Surface Modification of Hydroxyapatite Nanocrystallite by a Small

Jul 22, 2008 - As the main inorganic component of biological bone and tooth enamel, hydroxyapatite (HAP) is highly biocompatible and bioactive. Our re...
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J. Phys. Chem. C 2008, 112, 12219–12224

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Surface Modification of Hydroxyapatite Nanocrystallite by a Small Amount of Terbium Provides a Biocompatible Fluorescent Probe Ling Li,†,‡ Yukan Liu,§ Jinhui Tao,† Ming Zhang,§ Haihua Pan,† Xurong Xu,† and Ruikang Tang*,† Department of Chemistry and Center for Biomaterials and Biopathways, Zhejiang UniVersity, Hangzhou, 310027, China, School of Chemical and Material Engineering, Jiangnan UniVersity, Wuxi, 214122, China, and Department of Biology, Zhejiang UniVersity, Hangzhou, 310058, China ReceiVed: March 27, 2008; ReVised Manuscript ReceiVed: May 17, 2008

As the main inorganic component of biological bone and tooth enamel, hydroxyapatite (HAP) is highly biocompatible and bioactive. Our recent work has shown that 20 nm HAP can be readily internalized by living cells. However, HAP nanoparticles are not luminescent so that they are difficult to measure in living cells during in vitro experiments. A novel inorganic biological probe was suggested by doping 20 nm HAP with terbium. The calcium ions on the HAP particle surface could be partially replaced by Tb, which resulted in the 20 nm Tb-HAP particles. The Tb-doped HAP became luminescent and could be observed under a visible excitation of 488 nm. Compared with the usual probes, the modified HAP nanoparticles were more photostable and were almost nontoxic. It was important that the HAP characteristics remained after the surface modification with a small amount of Tb. After incubating with rabbit bone marrow mesenchymal stem cells (MSCs) in culture, the luminescence of the internalized HAP in the living cells was clearly observed under a fluorescent microscope. Transmission electron microscopy analysis also confirmed the uptake of the particles by MSCs. It was demonstrated that the modified HAP was a stable biological probe for cellular research. We suggested that the Tb-doped HAP should have a great potential to trace the evolvement of nanoparticles, which is a key topic in nanobiotechnology. 1. Introduction In studies of biological processes in living cells, it is important to investigate any change in situ at real time.1–3 Biological probes are utilized as the most effective tools for biological staining and diagnostics.4–6 Thus, the development of novel biological probes has recently received intensive attention. Usually, two kinds of probes have been widely studied. One is fluorescent organic molecules7–11 such as rhodamine, fluorescein, and acridine orange, etc. These molecules have cyclic structures so that their luminescence can be detected readily by using fluorescent microscopy. A wide variety of fluorescent organic molecules are currently used as different biological probes. But these organic probes have an obvious drawback of photobleaching, which is a phenomenon where the fluorescent intensity degrades quickly with time.12,13 Recently, the rapid growth of nanotechnology highlights that a kind of semiconductor nanoparticle, quantum dots such as CdSe, CdS, ZnS, InP, and InAs, etc., can be a novel probe.14 Compared with organic ones, these inorganic nanoparticles can be more stable in the biological milieu and can also provide steady fluorescent emission to reduce photobleaching. However, a serious problem of biological security or toxic nanosemiconductors is critically pronounced, which significantly prevents their application. The stable and nontoxic inorganic biological probes are explored. An alternative class of nanomaterial that can serve as a substitute for quantum dot is nanophosphor.15–17 Hydroxya* Corresponding author. E-mail: [email protected]. Phone: (86) 571-8795 3736. Fax: (86) 571-8795 3736. † Department of Chemistry, Zhejiang University. ‡ Jiangnan University. § Department of Biology, Zhejiang University.

patite (HAP, Ca10(PO4)6(OH)2) is considered a good candidate since it is the main inorganic component of biological hard tissues such as bone and enamel. This compound has been widely used as an implant biomedical material in orthopedic and dental treatments.18,19 Besides, it is suggested that 20-40 nm HAP may be the basic building blocks in biological constructions. The nano-HAP particles can be kinetically stable in biological fluids, which can be understood by a nanodissolution model.20,21 Bauer et al. have reported the internalization of hydroxyapatite nanoparticles in liver cancer cells.22 Our recent work also shows that 20 nm HAP can be readily internalized by living cells and we emphasize the importance of size control of HAP particles during the uptake.23 However, as a kind of biological probe, the luminescence of the 20 nm-sized nanophosphor should be observed when excited by visible domains since most cells are greatly affected under ultravisible (UV) lights. Thereby, the efforts are intensely devoted to tailoring phosphors that can be excited by visible light. Unfortunately, pure HAP cannot provide strong fluorescence under visible lights. It is well-known that Ca2+ does not have a useful luminescent property. When the native metal ion is devoid of some functional properties, it is often possible to substitute an ion with the specific characteristics.24 The ionic radii of the trivalent ions of the 14 stable elements from La3+ through Lu3+ have very close ionic radii ranging from slightly greater to slightly smaller than that of Ca2+.25 Previous studies have demonstrated some examples of the replacement of Ca2+ by the trivalent lanthanide ions.26,27 Thus, it is also suggested that HAP can display fluorescence under visible excitation if it is doped with lanthanide ions, in which Eu3+ and Tb3+ are the most strongly emitting elements.28 Europium-doped apatite has been studied by Doat et al.29 and colloidal Ln3+-doped calcium

10.1021/jp8026463 CCC: $40.75  2008 American Chemical Society Published on Web 07/22/2008

12220 J. Phys. Chem. C, Vol. 112, No. 32, 2008 phosphate has been reported by Lebugle et al.30 However, the particle sizes or the fluorescent emission in the cells of these materials are not well controlled, which limit their applications. In the study of Wang et al., hydroxyapatite nanoparticles were surface functionalized by depositing Y2O3:Eu nanoparticles.31 However, the resulting HAP nanoparticles agglomerate to form large clusters after an annealing treatment at high temperature, which may be an obstacle to the development of biological probes. Here, we suggest a convenient strategy to obtain nanoHAP whose luminescence can be excited by visible lights. The dimensions of the modified HAP are well controlled as their size distributions, 20 ( 5 nm, are relatively homogeneous. Different from the other methods, only the surface calcium ions on the HAP nanoparticles are partially replaced by a small amount of Tb3+. However, the presence of an extremely low concentration of the doped Tb3+ on the HAP surfaces can greatly improve the luminescence while the main physicochemical properties and bioactivity of HAP nanocrystallites are maintained. It is also confirmed experimentally that the Tb-HAP nanoparticles can provide a steady luminescence. Furthermore, these fluorescent particles can be internalized by living cells. Although the total content of Tb3+ in the particles is extremely low, it is still mentioned that terbium is relatively nontoxic since its LD50PO (lethal dose 50 per oral route, calculated for a substance orally adsorbed causing the death of 50% of an animal population) is more than 5000 mg/kg for terbium nitrate in rat.32 2. Experimental Section 2.1. Synthesis of Terbium-Doped Hydroxyapatite Nanoparticles. Terbium-doped hydroxyapatite nanoparticles were synthesized at room temperature. CaCl2 (6 mL 0.5 M) was added dropwisely into 294 mL of aqueous solution with 1.8 mmol of Na2HPO4 and 0.27 mmol of cetyltrimethylammonium bromide (CTAB), which was used as the size-controller.23,33 The final concentrations of CaCl2, Na2HPO4, and CTAB in the mixed solution were 10, 6, and 0.90 mM, respectively. The HAP nanocrystallites were precipitated in the mixed solution. During the reaction, the solution pH was maintained at 9.5 by using 0.1 M ammonia. After 1 h of reaction, 12.2 mL of a 5.0 mM Tb(NO3)3 solution was added to the HAP slurry. To reduce the hydrolysis of Tb, the solution pH was adjusted to 7.0 by using 1 M HCl prior to the addition of Tb. After the Tb addition, the solution pH was maintained at 7.0. The atomic ratio of Tb:(Ca +Tb) was 2:100 and the reaction was continued for another 23 h. It was suggested that the surface calcium ions on the HAP nanocrystallites can be partially replaced by Tb so that the modified HAP could be obtained. At the end of the experiment, the solids were collected by centrifugation (12 000 rpm) and filtration (Nucleopore N003 filter membrane) and then were washed thoroughly by using ethanol and triple-distilled water. The product was dried overnight at the vacuum condition at 30 °C. 2.2. Characterization. The solids were characterized by an X-ray diffraction (XRD, D/max-2550pc, Rigaku, Japan) with monochromatized Cu KR radiation (λ ) 0.15405 nm). The infrared (IR) spectra with a scanning range of 400-4000 cm-1 were obtained with a NEXUS 470 FT-IR spectrometer (Nicolet, U.S.A.). The particles were also examined by a transmission electron microscope (TEM, JEM200CX, JEOL, Japan) and a scanning electron microscope (SEM, S-4800, Hitachi, Japan). The excitation and emission spectra of the modified HAP particles were determined by a RF-5301pc spectrofluorometer (Shimadzu, Japan). 2.3. Biological Examination. The internalization of Tb-HAP by bone marrow mesenchymal stem cells (MSCs) was exam-

Li et al. ined. MSCs were obtained from rabbit bone marrow as described previously.23 After being washed twice with phosphate-buffered saline (PBS), the mononuclear cells were seeded at 3.0 × 105 cm-2 in Dulbecco’s Modified Eagle’s Medium (1 g/L glucose) containing 15% fetal bovine serum (Hyclone, Logan, UT) and were cultured in a 5% CO2 incubator at 37 °C. After 3 days, half of the culture medium was replaced and subsequently the medium was changed twice weekly. The MSCs were initially grown to 80% confluence in culture flasks and resuspended at 1.0 × 104 cm-2 after harvesting with 0.02% EDTA and 0.25% trypsin. Prior to in vitro experiments, the modified HAP nanoparticles were sterilized by steam autoclaving at 120 °C for 30 min. The sterilization did not change the characterization (size and morphology) of the Tb-HAP nanoparticles. The sterilized Tb-HAP nanoparticles were placed into 24 well plates with the cell suspension already in them. At the end of 7 days of incubation, the MSCs were washed with PBS, and then were detached with 0.25% trypsin and 0.05% EDTA for 5 min, prefixed with 2.5% glutaraldehyde and 2% paraformaldehyde solution, postfixed with 1% osmium tetroxide, dehydrated with a series of alcohols, and infiltrated with resin. The resin sample block was trimmed, thin-sectioned to a thickness of 70 nm, and collected on Formvar-coated copper grids. Before examination under the TEM, these grids were stained with uranyl acetate and lead citrate. The internalization of the particles was observed by a biological transmission electron microscope (TECNAL10, Philip, Holland), operated at 100 KV. The luminescence of Tb-HAP particles in MSCs was detected by a confocal laser scanning microscope (LSM 510 invert, Carl Zeiss, Jena, Germany). The parameters used for confocal laser scanning microscopy were the following: excitation provided by an argon laser adjusted to excitation of 488 nm, objective lens 63, and pinhole 30, and filter 50 nm. Confocal laser scanning microscopy was employed for its ability to make virtual cell sections and its high sensitivity. 3. Results and Discussions 3.1. Characterization of Terbium-Doped Nanoparticles. Our previous paper had demonstrated that CTAB could control the size distribution of the precipitated HAP.23 At the concentration of CTAB of 9.0 × 10-4 M, the resulting HAP particles had the relatively homogeneous size of 20 nm. There was no significant difference between the HAP particles with and without the Tb treatment. Under the TEM and SEM examinations, the nanoparticles were granular and their sizes were around 20 nm (Figure 1a). The result of the dynamic light scattering also confirmed that the size distribution of these particles was relatively homogeneous, which was 20 ( 5 nm. And these characters were exactly same as the previously prepared pure HAP. The XRD study (Figure 2a) also confirmed that the resulting Tb-HAP had the typical pattern of the pure hydroxyapatite nanoparticles. All diffraction peaks could be assigned to the standard one (JCPDS09-0432). The formation of the HAP structure was also confirmed by the IR spectrum (Figure 2b). The bands at 560, 600, and 1000-1100 cm-1 were the characteristic peaks of PO43- groups in an apatitic environment. The peaks of CO32- (1419 and 871 cm-1) implied that some carbonate ions incorporated into the particles. The incorporation of carbonate was a common phenomenon during the formation of biological apatites.34,35 In our experiment, the content of CO2 in air was not excluded during the preparation, which led to the formation of the carbonate-incorporated nanoHAP too. The peaks at 3426 and 1597 cm-1 corresponded to the remaining water. No signal of CTAB was detected,

Tb-Doped HAP Nanoparticles

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Figure 1. (a) TEM image of the HAP nanoparticles whose surfaces are partially Tb doped. Insert: The magnified image. (b) Dynamic light scattering result of the Tb-HAP nanoparticles.

indicating that the residual organic additives had been almost eliminated by the washing processes. Terbium(III) ion could be used as a luminescent probe in the bimolecular system.36 And Ca ions on the HAP surface could be replaced by the other metal cations with similar ionic radii, especially lanthanide ions.28 Since the added amount of terbium was relatively low and the adsorption of Tb onto the HAP only occurred on the surfaces, the presence and content of Tb on the HAP was hardly detected by electron energy dispersive X-ray spectroscopy (EDX). This phenomenon might be understood by the extremely low amount of the doped Tb, which was beyond the sensitivity of EDX. However, the surface modification could be detected by a fast dissolution of the particle surfaces. Ten milligrams of Tb-doped HAP solids was washed by 5 mL of 1.0 mM HCl for 3 s. The solution was collected and was then concentrated to 0.5 mL for an analysis of inductively coupled plasma (ICP) spectrometry. Terbium ions were found in the solution (its concentrations were 3-4 ppm) and the experimental atomic ratios of Tb:Ca were in a range of 1:247 to 1:135 (the concentration of Ca was 400-1000 ppm), which agreed with the suggested mode of a low amount of terbium on the surface. The low Tb:Ca ratio in the ICP examination could be explained by some bulk calcium ions of the Tb-HAP particles also being dissolved during the fast washing. It was important that the formation of Tb-doped HAP could be confirmed by the luminescence study. The luminescence spectra of pure HAP showed that they had no emission under the excitation at 272 nm. The Tb-doped HAP changed the spectra greatly although the amount of Tb was extremely low (Figures 3–5). The emission spectrum with the excitation of 272 nm (Figure 4) showed the luminescence at the wavelengths of 490, 544, 585, and 622 nm, which could be ascribed

Figure 2. (a) XRD of the pure nano-HAP and Tb-doped HAP. (b) IR spectrum of the terbium-doped HAP nanoparticles.

to 5D4-7F6, 5D4-7F5, 5D--7F4, and 5D4-7F3 transitions of Tb, respectively. These emission effects could not be observed in the pure HAP crystallites due to the absence of the featured Tb element. Thus, the presence of Tb in the HAP nanoparticles was confirmed. The maximum intensity was achieved at 544 nm, which corresponded to the 5D4-7F5 transitions of Tb. This result was in good agreement with the luminescence of Tb. Figure 4 was the excitation spectrum achieved by monitoring the luminescence at 544 nm. Although the highest intensity was obtained at an excitation of 272 nm, another excitation peak was recorded at 488 nm. Although the emission intensity was not as strong as that by the excitation at 272 nm, the luminescent peaks of 544 and 585 nm were sufficient for the biological application. It was important to note that the excitation light at 272 nm was in the domain of ultravisible and this UV light could destroy the activity of living cells in nature. However, the light at 488 nm wavelength is located in the blue region and could be acceptable for the living cells. Therefore, we suggested that the existence of the 488 nm as the excitation wavelength makes the observation of the terbium-doped hydroxyapatite particles in the living cells possible. It was mentioned that the stability of the fluorescent emission was an important parameter in the application of biological probes. The steady luminescence of Tb-HAP was demonstrated in Figure 6. Under persistent 488 nm excitation light, the fluorescent intensity of the nanoparticles degraded less than 10%

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Figure 3. Emission spectrum of terbium-doped HAP particles under excitation of 272 nm.

Figure 4. Excitation spectrum of Tb-doped HAP with the emission wavelength at 544 nm. Insert: The details in the range of 300-500 nm.

Figure 5. Emission spectrum of terbium-doped hydroxyapatite particles under excitation of 488 nm.

even within 1 week. This result implied that the Tb-HAP probe could be applied effectively for long investigations. Additionally, it should also be mentioned that both calcium and terbium can provide a similar coordination environment and hardness, ionic radii, and geometric characteristics. The terbium cation is so similar to the calcium cation that it can incorporate into the HAP particles readily.37 Such an incorporation also could be explained by the sector zoning model in minerals.38,39 This model suggested that the charge-compensating cation substitutions on the surface are not assumed to be necessary since the charge balance could be attained by adsorption (coordination) of additional anions, like OH-, from the surroundings.

Li et al.

Figure 6. The relative intensity of the green emission (544 nm) of the nanoparticles during the extended experimental time (under the persistent excitation light of 488 nm). The emission intensity, I, was measured at different times and the average value in the first 1 h was used as the initial emission intensity, I0.

3.2. Behavior of Tb-HAP in Cells. When the MSCs were cultured together with the Tb-modified HAP particles for 7 days, the uptakes of the nanoparticles were observed (Figure 7). These phenomena were exactly the same as those of pure HAP nanoparticles.23 Besides, the promotion effect of TbHAP on the cell proliferation was also detected. This confirmed that the featured biological functions of 20 nm HAP remained. The aggregates of the internalized HAP nanoparticles were detected in the cells (arrows, Figure 7a). Our previous study revealed that the uptake amount of the large-sized HAP was extremely low, only the nano-HAP could enter the cell readily.23 After the incubation, the size and morphology of the Tb-HAP were consistent with the original one (Figure 7b). The results of energy dispersive X-ray spectroscopy (EDX) confirmed that these nanoparticles in the cells were hydroxyapatite with a Ca:P ratio of 1.63 ( 0.06. The green fluorescent lights of the Tb-labeled HAP could be clearly detected in the cells by the confocal microscopy (Figure 8a). Under the excitation wavelength of 488 nm, the green-lighted particles in the image were just the internalized Tb-HAP particles. It could be concluded from the image that the HAP particles became detectable in situ in the cells after the modification. The result of confocal microscopy also indicated that a large number of nano-HAP particles were aggregated in the cell, which agreed with the TEM observation. It was important that the luminescence of these internalized HAP particles could be observed under confocal microscopy even after 24 h (Figure 8b). This phenomenon implied that there was no significant photobleaching in the in vitro experiment and these modified nanoparticles had the steady fluorescent characteristics in the living cells. In contrast, no fluorescence could be observed in the MSCs if the pure HAP nanoparticles were used (Figure 9). Our previous result had demonstrated that the conventional HAP could not be internalized by MSCs due to the large particle size.23 As a suitable biological probe, both the size control of HAP and the surface modification were essential. Furthermore, with respect to the value of LD50PO of the terbium,32 the terbium content used in this experiment was negligible and could be considered almost nontoxic. In the previous studies, we also showed that the nano-HAP could influence the proliferation and differentiation of MSCs cells.23,40 However, the mechanism was poorly understood and the Tb-labeled HAP would provide a useful tool to

Tb-Doped HAP Nanoparticles

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Figure 7. TEM images of the internalized Tb-HAP particles: (a) aggregates of a large number of nanoparticles in the cell (arrows) and (b) individual nanoparticles in the aggregates.

Figure 9. No fluorescent light in the cell can be detected when the 20 nm pure HAP particles are cocultured with MSCs.

the physicochemical characteristics and biological functions of nano-HAP were almost retained. However, a new property of fluorescence was conferred on the inorganic nanomaterials. It was important that the green-emission of the particles could be excited by a visible light beam at 488 nm. This feature could be used to trace the HAP in situ and at real time during the biological application. Besides, the ready internalization of the particles and its steady fluorescence suggested that the Tb-HAP is an ideal nanoinorganic probe for living cells with excellent biocompatibility. Acknowledgment. This work was supported by the National Natural Science Foundation of China (20571064, 20672145, and 20701032), the 973 Research Program of China (2007CB516806), the Zhejiang Provincial Natural Science Foundation (R407087), and the Cheung Kong Scholars Program (RT). Figure 8. (a) Green emission of the internalized Tb-HAP particles in the cells under confocal microscopy. (b) Emission of the particles remains good in the cells even after 24 h.

investigate the metabolism of the nanoparticle in the cells, which is a key issue in biotechnology. 4. Conclusions The surfaces of 20 nm-sized HAP nanoparticles were partially doped with a small amount of Tb. After such a modification,

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