Bioinspired mineralization of calcium carbonate in peptide hydrogel acting as a multifunctional three-dimensional template

ABSTRACT Biomineralization is the process by which biominerals, minerals composed of bioinorganic matter possessing a controlled structure and orientation and a biomacromolecular assembly with an ordered structure that acts as a 3D template, are formed. In this study, we investigated the fabrication of organic/inorganic hybrid gels by bioinspired mineralization in peptide hydrogels. An Ac-(VHVEVS)3-CONH2 peptide was used as a multifunctional template with a mineral source supply capability and structural controllability that facilitates the formation of hydrogels via self-assembly. Hydrogels with varying viscoelastic strengths were prepared from the designed peptide by controlling the concentration of calcium ions added as cross-linking agents. The peptide hydrogel supplied carbonate anions as the mineral source through the hydrolysis of urea and mineralized CaCO3 with controlled morphology on the peptide network. With increases in the concentration of calcium ions added, the morphology of the mineralized CaCO3 changed from a fibrous structure to a thin film. This implies that the nucleation and growth mechanisms of CaCO3 formed by bioinspired mineralization were affected not only by the morphology and supply rate of the mineral source by the peptide network acting as a multifunctional template, but also by the viscoelastic strength of the hydrogel that served as a 3D reaction field. Graphical abstract


Introduction
Biomineralization is a biosynthetic process by which biominerals (e.g. bones, teeth, and seashells) are formed under mild conditions at ordinary temperatures and pressures. Biominerals have a characteristic hierarchical nanohybrid structure that is composed of an inorganic component [1][2][3], which is a bioinorganic matter, and an organic component [4][5][6], which is a biomacromolecular assembly. Bioinorganic matter has a controlled crystal structure and orientation, while biomacromolecular assemblies with a threedimensional (3D) ordered structure act as templates for nucleation and crystal growth through biomineralization [7][8][9][10]. Among the various types of bioinorganic matter, calcium carbonate (CaCO 3 ) is one of the most studied [11][12][13][14]. The mechanisms of nucleation and crystal growth of bioinorganic matter in twodimensional reaction fields such as peptide monolayers [15,16], polymer brushes modified onto substrates [17,18], polymer solutions [19][20][21], and hydrogel/solution interfaces [22,23] have been extensively investigated. The hierarchical nanohybrid structure composed of bioinorganic matter with a biomacromolecular assembly endows biominerals with a wide variety of features, such as superior mechanical properties and structural color. Biomineralization has therefore attracted significant interest as a novel green method for the fabrication of functional hybrid materials. These hybrid materials are expected to be applied as catalysts, bone filling materials, and carriers for drug delivery systems. Hydrogel-based materials, contain water as a main component much like biological soft tissues, have been actively especially studied as base materials for use in several medical research areas. Among these studies, mineralization of CaCO 3 in hydrogels such as gelatin and agarose have been reported to have achieved control of the morphology and crystal phase of the mineralized CaCO 3 in the gel matrices [24][25][26][27]. These studies are therefore important to promoting understanding of structural control of biominerals in the biomineralization process. On the other hand, the physical properties of organic-inorganic hybrid materials utilizing mineralization have not been sufficiently evaluated. In addition, fabrication of a hybrid gel inspired by living organisms and obtained by the structural control of inorganic matter through a mineralization process using a multifunctional 3D template with a mineral-self-supplying capability and structural controllability [28] that facilitates the formation of hierarchical, ordered structures has not yet been reported. In this study, we call templates for mineralization with multiple functions, which are inspired by the biomineralization of CaCO 3 of nacrein, "multifunctional 3D templates". These templates have an ability to form af 3D reaction field by self-assembly and a self-supplied ability by hydrolysis of precursor utilizing enzyme-like catalytic activity. To design a multifunctional 3D template for bioinspired mineralization and hydrogel for based material, we focused on a peptide as a model protein molecule. Peptide molecules can assume different second-order structures (e.g. α-helix, β-sheet, and random coil) based on their primary structure and such external environmental factors as pH, temperature, and salt concentration. Peptides having specific second-order structures can not only control the spatial arrangement of functional groups of amino acid side-chain, moreover, but can also form 3D ordered assembly through self-assembly. We have previously reported the design of β-sheet peptides that can act as mineral source suppliers [8,29]. Peptide molecules are therefore superior building blocks for the formation of multifunctional 3D templates compared with those formed by typical synthesized polymers. In this study, we investigated fabrication of organic/inorganic hybrid gels by bioinspired mineralization in peptide hydrogels acting as multifunctional 3D templates and reaction fields.

Solid-phase peptide synthesis
We designed and synthesized a hydrophilic and hydrophobic amino acid alternating Ac-(Val-His-Val-Glu-Val-Ser) 3 -CONH 2 peptide (VHVEVS) 3 . The 18-mer (VHVEVS) 3 peptide was synthesized by solid-phase peptide synthesis on CLEAR-amide resin (CLEAR = crosslinked ethoxylate acrylate resin) by 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Fmoc-protected amino acids were polymerized in the order of in the sequence on the resin. To cap the N-terminal amino group of the synthesized peptide resin with an acetyl group, the peptide resin was stirred in a mixture of acetic anhydride and pyridine (1:2 v/v) for 4 h. To remove all the side-chain protecting groups and detach the peptide molecule from the resin, the peptide resin was added to a 95 vol% trifluoroacetic acid (TFA) aqueous solution. The peptide-containing TFA cocktail was then dropped into diethyl ether, and the precipitated peptide was recovered and lyophilized. The resulting peptide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF-MS) spectroscopy performed using a JEOL JMS-S3000 system.

Preparation of peptide hydrogel
First, 1.0 mL of (VHVEVS) 3 peptide solution (5.0 mM) was adjusted to pH 7.4 with 0.01 mM NaOH and HCl aqueous solutions. Then, a calcium acetate solution (50 μL) of different concentrations was added to the peptide solution, and the mixture was vigorously stirred using an ultrasonic homogenizer. The concentrations of the added calcium acetate solution were adjusted so that the molar ratios of the carboxyl group of the amino acid side-chain of the peptide to the and calcium ion ([Ca 2 + ]/[COO − ]) were 0.05, 0.125, 0.175, and 0.250.

Hydrolysis of urea in the peptide hydrogel and bioinspired mineralization
A urea hydrolysis reaction and bioinspired mineralization were carried out as follows: A urea aqueous solution (1.0 M, 20 μL) was injected into peptide hydrogels with different [Ca 2+ ]/[COO − ] molar ratios, and the sample hydrogels were left to stand at 25°C for 7 days. The hydrolysis activity of the peptides toward urea was measured by the indophenol method using a urease activity kit (BUN Kinos, Kinos Lab., Japan) according to previously reported procedures [8,29]. We determined the concentration of carbonate anions from the changes in the UV-vis absorption spectra caused by the indophenol generated by the reactions. UVvis spectroscopic measurements were performed using a quartz cell with a 1.0 mm path length in the wavelength range of 500−800 nm. The concentration of the generated carbonate anions was determined by halving the concentration of ammonium cations.

Rheological evaluation
Rheological evaluations of the pristine peptide hydrogels and CaCO 3 -peptide hybrid gels were conducted using a parallel-plate rheometer (MCR302, Anton Paar) with a diameter of 20 mm and a gap of 0.2 mm. One milliliter of hydrogel was placed between the plates using a micropipette, and the storage elastic modulus (G') was measured in the angular frequency range of 0.1−10.0 rad/s at 25°C.

Spectroscopic measurements
To analyze the second-order structure of the peptide, transmission Fourier transform infrared (TM-FTIR) spectroscopy (Perkin Elmer Spectrum 100) was performed in the wavenumber range of 1800 −1550 cm −1 at a resolution of 4 cm −1 with 512 scans. For the TM-FTIR spectroscopic measurements, the peptide hydrogel and CaCO 3 -peptide hybrid gel were lyophilized, and 1 wt% samples were mixed with KBr and pressed into pellets. The 1800 −1550 cm −1 spectral region was analyzed as the sum of the Gaussian−Lorentzian (9/1) composition of the individual bands, which was fitted to the experimental spectra. The ratio of the integrated peak intensities assigned to the individual secondary structures, obtained by deconvolution of the amide I band, yielded the percentage of each conformation of the peptide.

Electron microscopy
The morphology of mineralized CaCO 3 was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM, JEM-2100 F, JEOL). The SEM observation was performed at an acceleration voltage of 10 kV and an emission current of 15 μA (S4200, Hitachi). For the SEM observation, a small aliquot was taken from the CaCO 3 -peptide hybrid gel and placed on a mica substrate, and the hybrid gel was allowed time to adsorb onto its surface. After adsorption, the hybrid gel-adsorbed mica substrate was rinsed with H 2 O to remove unreacted urea and calcium acetate. The sample for SEM was coated with gold nanoparticles via ion sputtering. For TEM, a small aliquot was taken from the CaCO 3 -peptide hybrid gel and placed on an elastic carbon-coated scanning TEM (STEM) grid. The hybrid gel was allowed to adsorb onto its surface, and the grid was then rinsed with water to remove unreacted urea and calcium acetate. The crystal structure of the mineralized CaCO 3 was determined by high-resolution TEM (HR-TEM) and selected-area electron diffraction (SAED) analysis.
The morphology of the CaCO 3 -peptide hybrid networks in the hydrogel was observed under wet conditions by scanning-electron assisted dielectric microscopy (SE-ADM) performed using a fieldemission SEM (JSM-7000 F, JEOL) [30,31]. A sample solution (1.5 μL) was dropped into a hand-made liquid sample holder and sealed, and the holder containing the sample was placed on a sample stage with a preamplifier and loaded into the SEM. A low-acceleration voltage electron beam irradiated the tungsten-covered SiN film on top of the sample holder, causing a local potential change in the film. The electrical signal was amplified by the preamplifier in this stage and converted into a digital signal by an AD converter (AIO-163202FX-USB, CONTEC Co., Japan) after low-pass filtering (LPF). The LPF and EB scan signals were logged by a PC using an AD converter at a sampling frequency of 50 kHz. The SE-ADM signal data from the AD converter were transferred to a personal computer (Intel Core i7, 2.8 GHz, Windows 7), and SE-ADM images were composed from the LPF signal and scanning signal using MATLAB R2007b software with an image-processing toolbox (Math Works Inc., Natick, MA, USA).
SEM images (1280 × 960 pixels) were captured at a magnification of 80,000×, scanning time of 80 s, working distance of 7 mm, EB acceleration voltage of 3.0 kV, and current of 10 pA.

Molecular design of the peptide and structural analysis of the peptide hydrogel
In previous studies, we reported the synthesis of a αsheet peptide (Ac-VHVEVS-CONH 2 ) that supplies a mineral source through urea hydrolysis [8,29]. However, the VHVEVS peptide cannot form a hydrogel. Formation of stabile nanofibers by selfassembly of β-sheet peptide is important to achieving a hydrogelation ability. Stabilization of the β-sheet conformation of a peptide is induced by formation of intermolecular hydrogen bonding between peptide molecules, which is increased with increases in the chain length of the main chain of the peptide molecules. To endow the peptide with a hydrogelation ability, we designed and synthesized a new 18-mer Ac-(VHVEVS) 3 -CONH 2 peptide to serve as a multifunctional 3D template for bioinspired mineralization. The molecular weight of the synthesized (VHVEVS) 3 peptide was estimated as 2010.83 by MALDI-TOF-MS spectroscopic analysis. This value is in good agreement with the calculated value of 2011.28. MALDI-TOF-MS spectroscopy indicated the successful synthesis of the designed (VHVEVS) 3 peptide. We investigated the second-order structure of the (VHVEVS) 3 peptide by TM-FTIR spectroscopy. Figure 1 shows the TM-FTIR spectrum of the peptide under solution conditions (peptide concentration: 5.0 mM, pH: 7.4). The TM-FTIR spectrum shows the characteristic absorption peaks at 1630, 1698, and 1675 cm −1 , which are attributed to β-sheet amide I band, antiparallel β-sheet, and random coil conformation, respectively [32][33][34]. The ratios of the integrated peak intensities assigned to individual second-order structures were obtained by deconvolution of the TM-FTIR spectrum. These results indicate that the (VHVEVS) 3 peptide mainly assumed an antiparallel β-sheet conformation (β-sheet: 75%, random coil: 25%) in solution. β-Sheet peptides form a 3D structural network composed of self-assembled nanofibers, and crosslinking between the networks induces formation of peptide hydrogels [35]. To prepare the peptide hydrogels, we  (Figure 2a). The crosslinked structure formed by the Glu side-chain carboxyl groups and calcium ions were investigated by TM-FTIR spectroscopy (Figure 2b). The absorbances were normalized by calculating the area ratio of the amide I band in the TM-FTIR spectra of the peptide hydrogels. The absorption band corresponding to the -COO − stretching vibration appears at 1417 cm −1 [36]. This absorption band appeared in the TM-FTIR spectra of all the prepared peptide hydrogels with different [Ca 2 + ]/[COO − ] molar ratios, and the normalized absorbance increased with increases in the concentration of calcium ions that served as a cross-linking agents ( Figure  2c). The change in absorbance indicated that the crosslinking between the Glu side-chain carboxyl groups and calcium ions increased with increases in the concentration of calcium ions added. Interestingly, however, the -COO − stretching vibration band exhibited a constant normalized absorbance for the hydrogels with [Ca 2+ ]/[COO − ] molar ratios of over 0.125. This indicates that the Glu side-chain carboxyl group in the peptide hydrogel exists as COO − regardless of the increases in the calcium ion concentration at [Ca 2+ -]/[COO − ] molar ratios above 0.125. We believe that this phenomenon results not only in cross-linking of the Glu side-chain carboxyl groups and calcium ions but also in charge-shielding due to interaction between the His side-chain imidazole groups and Glu side-chain carboxyl groups. These results indicate that the prepared peptide hydrogels contain free calcium ions that can act as a mineral source for CaCO 3 mineralization.
Next, we investigated the viscoelastic strength of the prepared peptide hydrogels using a rheometer. Figure 3a shows the G' of the peptide hydrogels with different calcium ion concentrations and the peptide aqueous solution prepared with a [Ca 2+ ]/[COO − ] molar ratio of 0.05 (Non-gel). The viscoelastic strength of the peptide hydrogels increased with increases in the calcium ion concentration and measured over 700 Pa for all the peptide hydrogels (Figure 3b). In addition, similar to the normalized absorbance of the -COO

Self-supply of a mineral source through urea hydrolysis in peptide hydrogels
We determined the concentration of ammonium cations from the changes in the UV-vis absorption spectra caused by indophenol generated by the indophenol method. The concentration of the generated carbonate anions was determined by halving the concentration of ammonium cations. Figure 4a shows the time-dependent changes in the concentration of carbonate anions generated in the peptide hydrogels and peptide solution (Non-gel). A Non-gel system is an aqueous peptide solution that contains calcium ions but is not hydrogelled. In both the peptide hydrogels and peptide solution, the (VHVEVS) 3 peptide generated carbonate anions by hydrolyzing urea as a substrate. The changes in the concentration of the carbonate anions implies that the hydrolysis of urea by the (VHVEVS) 3 peptide occurs in two stages. The first stage is an induction period (0 − 2 days) in which the change in the concentration of carbonate anions generated by urea hydrolysis is extremely small; thus, the curve has a gradual slope. We believe that the induction period occurs because diffusion of the added urea in the peptide hydrogel takes some time. The second stage is the reaction period (3 − 7 days), in which on active period carbonate anions are generated by efficient urea hydrolysis. In the reaction period, urea diffused in the peptide hydrogel is efficiently hydrolyzed in the peptide network through enzyme-like activity; this significantly changes the concentration of carbonate anions in the peptide hydrogel. To investigate the relationship between viscoelastic strength and hydrolysis activity, we calculated the reaction rate of urea hydrolysis by the peptide in the hydrogels. The reaction rate was calculated from the slope of the active period curve. Figure 4b shows the relationship between the G' at 1.0 rad/s and the rate of urea hydrolysis on the peptide hydrogels for different [Ca 2+ -]/[COO − ] molar ratios. As can be seen, with increase in the [Ca 2+ ]/[COO − ] molar ratio, the G' increased, while the rate of urea hydrolysis decreased. Interestingly, the reaction rate and G' of the peptide hydrogel at [Ca 2 + ]/[COO − ] molar ratios above 0.125 did not change significantly. These results imply that diffusion of the substrate in the peptide hydrogel during the hydrolysis of urea on the (VHVEVS) 3 peptide networks is the ratelimiting step, and that the reaction rate is regulated.

Fabrication of CaCO 3 -peptide hybrid gel by bioinspired mineralization
Bioinspired mineralization of CaCO 3 was carried out by injecting 20 μL of urea solution (1.0 M) into peptide hydrogels with different [Ca 2+ ]/[COO − ] molar ratios at 25°C for 7 days. As the bioinspired mineralization proceeded, the appearance of the peptide hydrogels changed from transparent to cloudy (Figure 5a). In previous studies, we reported that in a non-peptide system, urea does not hydrolyze to form carbonate anions. These results indicate that the formation of CaCO 3 by bioinspired mineralization was triggered by the generation of carbonate anions through the hydrolysis of urea injected into the peptide hydrogels. We investigated the crystal structure of the CaCO 3 formed by bioinspired mineralization in the peptide hydrogels by electron microscopy. Energy dispersive X-ray (EDX) spectroscopic elemental mapping images of the CaCO 3 -peptide hybrid networks are shown in Figure  S1. The EDX elemental mapping images show the   presence of Ca arising from CaCO 3 and N originating from the peptide, thereby confirming the formation of a CaCO 3 -peptide hybrid network by bioinspired mineralization. In addition, we observed the morphology of the CaCO 3 -peptide hybrid networks by SEM. Figure 6 and S2 show SEM images of CaCO 3 formed by bioinspired mineralization in the peptide hydrogel and peptide solutions (Non-gel) with different [Ca 2+ ]/[COO − ] molar ratios. We observed that the mineralized CaCO 3 in the peptide hydrogels had different morphologies at different [Ca 2+ ]/[COO − ] molar ratios (Figure 6). At a low [Ca 2+ ]/[COO − ] molar ratio of 0.05, the mineralized CaCO 3 exhibited a fibrous morphology. Interestingly, with increases in the [Ca 2+ ]/[COO − ] molar ratio, the morphology of the mineralized CaCO 3 changed to a thin film. In contrast, the morphology of CaCO 3 mineralized in a Non-gel system remained the same, i.e. a fibrous structure, irrespective of the [Ca 2+ ]/[COO − ] molar ratio ( Figure S2). Samples of the EDX and SEM observations were prepared through lyophilization processing of the hybrid gels. Hybrid gel samples cannot maintain the morphology of hybrid networks under wet conditions due to lyophilization process. The morphology of the hybrid networks under wet conditions is therefore difficult to observe using a typical SEM system. We attempted to observe the mineralized CaCO 3 and peptide network in the peptide hydrogel under wet conditions by SE-ADM ( Figure S3). The CaCO 3 formed by bioinspired mineralization in the peptide hydrogel was observed only on the peptide network surface and at the cross-points of the networks. These results indicate that CaCO 3 was mineralized by selective nucleation on the peptide networks. Compared with peptide hydrogels, Non-gel systems with their lower G' have a higher ion diffusivity, which leads to formation of fibrous CaCO 3 similar to the morphology obtained in the peptide network by selective mineralization on the peptide network. The morphological changes of CaCO 3 formed by bioinspired mineralization in peptide hydrogels can be explained by two effects: (1) an increase in the concentration of calcium ions in the peptide, and (2) a decrease in the diffusivity of mineral sources in the reaction field due to hydrogelation. In the systems with a low [Ca 2+ ]/[COO − ] molar ratio (i.e. a low concentration of calcium ions), formation of fibrous CaCO 3 occurs through selective nucleation on the network surface and at the cross-points of the networks formed by self-assembly of the peptide. The concentration of calcium ions in the peptide hydrogel is, however, insufficient for mineralization of CaCO 3 in the spaces between the networks; thus, CaCO 3 cannot formed by bioinspired mineralization in the space between the peptide networks. In contrast, in systems with a high [Ca 2+ ]/[COO − ] molar ratio, CaCO 3 is formed not only on the peptide network surface, but also in the space between networks because of the sufficiently high concentration of calcium ions. In addition, bioinspired mineralization in the spaces between networks would require a reaction field with a high viscoelastic strength, which would inhibit diffusion of the generated carbonate anions as a mineral source. Hence, high viscoelastic strength facilitates the formation of thinfilm-like CaCO 3 due to an increase in the local concentration of mineral sources on the peptide network and in the spaces between the networks. Therefore, the morphology of the mineralized CaCO 3 changed with increases in the [Ca 2+ ]/[COO − ] molar ratio in the peptide hydrogel.
Next, we investigated the crystalline structure of mineralized CaCO 3 by conducting HR-TEM and SAED analyses ( Figure 7). As can be seen, peptide hydrogel with a low [Ca 2+ ]/[COO − ] molar ratio of 0.05 contains fibrous CaCO 3 ; however, the SAED pattern shows diffused rings with no well-defined spots. In addition, the HR-TEM image of mineralized CaCO 3 in the peptide hydrogel with a [Ca 2+ ]/[COO − ] molar ratio of 0.05 shows partial crystal lattice fringes. We also analysed the FT-IR spectra ( Figure S4). The FT-IR spectra showed the three characteristic peaks at around 1070 and 1450 cm −1 corresponding to amorphous CaCO 3 and around 856 cm −1 corresponding to aragonite, respectively [37][38][39]. These results indicate that the CaCO 3 formed by bioinspired mineralization in the peptide hydrogel with a [Ca 2+ ]/[COO − ] molar ratio of 0.05 was composed mainly amorphous and partial aragonite phases. Peptide hydrogel systems with [Ca 2+ ]/[COO − ] molar ratios above 0.125 did not be show obvious characteristic SAED pattern and FT-IR spectra similar to the CaCO 3 mineralized in a peptide hydrogel system with a molar ratio of 0.05. This suggests that the structure of mineralized CaCO 3 is significantly affected by the morphology and supply rate of the mineral source by the peptide network and the viscoelastic strength of the reaction field.
Finally, we measured the G' of the CaCO 3 -peptide hybrid gels to determine the effect of bioinspired mineralization in the hydrogels on the viscoelastic strength of the peptide hydrogels. The G' values of the CaCO 3 -peptide hybrid gels with different [Ca 2+ ]/[COO-− ] molar ratios were significantly higher than that of pristine peptide hydrogel (Figure 5b). In addition, the G' at an angular frequency of 1.0 rad/s increased exponentially with increases in the [Ca 2+ ]/[COO − ] molar ratios (Figure 5c). The increase in G' occurred because of the formation of a CaCO 3 -coated peptide hybrid network, which is stronger than the pristine peptide network, by the partial peptide network in the peptide hydrogel via bioinspired mineralization. Moreover, the exponential increase in G' with increases in the [Ca 2 + ]/[COO − ] molar ratio was caused by an increase in the proportion of the CaCO 3 -coated peptide hybrid network through mineralization due to an increase in the concentration of calcium ions in the peptide hydrogel.

Conclusion
We investigated fabrication of organic/inorganic hybrid gels by bioinspired mineralization in peptide hydrogels. The designed Ac-(VHVEVS) 3 -CONH 2 peptide formed by self-assembly acted as a mineral source supply template through urea hydrolysis, and the hydrogel acted as a 3D reaction field for bioinspired mineralization. With an increase in the [Ca 2+ ]/[COO − ] molar ratio to 0.125, the rate of hydrolysis of urea by the (VHVEVS) 3 peptide decreased, while the G' of the peptide hydrogel increased. At [Ca 2+ ]/[COO − ] molar ratios above 0.125, however, the hydrolysis rate and G' did not change significantly. The morphology of mineralized CaCO 3 changed from a fibrous structure to thin films with increases in the [Ca 2+ ]/[COO − ] molar ratio. The crystalline phase of the mineralized CaCO 3 , however, which is composed of amorphous CaCO 3 , remained the same. The morphological changes in the CaCO 3 formed by bioinspired mineralization in the peptide hydrogels can be explained by the increase in the concentration of calcium ions with increases in the [Ca 2+ ]/[COO-] molar ratio and the decrease in the diffusivity of mineral sources in the reaction field due to hydrogelation. The formation of a CaCO 3 -peptide hybrid gel increased the viscoelastic strength of the hydrogel because the partial peptide network in the peptide hydrogel formed a CaCO 3 -coated peptide hybrid network, which is stronger than the pristine peptide network, via bioinspired mineralization. The investigation on the formation of the hybrid gel during the bioinspired mineralization of CaCO 3 is expected to provide a better understanding of the biomineralization process and to pave the way toward the fabrication of soft matter with biomineral-like unique functionalities.