids without the hydrophobic phenyl group, such as inorganic H3PO4 and amino trimethylphosphonic acid (ATMP), were selected to take the place of PPA in the synthesis. Now the SEM images (Fig. 4a and b) show that the microsphere morphology cannot be observed in either case, which prompts us to reason this is because there may be no self-assembled aggregates (micelles or vesicles) to serve as template for the formation of products in those synthetic systems. In addition, another control experiment was conducted still using PPA with hydrophobic phenyl group, but using magnesium ions instead of calcium ions in the synthesis. As we expected, the magnesium phenylphosphonate exhibits microsphere morphology and hierarchical structure in SEM image as shown in Fig. 4c, similar to that of calcium phenylphosphonates. Furthermore, the diameter of magnesium phenylphosphonate microspheres is about 7 lm which is the same as that of calcium phenylphosphonate microspheres, meaning that the self-assembled aggregates acting as template for these microspheres is almost identical. The above two control experiments prove indirectly that the hydrophobic groups in phosphonic acids are necessary for c-PGA to exhibit the ‘‘schizophrenic” micellization behavior to form selfassembled aggregates which can induce and control the formation of microspheres. The control experiment using magnesium ions also suggests that only if the metal ions coordinate to not only the carboxyl groups in c-PGA but also the phosphonyl groups in phosphonates, c-PGA can exhibit the ‘‘schizophrenic” micellization behavior in the same way in other synthetic systems of metal phosphonates containing hydrophobic groups. In other words, this synthetic strategy can be extended to other systems, and therefore is broadly applicable for the morphosynthesis of metal phosphonates containing hydrophobic groups. 3.3.2. The mechanism of c-PGA addition amount on the morphology of calcium phenylphosphonates The amount of c-PGA, or the c-PGA concentration in the synthetic system, directly determines the morphology of calcium phenylphosphonates. At an initial pH value of 8, calcium phenylphosphonates exhibit a series of morphological change with the added amount of c-PGA increasing, which arises from the fact that c-PGA at different concentrations exerts a different effect on the formation of calcium phenylphosphonates in the synthetic system, as shown in Fig. 5. When c-PGA concentration is in the range of 0.33 to 0.67 mg mL 1 (5–10 mg c-PGA in 15 mL solution) which has not reached the CMC of Ca-c-PGA complex and PPA-Ca-c-PGA complex, unassembled c-PGA can provide the nucleation sites for calcium phenylphosphonate through binding and accumulating calcium ions using a-carboxyl groups in the structure. During the growth of calcium phenylphosphonate crystallites, the negatively

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charged c-PGA can attach onto the positively charged crystal faces of crystallites, inhibiting the subsequent growth of positively charged crystal faces and causing the different crystal faces to grow in different growth rates. The c-PGA at different concentrations inhibits the growth rates of positively charged crystal faces in different degrees, resulting in that the calcium phenylphosphonate crystallites grow into preliminary building blocks with different shapes, which can further self-assemble into irregular ellipsoids or flower-like microspheres. When the concentration of c-PGA is 1 mg mL 1 (15 mg c-PGA in 15 mL solution) which reaches the CMC of the Ca-c-PGA complex but does not reach the CMC of the PPA-Ca-c-PGA complex, the PIC micelles formed by the Ca-c-PGA complex act as template for the formation of hollow flower-like microspheres. The zeta potential of PIC micelles is measured to be 7.68 mV, which proves that PIC micelles exhibit negatively charged property. As a result, the anionic hydrophilic c-PGA corona chains of PIC micelles attach onto the positively charged crystal faces of calcium phenylphosphonate crystallites and inhibit the growth of the crystallites along certain orientations to form nanoflakes in the shape of narrow strip surrounding the PIC micelles. Under the influence of anionic hydrophilic c-PGA corona chains of the PIC micelles, these nanoflakes arrange along radial orientation perpendicular to the surface of sphere to form flower-like microspheres with PIC micelles as core. As the reaction proceeds, the phenylphosphonates can further capture calcium ions in the Ca-c-PGA complex so that the PIC micelles are destroyed and transform into free c-PGA to dissolve in the solution, which causes the flower-like microspheres to exhibit hollow cores. When the c-PGA concentration reaches the 3.33 to 5.33 mg mL 1 range (50–80 mg c-PGA in 15 mL solution) which exceeds the CMC of the PPA-Ca-c-PGA complex, it can self-assemble into aggregates and act as an template to induce and control the formation of cauliflower-like microspheres. This can be confirmed indirectly by the fact that calcium phenylphosphonates always exhibit uniform cauliflower-like microspheres whose diameter almost remains unchanged even when the amount of c-PGA is further increased from 50 mg to 80 mg. Especially when c-PGA concentration is 2 mg mL 1 (30 mg c-PGA in 15 mL solution), PIC micelles of the Ca-c-PGA complex and self-assembled aggregates of the PPACa-c-PGA complex emerge simultaneously in the solution, which exactly can be regarded as a strong evidence for c-PGA to exhibit ‘‘schizophrenic” micellization behavior in this synthetic system. As a result, the PIC micelles formed by the Ca-c-PGA complex act as templates for the formation of large hollow flower-like microspheres, whereas the self-assembled aggregates of the PPA-Ca-c-PGA complex serve as templates to form small cauliflower-like microspheres with solid structure. In summary, at low c-PGA concentrations, the unassembled c-PGA directly directs the formation of calcium phenylphosphonate, whereas at high concentrations, the PIC micelles formed by the Ca-c-PGA complex or/and the aggregates self-assembled by the PPA-Ca-c-PGA complex act as templates to control the formation of calcium phenylphosphonate microspheres. Only if the concentration of the PPA-Ca-c-PGA complex exceeds the CMC, the PPA-Ca-c-PGA

Fig. 4. The SEM images of control samples synthesized in the presence of c-PGA: (a) CaPO4; (b) CaATMP; (c) magnesium phenylphosphonate.

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X. Wang et al. / Journal of Colloid and Interface Science 556 (2019) 33–46

Fig. 5. The c-PGA with different addition amount (the c-PGA at different concentration) exhibits various statuses which exerts different effect on the formation of calcium phenylphosphonates.

complex can self-assemble into different aggregates (micelles or vesicles) at different initial pH to further act as template for the formation of calcium phenylphosphonate microspheres with solid or hollow structure, respectively. 3.3.3. The mechanism of c-PGA at different initial pH values on the morphology of calcium phenylphosphonate microspheres At different initial pH values, c-PGA self-assembles into different aggregates (micelles or vesicles) in presence of calcium ions and phenylphosphonic acids as mentioned above. These selfassembled aggregates can act as templates to induce and control the formation of calcium phenylphosphonate microspheres with solid or hollow structure, respectively, during which the crystallization of calcium phenylphosphonate occurs inside the template (more specifically, inside micellar hydrophobic core or inside vesicular hydrophobic bilayer) besides what occurs in the solution. (I) The self-assembled aggregates acting as templates in the formation of calcium phenylphosphonate microspheres Due to high mobility of the copolymer chains in the aggregates, the change of aggregation number and the morphology transitions are very fast [41]. As a result, the PIC micelles formed by the Ca-cPGA complex transform into the aggregates self-assembled by the PPA-Ca-c-PGA complex after the addition of PPA into the synthetic system, which is faster than the formation and growth of calcium phenylphosphonate crystallites in the solution. In other words, the formation of aggregates self-assembled by the PPA-Ca-c-PGA complex is prior to that of calcium phenylphosphonate crystallites.

Thus, the self-assembled aggregates (micelles or vesicles) act as templates whose anionic hydrophilic c-PGA chains on the surface can interact with calcium phenylphosphonate crystallites through electrostatic interaction, which induces calcium phenylphosphonate crystallites to grow around the selfassembled aggregates. A time-dependent experiment was expected to provide more details on the formation of microspheres to support the above argument, unfortunately, the formation of calcium phenylphophonate microspheres was an outburst process with a very short induction period. It is obvious that CaPP-c50/pH9 hollow microspheres have come into being after the synthesis proceeded for 1 min and 5 min in the SEM images (Fig. S3), however, the formation process of microspheres cannot be captured directly. Even so, the time-dependent experiment still indicates that once CaPP-c50/pH9 microspheres start to form, they exhibit hollow structure, which supports the argument that vesicles act as templates to control the formation of hollow microspheres. In addition, the deformation of hollow microsphere is another piece of strong evidence in support of our belief that the vesicles to act as templates for the formation of calcium phenylphosphonate microspheres with hollow structure. It can be observed from the SEM image of CaPP-c50/pH9 (Fig. S4) that the hollow microspheres with thinnest spherical shell deform seriously. The microsphere deformation can be ascribed to the fact that before the vesicles are destroyed by the crystallization inside vesicular hydrophobic bilayer, the large cavity full of solution in the vesicles causes the formed microspheres to be flexible and easy to deformation in the presence of external forces.

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X. Wang et al. / Journal of Colloid and Interface Science 556 (2019) 33–46

C1s Ca2p

P2p

1200

800

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O1s

b

C1s Ca2p N1s P2p

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Intensity (a.u.)

O1s

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Fig. 6. XPS survey spectra of (a) CaPP-c50/pH8; (b) CaPP-c50/pH9; (c) CaPP-c50/pH11.

The N content in the calcium phenylphosphonate microspheres can provide the relevant information of self-assembled templating aggregates (micelles or vesicles) in the synthetic system. Thus, the XPS and ICP data of three samples synthesized at initial pH value of 8, 9, and 11 were therefore measured. The results of XPS (Figs. 6 and S5) show that N atoms derived from c-PGA exists on the surface of hollow microspheres (CaPP-c50/pH9), however, it cannot be detected on the surface of solid microspheres (CaPP-c50/pH8 and CaPP-c50/pH11). The ICP result suggests that the N content for these three samples is almost the same in bulk phase, and the c-PGA content can be calculated to be 6.82 wt%, 6.84 wt% and 6.85 wt% for CaPP-c50/pH8, CaPP-c50/pH9 and CaPP-c50/pH11, respectively. The results of XPS and ICP show that the c-PGA exists in the calcium phenylphosphonate microspheres but distributes differently in the interior of the microspheres and on their surface. XPS analysis can only provide the element information on the surface within the maximum depth range of several hundred nanometers, whereas ICP analysis can give the element content of bulk phase. Additionally, it is common in the template synthesis of spherical materials that the diameter of spherical products is larger than that of templates. Thus, XPS can detect N element which distributes within spherical shells of c.a. 500 nm in thickness for CaPP-c50/pH9 hollow microspheres, however, it cannot be detected by XPS for solid microspheres (CaPP-c50/pH8 and CaPP-c50/pH11), which results from N element being distributed in the inner of microspheres with the diameter of several micrometer instead of on the surface of microspheres. Notably, the c-PGA contents in three samples are not as high as we expected, which may result from the fact that the micelles (or vesicles) are destroyed by the calcium phenylphosphonate crystallization inside micellar hydrophobic core (or vesicular hydrophobic bilayer) and transform into free c-PGA in the solution. The N element of three samples detected by elemental analysis might derive from the residual c-PGA adsorbed onto nanorods after the micelles or vesicles serving as template were disrupted. The observation of low c-PGA content in the calcium phenylphosphonate product leads us to speculate that the template self-assembled by biomacromolecules might be destroyed and transform into free biomacromolecules to leach from the biominerals, which is important for in-depth understanding of the template mechanism of biomineralization. (II) The crystallization of calcium phenylphosphonates As shown in Fig. 7, the calcium phenylphosphonate crystallization occurs simultaneously in the solution and inside the template (inside micellar hydrophobic core or inside vesicular hydrophobic bilayer) after the addition of PPA into the synthetic system. In the solution, the free calcium ions coordinate to phenylphosphonates to form nucleus for calcium phenylphosphonate crystallization. As the time goes on, the nucleus grow into crystallites, whose crystal faces with positive charge can be covered by anionic

hydrophilic c-PGA chains on the surface of aggregates (micelles or vesicles) so that the growth of crystallites along certain orientations is restricted. Thus, under the influence of anionic hydrophilic c-PGA chains on the surface of aggregates (micelles or vesicles), the calcium phenylphosphonate crystallites in the solution grow into nanorods which are radial arrangement perpendicular to the surface of sphere. Meanwhile, the crystallization of calcium phenylphosphonate also proceeds inside the template (inside micellar hydrophobic core or inside vesicular hydrophobic bilayer). The phenylphosphonates further capture calcium ions bound to a-carboxyl groups in the PPA-Ca-c-PGA block through the coordination substitution reaction, to form crystal nucleus of calcium phenylphosphonate and subsequently grow into crystallites. The a-carboxylates in the PPA-Ca-c-PGA block carry negative charge after losing calcium ions, and attach onto the positively charged crystal faces of calcium phenylphosphonate crystallites. Such an adsorption prevents the growth of calcium phenylphosphonate crystallites, and also confines these crystallites within the micellar hydrophobic core or vesicular hydrophobic bilayer. Finally, the cooperation effect of the calcium phenylphosphonate crystallization in the solution and that inside the template gives rise to the formation of calcium phenylphosphonate microspheres with solid or hollow structures. For solid microspheres, it is obvious from SEM image of CaPPc50/pH10.8 in Fig. 7A that there is a kernel different from the shell in the microsphere, meaning that when the micelles act as template (the initial pH value is 8, 8.4, 10.8 and 11), the calcium phenylphsophonate crystallization occurs inside micellar hydrophobic core to form the kernel, while that in the solution occurs under the influence of anionic hydrophilic c-PGA corona chains to form the shell. Similarly, when the vesicles serve as templates, the spherical shells of hollow microspheres consist of thick outer spherical shells and thin inner spherical shells as shown in SEM image of CaPP-c50/pH10 (Fig. 7B). The thick outer spherical shells constituted by nanorods in radial arrangement perpendicular to the surface of sphere is distinctly different from thin inner spherical shells, which can be observed from all of hollow microspheres obtained at the initial pH value from 8.6 to 10.6. The double spherical shell in hollow microspheres is formed by the calcium phenylphosphonate crystallization in the solution together with that inside vesicular hydrophobic bilayer. More specifically, the calcium phenylphosphonate crystallization occurs in the solution to form the thick outer spherical shell under the influence of anionic hydrophilic c-PGA chains covering the surface of vesicles, while that occurs inside vesicular hydrophobic bilayer to generate the thin inner spherical shell. Moreover, the thickness of thin inner spherical shell of hollow microspheres is in the range of nanoscale almost corresponding to that of vesicular hydrophobic bilayer, which exactly supports the argument that the crystallization of calcium phenylphosphonates occurs inside vesicular hydrophobic bilayer to form thin inner spherical shell of hollow microspheres.

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X. Wang et al. / Journal of Colloid and Interface Science 556 (2019) 33–46

A

B

calcium phenylphosphonate crystallite

Fig. 7. The schematic diagram of calcium phenylphosphonate crystallization occurs both in the solution and inside micellar hydrophobic core (or vesicular hydrophobic bilayer).

Fig. 8. The schematic diagram of hollow microspheres’ shell thickness varying with the initial pH value increasing.

When the initial pH is increased from 8.6 to 10.6, the shell thickness of hollow microspheres decreases and later increases again as shown in Fig. 8. As described above, the spherical shell of hollow microspheres is composed of calcium phenylphosphonate crystals formed both in the solution and inside vesicular hydrophobic bilayer. Obviously, the calcium phenylphosphonate crystallization inside vesicular hydrophobic bilayer cannot result in so conspicuous variation of hollow microspheres’ shell thickness, because vesicular hydrophobic bilayer is constituted by hydrophobic PPA-Ca-c-PGA blocks due to hydrophobic effect so that the thickness of vesicular hydrophobic bilayer is commonly in the range of nanometers. Thus, the shell thickness of hollow microsphere mainly depends on the outer spherical shell of hollow microspheres formed by the calcium phenylphosphonate crystallization in the solution. The formation of the outer spherical shell of hollow microspheres is under the control of the interaction between anionic hydrophilic c-PGA chains on the surface of vesicles and calcium phenylphosphonate crystallites. More specifically, the anionic hydrophilic c-PGA chains on the surface of vesicles

can adsorb onto positively charged crystal faces of calcium phenylphosphonate crystallites to inhibit the growth of these crystal faces. The interaction strength between anionic hydrophilic c-PGA chains on the surface of vesicles and calcium phenylphosphonate crystallites determines the inhibiting effect on the growth of positively charged crystal faces of calcium phenylphosphonate crystallites and also the length and aspect ratio of calcium phenylphosphonate crystallites. Thus, the interaction strength changes with pH, leading to the variation of the inhibiting effect on the growth of positively charged crystal faces of calcium phenylphosphonate crystallites and also the length of nanorods constituting of the outer spherical shell of hollow microspheres. Stronger interaction leads to stronger inhibiting effect so that the nanorods constituting of outer spherical shell of hollow microspheres shorten and the shell thickness become thinner, and vice versa; weaker interaction results in weaker inhibiting effect to form longer nanorods and thicker shells. As mentioned above, the Ca% of samples obtained at different initial pH values from ICP results has proved that the amount of calcium ions bound to

X. Wang et al. / Journal of Colloid and Interface Science 556 (2019) 33–46

a-carboxyl groups in c-PGA increases at first and reaches the maximum, then decreases with the increase of pH. Correspondingly, the amount of free calcium ions in the solution changes with the opposite trend when the initial pH is increased, namely, decreases at first and reaches the minimum, then increases. At an initial pH of 8.6 or 10.6, more free calcium ions in the solution interfere with the interaction between anionic hydrophilic c-PGA chains on the surface of vesicles and calcium phenylphosphonate crystallites, resulting in weaker inhibiting effect on the positively charged crystal faces of crystallites and the formation of longer nanorods and thicker shells. At an initial pH of 9, the interaction between anionic hydrophilic c-PGA chains on the surface of vesicles and calcium phenylphosphonate crystallites is the strongest due to the least interference from free calcium ions in the solution, leading to the strongest inhibiting effect and the formation of the shortest nanorods and the thinnest shell. In summary, the interaction strength between anionic hydrophilic c-PGA chains on the surface of vesicles and calcium phenylphosphonate crystallites varies with the initial pH value increasing from 8.6 to 10.6, which results in that the shell thickness of hollow microspheres decreases at first, and reaches the thinnest at initial pH value of 9, and then increases later. 3.3.4. The mechanism of c-PGA on the crystal phase of calcium phenylphosphonate microspheres In the control experiments without c-PGA, XRD results (Fig. 2C) show that the product synthesized at an initial pH of 8 is the Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O crystal phase, and the samples obtained at an initial pH of 9 and 11 are the Ca(C6H5PO3)
2H2O crystal phase [33–34]. However, all of CaPP-cm/pHn synthesized in the presence of c-PGA are mixed crystal phase of Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O and Ca(C6H5PO3)
2H2O as shown in Fig. 2B. The formation of mixed crystal phase in calcium phenylphosphonate microspheres may be closely related to the Ca-c-PGA complex and the PPA-Ca-c-PGA complex existing in the same system. The c-PGA exerts different effect on the crystal phase formation of calcium phenylphosphonates at different initial pH values. For CaPPc50/pH8, the Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O crystal phase always forms at an initial pH of 8, whether the calcium phenylphosphonate crystallization occurs in the solution or inside micellar hydrophobic core. Afterwards, the Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O crystal phase in solid microspheres can be partially transformed to the Ca(C6H5PO3)
2H2O crystal phase under the condition that Ca-c-PGA complex can provide additional calcium ions. Such a crystal phase transition from Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O to Ca(C6H5PO3)
2H2O has been found in the previous literatures, in which such a transformation happens under basic conditions in the presence of Ca2+ ions [33–34]. For CaPP-c50/pH9 and CaPPc50/pH11, the crystallization of calcium phenylphosphonates in the solution forms the Ca(C6H5PO3)
2H2O crystal phase, which is same as that in the absence of c-PGA at an initial pH of 9 or 11. Meanwhile, the calcium phenylphosphonate crystallization proceeds either inside vesicular hydrophobic bilayer or inside micellar hydrophobic core to generate Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O crystal phase through the coordination substitution reaction, more specifically, the phenylphosphonates further capture calcium ions which has been bound to a-carboxyl groups in PPA-Ca-c-PGA complex, to form Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O. Therefore, the simultaneous crystallization of calcium phenylphosphonates in the solution and inside vesicular hydrophobic bilayer (or inside micellar hydrophobic core) gives rise to a mixed crystal phase of Ca(C6H5PO3)
2H2O and Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O in hollow microspheres of CaPP-c50/pH9 (or solid microspheres of CaPP-c50/pH11). In short, c-PGA causes the crystal phase transition from Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O to Ca(C6H5PO3)
2H2O to occur for CaPP-c50/pH8 at an initial pH of 8, whereas c-PGA makes

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the calcium phenylphosphonate crystallization inside vesicular hydrophobic bilayer of CaPP-c50/pH9 and inside micellar hydrophobic core of CaPP-c50/pH11 to form Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O crystal phase instead of Ca(C6H5PO3)
2H2O crystal phase through coordination substitution reaction at an initial pH 9 or 11. The ICP measurement was originally expected to give more information on crystal phase composition of calcium phenylphosphonate microspheres, such as Ca/P ratio. The Ca/P ratio of Ca3(C6H5PO3H)2(C6H5PO3)2
4H2O crystal phase is 0.75, while that of Ca(C6H5PO3)
2H2O crystal phase is 1, resulting in the Ca/P ratio of calcium phenylphosphonate microspheres with mixed crystal phase being less than 1. However, the Ca/P ratios in bulk phase obtained from ICP results for CaPP-c50/pH8, CaPP-c50/pH9 and CaPP-c50/pH11 are greater than 1, which might arise from additional calcium ions derived from the Ca-c-PGA complex remaining within the inner of calcium phenylphosphonate microspheres. 3.4. The utilization of solid and hollow calcium phenylphosphonate microspheres with hierarchical structure in the removal of Pb2+ ions Phosphonates themselves are known to be effective flocculants for heavy metal ions. As such, the hierarchical calcium phenylphosphonate microspheres synthesized from phosphonates are expected to exhibit high adsorption capacity for heavy metal ions. The CaPP-c50/pH8 solid microsphere and CaPP-c50/pH9 hollow microsphere are selected and applied as adsorbents in the removal of lead ions, one of the most toxic heavy metal cations. As shown in Fig. 9A, the histogram of Pb2+ adsorption capacity at equilibrium vs. initial concentration of Pb2+ ions exhibits a sustained upward trend with the initial concentration of Pb2+ increasing from 50 mg L 1 to 600 mg L 1. After the initial concentration of Pb2+ ions exceeds 600 mg L 1, the Pb2+ adsorption capacity of CaPP-c50/pH8 almost remain unchanged whereas that of CaPP-c50/pH9 continues to increase slightly. The Pb2+ adsorption capacity of CaPP-c50/pH8 and CaPP-c50/pH9 is as high as 615 mg g 1 and 656 mg g 1 at the initial Pb2+ ion concentration of 1000 mg L 1, respectively. Likewise, the Pb2+ removal efficiency of these two adsorbents can always maintain at 100% until the initial concentration of Pb2+ ions exceeds 600 mg L 1. Afterwards, the Pb2+ removal efficiency of CaPP-c50/pH8 and CaPP-c50/pH9 obviously decline with the initial concentration of Pb2+ ions further increasing. It can be concluded that the calcium phenylphosphonate microspheres exhibit excellent performance on the removal of Pb2+ ions in water, especially when the initial concentration of Pb2+ ions is no more than 600 mg L 1. Because the Pb2+ removal performance of adsorbents is commonly related to their surface area, pore volume and pore size, the nitrogen adsorption-desorption measurement is conducted, and the results are showed in Fig. S6 and Table S1. The BET surface areas of both adsorbents are less than 72 m2 g 1, the larger pore volume of CaPP-c50/pH9 may be due to hollow microspheres possessing large cavities, and the pore size distributions are in a wide range of 2 to 70 nm, all of which could not explain the excellent performance of adsorbents on removal of Pb2+ ions. To understand adsorption process, the adsorption kinetics of these two adsorbents is investigated, shown in Fig. 9C. The adsorption behavior is analyzed using pseudo-first-order [42] and pseudo-second-order kinetic models [43], and the non-linear regression lines obtained by using these two kinetic models are presented in Fig. 9C. The corresponding kinetic parameters, including determination coefficients (R2) and chi-square (v2), obtained from the nonlinear regression by using these two kinetic models are listed in Table S2. It can be observed that the pseudo-secondorder kinetic curves exhibit a better fit to the experimental kinetic data for two adsorbents than pseudo-first-order kinetic curves, suggesting that the pseudo-second-order kinetic model describes the adsorption behavior of two adsorbents more appropriately

X. Wang et al. / Journal of Colloid and Interface Science 556 (2019) 33–46

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Fig. 9. (A) and (B): the adsorption capacity and removal efficiency of CaPP-c50/pH8 and CaPP-c50/pH9 for Pb2+ ions; (C) the adsorption kinetics data of two adsorbents, and the non-linear regression by using pseudo-first-order and pseudo-second-order models; (D) the Pb2+ ion removal efficiencies of CaPP-c50/pH9 after multiple recycles; (E) and (F): 1 the effect of competing cations (Mg2+ and Cd2+) at different concentration on the removal of Pb2+ ions (C2+ ) for CaPP-c50/pH8 and CaPP-c50/pH9. Pb = 50 mg L

[47–48]. To further clarify the diffusion mechanism, the WeberMorris intra-particle diffusion model [44] is used to analyze adsorption kinetic data, and the linear regression lines of qt versus t1/2 are shown in Fig. S7A and the corresponding kinetic parameters are listed in Table S3. It can be observed from Fig. S7A that the linear regression lines do not pass through the origin, suggesting that the intra-particle diffusion is involved but is not the sole rate-controlling step in the whole adsorption process, and the external mass transfer may also play a role to some extent due to the large intercepts of linear regression lines [46–48]. To further determine the rate-controlling step in adsorption process, the adsorption kinetic data are also analyzed by the Boyd model [45]. The plots of calculated Bt values against t and the linear

regression lines of Bt versus t are shown in Fig. S7B, and the corresponding kinetic parameters are listed in Table S4. The linear regression lines in Fig. S7B do not pass through the origin, confirming the involvement of external mass transfer in adsorption process [49]. From the results analyzed by the Weber-Morris and Boyd models, the diffusion rate is jointly controlled by external mass transfer and intra-particle diffusion [46–49]. The zeta potential is an important factor for the adsorbents in their removal of Pb2+ ions from water, as it presents the properties and degree of polarization for functional groups on their surface. The zeta potentials of the two adsorbents at different pH were measured (Fig. S8), from which it is clear that they always carry negative charge and have no zero charge point in the pH range

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from 2 to 11. In the removal of Pb2+ ions, the negatively charged surface of calcium phenylphosphonate adsorbents causes positively charged Pb2+ ions to gather around the adsorbents, followed by the ion exchange of Pb2+ ions with Ca2+ ions on the surface of calcium phenylphosphonates [51]. To investigate the adsorption mechanism of calcium phenylphosphoantes for Pb2+ ions, the CaPP-c50/pH9 adsorbent before and after adsorption of Pb2+ ions was characterized by powder XRD. The XRD pattern of CaPP-c50/pH9 after adsorption of Pb2+ ions in Fig. S9(b) exhibits three new diffraction peaks in comparison with that of fresh adsorbent as shown in Fig. S9(a). These new strong and sharp diffraction peaks are located at 2h of 6.4, 26.9 and 38.5°, which is consistent with that of Pb(C6H5PO3) in previously reported reference [52]. The Pb(C6H5PO3) phase appears in the adsorbent after adsorption of Pb2+ ions, suggesting that calcium phenylphosphonates on the surface of adsorbent partially dissolve, followed by the formation of lead phenylphosphonate precipitation in the adsorption process. It has been revealed that the adsorption of Pb2+ ions on calcium phenylphosphonate adsorbents is mainly based on the dissolution-precipitation mechanism [51,53]. The in situ multiple recycle experiment in column was conducted for the CaPP-c50/pH9 hollow microspheres, and the result of each recycle experiment is summarized in Fig. 9D. It is encouraging to find that the removal efficiency of Pb2+ ions are close to 100% throughout four recycles. The Pb2+ ions adsorbed onto the CaPP-c50/pH9 adsorbent can be desorbed by EDTA-2Na solution, and the desorption efficiency of Pb2+ ions is in the range of 99.2% to 97.6% during four desorption cycles. The effect of competing cations on the removal of Pb2+ ions for these two adsorbents was investigated using Mg2+ and Cd2+ as competing cations. The Pb2+ removal efficiencies of CaPP-c50/pH8 and CaPP-c50/pH9 in the presence of Mg2+ and Cd2+ cations at different concentration are shown in Fig. 9E and F, respectively. From Fig. 9E, the Pb2+ removal efficiency of CaPP-c50/pH8 is almost not influenced by Mg2+ in the whole concentration range. For CaPPc50/pH9, the Pb2+ removal efficiency is not affected by Mg2+ at a low concentration of 50 mg L 1, however, it decreases by 5% when the Mg2+ concentration is increased to 600 mg L 1, which is in agreement with the previously reported reference [50]. It is found that these two adsorbents are little influenced by Mg2+ cations in the removal of Pb2+ ions even in a broader range of Mg2+ concentration. When heavy metal Cd2+ acts as the competing cation, the Pb2+ removal efficiency of either CaPP-c50/pH8 or CaPPc50/pH9 is little influenced by Cd2+ cation at concentration of 50 mg L 1. With Cd2+ concentration increasing to 300 mg L 1, the Pb2+ removal efficiency of both CaPP-c50/pH8 and CaPP-c50/pH9 falls about 6%. With Cd2+ concentration further increasing to 600 mg L 1, the Pb2+ removal efficiency of CaPP-c50/pH8 drops to about 86% while that of CaPP-c50/pH9 falls to about 70%. In other words, the Pb2+ removal efficiency of two adsorbents is almost not affected by low concentration of Cd2+ cation, however it is influenced by high concentration of Cd2+ cation to a large extent. The results of competing cation experiment reveals that the calcium phenylphosphonate microspheres exhibit high selectivity towards Pb2+ ions in the presence of other metal cations.

4. Conclusions In summary, it has been found for the first time that c-PGA exhibits the ‘‘schizophrenic” micellization behavior, in that it induces and controls a series of interesting morphology transformations of hierarchical calcium phenylphosphonates. In particular, a rare, reversible morphology transformation between solid microspheres and hollow microspheres is observed with increasing pH. Whereas c-PGA alone does not exhibit the ‘‘schizophrenic”

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micellization behavior in the aqueous solution, in this work we found that it does form different self-assembled aggregates (micelles and vesicles) at various initial pH if it experiences structural and morphological changes. Spontaneous hydrophobic modification of c-PGA with phenylphosphonic acids and calcium ions is required. The self-assembled aggregates formed at different initial pH further act as templates for the formation of hierarchical calcium phenylphosphonate microspheres with different morphologies. With increasing pH, the self-assembled aggregates evolve from micelles to vesicles, and later from vesicles to micelles again, hence the reversible morphology transformation between solid and hollow microspheres. This work provides not only experimental evidence for the morphological change of biopolymers in biomimetic synthesis, but also gives an entirely new perspective for in-depth understanding of the structure-directing role and template mechanism of biomacromolecules in biomineralization. Furthermore, the synthetic strategy presented here provides a universally applicable method for morphosynthesis of metal phosphonates containing hydrophobic groups, because other metal ions can also cause c-PGA to exhibit ‘‘schizophrenic” micellization in a similar way as long as they can coordinate to both carboxyl groups of c-PGA and phosphonic acids containing hydrophobic groups. And last but not least, our results showed that the solid and hollow calcium phenylphosphonate microspheres with hierarchical structures are also promising candidates for efficient, reusable and selective adsorbents in the removal of Pb2+ ions. Acknowledgments Financial support of this work was provided by the Opening Foundation of State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China (N-17-02 and N-18-10) and Program for Innovative Talents of Higher Learning Institutions of Liaoning, China (2018). Declaration of Competing Interest The authors declare no completing financial interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcis.2019.08.039. References [1] A. Arakaki, K. Shimizu, M. Oda, T. Sakamoto, T. Nishimura, T. Kato, Biomineralization-inspired synthesis of functional organic/inorganic hybrid materials: organic molecular control of self-organization of hybrids, Org. Biomol. Chem. 13 (2015) 974–989. [2] S. Elsharkawy, A. Mata, Hierarchical biomineralization: from nature’s designs to synthetic materials for regenerative medicine and dentistry, Adv. Healthc. Mater. 7 (2018) 1800178. [3] S. Weiner, Biomineralization: a structural perspective, J. Struct. Biology 163 (2008) 229–234. [4] D. Hagmeyer, K. Ganesan, J. Ruesing, D. Schunk, C. Mayer, A. Dey, A.J.M.N. Sommerdijk, M. Epple, Self-assembly of calcium phosphate nanoparticles into hollow spheres induced by dissolved amino acids, J. Mater. Chem. 21 (2011) 9219–9223. [5] A.B. Krajina, C.A. Proctor, P.A. Schoen, J.A. Spakowitz, C.S. Heilshorn, Biotemplated synthesis of inorganic materials: an emerging paradigm for nanomaterial synthesis inspired by nature, Prog. Mater. Sci. 91 (2018) 1–23. [6] Y.R. Cai, J.M. Yao, Effect of proteins on the synthesis and assembly of calcium phosphate nanomaterials, Nanoscale 2 (2010) 1842–1848. [7] C. Qi, Y.J. Zhu, B.Q. Lu, X.Y. Zhao, J. Zhao, F. Chen, Hydroxyapatite nanosheetassembled porous hollow microspheres: dna-templated hydrothermal synthesis, drug delivery and protein adsorption, J. Mater. Chem. 22 (2012) 22642–22650. [8] F. Ridi, I. Meazzini, B. Castroflorio, M. Bonini, D. Berti, P. Baglioni, Functional calcium phosphate composites in nanomedicine, Adv. Colloid Interf. Sci. 244 (2017) 281–295.

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