Biomineralized organic–inorganic hybrids aiming for smart drug delivery

Introduction

Controlled drug delivery systems have been investigated extensively in the field of modern pharmaceutical and medication in recent years. Compared with conventional formulations, controlled drug release undoubtedly has many advantages, such as controlled release rate, improved efficacy and reduced poisonous side effects [1, 2]. Much research has been done to associate biopolymers, such as alginate and chitosan, with thermo-sensitive macromolecules to prepare smart drug carriers with pH and temperature sensitivity [3, 4].

It has also demonstrated that organic–inorganic hybrid materials have received great interest in the last 10 years in controlled drug delivery because of their excellent biocompatible, biomimetic, and pH-sensitive properties [5, 6], and in this regard, synthetic approaches based on mimicking natural process such as biomineralization should offer much promise. Biomineralization often involves a diffusion-controlled deposition of inorganic minerals within porous organic polymeric matrices. It has been reported that biomineralized polysaccharide capsules have potential applications for cell growth [7], release of functional biomolecules [8, 9], gene delivery [10, 11] and tissue engineering [12]. For example, Green et al. demonstrated that both in vitro and in vivo a number of human cell types and immortalized cell lines had been encapsulated within mineralized polysaccharide capsules [7]. Oreffo et al. demonstrated that mineralized polysaccharide microcapsules could be used successfully for temporal and spatial delivery of cells and biomolecules for skeletal tissue engineering [10].

Biomineralization is a biomineral-inspired route to prepare novel organic–inorganic hybrids, which involves a diffusion-controlled deposition of inorganic minerals within porous polymeric matrices [13–15]. The diffusion-controlled deposition of inorganic minerals within porous organic polymeric matrices could hinder the permeation of the encapsulated drug and reduce the drug release effectively. Previous studies have shown that biomineralized organic/inorganic hybrid structure could improve the mechanical strength and controlled release behavior of the polymer matrix. Calcium carbonate (CaCO₃) [16, 17] and calcium phosphate (CaP) [18, 19] have shown promising potential in the design of organic–inorganic hybrid drug carriers because of their ideal biocompatibility, biodegradability, and pH-sensitive properties. For example, Perkin et al. [16] prepared hybrid nanocapsules by CaP mineralization of shell cross-linked polymer micelles and nanocages. Tang et al. [17] fabricated poly(ethyleneglycol)-b-polylactide (PEG-PLA)/CaP nanocomposites at room temperature. Wei et al. [18] fabricated hierarchical hollow CaCO₃ particles for pH-sensitive anticancer drug carrier. However, few reports have been found concerning the rational design of inorganic component and polymer matrix to prepare pH and temperature dual-responsive hybrid drug carriers for controlled drug delivery.

In the past few years, we intend to combine the controlled biomineralization technique with the rational choice of polymer templates to prepare biomineralized organic–inorganic hybrids with pH and temperature dual-responsive drug delivery properties. The present work will mainly summarize our recent work about the biomineralized organic–inorganic hybrids aiming for smart drug delivery including hybrid beads, membranes and micro/nano gels. Prospect for future development of the smart organic–inorganic hybrids is also discussed.

Organic–inorganic hybrid beads

Poly(N-isopropylacrylamide) (PNIPAAm) is one of the most widely studied thermal sensitive polymers, exhibiting a temperature-dependent volume phase transition at lower critical solution temperature (LCST) around 32 °C. Alginate is a pH sensitive and biocompatible natural hydrogel with relatively low cost, and its dissolution and biodegradation under normal physiological conditions enable it to be a suitable matrix for the entrapment and delivery of proteins, drugs and cells. To overcome the rapid erosion and high release rate in a neutral pH condition of alginate beads, a biomineralized polysaccharide layer has been introduced to the dual-stimuli-responsive (pH and temperature) alginate drug delivery system with a one-step method [13]. The deposition of an alginate-chitosan layer around droplets of sodium alginate is coupled with the controlled precipitation of calcium phosphate arising from the counter-diffusion of ions across the polysaccharide interface. The beads can be further strengthened with a semi-permeable organic–inorganic hybrid outer membrane that spontaneously formed around alginate droplets when placed in a chitosan solution containing Ca²⁺ ions owning to the interfacial charge matching at a suitable pH value between the anionic and the cationic polysaccharide.

The equilibrium swelling of the developed beads was found to be pH- and thermo- responsive. The swelling behavior of alginate beads is affected by the biomineralized polysaccharide coating at pH 7.4: the swelling ratio decreased from 48 % for unmineralized ones to 17 % for biomineralized ones at 25 °C, and from 53 % for unmineralized ones to 18 % for biomineralized ones at 37 °C. Indomethacin release was found to be retarded in a neutral condition when the beads were reinforced through biomineralized polysaccharide coating, especially at 25 °C, the release amount reached 92 % within 600 min for the unmineralized beads, while a drug release of only 44 % took 600 min for the biomineralized beads, indicating that the mineralized polysaccharide membrane could prevent the permeation of the encapsulated drug and reduce the drug release effectively below the LCST of PNIPAAm. A significant change in drug release was achieved for the biomineralized polysaccharide coated beads. For the beads prepared with 250 mM phosphate, the drug release was higher at 37 °C (70 %) than that at 25 °C (44 %) due to the precipitation of PNIPAAm and the higher swelling ratio above LCST. At pH 7.4 the maximum value attained for drug release is 70 % (at 37 °C), whereas the maximum drug release is only 3 % at pH 1.2.

From the polarized microscope images of the biomineralized polysaccharide beads [13], a transparent membrane around the beads could be observed clearly, which was derived from the calcium phosphate mineralized alginate/chitosan membrane. Additionally, the beads was reduced in size with increasing temperature from 25 to 55 °C, indicating that PNIPAAm inside the beads has changed from hydrophilic to hydrophobic when temperature increased above the LCST. The LCST was found in between 28.6 and 29.6 °C for all the studied beads, which is very close to the LCST of neat PNIPAAm.

Poly(NIPAAm-co-AAm) with an LCST of 37.5 °C, which is near human body temperature, was also employed as the thermal-responsive component to prepare biomineralized polysaccharide alginate beads with pH- and thermo-responsibility via a one-step method [20]. SEM and EDS results demonstrated that calcium phosphate could be observed not only in the surface but also in the cross section of the biomineralized polysaccharide beads. The equilibrium swelling and the release profile of the developed materials were found to be pH- and thermo- responsive. Indomethacin release results also demonstrated that the release profile could be sustained with the organic–inorganic hybrid layer: the release amount reached 96 % within 240 min for the unmineralized beads, while a drug release of only 64 % was obtained after subjecting the biomineralized polysaccharide beads to the same treatment.

Chemical grafting is another approach to combine biopolymers with thermo-sensitive macromolecules, such as PNIPAAm. In the former studies concerning PNIPAAm/alginate dual responsive system, PNIPAAm exist in the alginate semi-IPN network. Therefore, the squeezing of PNIPAAm at 37 °C can break the balance of the semi-IPN network and accelerate the disruption of alginate beads. In the succeeding work, PNIPAAm is grafted onto the biodegradable alginate beads to prepare thermo-responsive “smart” polysaccharide materials [21]. To overcome the rapid erosion and high release rate in a neutral pH condition, the alginate beads are introduced with a biomineralized polyelectrolyte layer as illustrated in Fig. 1. The biomineralized layer formed between Ca²⁺ and HPO₄^2- and the polyelectrolyte layer formed from chitosan (positive charge), poly(sodium acrylate) (PAANa) and alginate (negative charge) both exist in the alginate beads, which could result in the enhancement of the mechanical strength of the alginate beads. PAANa with ultra high molecular weight (M_w > 10⁷) has good flexibility and hydrophilicity [22]. With the assistance of biomineralized polyelectrolyte layer, the alginate beads could be kept intact and flexible when being grafted with PNIPAAm. Additionally, the alginate beads would be in a swollen state during the course of grafting because of the good swelling property of alginate in water. Therefore, it is easy for NIPAAm molecules to permeate into the inner pores of alginate beads to finish the grafting reaction.

Fig. 1

Schematic illustration for the formation of smart alginate beads. Biomineralized polyelectrolyte bead (a), PNIPAAm-grafted hybrid alginate bead (b) and digital photos (c) of PNIPAAm-grafted alginate beads at 25 and 37 °C [21].

Instead of precipitating from the alginate network as discussed in the semi-IPN PNIPAAm/alginate dual responsive system, the shrinkage of PNIPAAm will not break the polymeric network in PNIPAAm-grafted alginate beads, because PNIPAAm is mostly attached in the surface of the pores. The thermo-responsive swollen/shrunken characteristic of PNIPAAm gates grafted in the pores of the alginate beads is presented here: the pores of the beads are covered by the stretched PNIPAAm to delay indomethacin release at temperatures below LCST, while opened to accelerate the drug release because of the shrinking of PNIPAAm at temperatures above LCST. The reversible temperature-dependent “on/off” characteristics of PNIPAAm-grafted alginate beads enable the smart drug release in a controlled way by simply adjusting the environmental temperature as presented in Fig. 2.

Fig. 2

Reversibility of drug release in response to temperature for the PNIPAAm-grafted beads measured at pH 7.4 [21].

As we know, PNIPAAm is one of the most widely studied thermal responsive polymers. However, its poor degradability and potential cytotoxicity should not be ignored for biomedical applications. Aliphatic poly(urethane-amine) (PUA), which is obtained from the copolymerization of CO₂ with aziridines under supercritical CO₂ and consists of both hydrophobic urethane and hydrophilic amine unites, has been reported to have a thermally induced reversible transition in aqueous solution at its LCST [23, 24]. Moreover, the amine group of aliphatic PUA would change to the protonated amino forms with the decrease of solution pH value and its LCST can be adjusted by changing the hydrophilicity/hydrophobicity balance of hydrophobic urethane and hydrophilic amine unites in the copolymer, as illustrated in Scheme 1. As we have been known that aliphatic PUA has attracted a great deal of interest for the potential applications in drug delivery systems, microactuators, and gene-transfection agents due to their biodegradability, biocompatibility and noncytotoxicity. Thus, we have attempted to fabricate hybrid vehicles composed of alginate/CaCO₃/PUA in the presence of Ca(OH)₂ and poly(acrylic acid) (PAA) under compressed CO₂ by using aliphatic PUA as the thermal-/pH- dual responsive component as illustrated in Fig. 3 [25].

Scheme 1

Chemical structure of aliphatic PUA [23].

Fig. 3

Schematic illustration of the interaction among alginate, aliphatic PUA and PAA within alginate/CaCO₃ hybrid bead under compressed CO₂ reaction [25].

It is evident that the understanding and ultimately mimicking of the biomineralization processes may provide new approaches to the fabrication of specialized organic–inorganic hybrid materials. Organic matrix plays an important role in the fabrication of CaCO₃ microparticles. As illustrated in Fig. 4, the distinct difference from the three kinds of beads may be explained by the interaction of alginate, aliphatic PUA and PAA during the course of compressed CO₂ reaction. As discussed in literatures, PAA is an effective additive to promote CaCO₃ nucleation and growth in the surface of alginate beads. In the present study, the electrostatic force between PAA (with negative charge) and PUA (with positive charge) could fix PAA tightly in the surface of the alginate beads, which leads to a tight adsorption of Ca(OH)₂ around the beads. Therefore, for the alginate/CaCO₃/PUA/PAA beads, CO₂ reacts with Ca(OH)₂ immediately to form the compact CaCO₃ shell around the beads during the course of reaction under compressed CO₂. The shell may hinder the diffusion of CO₂ into the inside of alginate beads, resulting in a relatively compact inner structure. Indomethacin release behaviors were found to be pH- and thermal-responsive. In addition, the release profile was sustained with the introduction of CaCO₃ microparticle shell, indicating that the organic–inorganic hybrid structure could hinder the permeation of the encapsulated drug and reduce the drug release effectively.

Fig. 4

SEM micrographs of the hybrid beads. 1, 2 and 3 refer to alginate/CaCO₃, alginate/CaCO₃/PAA and alginate/CaCO₃/PUA/PAA bead, respectively. A and B refer to the cross section and the high magnification of the cross section. The inset of 3A is a magnifacation of the outer surface of 3 [25].

Organic–inorganic hybrid membranes

Polyelectrolyte membranes, obtained by alternate deposition of cationic and anionic polymers, have been found wide applications as a convenient way for surface modification. The polyelectrolyte complexes of alginate with other polysaccharides have also been found many applications in biotechnological and pharmaceutical fields. Much research has been done to associate biopolymers, such as alginate and chitosan, with thermo-sensitive macromolecules to prepare stimulus-responsive membranes, especially with pH, temperature or ionic strength sensitivity [26, 27].

Biomineralized polysaccharide alginate membranes with multi-responsive drug release characteristics also can be prepared via a one-step method, in which the deposition of the porous alginate/chitosan polyelectrolyte around alginate membranes is coupled with the controlled precipitation of calcium phosphate arising from counter-diffusion of ions across the polysaccharide interface as illustrated in Fig. 5 [28]. Both the biomineralized component (CaHPO₄) formed between Ca²⁺ and HPO₄^2- and the polyelectrolyte formed between positively charged chitosan and negatively charged alginate exist in the resulting membranes. This outer shell hardens with time due to the deposition of calcium phosphate but remains permeable to Ca²⁺ ions, therefore the membranes are also internally stabilized due to the cross-linking of the alginate network with Ca²⁺ ions. Indomethacin release also demonstrates that the hybrid membranes exhibit pH/temperature responsive drug release properties.

Fig. 5

Schematic illustration of biomineralized polysaccharide membrane. Nucleation of calcium phosphate occurs not only in the surface but also in the cross section of the biomineralized polysaccharide membrane [28].

Sodium palmitate is a non-toxic fatty acid sodium salt with an appropriate alkyl chain length (–(CH₂)₁₄–), which can effectively inhibit water penetrating into the beads and decrease the water uptake. Thus, hydrophobically modified biomineralized polysaccharide alginate membranes with sustained smart drug release were prepared via a one-step method as illustrated in Fig. 6, in which the deposition of the porous alginate/chitosan polyelectrolyte around alginate membranes containing hydrophobic component is coupled with the controlled precipitation of calcium phosphate [29]. The biomineralized component CaHPO₄ formed between Ca²⁺ and HPO₄^2– and the polyelectrolyte formed between positively charged chitosan and negatively charged alginate exist in the resulting membranes. The equilibrium swelling behavior of the modified membranes as well as their controlled delivery performance was investigated as a function of pH and temperature.

Fig. 6

Schematic illustration of hydrophobically modified alginate membrane [29].

Alginate/CaCO₃/PUA/PAA hybrid membranes were then prepared in the presence of Ca(OH)₂ suspension under compressed CO₂ using PAA as a crystal growth additive to control the nucleation of micro-scale CaCO₃ microparticles (Fig. 7) [30]. The key advantages here are to combine a novel biocompatible thermal-responsive polymer with alginate membranes and the controlled deposition of CaCO₃ microparticles within porous polymeric matrix. By adjusting the pressure and reaction time of the biomineralization process, sustained drug release could be achieved from the hybrid membranes. Moreover, the thermal-sensitivity of aliphatic PUA and pH-sensitivity of alginate matrix would be preserved after the biomineralization process.

Fig. 7

Schematic illustration of the interaction among alginate, aliphatic PUA and PAA within alginate/CaCO₃/PUA/PAA membrane under compressed CO₂ reaction [30].

The electrostatic interaction between PAA (with negative charge) and aliphatic PUA (with positive charge), as well as hydrogen bonding between the carboxyl group of PAA and the urethane group of aliphatic PUA, could fix PAA tightly with alginate matrix and lead to a tight adsorption of Ca²⁺ (derived from Ca(OH)₂) with PAA (trapped in alginate matrix) via the electrostatic interaction. CO₂ reacts with Ca²⁺ immediately to form numerous CaCO₃ microparticles around and inside the alginate membranes. Therefore, the interaction between PAA and aliphatic PUA contributed the formation of compact CaCO₃ microparticles and the high drug loading efficiency of the hybrid membranes. By adjusting the pressure and reaction time of the biomineralization process, sustained drug release property could be achieved from the hybrid membranes. Drug release results indicated that the compact CaCO₃ microparticles could hinder the permeability of the encapsulated indomethacin and reduce the drug release effectively.

Biomimetic self-assembly of micro/nano hybrids

Recent advances in supermolecular assembly illustrated that nano-structured polymeric/inorganic hybrid materials can be fabricated with ease under ambient conditions [31, 32]. It has been demonstrated that the rational mixing of polymeric nanogels with inorganic particles in a micro/nano-scale may achieve the novel hybrid materials with unusual properties [33]. Moreover, the combination of controlled mineralization technique with the rational choice of polymer templates would lead to successful development of robust and smart self-assembled nanocarriers for drugs. Such polymeric/inorganic hybrid materials exhibit structural hierarchy and offer a unique combination of properties with potential applications in drug delivery and tissue engineering.

Calcium phosphate (CaP), a major mineral component of native bones and teeth, is superior to other inorganic species such as silica in terms of biocompatibility and pH-sensitive property. In addition, CaP is absorbable in specific cellular enviroments (endosome/lysosome) as non-toxic ionic species. Nano-structured CaP/polymer hybrid materials can be fabricated with ease under ambient conditions [11]. To combine the controlled CaP mineralization technique with the rational choice of polymer templates, a novel method for preparing nanohybrid PNIPAAm/CaP composites with sustained dual-responsive drug delivery has been proposed [34]. As described in Fig. 8, PNIPAAm micelle nanotemplates were firstly prepared from NIPAAm monomer via surfactant-free emulsion polymerization. PAA was employed as an effective crystal growth additive to control the nucleation and growth of CaP nanocrystallines. Negatively charged PAA assembled around the PNIPAAm micelles would combine with Ca²⁺ and finally CaP nanocrystallines were self-assembled around or in the near-surface regions of PNIPAAm micelle nanotemplates. CaP enhanced the structural robustness of the hybrid nanocomposites, at the same time served as a diffusion barriers for drugs, thereby producing stable nanocarriers with controllable drug release. Such an experimental design was based on the consideration that the diffusion-controlled deposition of inorganic minerals around the porous polymeric matrices could hinder the permeation of the encapsulated drug and endow the resulting hybrid nanocomposites with controlled release properties, and at the same time does not ruin the thermo- and pH-sensitivity of the hybrid nanocomposites.

Fig. 8

Schematic illustration for the formation of the nanocomposites. CaP nanocrystallines nucleate on the outer surface of PNIPAAm microgels (a) and scatter homogeneouly within near-surface regions of the polymer nanostructures (b) [34].

The successful combination of PNIPAAm nanogels with CaP was further examined by TEM analysis. Fig. 9a and 9b show the TEM images of PNIPAAm/CaP nanocomposites. It can be clearly observed that the size of PNIPAAm/CaP nanocomposite is around 200 nm. CaP nanocrystals were scattered on the surface of the PNIPAAm nanogels (black part on the edge of the nanogels). Fig. 9c and 9d show the EDX spectra of the surface and the surrounding of nanogels, respectively. Signals for P and Ca elements were observed from both surface and surrounding of the nanogels, which could not be found from the neat PNIPAAm nanogels. The results indicated that CaP nanocrystals nucleated both within the near-surface regions and on the outer surface of PNIPAAm nanogels.

Fig. 9

TEM micrographs of PNIPAAm/CaP nanocomposites (a) and a magnification (b). TEM-associated EDX spectra of the surface (c) and the near-surface regions (d) of nanogels [34].

PNIPAAm/CaP nanocomposites are superior to pH- and thermal-responsive polymeric nanogels in terms of easy preparation and biocompatibility. Moreover, CaP nanocrystallines in the nanocomposites could decrease the permeability of encapsulated drug and then the drug release effectively. The pH- and thermal-responsive drug release properties using vitamin B₂ (VB₂) as a model drug also indicated that the PNIPAAm/CaP nanocomposites could possess sustained drug release, and at the same time, preserve the stimuli-responsive properties after the biomineralized reaction.

In addition to CaP, calcium carbonate is another important inorganic material to be employed in the smart hybrids. Thus, a novel approach for preparing hierarchical PNIPAAm/CaCO₃ hybrid composites with controlled drug delivery was proposed as illustrated in Fig. 10. PNIPAAm micelle nanotemplates were firstly prepared from NIPAAm via surfactant-free emulsion polymerization [35]. Sodium poly(styrene sulfonate) (PSS) was employed as an effective crystal growth additive to control the nucleation of micro-scale vaterite CaCO₃ microparticles. PNIPAAm/CaCO₃ micro/nano hybrids were formed via the self-assembly of nano-scale PNIPAAm micelle around the surface of vaterite microparticles. Vaterite microparticles could serve as a diffusion barrier for the encapsulated drugs, thereby producing robust micro/nano carriers with controllable drug release property.

Fig. 10

Schematic illustration of PNIPAAm/CaCO₃ hybrid composites [35].

An interesting phenomenon could be found from FESEM micrographs of the hybrid composites (Fig. 11) that the surface of CaCO₃ microparticles was covered by a layer of PNIPAAm nanogels. The size of PNIPAAm nanogels was around 200 nm. Then the EDX analyses were performed to confirm the composition of the resulting spherical composites (Fig. 11C and 11D). It can be found that no signal for Ca element could be found and the signal of N element was observed in the surrounding area of the spherical structure (location D in Fig. 11A), suggesting that the nanogels scattering around the spherical structure are PNIPAAm micelles. While a strong signal for Ca element was observed from the outer layer of the spherical structure (location C in Fig. 11A), indicating that main component of the inner spherical was CaCO₃ microparticles and CaCO₃ microparticles were covered by PNIPAAm nanogels. Therefore, the successful combination of PNIPAAm nanogels with CaCO₃ microparticles could be demonstrated by FESEM and EDX analysis.

Fig. 11

FESEM micrographs of PNIPAAm/CaCO₃ hybrid composites using PSS as crystal growth additive prepared with 2.37 mM of Ca²⁺ (A), PNIPAAm nanogels in the surface of vaterite microparticles with high magnification (B). FESEM-associated EDX spectra of the vetarite microparticle surface (C) and FESEM-associated EDX spectra of PNIPAAm nanogels in the near-surface of vetarite microparticles (D) [35].

In the succeeding work, organic–inorganic hybrid CaCO₃ microparticles (with diameter around 4.0 μm) coated by PSS and aliphatic PUA multilayers were prepared via LbL technique (Fig. 12) [36]. Microparticles are of especial interest for oral drug delivery because of their small size and large surface area compared to larger carriers. In addition, microparticles with diameters of 1–5 μm would be ideal for passive targeting of professional antigen-presenting cells. The electrostatic interaction under weak-acid condition between aliphatic PUA and PSS contributes to the successful LbL assembly of the multilayers. Doxorubicin hydrochloride (DOX) release behaviors revealed that the release profiles of the multilayer-coated CaCO₃ microparticles were sustained with the introduction of PUA/PSS multilayers, indicating that PUA/PSS multilayers could hinder the permeation of the encapsulated drug and assuage the initial burst release of DOX [36]. In addition, the drug release of the multilayer-coated CaCO₃ microparticles was thermal-/pH- dual responsive due to the shrinkage of aliphatic PUA above its LCST and the dissolutions of the CaCO₃ core at acid condition.

Fig. 12

Schematic illustration of the PUA/PSS-coated CaCO₃ microparticle fabrication via LbL deposition and subsequent pH/thermal-controlled release of DOX [36].

Outlook

This paper systematically summarized our efforts in the fabrication of biomineralized organic–inorganic hybrids aiming for smart drug delivery, including hybrid beads, membranes and micro-nanogels. The simplicity and adaptability of the biomineralized organic–inorganic hybrids provides their significant potential applications in human cell, gene and pharmaceutical drug-based therapies. Our work demonstrated that the morphology and structure of the hybrids could be easily controlled by reaction condition and the biomineralized organic–inorganic structure could hinder the permeation of encapsulated drug and reduce the drug release effectively. Moreover, the thermal- and pH-sensitivity of polymeric matrix could be preserved after the biomineralization process. However, the big size of the biomineralized organic–inorganic beads and membranes may limit their extensive application. Therefore, decreasing the size of drug carriers will be the efforts of our future work.

Nano/micro polymeric multilayer hybrid capsules have received much attention in recent years, which may be considered as an excellent candidate for smart drug carriers [37, 38]. The size of drug carriers plays an important role in the controlled delivery area. For example, the well-known enhanced permeability and retention effect of the blood vessels in cancerous tissues allows particles with a size of up to 200 nm to escape from the blood vessel, but this is much smaller than the sizes of most polymeric multilayer hybrid capsules [39, 40]. Therefore, exciting future development of smart organic–inorganic hybrids will without doubt focus on the design of capsules with a size of < 500 nm [41–44]. On the other hand, the biomineralization mechanism during the hybrid process and the interaction between inorganic microparticles and polymeric matrix are still not clear [42]. Therefore, thorough understanding concerning the biomineralization mechanism, together with the interface interaction between inorganic and organic components, is another challenge.