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

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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 Na2HPO412H2O 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 1137

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 increasing concentration of KMnO4 until it was totally
because of efficient energy transfer from the broad emitter quenched (fluorescence off, Fig. 3a), and subsequent
(i.e., Ce3?) to the narrow-line emitter (i.e., Tb3?) being reduction of Ce4? by addition of aqueous ascorbic acid
possible only between nearest neighbors in the crystal solution to the oxidized solution induced an increase in the
lattice and only when there is optimal spectral overlap at luminescence (fluorescence on, Fig. 3b). These results
the same time [25]. In CTHA nanoparticles, the energy indicate that the luminescence can be switched on and off
levels of Tb3? (4fn) are suitable to accept energy from by the alternate addition of KMnO4 and ascorbic acid to a
the excited state of Ce3?, accompanied by the allowed colloidal dispersion of CTHA nanoparticles. The redox
f–d transition upon its excitation by UV light [20, 26]. As a luminescence switching behavior of the nanoparticles is
result, the as-prepared CTHA nanoparticles exhibit a strong shown in Fig. 3c. It is suggested that the redox switch
green emission under a UV lamp just as shown in the insert ability of CePO4:Tb nanoparticles is maintained after HA
in Fig. S3. The total luminescence quantum yield of the has been doped with them, which is probably attributable
CTHA nanoparticles was 12.40%. The emission decay to the porous structure of HA. Thus, CePO4:Tb dispersed
curves can be fitted by multiexponential functions, and the into the ‘‘cavities’’ of HA that the redox reagents can easily
mean lifetime is 2.617 ls. enter.
The luminescence switching behavior of the CTHA An essential feature of a biomaterial is its surface area,
nanoparticles was tested by measuring the emission which is an important parameter in drug storage, biocata-
intensity of Tb3? at 542 nm after oxidation and reduction lytic devices, and sensors. The surface areas of the
of the Ce species. Ascorbic acid and KMnO4 were used as CePO4:Tb, HA, and CTHA nanoparticles measured from
redox reagents [27–29]. The emission intensity of the Brunauer–Emmett–Teller adsorption isotherms are 93.4,
nanoparticles (kex = 274 nm) decreased gradually with 117.0, and 153.0 m2 g-1, respectively. Thus, we can see

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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
Fig. 3 The emission (kex = 274 nm) intensity of CTHA nanoparti-
cles a decreases gradually with increasing concentration of KMnO4 CTHA also confirmed biocompatibility of this hybrid
(0–4 lM) and b increases with increasing concentration of ascorbic material (Fig. S5).
acid (0–5 lM). c Photographs of the redox luminescence switch In summary, luminescent CTHA nanoparticles with
based on CTHA nanoparticles large surface area were successfully synthesized and
characterized. The biocompatibility was evaluated
using the MTT assay. The luminescence properties of
that the surface area of the CTHA nanoparticle is larger the as-prepared nanoparticles mean they could act as a
than that of pristine CePO4:Tb and HA. So increased redox switch. In addition, the large surface area of the
bovine serum albumin (BSA) adsorption was probably CTHA nanoparticles will result in high biological
produced, which is beneficial for drug delivery [11]. affinity and more applications in biomedical diagnos-
Therefore, the as-prepared CTHA nanoparticles were tics and analysis. This study is relevant for developing

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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

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