Materials Letters 161 (2015) 128131

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Materials Letters
journal homepage: www.elsevier.com/locate/matlet

Ultrasound-induced albumin gelation method for the preparation of

nanostructured hydroxyapatite
Xiaofei Lu a,b,1, Yunfei Xie a,b,1, Yingchao Han a,b,n, Xinyu Wang a,b, Honglian Dai a,b,
Shipu Li a,b
a
b

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, PR China
Biomedical Materials and Engineering Research Center of Hubei Province, Wuhan University of Technology, Wuhan 430070, PR China

art ic l e i nf o

a b s t r a c t

Article history:
Received 7 May 2015
Received in revised form
13 July 2015
Accepted 19 July 2015
Available online 18 August 2015

An ultrasound-induced albumin gelation method was utilized to prepare nanostructured hydroxyapatite

(HAP). After ultrasound irradiation, hybrid colloids of bovine serum albumin (BSA) and amorphous
calcium phosphate (ACaP) were formed, where the nanosized ACaP was homogenously distributed in the
bril-like BSA matrix. The freeze-dried BSA/ACaP sponge showed an oriented pore structure and the
ACaP nanoparticles were transformed into nanostructured HAP after calcinations at 400600 C. With
the aid of BSA/ACaP sponge, HAP nanocrystals were aggregated into rod-like nanostructures, which were
merged into large rod-like HAP crystals at 700 C. This study demonstrates that ultrasound-induced
albumin gelation method can be successfully used to fabricate nanostructured HAP.
& 2015 Elsevier B.V. All rights reserved.

Keywords:
Biomaterials
Hydroxyapatite
Nanocrystalline materials
Ultrasound
Albumin
Gelation

1. Introduction

2. Experimental

Hydroxyapatite (Ca10(PO4)6(OH)2, HAP) has attracted much

attention in the last several decades due to its good biocompatibility, bioactivity, osteoconductivity, non-toxicity and non-inammatory [1,2]. Recently nanostructured HAP shows special
properties such as improved osseointegrative property, higher
inhibitory effect on cancer cells than on normal cells, good candidates for cell uorescent labels and drug carriers [36]. So far,
lots of methods have been developed to prepare nanostructured
HAP [612].
Recently, ultrasound is successfully utilized to induce the gelation of organic liquids to form new products and novel architectures [13,14]. Sonication also can induce aggregation of protein
similar to gelation [15]. In our previous study, we utilized ultrasound irradiation to prepare bovine serum albumin (BSA)/ amorphous calcium phosphate (ACaP) hybrid colloids [16]. Herein, we
utilize this ultrasound-induced albumin gelation method to prepare nanostructured HAP.

Ultrasound-induced albumin gelation method was performed

in the following steps. First, according to the Ca/P molar ratio of
1.67, Ca(H2PO4)2 solution was rapidly mixed with Ca(OH)2 solution
under strong stirring. BSA was added with a concentration of
5 mg/mL. After ultrasound irradiation of 8 min using a highintensity ultrasonic probe, the obtained colloids were dried by
freeze drying. Finally, the as-dried sample was calcined for 1 h in
air to get products.
Samples were measured by X-ray diffraction (XRD, D/Max-IIIA,
Japan) and fourier transform infrared spectroscopy (FT-IR, Nexus,
Thermo Nicolet, USA). The crystallite size (D002, D310) [17] and
crystallinity degree (Xc) [18] were calculated. Size distribution of
colloids was determined using Zetasizer 3000HS (Malvern instruments, UK). Colloids on mica were determined by atomic force
microscopy (AFM, DI Nanoscope IV, USA). Samples were determined by transmission electron microscopy and selected area
electron diffraction (TEM and SAED, JEM2010, Japan) and scanning
electron microscopy (SEM, JSM-5610LV, JEOL, Japan). The morphology of freeze-dried sample was observed by 3D digital microscope (KH1300, HIROX). The cytotoxicity of HAP synthesized at
different temperature was determined to human normal liver cells
(L02). Bel-7402 human liver cancer cells were cultured with HAP
nanocrystals (500 C) for TEM observation.

Corresponding author. Fax: 86 27 87880734.

E-mail address: hanyingchao@whut.edu.cn (Y. Han).
1
Co-rst author.

http://dx.doi.org/10.1016/j.matlet.2015.07.102
0167-577X/& 2015 Elsevier B.V. All rights reserved.

X. Lu et al. / Materials Letters 161 (2015) 128131

3. Results and discussion

The XRD patterns of freeze-dried sample and calcined samples
are shown in Fig. 1. Fig. 1(a) indicated that the freeze-dried sample
was amorphous. At 400 C, the broadening diffraction peaks (Fig. 1
(b)) of HAP (JCPDS no. 74-0565) were observed, showing the nanosized nature. The calculated crystalline sizes were 16.6 nm
(D002) and 13.7 nm (D310) respectively, indicating the formation of
near spherical nanocrystals. However, sample showed a low
crystallinity degree (0.26 of Xc). At 500 C, the diffraction peaks
(Fig. 1(c)) became sharper and displayed an enhanced degree of
crystallinity (0.68 of Xc) while remaining the nanosized nature.
The calculated crystallite sizes were 27.5 nm (D002) and 19.7 nm
(D310) respectively. The further calcination at 600 C resulted in a
little increase of crystallinity degree (0.71 of Xc) (Fig. 1(d)).
Moreover, the calculated crystallite sizes of D002 and D310 were
60.7 nm and 25.8 nm, demonstrating a preferable growth of HAP
crystal along its c-axis. At 700 C, XRD pattern (Fig. 1(e)) showed
the main diffraction peaks of (211) and (112) of HAP were more
separated, indicating the further growth of HAP crystals.
The FT-IR spectra of samples are shown in Fig. S1. The freezedried sample displayed the characteristic vibrational modes of BSA
and phosphate group (Fig. S1b) and the characteristic vibrational
modes of HAP were not detected, also indicating that the sample
was amorphous. After calcinations at 400600 C, the characteristic vibrational modes of HAP with carbonate substitution were
observed (Fig. S1cf), for example, phosphate group (962 cm  1,
473 cm  1, 1092 cm  1, 1062 cm  1 and 1032 cm  1, 603 cm  1 and
563 cm  1), carbonate substitutions (1544 cm  1, 1458 cm  1,
1416 cm  1 and 877 cm  1), OH stretching and libration
(3571 cm  1 and 633 cm  1). Along with the increase of calcinations temperature, the characteristic vibrations assigned to HAP
were gradually enhanced. In addition, the vibrations due to the
combustion residues of BSA molecules (29752845 cm  1 and
18001400 cm  1) were gradually declined.
Fig. 2 shows the characteristics of ultrasound-induced hybrid
colloids. As shown in TEM image (Fig. 2(a)), colloids were composed of a large number of brous particles (1020 nm  80
100 nm) and some 1080 nm spherical nanoparticles (black arrows). The intensity-averaged particle size (ZAve) of colloids was
78.8 nm with a polydispersity index of 0.228 (PDI) (Fig. 2(b)). The
zeta potential was  19.3 mV. AFM observation further showed

Fig. 1. XRD patterns (a) Freeze-dried sample, (b) 400 C, (c) 500 C, (d) 600 C and
(e) 700 C.

129

the spherical and short rod-like nanoparticles (Fig. S2). After removal of ice by freeze drying, the dried sponge displayed a berlike porous structure with a sort of spatial orientation in Fig. S3
and 3D digital microscope video (Supplementary Video).
Supplementary material related to this article can be found
online at 10.1016/j.matlet.2015.07.102.
The calcinations resulted in the formation of nanostructured
HAP. At 400 C, 1020 nm near spherical nanoparticles were observed and SAED pattern proved the product was HAP nanocrystals (Fig. 3(a)). At 500 C, short rod-like nanoparticles (35
60 nm  80140 nm) were aggregated into about 0.4 m  2.0 m
rod (Fig. 3(b)), which were veried to be crystallographic HAP by
SAED pattern. At 600 C, TEM image and SAED pattern (Fig. 3(c))
demonstrated that the sample was HAP rod-like nanocrystals,
which were also aggregated into large rod-like agglomeration.
With the further rising of temperature to 700 C, a large number of
long rod-like particles with about 0.20.8 m in diameter were
formed in the large sheet-like particles (Fig. S4), which were HAP
single crystals as shown in TEM image and SAED pattern (Fig. 3
(d)). Clearly, it could be indicated that the long rod-like HAP
crystal was transformed from HAP nanocrystals aggregation by the
merging and growing of HAP nanocrystals.
The formation of nanostructured HAP by the ultrasound-induced albumin gelation method could be described as the following three stages of (i) formation of the BSA/ACaP colloidal
precursors, (ii) constructing of the oriented porous structure and
(iii) growing of ACaP nanoparticles into nanostructured HAP (Fig.
S5). Ultrasonic treatment can generate sonochemistry effect and
activate the water molecules to produce HO2 radicals, which can
easily oxidize the SH groups of the cysteine residues of BSA and
result in the cross-link of BSA molecules [19,20]. Moreover, BSA
can increase the diffusion resistance of Ca2 and PO43- ions and
inhibit the crystallization of amorphous CaP nanoclusters [21]. As a
result, the BSA/ACaP hybrid colloids can be obtained by ultrasound-induced albumin gelation. ACaP nanoparticles are well
distributed and adsorbed on cross-linked BSA molecules through
the electrostatic attraction force between the negative charged
 COO groups of BSA and the positive charged Ca2 sites of ACaP
[22]. During the freeze procedure, due to the temperature gradient
from the surface to the bottom layer of liquid, the bottom water is
rst supercooled to form dendritic ice, then up the temperature
gradient the dendritic ice further grows [23]. Meanwhile, the BSA/
ACaP hybrid solids are pushed by ice crystals before they are included in ice, resulting in the re-arrangement of BSA/ACaP hybrid
solids between the growing ice crystals. The uniformly distributed
BSA/ACaP hybrid solids are compacted into lamellar walls around
the ice crystals. In the subsequent lyophilization, there is no
structural disruption due to the diffusion based drying process and
the BSA/ACaP sponge with oriented pore structure is obtained similar to the preferred orientation of ice crystals. During the calcination procedure, the ACaP nanoparticles are transformed into
nanostructured HAP along with the burning of BSA under the
space control of oriented pore structure in the freeze-dried
sponge. The resulting HAP nanocrystals are further merged and
grew into large rod-like HAP crystal.
Cytotoxicity experiment demonstrated that the prepared nanostructured HAP through the ultrasound-induced albumin gelation method showed no cytotoxicity to L02 cells after one day of
coculture (Fig. S6). Moreover, the endocytosis of nanostructured
HAP (500 C) by cancer cells was studied. As shown in Fig. S7, the
nanostructured HAP was transported into Bel-7402 human liver
cancer cells. These results proved that the prepared nanostructured HAP in the present study possessed good biocompatibility and potential ability as carrier.

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X. Lu et al. / Materials Letters 161 (2015) 128131

Fig. 2. (a) TEM image and (b) size distribution curve.

Fig.3. TEM images and SAED patterns (a) 400 C, (b) 500 C, (c) 600 C and (d) 700 C.

4. Conclusions

Acknowledgment

The ultrasound-induced albumin gelation method was successfully introduced to prepare nanostructured HAP. During ultrasound-induced gelation procedure, BSA molecules were crosslinked through disulde bonds due to sonochemistry effect and
ACaP nanoparticles were homogeneously dispersed in the brillike BSA matrix, resulting in the formation of stable BSA/ACaP
hybrid colloids. The freeze-drying process generated the oriented
pore structure in the dried BSA/ACaP sponge. During the calcination procedure, the ACaP nanoparticles were transformed into
nanostructured HAP at 400600 C. Especially, HAP nanocrystals
were aggregated into rod-like agglomerations and the nanocrystals agglomerations were further transformed into large HAP single crystals in rod-like shape at 700 C via the merging and
growing of nanocrystals.

This work was supported by the National Natural Science

Foundation of China (51002109, 81190133, 51172171), the Keygrant
Project of Chinese Ministry of Education (313041), the HongKong,
Macro and Taiwan Science & Technology Cooperation Program of
China (2015DFH30180), the Fundamental Research Funds for the
Central Universities (WUT: 2014-IV-121, 2014-VII-028, 2015-zy017), and the project supported by State Key Laboratory of Advanced Technology for Materials Synthesis and Processing (Wuhan
University of Technology).

Appendix A. Supplementary material

Supplementary data associated with this article can be found in
the online version at 10.1016/j.matlet.2015.07.102.

X. Lu et al. / Materials Letters 161 (2015) 128131

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