Biomimetic mineralization of calcium carbonate mediated by a polypeptide-based copolymer

Article abstract of DOI:10.1039/c2tb00182a

A novel copolymer, β-cyclodextrin-b-poly(l-glutamic acid) (β-CD-b-PLGA), was synthesized by ring-opening polymerization and subsequent hydrolysis reaction. The β-CD-b-PLGA copolymer possesses an oligosaccharide β-CD segment and a polypeptide PLGA segment, with chemical structure resembling natural glycoprotein. The copolymers were applied in regulating the crystallization of calcium carbonate. The effects of the concentration of copolymers and calcium ions were systemically investigated. Various morphologies, including rhombohedra, rod, pseudo-dodecahedra and rosette-like structures, were obtained by adjusting the polymer and Ca ²⁺ concentrations of the initial solution. Investigation of the pseudo-dodecahedra growth mechanism indicates that the copolymers mediate amorphous calcium carbonate formation initially, and then regulate the meso-scale self-assembly of CaCO₃ subunits. The morphology variation is influenced by the binding of β-CD-b-PLGA chains on specific crystal faces combined with the steric repulsive force of β-CD-b-PLGA chains. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2tb00182a

Journal of
Materials Chemistry B
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PAPER
Biomimetic mineralization of calcium carbonate
mediated by a polypeptide-based copolymer†
Cite this: J. Mater. Chem. B, 2013, 1,
41
8
Wenjie Zhu, Jiaping Lin,* Chunhua Cai and Yingqing Lu
A novel copolymer, b-cyclodextrin-b-poly(L-glutamic acid) (b-CD-b-PLGA), was synthesized by ring-opening
polymerization and subsequent hydrolysis reaction. The b-CD-b-PLGA copolymer possesses an
oligosaccharide b-CD segment and a polypeptide PLGA segment, with chemical structure resembling
natural glycoprotein. The copolymers were applied in regulating the crystallization of calcium
carbonate. The effects of the concentration of copolymers and calcium ions were systemically
investigated. Various morphologies, including rhombohedra, rod, pseudo-dodecahedra and rosette-like
2+
structures, were obtained by adjusting the polymer and Ca concentrations of the initial solution.
Investigation of the pseudo-dodecahedra growth mechanism indicates that the copolymers mediate
amorphous calcium carbonate formation initially, and then regulate the meso-scale self-assembly of
Received 28th September 2012
Accepted 30th November 2012
DOI: 10.1039/c2tb00182a
3
CaCO subunits. The morphology variation is influenced by the binding of b-CD-b-PLGA chains on
www.rsc.org/MaterialsB
specific crystal faces combined with the steric repulsive force of b-CD-b-PLGA chains.
and covalently attached oligosaccharide chains has received
increasing interest due to its effect in the nucleation and growth
1
Introduction
It is widespread in biological systems that living organisms of crystallization.7–10 Takagi et al. studied the effects of a glyco-
synthesize inorganic minerals with complex shapes, hierar- protein aggregate of sh otolith matrix and its separate
chical structures and fascinating properties. Biomineralization
components on CaCO
the aggregate and the separated components induced different
polymorphs and morphologies of CaCO crystals under the
3
crystallization in vitro. It was found that
is regulated by the cooperation of soluble and insoluble organic
1
components in biological systems. However, the exact mecha-
3
8
nism of the interaction between organics and minerals is same conditions. Addadi et al. extracted glycoproteins from
difficult to explore because the organic components are oen sea-urchin spines and cleaved the glycoproteins to several
structurally complex and difficult to individually isolate from fractions, and studied their respective effects on CaCO crys-
3
2
the biominerals. Recently, increasing attention has been given tallization. The result shows that the polysaccharide moieties as
to the biomimetic synthesis of inorganic materials by using well as the proteins are involved in certain types of interactions
9
various biopolymers and synthetic polymers to explore the
fundamental mechanism in biomineralization.
with calcite crystals. Mcgrath et al. studied the mineralization
of CaCO
3
3
in the presence of acidic glycoproteins isolated from
Calcium carbonate (CaCO ) has been of considerable
3
adult spines of the sea urchin Evechinus chloroticus. It was found
interest because it is one of the most abundant biominerals and that the cleaved oligosaccharides provide a similar growth
used industrially in vast quantities. Hydrophilic polymers, environment for the CaCO as the parent glycoproteins, while
3
10
including proteins, polysaccharides and glycoproteins, exist in the deglycosylated protein does not produce this effect.
CaCO biominerals and guide the development of the mineral Despite these observations, the roles of glycoproteins in bio-
3
4
phase. Acidic groups (carboxylate, sulfate, phosphate, etc.) in mineralization are still far from fully understood due to their
2
+
these copolymers could bind Ca and crystal faces to control
complex components and complicated three-dimensional
structures.
Inspired by biomineralization process, various synthetic and
natural polymers which have a simple structure have been
applied in bio-inspired crystallization. Polysaccharides
including pectin, k-carrageenan, and sodium alginate have
5,6
the nucleation and growth of CaCO3. Among the biomineral
polymers, glycoprotein which comprises of protein segment
Shanghai Key Laboratory of Advanced Polymeric Materials, State Key Laboratory of
Bioreactor Engineering, Key Laboratory for Ultrane Materials of Ministry of
Education, School of Materials Science and Engineering, East China University of
3
been utilized for directing the CaCO growth, and intriguing
11
Science and Technology, Shanghai 200237, China. E-mail: jlin@ecust.edu.cn; Fax: hollow crystals were obtained. Because proteins in bio-
+
86-21-64251644; Tel: +86-21-64253370
minerals oen contain acidic amino acid residues, to mimic the
protein-mediated biomineralization, acidic polypeptides which
can be synthesised by living polymerization are usually used as
†
Electronic supplementary information (ESI) available: GPC traces of
b-CD-b-PBLG, thermal analysis of the CaCO
3 3
sample and CaCO crystals
controlled by CG2, CG3, b-CD and PLGA. See DOI: 10.1039/c2tb00182a
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 841–849 | 841

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1
2–15
17
1
2
the crystallization modier.
Guo and Yu et al. prepared the literature. H NMR (500 MHz, DMSO-d
6
): d ¼ 7.75 (d, H ),
2
14
6
various kinds of porous CaCO spheres by using poly(ethylene d ¼ 7.43 (d, H ), d ¼ 5.86–5.61 (m, H ), d ¼ 4.89–4.73 (d, H ),
3
13
3
glycol)-b-poly(aspartic acid) as a crystal growth modier. In d ¼ 2.43 (s, H ) ppm.
recent work, we studied the inuence of a thermo-responsive
Synthesis of mono[6-(2-aminoeythylamino)-6-deoxy]-b-
polymer, poly(N-isopropyl acrylamide)-b-poly(L-glutamic acid), cyclodextrin (EDA-b-CD). EDA-b-CD was prepared by heating
1
4,15
on the crystallization of CaCO
3
and BaCO
3
.
By tuning the 6-OTs-b-CD (2 g) in excess anhydrous ethylenediamine (15 mL)
ꢀ
18
temperature, we obtained rosette-like calcite in the unimer at 70 C for 7 h. Aer the reaction was completed, the mixture
solution and aragonite bers in the polymer micelle solution in was allowed to cool to room temperature, and then, 50 mL of
the CaCO crystallization. It is anticipated that a type of cold acetone were added. The precipitate was repeatedly dis-
3
14
copolymers, which comprises both polypeptide and sugar solved in 10 mL of water–methanol mixture, and poured into
moiety, can be applied as a model compound for imitating 50 mL of acetone several times for the removal of unreacted
ꢀ
glycoprotein-controlled mineralization.
EDA. Finally, the sample obtained was dried at 50 C for 3 days
1
7
To better understand the glycoprotein-controlled minerali- in a vacuum oven. H NMR (500 MHz, D
2
14
O): d ¼ 5.01 (s, H ), d ¼
2
8
2
zation, we design and construct a simple model by utilizing a 4.01–3.88 (m, H ), d ¼ 3.67–3.45 (m, H ), d ¼ 2.86–2.76 (m, H )
ꢁ
1
ꢁ1
novel copolymer, b-cyclodextrin-b-poly(L-glutamic acid) (b-CD-b- ppm; IR (KBr): 3345 cm
(s, OH), 2928 cm
2
(w, CH ),
ꢁ
1
PLGA), as the CaCO
3
crystallization modier. The b-CD is a 1030 cm (s, C–OH).
Synthesis of b-cyclodextrin-b-poly(g-benzyl-L-glutamate). b-
common cyclic oligosaccharide, which is a truncated conical
molecule with a hollow cavity constituted by seven glucopyr- Cyclodextrin-b-poly(g-benzyl-L-glutamate) (b-CD-b-PBLG) was
anoside units. The copolymer can be considered as a simple synthesized by ring-opening polymerization of BLG–NCA,
model of glycoprotein since the two segments resemble oligo- initialized by the primary amino group of EDA-b-CD. In a typical
saccharide and protein respectively. It was discovered that the procedure, EDA-b-CD (0.2 g) and BLG–NCA (1.6 g) were dis-
copolymers have remarkable effects on the control of CaCO
3
solved in DMF separately in two dry bottles. Then they were
mineralization. A series of calcite morphologies, including mixed in a dry three-neck round-bottom ask. The reaction was
rhombohedra, rods, rosettes and pseudo-dodecahedrons, were performed at room temperature in nitrogen atmosphere. The
obtained by adjusting the concentration of the polymer and concentration of BLG–NCA was xed at ꢂ5%. Aer 72 h of
calcium ion. The mechanism underlying the polymer-mediated stirring, the mixture was dialyzed against deionized water and
1
crystallization behavior was elucidated.
lyophilized to get b-CD-b-PBLG powder. The H NMR spectra are
1
provided in Fig. S1 in the ESI.† H NMR (500 MHz, DMSO-d ):
6
7
.32 (Ar–HPBLG), 5.04 (CH2PBLG), 4.48 (Hb-CD-1), 3.72–4.17
2
Experimental section
(OHb-CD), 3.33 (CHPBLG), 1.75–2.74 (Hb-CD-2, Hb-CD-3, Hb-CD-4,
2.1 Materials
H
b-CD-5, Hb-CD-6).
Synthesis of b-cyclodextrin-b-poly(L-glutamic acid). b-Cyclo-
b-CD was purchased from Sinopharm Chemical Reagent Co.,
Ltd., and used aer drying in a vacuum oven for 24 hours.
p-Toluenesulfonyl chloride (p-TSCl) was puried by standard
dextrin-b-poly(L-glutamic acid) (b-CD-b-PLGA) block copolymers
were prepared by hydrolyzation of b-CD-b-PBLG with potassium
hydroxide (KOH). As a brief, 1 g b-CD-b-PBLG was suspended in
50 mL tetrahydrofuran (THF). Then an aqueous solution of
KOH (3 mol equiv. with respect to the benzyl group) was added
to the suspension. Aer 4 h of stirring, the mixture was
neutralized with hydrochloric acid and dialyzed against water to
remove organic solvent and other small molecule impurities. A
white powder was obtained by lyophilizing the dialyzed solu-
tion. The benzyl groups were completely removed aer hydro-
lyzation as indicated by the disappearance of methylene proton
0
procedures. Ethylenediamine (EDA) and N,N -dimethylforma-
˚
mide (DMF) were dried over 4 A molecular sieves for at least 24 h
and vacuum distilled before use. Deionized water (DIW) was
made in a Millipore Super-Q Plus Water System to a level of
18.2 MU cm resistance. g-Benzyl-L-glutamate–N-carboxyanhy-
dride (BLG–NCA) was synthesized by the reaction of g-benzyl-
L-glutamate with triphosgene according to the procedures
16
reported in the literature. CaCl , (NH ) CO and other
2
4 2
3
reagents were of analytical grade and used without further
purication. All glassware (glass bottles and small pieces of
glass substrates) for the crystallization experiments was cleaned
and sonicated in ethanol for 5 min. It was then rinsed with DIW,
1
peak (5.04 ppm) from the H NMR spectra (Fig. S1†).
2
.3 Mineralization
2 3 2 2
further soaked in a H O–HNO (65%)–H O (1 : 1 : 1, v/v/v)
The b-CD-b-PLGA solution was rst prepared by directly dissolv-
ing b-CD-b-PLGA in basic solution. In a typical procedure, 50 mg
b-CD-b-PLGA was dissolved in 40 mL DIW in a 100 mL bottle.
solution, and then rinsed with DIW. Aerward, it was dried in
air with acetone. A dialysis bag (Membra-cel, 3500 molecular
weight cutoff) was provided by Serva Electrophoresis GmbH.
1
1.1 mg (0.1 mmol) CaCl
CaCO . Then, the pH value of the solution was carefully adjusted
to 9.0 (ꢃ0.1) by adding a small quantity of 1 M hydrochloric acid
Synthesis of mono[6-O-(p-toluenesulfonyl)]-b-cyclodextrin (HCl)or1Msodiumhydroxide(NaOH). Thenalsolutionvolume
6-OTs-b-CD). Mono[6-O-(p-toluenesulfonyl)]-b-cyclodextrin (6- was xed at 50 mL by adding water. Aer then, the solution was
OTs-b-CD) was prepared by the reaction of p-TSCl with b-CD in ltered by using a 0.22 mm Millipore to remove impurities. The
alkaline aqueous solution according to the method reported in synthesis of CaCO was performed by the gas diffusion method
2
was also added as the calcium source of
3
2.2 Synthesis of the polymer
(
3
8
42 | J. Mater. Chem. B, 2013, 1, 841–849
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Journal of Materials Chemistry B
9
described by Addadi and co-workers. The reaction vessel with
0 mL b-CD-b-PLGA solution was covered with paralm punched
3
Results and discussion
1
3.1 Synthesis and characterization of b-CD-b-PLGA
with 4 holes, and then placed in a closed desiccator at room
ꢀ
temperature (22 ꢃ 1 C). A glass bottle containing crushed The synthesis of b-CD-b-PLGA block copolymers was illustrated
ammonium carbonate, also covered with pierced paralm, was in Scheme 1. The initiator EDA-b-CD, which has only one amino
placed in the same desiccator as the source of CO
2
. The glass group as the active initiating site, was prepared by modifying b-
slides were taken out from the vessels at different times, rinsed CD with two reaction steps, etherifying with p-TSCl and ami-
with distilled water and allowed to dry in the air for further nolysis with an excess amount of EDA. Then b-CD-b-PBLG
analysis. All experiments were repeated several times.
diblock copolymer was synthesized via ring-opening polymeri-
zation of BLG–NCA initiated by primary amino groups of EDA-b-
CD. The length of PBLG segment was controlled by adjusting
the feeding ratio of BLG–NCA to macroinitiator. Finally, the b-
2.4 Measurements
1
1
1
H Nuclear Magnetic Resonance ( H NMR). H NMR spectra CD-b-PLGA was obtained aer alkaline hydrolysis for depro-
measurements were performed on a Bruker Avance 550 spec- tection of benzyl ester.
trometer using dimethyl sulfoxide-d , D O or other deuterated
The number-average molecular weight (M
solvent and tetramethylsilane as an internal standard at room dispersity index of the molecular weight distribution (PDI ¼
temperature.
/M ) were measured by gel permeation chromatography
6
2
n
) and the poly-
M
w
n
Gel Permeation Chromatography (GPC). The molecular (GPC) using DMF/LiBr as eluent. GPC traces of the b-CD-b-PBLG
weight and polydispersity of b-CD-b-PBLG copolymers were block copolymer samples with various PBLG lengths are pre-
measured by GPC (Polymer Lab, PL-GPC50 plus) using PBLG sented in Fig. S2 (see ESI†). Each sample shows a monomodal
with narrow molecular weight distribution as standard, per- distribution, indicating a successful synthesis of copolymers.
formed in LiBr/DMF solution.
The PDIs are 1.19, 1.24 and 1.27 for b-CD-b-PBLG55, b-CD-b-
Field Emission Scanning Electron Microscopy (FE-SEM). The PBLG36, and b-CD-b-PBLG17 respectively (the subscripts refer to
crystals on the glass wafers were observed by scanning electron the PBLG repeating units). The hydrolysis products b-CD-b-PLGA
microscopy (SEM) (HITACHI-4800) operated at an accelerating copolymers are denoted as CG1, CG2 and CG3 respectively. The
voltage of 10 kV. Before the observation, the samples were detailed information is provided in Table 1. The b-CD-b-PLGA
sputtered with gold.
copolymers can be directly dissolved in water without using any
High-Resolution Transmission Electron Microscopy (HR- NaOH, indicating that the copolymers have a good solubility.
TEM) and Selective Area Electronic Diffraction (SAED). The HR-
TEM image and SAED were performed on a JEM-2100F HR-TEM
operated at an accelerating voltage of 200 kV. The crystal in the
glass bottle was collected and dispersed in water. And then, the
3
.2 Effect of polymer concentration on morphosynthesis of
CaCO crystals
3
sample was transferred to a copper grid with carbon lm and The calcium carbonate crystallization was performed by using a
dried in air before the observation. SAED was also performed gas diffusion method. The gas diffusion method is a commonly
9
using this instrument.
used method for mineralization. In such a method, the
Atomic Force Microscopy (AFM). The surface of the crystals ammonium carbonate decomposes slowly and releases CO2,
on the glass wafers was directly observed by a multimode which further gradually diffuses into the solution, generates
2
ꢁ
atomic force microscopy (XE-100, PSIA), using the non-contact CO3 and reacts with CaCl to form CaCO . In such processes,
2
3
mode.
the supersaturation in the solution is not very high. Therefore,
X-ray Diffraction (XRD). XRD was used to analyze the crys- the additives can control the crystallization properly. As is well
talline structure. The XRD patterns were recorded on Rigaku known, rhombohedral shaped calcite crystals consisting of six
ꢀ
D/Max-2550V with Cu Ka radiation in the range 2q ¼ 10–80 .
Fourier Transform Infrared Spectra (FT-IR). For FT-IR anal-
ysis, the samples were pressed with KBr into pellets. The spectra
of the samples were recorded on a Nicolet 5700 FT-IR spec-
trometer in transmission mode.
Thermogravimetry analysis (TG). TG was performed with a
thermogravimetric analyzer (NETZSCH STA 409 PC/PG) at a
ꢀ
ꢁ1
ꢀ
heating rate of 10 C min from room temperature to 800 C
under nitrogen ow.
Conductivity measurement. The interactions between poly-
2
+
mers and Ca were tested by using conductivity measurement.
2
+
At a xed Ca concentration of 50 mM, the concentration of
ꢁ1
polymers was varied from 1.0 to 5.0 g L . The conductivities for
the polymer and polymer–CaCl hybrid systems were measured
2
with a DDSJ-308A conductometer (Shanghai REX Instrument
ꢀ
Factory) at 25 C.
Scheme 1 Synthetic route for the b-CD-b-PLGA copolymers.
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Table 1 Characteristics of b-CD-b-PLGA copolymers
a
a
b
Sample
Copolymer
M
n
PDI
CG1
CG2
CG3
b-CD-b-PLGA55
b-CD-b-PLGA36
b-CD-b-PLGA17
8316
5730
3311
1.19
1.24
1.27
a
n
The number-average molecular weights (M ) were calculated by GPC of
the precursor b-CD-b-PBLG using PBLG with narrow molecular weight
b
distribution as standard, LiBr/DMF as eluent. Polydispersity indexes
w n w n
(PDI), M /M (M is the weight-average molecular weight and M is
the number-average molecular weight) of the precursor b-CD-b-PBLG
are determined by GPC.
{
104} faces can be produced under equilibrium conditions in
6
the absence of polymers. The CG1 was rst presented as the
modier of CaCO crystallization. The concentration of Ca
2
+
3
was xed at 2 mM. The effect of polymer concentration on the
mineralization was investigated by adjusting the polymer
ꢁ
1
concentration from 0.001 to 2.0 g L . XRD was used to identify
the crystalline phase of the as-prepared samples (Fig. 1). For all
samples prepared, the XRD pattern exhibits the characteristic
˚
reection of calcite (d-spacings/A: 3.87, 3.04, 2.84, 2.50, 2.29;
corresponding to hkl: 012, 104, 006, 110, and 113 respectively).
The evolution of CaCO morphology as the polymer concen-
tration varied was displayed in Fig. 2. Fig. 2a shows the SEM
3
Fig. 2 SEM images of CaCO
3
crystals obtained in the presence of CG1 with
various polymer concentrations, (a) 0.001, (b) 0.003, (c) 0.01, (d) 0.3, (e) 1.0, (f)
images of CaCO
of 0.001 g L polymer solution. The CaCO products consist
3
crystals aer 3 days of reaction in the presence
ꢁ1
2+
2
.0 g L . The concentration of Ca is 2 mM.
ꢁ
1
3
almost entirely of rhombohedra structures, which are similar to
typical calcite rhombohedra crystals formed in the absence of
ꢁ1
polymers. The magnied image in the inset shows the etching the sample prepared in 0.3 g L polymer solution. The rods
pits in {104} faces and truncated edges between the faces. At a elongated along the c-axis indicating that the polymers express
ꢁ1
CG1 concentration of 0.003 g L , the truncated edges can be nonspecic inhibition of growth in the envelope of planes {hk0}.
seen more obviously (Fig. 2b). When the concentration of CG1 This is because the carbonate ions align almost perpendicular
ꢁ
1
further increases to 0.01 g L , the morphology of CaCO
tals changes to entire rods (Fig. 2c). The rods range from 16 to the crystal planes in this orientation. In this case, {104} faces
4 mm in length and from 8 to 10 mm in width. A similar can only partially be expressed at the tips of a rod crystal.
structure can also be produced in CG1 copolymer solution with
As the concentration of CG1 was increased to 1.0 and 2.0 g
3
crys- to the {hk0} planes and acidic polymers can easily interact with
19
2
ꢁ1
ꢁ1
the concentration ranging from 0.01 to 0.8 g L . Fig. 2d shows
L , calcite crystals with a pseudo-dodecahedral shape were
produced, as shown in Fig. 2e and f. The crystals have a rela-
tively uniform size, about 6–8 mm. Each pseudo-dodecahedron
contains six smooth and six coarse faces. However, the coarse
faces cannot be viewed as real faces since they are highly curved.
From the magnied image, we can nd that the curved faces
show a nanogranular surface structure (Fig. 2f, inset). The six
20
rough faces could be indexed as {011} faces. The pseudo-
dodecahedral shapes, which are characterized by their barrel-
shaped habit with triplanar faceted ends, have been obtained in
2
+
2+ 21
the presence of inorganic ions (Mg and Co ), small organic
22
23
20,24
molecules, biopolymers, synthetic polymers
and in some
25
gel systems. Especially, such shapes can be obtained by using
biopolymers extracted from sea-urchin spines and nacre
proteins. Moreover, the crystal morphology has a high degree of
similarity to that of biogenic calcite otoconia. Otoconia in
mammals, which act as gravity receptor organs in the inner ear,
consist of microcrystals joined together by a lamentous
organic matrix of glycosylated proteins to form composite
Fig. 1 XRD pattern of CaCO
3
samples obtained in the presence of CG1 with
ꢁ1
various of polymer concentrations, (a) 0.001, (b) 0.01, (c) 0.1, (d) 2.0 g L . The
2+
26
concentration of Ca is 2 mM.
crystals. However, little is known about the mechanism of the
8
44 | J. Mater. Chem. B, 2013, 1, 841–849
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otoconial biosynthesis. The study of the CaCO
Journal of Materials Chemistry B
27
3
crystallization
under the control of CG1 may be helpful for studying glyco-
protein-mediated mineralization.
The pseudo-dodecahedron structure was further studied by
using AFM and HR-TEM (Fig. 3). Fig. 3a shows the AFM image of
the upper surface of a typical pseudo-dodecahedron. The rough
and curved feature of the coarse face is in line with the SEM
observation. It was calculated that particles on the curved
surface are about 100 nm in size. Because the particle may be an
aggregate of more than one primary particle, the result indi-
cates that the dimension of primary particles is in the nano-
scale. Fig. 3b shows a typical TEM image of the dodecahedron. A
thin part of the pseudo-dodecahedron shows lattice fringes with
spacing of 0.306 nm, corresponding to an interplanar distance Fig. 4 FT-IR spectra of the CG1 polymer and CaCO3 crystals formed in the
of the (104) plane (Fig. 3c). The image also displays interstices polymer solution with various polymer concentrations, (a) 0.01, (b) 0.1, (c) 0.3, (d)
ꢁ
1
2+
1
.0, (e) 2.0 g L . The concentration of Ca is 2 mM.
embedded in the crystal matrix (as the white arrow shows).
Similar defects can be widely observed in the lattice fringe
image (Fig. S3†). The existence of the interstices indicates that
the crystallization follows an intermediate mesocrystal
pathway. Mesocrystals are a special type of colloidal crystals
composed of individual crystallographically aligned nano-
crystalline building blocks. In the mesocrystals, the nano-
crystals are in perfect crystallographic orientation. However,
planar stacking defects between the nanocrystals are inevitable
and transform into interstices with polymers upon the ripening
process.
of remained CG1 is calculated to be about 1.68% in the
pseudo-dodecahedra sample obtained in 2.0 g L
solution (Fig. S4†). And lowering the CG1 concentration leads to
a lower weight loss, which is consistent with the FT-IR results.
The above results reveal that the polymer plays a crucial role in
the morphosynthesis of the calcite crystals.
ꢁ
1
28
polymer
The inuence of CG2 and CG3 copolymers on CaCO crys-
tallization was also studied respectively. Both the CG2 and CG3
3
3
copolymers have similar controlling effects to CaCO crystalli-
zation with CG1 as shown in Fig. S5.† Rod structures were
formed in the presence of CG2 and CG3 at a lower concentration
The FT-IR spectra were also recorded to characterize the
rhombohedra, rods and pseudo-dodecahedra (Fig. 4). For each
ꢁ
1
sample, the vibration bands at ca. 712 and 874 cm can be
2
ꢁ
(
Fig. S5a and S5c†). While in the presence of CG2 and CG3 at a
higher concentration, pseudo-dodecahedra were generated
Fig. S5b and S5d†).
attributed to the n4 and n2 modes of calcite CO
3
. As the
polymer concentration increases, a more evident characteristic
ꢁ
1
(
absorption band in 1654 cm which corresponds to the typical
amide I band of CG1 in the crystals can be distinguished (as
indicated by the blue dashed line). The results suggest that the
CG1 have been gradually incorporated into the crystals. The
weight amount of polymers included in the crystals was
3
.3 Morphological evolution of CaCO mineral with various
3
2
+
Ca concentrations
2
+
determined by thermalgravimetric analysis (TG). The percent In the mineralization, the polymer and Ca concentrations are
two important factors inuencing the nal CaCO3 crystal
structures. We further investigated the mineralization behavior
2
+
of CaCO
3
crystal at Ca concentrations of 1, 3 and 4 mM
2
+
respectively. At a xed Ca concentration of 1 mM, the CaCO
3
morphology changes from rhombohedral structures (Fig. 5a
and b) to rods (Fig. 5c) and then to pseudo-dodecahedral
structures (Fig. 5d and e) with increasing the polymer concen-
tration. The behavior is similar to that at 2 mM calcium ions
(Fig. 2). It is worth noting that the structure displayed in Fig. 5d
shows an intermediate morphology between rods and pseudo-
dodecahedrons. The structure is oval-shape and has {104} caps
at both ends, which suggests a continuous structural transition
in the product particles with the variation of copolymer
concentration.
Fig. 5f–i display the morphological evolution of CaCO
3
2
+
crystals at a xed Ca concentration of 4 mM. The crystals show
a rhombohedra appearance indicating a less marked inuence
ꢁ
1
of polymers at a concentration of 0.001 g L . When the polymer
Fig. 3 (a) AFM image of a pseudo-dodecahedral CaCO
3
crystal prepared in the
crystal. (c) HR-
ꢁ
1
ꢁ1
concentration was in the range of 0.01 g L to 1.0 g L , rosette-
like spheres were generated which are constructed from
polymer solution. (b) TEM image of a pseudo-dodecahedral CaCO
3
3
TEM image of an ultra thin section of the pseudo-dodecahedral CaCO crystal.
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2+
Fig. 6 CaCO
3
crystal morphology diagram as functions of Ca and polymer
concentrations.
3.4 Mechanism study of the b-CD-b-PLGA controlled
mineralization
Fig. 5 SEM images of CaCO
3
crystals obtained in the presence of CG1 with
2+
The interaction between b-CD-b-PLGA and Ca
was
2+
ꢁ1
2+
various of polymer and Ca concentrations, (a) [CG1] ¼ 0.001 g L , [Ca ] ¼ 1
ꢁ1
2+
ꢁ1
2+
investigated to unveil the mechanism behind the mineraliza-
mM, (b) [CG1] ¼ 0.003 g L , [Ca ] ¼ 1 mM, (c) [CG1] ¼ 0.1 g L , [Ca ] ¼ 1 mM,
ꢁ
1
2+
2+
ꢁ1
ꢁ1
2+
2+
(
[
[
d) [CG1] ¼ 0.3 g L , [Ca ] ¼ 1 mM, (e) [CG1] ¼ 1 g L , [Ca ] ¼ 1 mM, (f) tion behavior. A conductivity measurement was applied to verify
ꢁ1
2+
29
CG1] ¼ 0.001 g L , [Ca ] ¼ 4 mM, (g) [CG1] ¼ 0.3 g L , [Ca ] ¼ 4 mM, (h) the interaction between Ca and CG1 (Fig. 7). The conduc-
CG1] ¼ 1.0 g Lꢁ , [Ca ] ¼ 4 mM, (i) [CG1] ¼ 2.0 g L , [Ca ] ¼ 4 mM.
1
2+
ꢁ1
2+
tivity of the polymer solution (LCG1) and Ca–CG1 hybrid solu-
tion (LCa–CG1) was recorded respectively. As the complexion of
Ca and acidic polymer chains connes the free migration of
2
+
ꢁ
1
micrometer crystalline units. In the presence of 2.0 g L CG1,
rod-shaped calcite crystals were the major product.
The above results indicate that the Ca concentration has
a remarkable inuence on the mineralization behavior. At a
2
+
Ca , increase in the polymer concentration makes the value of
2
+
L
Ca–CG1 ꢁ LCG1 decrease markedly. For comparison, the
conductivity experiments for PLGA99 homopolymer and b-CD
solution were also carried out respectively. For the PLGA99
homopolymer, the changing trend of LCa–PLGA ꢁ LPLGA is
ꢁ
1

xed polymer concentration of 0.3 g L , the morphology of
CaCO3 crystals transforms from pseudo-dodecahedrons
2
+
2+
2
similar to that observed in CG1/CaCl solution. The value of
(
Fig. 5d, Ca is 1 mM) to rods (Fig. 2d, Ca is 2 mM) and
2
+
L
Ca–b-CD ꢁ Lb-CD has no obvious uctuation with the variation
then to rosettes (Fig. 5g, Ca is 4 mM) with increasing the
2
+
of the b-CD concentration, which indicates the b-CD has
Ca concentration. Since the disorder degree of the arrange-
ment of crystalline units gradually increases with the
morphology transformation from pseudo-dodecahedrons to
rods and then to rosettes, it can be concluded that a higher
2
+
negligible affinity for Ca . The above results indicate that the
PLGA block in the copolymer is the functional segment that
2
+
binds with the Ca .
2
+
As control experiments, b-CD and PLGA99 homopolymers
were used individually as additives for modifying CaCO
Ca concentration could lead to a lower efficiency of polymer
control action.
3
The observed structures are summarized in a morphology
2
+
diagram with respect to the concentration of Ca and CG1
Fig. 6). Each point in the phase diagram corresponds to a
(
mineralization result, and the lines were drawn to identify the
resulting morphology boundaries. The morphology diagram is
divided into four regions, rhombohedra, rosettes, rods and
pseudo-dodecahedrons. At a CG1 polymer concentration below
ꢁ
1
0
.003 g L , the copolymers have little effect on the minerali-
zation, leading to the formation of rhombohedra. The etching
of the rhombohedra was due to face selective polymer adsorp-
2
+
tion in a classical growth process in these cases. At a higher Ca
concentration (above 3 mM) and lower polymer concentration,
the crystals exhibit a rosette-like appearance, showing a disor-
dered organization of the building units. In the other two
regions, the crystal morphologies are relevant to the concen-
Fig. 7 The conductivity discrepancy curves of the polymer and polymer–CaCl
2
2
+
tration ratio of polymer and Ca . Rods were formed with lower
hybrid systems as a function of polymer concentration. The Lb-CD, LCG1 and LPLGA
2
+
CG1/Ca ratios and pseudo-dodecahedrons were obtained with
are conductivities of b-CD, CG1 and PLGA. The LCa–b-CD, LCa–CG1 and LCa–PLGA are
2
+
2+
higher CG1/Ca ratios.
conductivities of b-CD, CG1 and PLGA in the presence of 50 mM Ca .
8
46 | J. Mater. Chem. B, 2013, 1, 841–849
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mineralization (Fig. S6†). In the case of b-CD, rhombohedral pseudo-dodecahedral calcite and does not change obviously aer
calcite was obtained with the b-CD concentration ranging from more time (Fig. 8f).
ꢁ
1
0
.01 to 5.0 g L (see Fig. S6a†). The morphology appears to be
Based on the above analysis, a possible formation mecha-
identical to that prepared in the absence of additives. In the nism for the CaCO3 crystals is proposed as displayed in
case of PLGA99, most of the formed crystals are calcite rods at a Scheme 2. At the beginning, the solution contains complexes
ꢁ
1
polymer concentration of 2.0 g L , as shown in Fig. S6b (see of polymers and calcium ions due to the binding interactions
ESI†). The above results certify that the PLGA segment is a between carboxyl groups and calcium ions. Meanwhile, the b-
binding block, while the b-CD is a solvating block. On the other CD serves as a solvating segment because the hydroxyl groups
2
+
hand, the b-CD component plays a signicant role in the mor- of b-CD cannot attract Ca efficiently. In the early stage, the
phosynthesis of the minerals as indicated by the difference of mineralization of CaCO is initialized by the decomposition of
3
PLGA and CG1 controlled mineralization.
ammonium carbonate and subsequent diffusion of the CO2
The time-dependent growth of pseudo-dodecahedra calcite into the solution (Scheme 2a). ACC particles were deposited as
ꢁ1
was studied for the mineralization system with 2.0 g L polymer a kinetic product and transiently stabilized by the b-CD-b-
2
+
30
3
and 2 mM Ca . The evolution of CaCO morphologies at various PLGA for the carboxyl groups. And the ACC particles tended
intervals is shown in Fig. 8. Aer 2 h reaction, the agglomeration to conglomerate due to van der Waals forces (Scheme 2b).
of particles was identied from SEM observation (Fig. 8a). TEM Aer that, the crystallization of calcite occurs in the ACC
andSAED studiesshow that theseparticleswere inanamorphous particles because of the instability of ACC. As discussed above,
state (Fig. 8b). Aer mineralization for 5 hours, the amorphous the copolymer prefers to adsorb to {hk0} faces (Scheme 2c).
calcium carbonate (ACC) particles tended to aggregate with The b-CD can trap surrounding water molecules within its
increasing population, building larger aggregates (Fig. 8c). Over hydrophobic cavity and in the hydration shell, leading to a
time the aggregates exhibited some crystal faces, indicating that volume occupancy of the b-CD segments with surrounding
31
the subunits which are transformed from ACC particles could water molecules on the faces. And thus the calcite units are
regulate their positions and self-assemble into oriented aggre- limited to orienting along the crystallographic c-axis and fuse
gation (Fig. 8d). With more ACC particles coalescing into the together to minimize energy, resulting in the formation of
2
+
aggregates and transforming to calcite, a much larger crystal elongated rods with a lower ratio of CG1/Ca (Scheme 2e). At a
which exposed some crystal faces was obtained at 14 hours higher calcium ion concentration, the supersaturation of
(
Fig. 8e). Aer 24 hours of reaction, the morphology is mainly CaCO is much higher and thus aggregation of the subunits
3
proceeds rapidly with either no or a low degree of orientation.
The poorly controlled process leads to rosette-like crystal
formation (Scheme 2f).
2
+
While in the case of a higher ratio of b-CD-b-PLGA/Ca ,
other crystal faces of a calcite unit may also be covered with
extended b-CD segments (Scheme 2d). The dominant growth
along the c-axis is partly suppressed and {104} faces with lowest
energy are well expressed at tip of rods again, resulting in the
formation of otoconia crystals (Scheme 2g).
The polymers inuence the crystallization process in every
period including nucleation, growth and aggregation. In the
present study, the presence of amorphous precursors in the
initial stage indicates a nucleation inhibition by the b-CD-b-
PLGA copolymers, which results from the strong interaction
2
+
ꢁ
between Ca and COO . In the crystal growth process, the
polymers adsorb on the surface of crystals and prevent the
further growth along specic crystal faces. On the other hand,
Fig. 8 The morphology evolution of pseudo-dodecahedral calcite crystals. (a)
SEM image of particle aggregates for 2 h reaction. (b) TEM image of the particle
aggregates and the inset is the SAED pattern of the aggregates. SEM image of
CaCO
3
sample for various times of reaction, (c) 5 h; (d) 9 h; (e) 14 h; (f) 24 h.
3
Scheme 2 Scheme showing the growth mechanism of the CaCO crystals.
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2
+
the polymers can decrease the free Ca concentration and thus
reduce the rate of ionic growth because of the complex forma-
tion. A high b-CD-b-PLGA concentration leads to a slow nucle-
ation and growth speed, which is favorable for the
reorganization of the building units. It can be inferred that the
slow nucleation, slow growth and well-organized aggregation
are important factors for the formation of pseudo-dodecahedral
crystals in a high polymer concentration.
Oxford,
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Dey,
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de
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Compared with glycoproteins, the b-CD-b-PLGA has a denite
chemical structure and mediates a glycoprotein-like mineraliza-
tion behavior of CaCO . Therefore, the present work provides a
3
simple method to explore the fundamental mechanism of glyco-
protein controlled mineralization. The crystallization pathway via
the transient ACC and meso-scale assembly was also found in a
ovalbumin-controlled CaCO
of glycoprotein found in egg white), implying that similar pattern
may widely exist in glycoprotein-mediated biomineralization.
From the experiments, we learned that both the b-CD and PLGA
segments play important roles in the morphosynthesis of CaCO3.
The results agree with the ndings that both polysaccharide and
peptide moieties are involved in controlling aspects of crystal
3
mineralization (ovalbumin is a kind
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32
8–10
growth.
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The function of the acidic polypeptide is to bind
3
ˇ
exist in glycoprotein-controlled mineralization, in which one part
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9
6
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4
Conclusions
2
In summary, a novel glycoprotein-like copolymer b-CD-b-PLGA
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2
+
zation. By adjusting the Ca and b-CD-b-PLGA concentrations,
structures including rosettes, rods, and pseudo-dodecahedrons
can be controllably prepared. Both the b-CD and PLGA
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Acknowledgements
This work was supported by National Natural Science Founda-
tion of China (50925308, 21234002), Key Grant Project of
Ministry of Education (313020) and National Basic Research 13 X. Guo, L. Liu, W. Wang, J. Zhang, Y. Wang and S. H. Yu,
Program of China (no. 2012CB933600). Support from project of
Shanghai municipality (10GG15) is also appreciated.
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