Structural evolution of aragonite superstructures obtained in the presence of the siderophore deferoxamine

The effect of the amphoteric siderophore deferoxamine on the crystallization behavior of calcium carbonate was investigated under bioinspired conditions. Amphoteric siderophore deferoxamine possesses the ability to self-organize, surface activity and ionchelating properties. It induces in the present case the formation of unusual, highly organized aragonite mesocrystals during the precipitation of calcium carbonate from the liquid phase. A detailed investigation of the structure and growth of the particles provides an insight on the role of the deferoxamine and its function in the crystallization process. Page 1 of 23 CrystEngComm C ry st E ng C om m A cc ep te d M an us cr ip t View Article Online DOI: 10.1039/C5CE00186B


INTRODUCTION
Biominerals in nature, as for example diatoms or coccoliths, inspire researchers to elaborate on the construction of new materials. The mechanisms of the biological processes, performed by living organisms to synthesize their inorganic components, are far from being understood, leading researchers to use chemical approaches to mimic them. The motivation is to obtain and understand complex morphologies in simpler model systems. In addition, this field of research is also driven by industrial interest, e.g. motivated by the very favourable mechanical properties of biominerals 1 and their application, for instance as organized catalyst supports 2 . Their synthesis in aqueous environment at ambient conditions from non-toxic, cheap and commonly available compounds is also quite attractive. However, the hierarchical organization found in nature is very difficult to reproduce in vitro. Calcium carbonate is one of the most abundant biominerals, and among them, the most studied one due to its scientific and industrial relevance 3 . Calcium carbonate precipitation represents a relatively complex mineralization process, due to the fact that it starts from prenucleation clusters 4 and can proceed via liquid or amorphous precursors 5 and finally often involves the coexistence of three stable anhydrous polymorphs: vaterite, aragonite and calcite 6 . Calcite is the thermodynamically stable polymorph under ambient conditions, while aragonite is known to be metastable 7 . The least stable polymorph, vaterite, is the standard kinetic intermediate, but rapidly recrystallizes towards calcite 8, 9 . Especially aragonite attracts much interest mainly due to its presence in nacre. 10 Nevertheless, the described routes for aragonite synthesis depend on an intricate set-up or well-defined reaction conditions. So far, aragonite was synthesized under high concentration of Mg 2+ ions 11,12 , and with additives such as extracted glycoproteins 13 which are associated to a β-chitin-silk matrix 14 , specific imidazolium based ionic liquids 15 or under harsh conditions such as elevated temperatures 16, 17 , high-power ultrasonic 18 or microwave irradiation 19 . Moreover, aragonite could be synthesized at the air-water interface under compressed monolayers 7,20 via an amorphous precursor route 21 , at the liquid-liquid interface in a radial Hele-Shaw Cell 22 or by using block copolymer 3 microgels 23, 24 where aragonite bundles are obtained. A synthesis in alcoholic medium in the presence of sodium stearate was also proposed to obtain lamellar aragonite 25 .
In this work, a novel model system for complex morphosynthesis is presented. More specifically, the influence of deferoxamine on the crystallization behavior of calcium carbonate is reported. Deferoxamine belongs to the family of siderophores, a class of low-molecular weight, iron-coordinating agents produced by plants and microbial organisms. It is clinically used for the treatment of iron overload disease 26 . With its three hydroxamic acid groups (RCONR'OH) interspaced by two amide groups and terminated with an amine, this biomolecule is a chelating agent 27 with a rich coordination chemistry 28 . Applied to the nonaqueous synthesis of metal oxides, deferoxamine can also provide intra-and intermolecular amide-amide interactions with the potential to form supramolecular hybrid nanostructures 29 . Our intention to use deferoxamine was to have a clean model system for a siderophore which is able to bind weakly and reversibly to Ca 2+ to provide a high local availability of non-clustered Ca 2+ , an interesting limit also for natural mineralization processes.

Phase identification and morphology.
Identification of the synthesized product was carried out by powder X-ray diffraction (XRD) analysis. The XRD pattern reveals aragonite as the main phase (diffraction peaks 2θ correlated respectively to the (hkl)  Figure 1a) as a minority phase. Their morphology is typical for calcium carbonate that precipitates in the absence of additives (S.I. 1). This suggests that some crystals grow from the continuous phase without being controlled by deferoxamine.
At higher magnification, the SEM images reveal further details of the peculiar crystal superstructure of the obtained particles (Figure 1b, c and d). Their morphology is characterized by two parts of a distinct aspect. The main part is formed by a globular body of 20-50 µm in diameter. It is characterized by striation lines that are running from one pole towards the other. The other characteristic feature is formed by a bundle of outgrowing structures that generally appear at one of the poles. The length of these outgrowing structures varies from particle to particle, but generally reaches a length that is comparable to the diameter of the globular body. Besides a majority of isolated particles, there are some that are connected to each other, either at a common plane or Crystal growth. In order to understand the formation mechanism of these highly organized aragonite crystals, samples were investigated by electron microscopy after 6 different times of growth ( Figure 2). After 5 hours, spherical particles with a diameter of about 10 µm were found on the bottom of the reaction flasks. After 9 hours, the particles almost reached their final diameter of about 20 -50 µm. They are characterized by a roundish shape that is slightly deformed by a bulge that forms on one of the poles ( Figure   2a). At higher magnification, a rough, particulate surface morphology is revealed ( Figure   2b). After 13 h of growth, thin lamellas of different length can be observed at the surface.
They are aligned and give rise to striations that are running from one pole towards the other (Figure 2c). At one of the two poles, outgrowth of structures can now be clearly Each individual outgrowth appears as a stack of thin platelets or pillars that are aligned and oriented towards the tip of the outgrowth. The diameter of each individual outgrowth shrinks through a progressive termination of the outmost crystallites at their growth front.
Stepwise, deeper lying platelets are exposed until the axis is reached at the tip of the outgrowth. Looking at tips of the outgrowing structures from above reveals a welldefined six-fold symmetry ( Figure 2h). Similar pseudo-hexagonal crystals were previously obtained using lithium niobate as carbonate free single-crystal substrate. In that case, epitaxial aragonite growth occurred through the arrangement of cations on the substrate 30 . 7 Figure 2. Overview (left) and high magnification SEM images (right) recorded from particles at different growth stages show the progressive development of the complex morphology. a), b) were recorded after 9h, c), d), after 13h and e), f) after 24 hours of growth, respectively. g) and h) show, respectively, details of the characteristic outgrowth and the apex of an outgrowth viewed from the top, revealing a hexagonal assembly of platelets.

Aragonite-Siderophore Interactions.
In order to investigate the role of the siderophore deferoxamine in the formation of the aragonite structures, dynamic light scattering (DLS) investigations were performed on two CaCl 2 /deferoxamine solutions with a molar ratio of 1:1 and 2:1, which were previously gently sonificated. DLS analysis of both these primary CaCl 2 /deferoxamine solutions indicates deferoxamine-Ca-aggregates of about 0.5 -2.7 nm, with an average hydrodynamic radius of ~1 nm, but no other species. This supports our point of view that 8 the siderphore essentially improves the availability of single Ca 2+ ions or very small clusters, but no biiger clusters or colloids instead. An excess of CaCl 2 favors calcite crystallization, as seen by the higher number of rhombohedral calcite particles that formed in presence to a molar ratio of 2:1 ( Figure 1a) in comparison to a ratio 1:1. This observation is further supported by previous studies 31 , which showed that deferoxamine only allows the monochelation of calcium(II). Interestingly, the monochelation of calcium(II) by deferoxamine has a low stability constant due to the large radius of the cation, which is nearly twice as large as that of iron, and the spacers that bridge the three hydroxamic acid groups are comparably short 31 . The defined small aggregates could only be detected after weak sonication of the CaCl 2 /siderophore solution, suggesting that the siderophoreneeds some activation to dissolve homogeneously. This is potentially due to hydrogen bridges between single deferoxamine entities in a peptide like fashion.
Crystallization events simply become more homogeneous when the starting situation is more defined. . Although interactions between calcium(II) and deferoxamine are expected to be very weak until reaching a pH of around 7, an effect on the crystal growth could be observed already at pH 5.8 in our study. Starting from this pH, the presence of the siderophore increases the concentration of calcium in the solution, due to the binding equilibrium.
The primary crystal seed is known to play a very important role in the determination of whether calcite or aragonite forms and the polymorph can even be encoded in an amorphous CaCO 3 precursor phase 32 . Indeed, our data shows that once the growth of a calcite particle is initiated, it would just keep on growing while ignoring the presence of the siderophore. It is probable that siderophore molecules interact differently with the growing structures in a time-dependent manner. Indeed, the total charge of deferoxamine at different pH may define its binding properties and thus, control the mode of aragonite growth. During the mineralization protocol, ammonia and CO 2 diffuse into the CaCl 2 solution, leading to an increase of pH from 5.8 to 9.5 throughout the crystallization process. With dissociation constants equal to pH 8.30, 9.00, 9.46 and 10.4 that respectively correspond to that of the three hydroxamic acids and the amine group, 31 deferoxamine is positively charged at pH 5.8 and may electrostatically bind to hydrogen 9 carbonate (HCO 3 -) via the ammonium group but not to calcium(II) at the beginning of the crystallization process. During the increase of pH, although the total charge of deferoxamine becomes neutral and then negative, the monochelation of calcium(II) to this siderophore remains rather weak, as the stability constant of the complex is not significantly changed. 31 In spite of the rather high deferoxamine concentrations, it maybe comes as a surprise that the organic content of the final structures is very low, as determined by TGA. This just means that the siderophore is only active as a growth modifier, but is effectively released at final pH and within the final structure. This low consumption makes such a modifier also useful for technical mineralization recipes.

Crystallographic Investigation of the Aggregates.
A more detailed insight on the early stages of the structural evolution was obtained by crushing samples and sampling some of the obtained fractions. The SEM image in Figure   3a shows a random agglomeration of nanoparticles that was abstracted from a fullygrown structure. A TEM image of a slightly more ordered structure that was abstracted from a sample after 9h of growth is shown in Figure 3b. It reveals nanoparticles that are assembled in a branched, dendritic like manner. As indicated by the distribution of the (111) spots in the corresponding electron diffraction pattern, the nanoparticles however show a comparably high crystallographic alignment with only some minor distortions, i.e. they obviously grow and arrange via "vectorial alignment". Similar structures, however, slightly more dense and with clearly developed pillar-like structures, were also observed in the fragments of fully-grown particles (Figure 3c).
10 Figure 3. SEM images recorded from fractions obtained by crushing particles after 24 h of growth are shown in a) and c). In b) a TEM image of a fraction obtained by crushing a particle after 9 h of growth is shown. The corresponding diffraction pattern is shown as an inset.
Putting the crushed pieces in context with complete particles was, however, quite challenging and could only be achieved by analysing distorted growth events. Some of the particles that nucleated at the wall of the reaction flask grew such that the glass cut a plane through their center. Particles "cut" in this manner reveal their inner structure and allow studying changes in the growth modus from the center towards the surface. In

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Interestingly, in both cases, the shape of the final particle seems to be unaffected by the presence of the glass substrate on which they grew. Indeed, the usual morphology is obtained, such as if the particles were cut after growth. The presence of both, longitudinal and equatorially cut particles at the glass surface could be a hint on the role that the inner, central region of the particles play in defining the growth directions and crystallographic arrangement of crystalline domains of the complex shape and thus, the final shape. In the very center, the nanoparticle aggregate does not show any sign of pre-determined directionality or anisotropy. From the center towards the outside, the structural density gradually increases and orientation relationships emerge. This can clearly be seen in Figure 4b. The aspect of the central region, marked as square I in Figure 4b is similar to the one of the agglomeration shown in Figure 3a. Further away from the center, in the region marked with square II, a pillar like, dendritic structure, similar to the one in Figure   3c, can be identified. Hence, the crushed samples shown in Figure 3 are indeed from the inner volume and resemble the first growth stages of the particles. Formation of the particles therefore starts with a loose agglomeration of nanoparticles at the center. It is followed by the formation of nanoparticulate, dendritic structures that radiate outwards. This is the region where first signs of local crystallographic alignment can be observed ( Fig. 3b), presumably following the so-called oriented attachment. The can finally result in the formation of a so-calledf mesocrystal, which however cannot be proven in the present case 33 .
As ordinary Aragonite nanoparticles hardly show oriented attachment, we assume that the nanoparticles observed at this growth stage are still stabilized by the siderophore, locally liberated throughout the consumption of Ca 2+ by the precipitating nanoparticles.     Once the crystallographic relationships have been revealed, the initial reason for the formation of the outgrowth has to be elaborated.. On a sphere, radial growth with an alignment of the crystallographic axis faces some constraints (similar to the "hairy ball theorem" 38 ). Figure 9. left shows a montage made from half spheres shown in Fig. 4a and 4c. Crystallographic directions as well as crystalline domains and striation lines are indicated. Aragonite grows radially from the center, with the c axis points outwards and the b axis pointing longitudinally towards the poles. Hence, crystalline domains are oriented parallel at the equatorial belt, but crossing each other in the region close to the poles, as schematically shown in the right diagram Small pictures show that polycyclic twinning occurs at the poles.
It has already been shown by SEM images recorded at an early growth stage (Figure 2) that the same lamellas that give rise to the striations at the surface of the globular body are also found at the outgrowth. However, while they are running parallel at the equatorial belt, with their b axis pointing in longitudinal direction, they are crossing each other at the pole, as indicated in Figure 9. Aragonite is known to form polycyclic twins at the (110) planes. This condition is met when [010] or [100] directions cross at 120º.

CrystEngComm Accepted Manuscript
View Article Online Indeed, outgrowing structures are formed specifically at positions where lamellae cross at 120º. Under such conditions, a twin is formed. This is a straightforward explanation for the six-fold symmetry that is revealed when the outgrowing structures are viewed from the top as shown in the Figures 2 h and 9c. Therefore, the outgrowing structures are formed as a consequence of the radial growth and crystallographic alignment combined with the ability of aragonite to form twins.
Looking at Figure 4a, one can see that the outgrowth on the left shows its roots deep inside the particle. The first signs of the formation of outgrowing structures are therefore manifested in a relatively early growth stage (i.e., after less than 9h, Figure 2a). The corresponding region, which is characterized by intergrowth and twinning, expands faster due to an increased growth speed and hence, requires more space. Presumably, the attachment of the growth carrying nanoparticles adds up at the point where lamellae intercept in a preferred angle and where a cyclic twin is formed. This is possibly due to a combination of a rather good stabilization of the nanoparticles by the siderophore and field effects which allow the particles in later stages of reaction to discriminate between different positions of aggregation, thus preferring twinning positions where the fields are not compensated.
A strong indication for the presence of field effects is found in the fact that the outgrowth is exclusively formed on only one of the poles. As seen in Figure 5c, aragonite grows preferably along [001], which is the fast growth direction. The {001} are, however, highly polar faces and not stabilized by the siderophore. Consequently, the surfaces on the globular body are mainly defined by (101) and some (100) planes (see Fig. 4d).
However, the faces of outgrowing structures at the pole are constituted mainly by (100) planes, defining the thin direction of the aragonite slices (thereby most stabilized and also loosing against [001] in the growth competition). The pole-to pole direction of the striation lines is the [010] direction, associated to polar surfaces. As the siderophore binds to carbonate especially at the more acidic pH at the beginning of the crystallization reaction but not or only weakly to calcium ions, 31 one of the (010) faces of the primary nanoparticles, the negative side, is hindered in growth causing a dipole field along [010]).
If we define the equatorial cut through the superstructure to be along the [100] directions, this gives an exact cycle, i.e. all [100] directions sum up into the same circular direction.

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The (010)-cut is more an ellipse, and the structure must be twinned (as already all the [100] directions are aligned). Indeed, the "cut through the particle" reveals this pole-topole distortion by its lower structural density (see Fig.4a) along the pole-to-pole axis.
This means that all meridians are oriented in the same direction, supported by twinning structures along the axis. By superstructure formation, this sums up to the discussed fields at the poles where the "negative pole" does not outgrow, while the "positive pole" attracts further nanoparticles and leads to the found pronounced structured outgrowth.
Such inner field effects were found for CaCO 3 before and can lead to very specific superstructures of remarkable beauty 39,40 . The fact that the siderophore, contrary to the previously reported cases, binds here mostly to the (100) and (010) faces of aragonite (instead of the (001) of calcite) is attributed to appropriate charge distances in the surface and ligand and pattern recognition.

CONCLUSION
This study shows the formation of aragonite superstructures through a non-classical crystallization process. In contrast to the well-organized natural aragonite crystals that are intimately associated with an organic matrix in mollusk shells, the crystal superstructure here is achieved only with the use of a soluble small molecule. Nevertheless, the siderophore as amphoteric additive with time/pH dependent interaction with the crystals is close to the soluble part of natural biomineralization proteins, which are also amphoteric. We identified its action in an at least threefold fashion, stabilizing nanoparticles, controlling their alignment by stabilization of specific crystal faces, and controlling a complex dynamic self organization process which finally results in a delicate crystal superstructure where primary crystals are oriented along the radial growth axis, along the equator or latitudes, and along the meridians in three well defined fashions. Hence, a great complexity can be achieved by synergy between pH dependent polymer/crystal interaction and crystallization. This process, which should be adaptable to other systems, may help to better understand the presence of additives and especially small molecules that could influence the kinetics of crystal growth as well as provide an interesting aspect of aragonite crystal growth in natural media.

EXPERIMENTAL PROCEDURE
Synthesis. Calcium carbonate polycrystals were grown by diffusion of carbon dioxide into calcium chloride solutions according to the gas diffusion method 34  Sigma-Aldrich (3.2 and 6.4 mg/mL ; MW=656.79 g/mol, i.e. corresponding to a molar ratio of 2:1 and 1:1 calcium to deferoxamine respectively) and fresh ammonium carbonate (2 g) were placed into a closed chamber (1000 cm 3 ). The aqueous solutions of siderophore deferoxamine/CaCl 2 were prepared in doubly distilled water, sonicated for 10-15 minutes and bubbled with N 2 overnight before use. The decomposition of ammonium carbonate produced ammonia and carbon dioxide diffusing through 5 needle holes pierced into the parafilm cover of the flasks. The initial solution was slightly acidic (pH 5.8) but the pH value rose to 9.5 because of the dissolved NH 3 . After different ageing times (5,9,13 and 24 hours), the sample was removed from solution, rinsed with filtered water and acetone then air-dried.
Crystal characterization. The crystals were washed with distilled water then ethanol and dried in air for further characterization. Powder X-ray diffraction (XRD) patterns were recorded on a PDS 120 diffractometer (Nonius GmbH, Solingen) with Cu-K α -radiation.
The SEM measurements were performed on a LEO 1550 -GEMINI with the gold-coated samples. FT-IR spectra were recorded either with a Nicolet Impact 400 or a BioRad yields an average value for the hydrodynamic radius and the polydispersity index, which are characteristic for the particle size distribution. DLS measurements were triplicated for each sample to ensure the reproducibility of the preparation conditions.

TOC
A synthetic siderophore, deferoxamine, is used to control the crystallization behavior of calcium carbonate. It induces the formation of unusual, highly organized aragonite superstructures with elliptical shape and pronounced outgrowth on only one pole. A detailed investigation of the structure and growth of the particles provides an insight on the role of the deferoxamine and its function in the crystallization process.