Biomimetic Crystallization of Unusual Macroporous Calcium Carbonate Spherules in the Presence of Phosphatidylglycerol Vesicles Xiaohua Liu,†,‡ Lixue Zhang,†,‡ Yuling Wang,†,‡ Cunlan Guo,†,‡ and Erkang Wang*,†
CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 3 759–762
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, P. R. China, and Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China ReceiVed NoVember 16, 2006; ReVised Manuscript ReceiVed December 30, 2007
ABSTRACT: We report the interesting finding that crystallization of calcium carbonate (CaCO3) in the presence of dimyristoylphosphatidylglycerol (DMPG) vesicles by a simple gas diffusion method results in the formation of unusual microscopic CaCO3 spherules. The experimental results indicate that the as-prepared CaCO3 spherules, which have a complex macroporous structure, are predominantly vaterite. It is believed that DMPG vesicles play an important role in the process of crystallization, and the possible formation mechanism is proposed. Advanced inorganic materials with complex patterns hold promise for use in some fields, such as catalysis, separation, biomedical implants, drug delivery and release, gene vectors, etc.1 Because of its abundance in nature and its important applications, calcium carbonate (CaCO3), which exists in three main crystal polymorphs: calcite, aragonite, vaterite, has been considered as one of the standard model systems for studying biomimetic mineralization. A wealth of papers has been reported on the biomimetic crystallization of CaCO3. In particular, bioinspired morphosynthesis strategies using a variety of organic additives and/or templates, such as organic acids,2–4 polymers,5–12 surfactants,13–15 etc, to control CaCO3 crystallization with different morphologies and complex textures have attracted considerable attention. On the basis of these strategies, various crystal morphologies have been obtained, such as calcite rhombohedra, urchin-shaped aragonite,14 vaterite sphere,11 calcite sphere,8,10 hollow shell,2,5,13 rod,5,15 ellipsoide,5 dumbbell,5 disc,6 etc. Phospholipid, often in the form of bilayer vesicles, has been commonly involved in natural biomineralization processes.16 Thus, the bilayer vesicles can be used as ideal models to study biomimetic mineralization and provide not only a confined, organized microenvironment but also an organic matrix for biomimetic mineralization.17–21 During crystal growth, complex crystal shapes and textures can be produced by altering the shape of the lipid matrix.21 Here, we report that microscopic scale, macroporous CaCO3 spherules, which are predominantly vaterite, can be prepared by a simple gas-diffusion method22 in the presence of dimyristoylphosphatidylglycerol (DMPG) vesicles at around 18 °C. In a typical synthesis, the mixture of calcium chloride and DMPG vesicles (cDMPG ) 1.5 mM) with a [Ca2+]/[DMPG] molar ratio of 1:2 was vigorously stirred for several minutes. The bottle of this mixture and a glass bottle of fresh ammonium carbonate ((NH4)2CO3) powder were covered with parafilm punched with three needle holes respectively and then placed in the same desiccator simultaneously. After 5 days, the precipitates were separated and rinsed with doubly distilled water thoroughly and allowed to dry in the atmosphere. The morphology of the resulting precipitates placed on the top of Si substrate (sputter coated with gold film) was characterized with a XL 30 ESEM FEG scanning electron microscope (SEM). The typical SEM images are shown in Figure 1. The lower magnification image (Figure 1A) indicates that the precipitates consist of a large quantity of big spherules with a size in the range of 20–80 µm. (We also find few calcite rhombohedra. Data are not * To whom correspondence should be addressed. Fax: 86-431-85689711. E-mail:
[email protected]. † Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. ‡ Graduate School of the Chinese Academy of Sciences.
Figure 1. (A) Lower magnification SEM image of the resulting precipitate deposited on Si substrate (sputter coated with gold film); (B) higher magnification SEM image of a CaCO3 spherule.
shown.) The high magnification image (Figure 1B) reveals that these spherules are like honeycomb with a complex macroporous network structure on their surfaces. The control experiment was conducted in the absence of DMPG vesicles. Under this condition, we found that the majority of the products were thermodynamically stable calcite rhombohedra (see Figure S1, Supporting Information), which is a common phenomenon when calcium carbonate precipitation is obtained by this method without additives. Thus, it can be concluded that DMPG vesicles play a critical role in the formation of the unusual CaCO3 crystals.
10.1021/cg060812s CCC: $40.75 2008 American Chemical Society Published on Web 02/09/2008
760 Crystal Growth & Design, Vol. 8, No. 3, 2008
Figure 2. (A) The XRD of the resulting precipitates and (B) the Raman spectrum of single CaCO3 spherule.
The X-ray diffraction (XRD) analysis of the resulting precipitates was carried out on a D/MAX 2500V/PC X-ray diffractometer using Cu radiation. Figure 2A shows the XRD pattern of the CaCO3 spherules, with the principal peaks corresponding to vaterite (V) and calcite (C) phases highlighted. It indicates the coexistence of vaterite and calcite. According to XRD results, we can calculate the precipitates to be composed of 88% mol vaterite and 12% mol calcite.23 This phenomenon is common in biomimetic crystallization experiments,24 which can be ascribed to the phase transformation of the sphere or some nucleation of CaCO3 crystal in the aqueous phase as opposed to the surface of phospholipid vesicles.25 To characterize the single CaCO3 spherule, the Raman spectrum of the resulting precipitates was recorded with a Renishaw 2000 model confocal microscopy Raman spectrometer with a CCD detector and a holographic notch filter. The laser beam was focused onto a spot on single spherule of approximately 1 µm in diameter and the radiation of 514.5 nm from an air-cooled argon ion laser was used for excitation. The spectrum of CaCO3 crystal contains several discernible bands between 100 and 1600 cm-1. The strongest band at about 1000 cm-1 for CaCO3 crystal overlaps unfortunately for three polymorphs of calcite, aragonite, and vaterite. The Raman band at about 700 cm-1 is too weak to be identified sometimes.26 Thus, the Raman bands between 100 and 400 cm-1 were used to distinguish the polymorphs of calcium carbonate because the three polymorphs have different typical peaks in this range. As shown in Figure 2B, the bands at 301 and 268 cm-1 associated with the vibrations of the carbonate group relative to calcium ions are distinctive features of vaterite.23,27 Thus, it can be concluded that the as-prepared CaCO3 products were predominantly vaterite according to the XRD data and Raman spectrum.
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Figure 3. (A) The TEM image of the intermediate products for 10 h growth and (B) SEM of the resulting precipitates after 1 day growth.
Figure 4. FTIR spectra of the CaCO3 products (a) and pure DMPG (b).
To determine the formation mechanism, first, the CaCO3 products in the early stage of crystallization have been monitored by quenching the reactions when the reaction times were 10 h and 1 day, respectively. As shown in Figure 3A, when the reaction time was 10 h, the intermediate products were nanoparticles with diameters of tens of nanometers, and many CaCO3 nanoparticles were hollow spheres. It can also be found that many CaCO3
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Crystal Growth & Design, Vol. 8, No. 3, 2008 761 Scheme 1. The Possible Formation Mechanism of CaCO3 Spherules
nanoparticles aggregated together into a network structure. When the reaction time was 1 day (Figure 3B), the spherules with a diameter of about 15–30 µm can be observed. These spherules had a porous network structure similar to the final products. Second, the role of DMPG vesicles in the CaCO3 crystallization was investigated since the interaction between Ca2+ and DMPG vesicles is the precondition of the CaCO3 crystallization experiment. A Bruker Vertex 70 FTIR spectrometer was used to acquire the FTIR spectra of the CaCO3 products after they were thoroughly washed. From the FTIR spectra, it can be seen that the CaCO3 products indeed contain DMPG (Figure 4). The interaction of Ca2+ with anionic phospholipid has been studied widely, and large numbers of papers and reviews have been published.28–36 Ca2+ can bind to anionic phospholipid through an electrostatic effect and induce anionic phospholipid vesicles aggregation and fusion, which have been proven by different techniques such as differential scanning calorimetry, electron spin resonance, freeze-fracture electron microscopy, etc.28–31 However, there is a threshold concentration at which Ca2+ becomes effective in inducing vesicles aggregation and fusion, and the threshold concentration varies for different anionic phospholipids.32,33 As for DMPG vesicles, the threshold concentration of Ca2+ for inducing its aggregation and fusion is 5 and 15 mM, respectively.32 Thus, the DMPG vesicles did not aggregate and remained stable before the CaCO3 crystallization because the concentration of Ca2+ (0.75 mM) used was very low in our typical experiment. When the concentration of Ca2+ was 28 mM, the CaCO3 products were mainly normal prisms and spheres (as shown in Figure S2, Supporting Information). This phenomenon should be attributed to the higher concentration of Ca2+, which led to the rapid crystallization and the disruption of templates (i.e., the aggregation and fusion of DMPG vesicles). We also investigated the effect of reaction temperature on the CaCO3 products. When the reaction was performed at 8 °C, most of the CaCO3 products were semispheres, and they were a mixture of vaterite and calcite (Figure S3A,B, Supporting Information). When the reaction was performed at 28 °C, parts of the CaCO3 products were flower-like and the other parts were quasi-spheres, and they also were a mixture of vaterite and calcite (Figure S3C,D, Supporting Information). The effect of reaction temperature on the CaCO3 products may be mainly related to the kinetics of nucleation and growth of CaCO3, and the higher temperature brings on a higher reaction speed. Moreover, the temperature would also change the phase of DMPG vesicles. Because the main phase transition temperature of DMPG is about 24 °C,37 the DMPG vesicles were in gel phase when the reaction temperature was at 8 and 18 °C, and in liquid-crystalline phase when the reaction temperature was at 24 °C. It is well-known that the vesicles in gel phase are more stable than that in the liquid-crystalline phase, but the changes of temperature would not change the size of DMPG vesicles. Thus, considering the fast dynamics of the reaction and the lower stability of DMPG vesicles at 24 °C, the CaCO3 nanoparticles would form and aggregate into flower-like or quasispheres with a diameter of micrometers. When the reaction was performed at 8 °C, it was similar to the reaction
at 18 °C except that the nucleation occurred both in the solution and at the interface of the solution and the glass vessel, which led to the formation of many semispheres. However, the exact reason for the effect of reaction temperature on the CaCO3 products still remains unclear and is under further study. Thus, on the basis of the experiment results above and according to the interaction between Ca2+ and DMPG vesicles, the formation mechanism of the CaCO3 spherule in the typical experiment was proposed as illustrated in Scheme 1: Because of the electrostatic effect, there is a high local concentration of Ca2+ at the surface of vesicles, which leads to the nucleation of CaCO3 on the surface of DMPG vesicles. Then the CaCO3 nanoparticles form, and many of them are hollow spheres.17 In addition, during this process, DMPG vesicles undergo microscopic phase separation together with fusion, fission, reshaping, and collapse affected by the change of ionic strength and pH of the solution and/or the mechanical stress of the CaCO3 layer. The CaCO3 nanoparticles tend to aggregate together and then the porous network structure of CaCO3 spherules forms through self-assembly of the CaCO3 nanoparticles.2,38,39 It is believed that the formation of a porous network structure should be ascribed to the existence of DMPG vesicles. Finally, the smaller spherules grow into bigger spherules after a long time ripening. In conclusion, we present here an easy route for the fabrication of microscopic CaCO3 spheres with a unusual macroporous network structure using DMPG vesicles as additives. The XRD data and Raman spectra indicated that such CaCO3 products were predominantly vaterite, and the formation mechanism was proposed. Our experiments show that DMPG vesicles play important roles of control on the nucleation, growth, and pattern of calcium carbonate particles in the formation of such CaCO3 crystals. The reported results here may be helpful in understanding the complex process in the natural biomineralization, and the produced calcium carbonate crystals with a well-defined morphology, size, and special macroporous network structure could be a promising candidate for materials due to the significance of shape and texture in determining the properties of materials. It is expected that various phospholipid vesicles could be similarly applied to different carbonate systems (BaCO3, MgCO3, PbCO3, CdCO3, etc.) as effective crystal additives due to the interaction between the divalent metal ions and the phospholipid.
Acknowledgment. This work is supported by the National Natural Science Foundation of China with Grant No. 20575063 and the Chinese Academy of Sciences KJCX2-YW-H09. Supporting Information Available: The data of CaCO3 products prepared under other conditions. This material is available free of charge via the Internet at http://pubs.acs.org.
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