J Biol Inorg Chem (2011) 16:1135–1140
DOI 10.1007/s00775-011-0815-6
REPORT
Biocompatible hydroxyapatite nanoparticles as a redox
luminescence switch
Hongyan Liu • Pinxian Xi • Guoqiang Xie •
Fengjuan Chen • Zhenpeng Li • Decheng Bai •
Zhengzhi Zeng
Received: 5 April 2011 / Accepted: 27 June 2011 / Published online: 19 July 2011
Ó SBIC 2011
Abstract A redox luminescence switch was prepared by
doping hydroxyapatite nanoparticles with CePO4:Tb. The
resulting multifunctional material exhibits good biocom-
patibility, biological affinity, and potential drug-carrying
capability. The luminescent hydroxyapatite nanoparticles
may find important applications in biomedical diagnostics,
drug delivery, and biological sensors.
Keywords Redox luminescence switch

Hydroxyapatite
Protein adsorption
Biocompatibility
In recent years, hydroxyapatite (HA)— Ca10(PO4)6(OH)2—
has attracted great interest in modern materials chemistry
because of its high potential as an advanced functional
biomaterial. It exhibits excellent biocompatibility and
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-011-0815-6) contains supplementary
material, which is available to authorized users.
H. Liu
P. Xi
G. Xie
F. Chen
Z. Li
Z. Zeng (&)
Key Laboratory of Nonferrous Metals Chemistry and Resources
Utilization of Gansu Province and
State Key Laboratory of Applied Organic Chemistry,
College of Chemistry and Chemical Engineering,
Lanzhou University,
Lanzhou 730000,
People’s Republic of China
e-mail: zengzhzh@lzu.edu.cn
D. Bai (&)
School of Basic Medical Sciences,
Lanzhou University,
Lanzhou 730000,
People’s Republic of China
e-mail: bdc@lzu.edu.cn
bioactivity [1]. HA has been widely used as an implantable
biomedical material in orthopedic and dental treatments
[2, 3]. HA is also used to improve the biofunction of other
biomaterials [4]. It has been found that the HA particle
surface is porous, so it can be used for drug storage [5–7].
The internalization of HA nanoparticles in cancer cells and
normal cells has been reported [8–11]. Because of their
biodegradable properties, HA nanoparticles may serve as
an ideal candidate for both cancer diagnosis and drug
delivery.
Lanthanide ions as dopants in nanoparticles of different
inorganic hosts have potential applications as phosphor
materials and biolabels [12–16]. As a consequence of their
unique electronic structures and the numerous well-defined
transition modes involving the 4f shell of their ions, lan-
thanide-doped phosphates with outstanding physical and
chemical properties constitute an important domain of
nanostructure families. Among them, Tb-doped CePO4
nanostructures have attracted great interest because of their
high luminescence efficiency [17–19]. Recently, it was
reported that Tb-doped CePO4 has been used as a redox
luminescence switch based on the reversible switching of
the Ce4?/Ce3? redox couple [20, 21]. Owing to its low
toxicity, this material has great potential applications in
biomedical diagnostics, biomedical analysis, and biological
and chemical sensors. However, such specific applications
often require multifunctional nanoparticles with fluorescent
signaling, biocompatibility, biological affinity, and drug-
carrying capability [22]. Therefore, searching for lumines-
cent biodegradable biomarkers and drug-delivery vehicles is
of fundamental importance.
We synthesized CePO4:Tb-doped HA (CTHA) nanopar-
ticles, which exhibited a strong luminescence and acted as a
redox luminescence switch. The hybrid nanoparticles dem-
onstrated high surface area, good protein adsorption, and
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1136 J Biol Inorg Chem (2011) 16:1135–1140

excellent biocompatibility. This hybrid technique permits a

perfect contact between biomaterials and bioactive mole-
cules such as proteins and cells. Furthermore, they may be
loaded with drugs and growth factors for regional delivery.
The CePO4:Tb nanoparticles were synthesized as repor-
ted [20]. The emission intensity of CePO4:Tb varies as a
function of Tb3? concentration. The optimum concentration
of Tb3? was found to be 10% (molar ratio), which was
employed in our research. CTHA nanoparticles were syn-
thesized from codeposition of Na2HPO4
12H2O and CaCl2
in the presence of CePO4:Tb at room temperature (see the
electronic supplementary material). The structures of the
products were examined by X-ray diffraction. Figure 1a
displays the X-ray diffraction patterns of CePO4:Tb, HA, and
CTHA nanoparticles. For CePO4:Tb, all the diffraction
peaks can be well assigned to a hexagonal phase known from
bulk CePO4 (JCPDS card no. 34-1380, Fig. S1), indicating
that the CePO4 crystalline lattice was successfully doped
with Tb. For CTHA, the diffraction pattern shows charac-
teristic lines of the HA hexagonal phase (JCPDS card no.
09-0432) combined with the CePO4 hexagonal phase. The
elemental composition of the CTHA nanoparticles was
investigated by energy-dispersive X-ray spectrometry
(Fig. 1b). Significant Ca and P peaks observed in the spec-
trum of the particles can be attributed to the contribution
from the HA. Strong signals of Ce and Tb were observed in
the energy-dispersive X-ray spectrometry spectrum. The
chemical composition of the nanoparticles showed a molar
ratio of Ca2? to Ce3? to Tb3? of 46.78:9.48:1, yielding
doping molar ratios [Tb3?]/([Ca2?] ? [Tb3?]) of 0.021 and Fig. 1 a X-ray diffraction patterns for A hydroxyapatite (HA),
B CePO4:Tb-doped HA (CTHA), and C CePO4:Tb; b Energy-
[Tb3?]/([Ce3?] ? [Tb3?]) of 0.095, values consistent with dispersive X-ray spectrometry spectra acquired from the CTHA
those employed in the synthesis. These results suggest that nanoparticles
the HA nanoparticles are doped with all the CePO4:Tb
nanoparticles without any loss.
The morphology and size of the nanoparticles were To understand the optical properties of this material,
investigated by transmission electron microscopy (TEM). the absorption and emission spectra were investigated.
The TEM image of a representative CTHA sample is The UV/vis absorption spectrum of CTHA nanoparticles
shown in Fig. 2a. It can be clearly seen that the resulting shows several peaks between 200 and 350 nm, with a
structures are needlelike nanoparticles of 50–100 nm maximum at 274 nm (Fig. S2), which is consistent with
length and 5–10 nm width. A high-resolution TEM image the reported data for 4f–5d electronic transitions for Ce3?
(Fig. 2b) shows the nanocrystalline feature of HA and [23]. The emission spectrum of CTHA in ethanol
CePO4:Tb particles with random orientations. The spacings (kex = 274 nm) is shown in Fig. S3. The characteristic
of 2.810 and 4.071 Å between the lattice fringes match emission bands at 490, 542, 585, and 620 nm are attributed
well the distances between the (211) and (200) planes of to the transitions from 5D4–7F6, 5D4–7F5, 5D4–7F4, and
5
the hexagonal structure of HA. The spacing of 3.055 Å D4–7F4, respectively [24]. The broad peak centered around
between the lattice fringes is characteristic of the distance 350 nm is characteristic of the 5d–4f transition of Ce3?
between the (200) planes of the hexagonal structure of ions. The emission spectra are similar to the emission
CePO4. The selected-area electron diffraction patterns from spectrum observed for CePO4:Tb (Fig. S4), suggesting that
the nanoparticles (Fig. 2c) can be indexed by structural CePO4:Tb nanoparticles are quite stable and the energy
data of the CePO4:Tb hexagonal phase. The lattice image transfer is not affected after HA nanoparticles have been
(Fig. 2d) shows that the synthesized nanoparticles were doped with them. The presence of both Ce3? emission and
crystallized with some defects. Tb3? emission from these samples suggests that the energy

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J Biol Inorg Chem (2011) 16:1135–1140
Fig. 2 Transmission electron
microscopy (TEM) images of
CTHA nanoparticles. a Bright-
field TEM image showing the
typical morphology, b high-
resolution TEM images,
c selected-area electron
diffraction pattern, and d the
lattice fringes present in the
sample showing some defects
transfer between CePO4 and Tb3? is incomplete, probably
because of efficient energy transfer from the broad emitter
(i.e., Ce3?) to the narrow-line emitter (i.e., Tb3?) being
possible only between nearest neighbors in the crystal
lattice and only when there is optimal spectral overlap at
the same time [25]. In CTHA nanoparticles, the energy
levels of Tb3? (4fn) are suitable to accept energy from
the excited state of Ce3?, accompanied by the allowed
f–d transition upon its excitation by UV light [20, 26]. As a
result, the as-prepared CTHA nanoparticles exhibit a strong
green emission under a UV lamp just as shown in the insert
in Fig. S3. The total luminescence quantum yield of the
CTHA nanoparticles was 12.40%. The emission decay
curves can be fitted by multiexponential functions, and the
mean lifetime is 2.617 ls.
The luminescence switching behavior of the CTHA
nanoparticles was tested by measuring the emission
intensity of Tb3? at 542 nm after oxidation and reduction
of the Ce species. Ascorbic acid and KMnO4 were used as
redox reagents [27–29]. The emission intensity of the
nanoparticles (kex = 274 nm) decreased gradually with
1137
increasing concentration of KMnO4 until it was totally
quenched (fluorescence off, Fig. 3a), and subsequent
reduction of Ce4? by addition of aqueous ascorbic acid
solution to the oxidized solution induced an increase in the
luminescence (fluorescence on, Fig. 3b). These results
indicate that the luminescence can be switched on and off
by the alternate addition of KMnO4 and ascorbic acid to a
colloidal dispersion of CTHA nanoparticles. The redox
luminescence switching behavior of the nanoparticles is
shown in Fig. 3c. It is suggested that the redox switch
ability of CePO4:Tb nanoparticles is maintained after HA
has been doped with them, which is probably attributable
to the porous structure of HA. Thus, CePO4:Tb dispersed
into the ‘‘cavities’’ of HA that the redox reagents can easily
enter.
An essential feature of a biomaterial is its surface area,
which is an important parameter in drug storage, biocata-
lytic devices, and sensors. The surface areas of the
CePO4:Tb, HA, and CTHA nanoparticles measured from
Brunauer–Emmett–Teller adsorption isotherms are 93.4,
117.0, and 153.0 m2 g-1, respectively. Thus, we can see
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Fig. 3 The emission (kex = 274 nm) intensity of CTHA nanoparti-
cles a decreases gradually with increasing concentration of KMnO4
(0–4 lM) and b increases with increasing concentration of ascorbic
acid (0–5 lM). c Photographs of the redox luminescence switch
based on CTHA nanoparticles
that the surface area of the CTHA nanoparticle is larger
than that of pristine CePO4:Tb and HA. So increased
bovine serum albumin (BSA) adsorption was probably
produced, which is beneficial for drug delivery [11].
Therefore, the as-prepared CTHA nanoparticles were
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J Biol Inorg Chem (2011) 16:1135–1140
tested under in vitro conditions to characterize the amount
of protein adsorbed, which was measured by a batch
method according to a previously published procedure
[30]. Adsorption isotherms of BSA for the CePO4:Tb, HA,
and CTHA nanoparticles are shown in Fig. 4a. All of the
adsorption isotherms of BSA from 1 9 10-4 mol L-1 KCl
solution are of the Langmuirian type [30]. The saturated
amount of adsorbed BSA (nBSA s ) for CTHA nanoparticles
was 1.71 mg m-2. This level was considerably higher
than for pristine CePO4:Tb (0.76 mg m-2) or HA
(1.30 mg m-2), most likely owing to the increase in the
available surface area and the positive charge. Therefore,
we can deduce a possible protein adsorption model as
shown in Fig. 4b. The CePO4:Tb nanoparticles have
chrysanthemum-like architecture [20] so the available
surface area for adsorption is decreased (Fig. 4b, top).
When HA is doped with them, they are all embedded
within or dispersed in the ‘‘cavities’’ of the HA, which
increases the available surface area for the BSA
adsorption (Fig. 4b, bottom). On the other hand, Ca
atoms and Tb atoms exposed on the surface produced
rich positively charged sites to bind to acidic groups of
proteins.
Biocompatibility is a prerequisite for the use of mul-
tifunctional materials in biological or medical applica-
tions. Thus, the biocompatibility of the prepared
nanocomposite was further investigated on L929 fibro-
blast cells. The cell viability was evaluated using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay (see the electronic supplementary
material). We added solutions of CTHA to the culture
medium at different concentrations for 24, 48, and 72 h to
investigate the effects of the amount of CTHA. Figure 5
shows the results, which indicate that the cells with the
CTHA nanoparticles showed more than 90.2% L929 cell
viability over the Ca2? concentration range tested, indi-
cating that the CTHA nanoparticles exhibit no cytotoxicity
to L929 fibroblast cells. The bright-field microscopy ima-
ges of cells grown in the presence and absence of the
CTHA also confirmed biocompatibility of this hybrid
material (Fig. S5).
In summary, luminescent CTHA nanoparticles with
large surface area were successfully synthesized and
characterized. The biocompatibility was evaluated
using the MTT assay. The luminescence properties of
the as-prepared nanoparticles mean they could act as a
redox switch. In addition, the large surface area of the
CTHA nanoparticles will result in high biological
affinity and more applications in biomedical diagnos-
tics and analysis. This study is relevant for developing
J Biol Inorg Chem (2011) 16:1135–1140
Fig. 4 a Adsorption isotherms of bovine serum albumin (BSA) for CePO4:Tb, HA, and CTHA nanoparticles. b The assumed models of the
adsorption of BSA
Fig. 5 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay of L929 cells cultured for 24, 48, and 72 h in
medium containing CTHA nanoparticles
a new system that may be applied in studying
enzyme or protein activities based on luminescent HA
nanoparticles.
Acknowledgments This study was supported by the Foundation of
Key Laboratory of Nonferrous Metals Chemistry and Resources
Utilization of Gansu Province, State Key Laboratory of Applied
Organic Chemistry, and the NSFC (20171019).
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