Biocompatible Fluorescent Hydroxyapatite: Synthesis and Live Cell

Aug 24, 2011 - Various fluorescent nanoparticles have been applied in imaging of tissues or intracellular structures. Searching for biocompatible nano...
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Biocompatible Fluorescent Hydroxyapatite: Synthesis and Live Cell Imaging Applications Hongyan Liu,† Fengjuan Chen,† Pinxian Xi,† Bin Chen,‡ Liang Huang,† Ju Cheng,§ Changwei Shao,|| Jun Wang,|| Decheng Bai,§,* and Zhengzhi Zeng†,* †

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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, PR China ‡ School of Dentistry, Lanzhou University, Lanzhou 730000, PR China § School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, PR China State Key Laboratory of Advanced Ceramic Fibers & Composites, College of Aerospace and Materials Engineering, National University of Defense Technology, Changsha 410073, PR China

bS Supporting Information ABSTRACT: Various fluorescent nanoparticles have been applied in imaging of tissues or intracellular structures. Searching for biocompatible nanoparticles that can be observed under visible light is of fundamental importance for the applications in living cells. In this study, a simple, template-based route for the synthesis of fluorescent hydroxyapatite (HA) nanoparticles has been developed. Well-dispersed HA nanoparticles prepared by using SiO2 as templates (THA) were surface-functionalized with fluorescein isothiocyanate (FITC), resulting in bright and stable fluorescent particles (THA-FITC). These fluorescent nanoparticles were demonstrated to be internalized into Hela cells and have no apparent cytotoxic effects on fibroblast cells, which confirmed the biocompatibility and cell labeling capability of the nanoparticles. Due to the large surface areas, porous nature, and visible light excited luminescence characters, THA-FITC nanoparticles will have applications in fields such as cell labeling, whole animal imaging and therapy, and drug delivery and release.

’ INTRODUCTION Imaging of tissues or intracellular structures is an important issue in medicine.1 4 Radioactive molecular imaging,5,6 magnetic resonance imaging (MRI),7,8 and optical imaging9 have been recognized as three powerful techniques in this field. For radioactive molecular imaging, the radiation dose is not negligible, which can limit the reproducible imaging of the same animal. MRI does not induce harmful effects and is a noninvasive imaging technique. However, low sensitivity, high cost, time-consuming scan, and processing are limiting factors for its use.10 Otherwise, low cost, easy manipulation, and high sensitivity make optical imaging the most widely used technique. Multiple probes with different spectral characteristics could potentially be used for multichannel imaging.11 Optical imaging also allows for a relatively low-cost study of reporter gene expression in small animal models. In optical imaging, two kinds of probes have been widely studied. One is fluorescent organic molecules, and the other is fluorescent nanoparticles. For traditional fluorescent dyes, low fluorescence intensity and low photostability severely limit the use of them as high-sensitivity detectors and real-time monitors.12,13 Compared with organic dyes, fluorescent nanoparticles are more stable in the biological, so they turn out to be more useful than r 2011 American Chemical Society

fluorescent dyes for labeling cells. Recently, new fluorescent nanoparticles, including quantum dots, fluorescent latex particles, and fluorescent silica particles, have been developed and have shown great utility. Quantum dots such as CdSe, CdS, ZnS, InP, and InAs, etc., have been applied as novel cell labeling agents.14 However, poor solubility, poor toxicity, and disposal issues make the use of quantum dots impractical in some cases.15,16 In addition, the “blinking” characteristic is a limiting factor for raster scanning systems such as confocal microscopy and flow cytometry. Fluorescent latex polymer or nanoparticles have also been employed in some biological applications.17,18 However, for their agglutination, large size, hydrophobicity, and dye leakage, these latex nanoparticles are not suitable for bioapplication.19 Otherwise, silica nanoparticles with encapsulating or doping dye molecules have higher brightness and better photostability than the constituent fluorophore and have been the focus of recent studies.20 22 However, poor biocompatibility limited the usefulness of these nanoparticles for in vivo imaging. It has been found that the interaction of silica particles with cells causes Received: July 18, 2011 Revised: August 23, 2011 Published: August 24, 2011 18538

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The Journal of Physical Chemistry C inflammation that may be involved in the initiation of various diseases including systemic sclerosis, rheumatoid arthritis, lupus, and chronic renal disease.23,24 Furthermore, silica and polymeric aggregation in physiological solutions causes an increase in the particle size that can result in obstruct blood flow and blood capillary blockage.25,26 Polymeric nanoparticles may be suboptimal carriers of organic cargo since they often suffer from swelling and dye leakage.25 Biocompatibility is a common characteristic of materials that should be considered when used in biomedicine. As we known, nanoparticles interacting with proteins, membranes, cells, DNA, and organelles establish a series of nanoparticle/protein interfaces. These interactions lead to the formation of protein coronas, particle wrapping, intracellular uptake, and biocatalytic processes that could have biocompatible or bioadverse outcomes.27 On this basis, we should use biocompatible nanoparticles to meet the need for safety assessment or tailor the physicochemical properties of nanoparticles to predetermine the nature and conformation of the proteins that adsorb, to acquire a specific, desired biological identity via the protein (biomolecule) corona. Also, in biocompatibility assessment, nanoparticles with saturated surfaces should be employed.28 In recent years, hydroxyapatite (Ca10(PO4)6(OH)2, HA) nanoparticles have gained increasing interest in medicine because of their high biocompatibility and good biodegradability which is due to the fact that calcium phosphate is the inorganic mineral of human bone and teeth.29 31 It has been found that the HA particle surface is porous, so it can be used for drug storage.32,33 Because of their excellent biodegradable properties, HA nanoparticles may serve as an ideal candidate for both biological image and drug delivery. Previous studies have suggested that calcium phosphate nanoparticles can be used as fluorescing probes after doping with lanthanides.34 36 However, the fluorescence of lanthanide ions requires some degree of crystallinity, i.e., it is very weak in amorphous nanoparticles, and a compromise must be found between small particle size and sufficient crystallinity. Furthermore, lanthanide-doped HA nanoparticles could not be observed under a visible excitation, which limits their biomedical applications in living cells to investigate any change in situ at real time. In addition, minimization of particle size to the range for intracellular delivery is critical in the biological usage of HA. Therefore, searching for visible light excited HA nanoparticles with small size is of fundamental importance. Fluorescein isothiocyanate (FITC), which can be excited at 488 nm, has been extensively utilized in the design of fluorescent sensors,37 activity of enzymes,38 as well as cell cycle assay.39 Herein, we synthesized THA nanoparticles with sizes around 50 nm by using SiO2 as templates. The obtained nanoparticles were further surface functionalized with FITC, resulting in green emitting THA-FITC nanoparticles. From the demonstrated biocompatibility and cell labeling capability of these nanoparticles, it is suggested that these fluorescent HA nanoparticles with large internal space will serve as a carrier for further studies in biological applications.

’ EXPERIMENTAL SECTION Chemicals. The reagents and solvents were purchased commercially and are of analytical grade. Tetraethyl orthosilicate (TEOS), (3-aminopropyl) triethoxysilane (APTES), Na2HPO4 3 12H2O, and CaCl2 were obtained from Guangfu Chemical Co. (Tianjin, China). Fluorescein isothiocyanate (FITC) was purchased from Alfa Aesar. Kaiser test reagents were prepared according to the

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literature.40 Deionized (DI) water was used throughout the experiments. Synthesis of THA Nanoparticles. SiO2 particles were performed in accordance with the literature procedure (see Supporting Information).41 Typically, 60 mg of dried SiO2 templates was dispersed in 20 mL of DI water with ultrasonication. Na2HPO4 3 12H2O (0.6 mmol, 80 mL) was added, and the mixture was stirred for 1 h. Then CaCl2 (20 mL, 0.05 M) was added dropwise into this mixture. During the reaction, the solution pH was maintained at 9.5 by using 0.1 M ammonia. The HA nanocrystallites were precipitated in the mixed solution. The reaction was continued for 24 h. At the end of the experiment, the solids were collected by centrifugation (8000 rpm) and then were washed thoroughly by using ethanol and DI water. The product (SiO2 HA) was dried at the vacuum condition at room temperature and collected for identification and characterization. To remove the silica cores, the SiO2 HA particles were dispersed in 5 M NaOH for 2 days at room temperature (20 °C) in a 50 mL polypropylene centrifuge tube, with a fresh solution of NaOH (10 mL) exchanged after 1 day. The THA nanoparticles were isolated by successive centrifugation (3200 RCF, 10 min), redispersed in water and then ethanol, and dried in air. HA nanoparticles were synthesized in the same way without using SiO2 templates. Synthesis of THA-FITC Nanoparticles. THA nanoparticles were first functionalized by APTES. An amount of 200 mg of THA nanoparticles in 50 mL of toluene mixing with 1 mL of APTES was stirred and refluxed for 8 h. The solid was then separated by centrifugation, washed with ethanol 5 times, and dried at room temperature, resulting in THA-APTES. The amino terminal groups on the surface or in the “channel” of THA then reacted with FITC. THA-APTES nanoparticles (100 mg) were added to 30 mL of ethanol solution of FITC, and this mixture was stirred for 2 days. The as-synthesized products were washed and redispersed with deionized water and ethanol several times. THA-FITC nanoparticles were obtained by centrifuge and dried overnight, followed by dialysis for 5 days to remove the free dyes. The FITC adsorption HA fluorescent nanoparticles were prepared in the same way without functionalization by APTES. Cell Culture. The Hela cell line and L929 mouse fibroblast cell line were provided by the Institute of Biochemistry and Cell Biology (China). Cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (Fetal Bovine Serum) in an atmosphere of 5% CO2 and 95% air at 37 °C. Assessment of Biocompatibility. The biocompatibility was determined in L929 mouse fibroblast cell lines. The cell viability was evaluated using the modified MTT assay.42,43 Basically, cells were plated at a density of 1  105 in 96-well plates 24 h prior to exposure to the materials. Different concentrations of THAFITC nanoparticles with saturated surfaces (via interactions with DMEM for 24 h before use) were added to the wells, and the cells were further incubated for for 24, 48, and 72 h. After treatment, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg mL 1 in PBS) was added into each well. After 4 h of incubation, culture supernatants were aspirated, and purple insoluble MTT product was redissolved in 150 μL of dimethyl sulfoxide (DMSO) in 10 min. The concentration of the reduced MTT in each well was determined spectrophotometrically by subtraction of the absorbance reading at 630 nm from that measured at 570 nm using a microplate reader. All MTT experiments were performed five times, and the maximum and minimum were deleted. The results were expressed as the mean ( 18539

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The Journal of Physical Chemistry C standard deviation. Cell viabilities were presented as the percentage of the absorbance of material-treated cells to the absorbance of control cells. Ca2+ concentration was measured (after etching the THA nanoparticles and releasing all the Ca2+ ions) by ICP analysis. Flow Cytometry. Flow cytometry analysis was performed on FACS Calibur flow cytometers (Becton Dickinson, San Jose, CA) with 488 nm excitation lasers. THA-FITC nanoparticles were analyzed, and data were obtained without compensation. For cell analyses, HeLa cells after being exposed with THA-FITC particles at several concentrations were dissociated with nonenzymatic cell dissociation solution (Sigma) for 10 min and then analyzed. Data analysis was carried out using CellQuest software. In Vitro Confocal Fluorescence Microscopy Imaging. HeLa cells were grown on coverslips for 24 h, and then THA-FITC nanoparticles were added. After incubation at 37 °C and in 5% CO2 atmosphere for 2 h, cells were washed with PBS three times before analysis to remove free THA-FITC nanoparticles. Multilabeling laser scanning confocal microscope studies were also performed. After incubation of THA-FITC nanoparticles with HeLa cells for 2 h at 37 °C (5% CO2), the cytoplasm of cells was stained red by rhodamine 6G. The confocal microscopy optical setup was in multichannel mode. All confocal images were collected with a Zeiss Leica inverted epifluorescence/reflectance laser scanning confocal microscope. Characterization. XRD measurements were performed on X-ray diffraction (D/max-2400pc, Rigaku, Japan) with Cu Kα radiation (λ = 1.54178 Å), with the operation voltage and current Scheme 1. Template-Based Route for Synthesis of Fluorescent Hydroxyapatite (HA) Nanoparticles

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at 40 kV and 60 mA, respectively. The 2θ range was from 10 to 90° in steps of 0.02°. The scanning electron microscope (SEM) microscopy was obtained on a Hitachi S-4800 field emission scanning electron microscope. The transmission electron microscopy (TEM) and the energy-dispersive spectrometry (EDS) were obtained on a Tecnai-G2-F30 (FEI) transmission electron microscope at an acceleration voltage of 300 kV. Samples were prepared by placing a drop of a dilute alcohol dispersion of the products on the surface of a copper grid. Samples for fluorescence measurements were prepared by ultrasonic dispersion of the powders in ethanol to give transparent colloidal solutions. The solutions were immediately employed for the recording of the spectra. The excitation and emission spectra were recorded on a Hitachi RF-4500 spectrofluorophotometer at room temperature. Inductively Coupled Plasma Spectroscopy (ICP) analysis was done on an IRIS ER/S (Thermo Electron, American) meter. The specific surface area of the samples was determined by Brunauer Emmett Teller (BET) nitrogen gas adsorption using a Chemisorb2750 surface area and pore size analyzer. Approximately 100 mg samples were placed into the sample cell, weighed, and outgassed under vacuum at a temperature of 150 °C for 5 h prior to sample measurement. Duplicate measurements were taken, and the mean specific surface area was calculated. All pH measurements were made with a pH-10C digital pH meter.

’ RESULTS AND DISCUSSION Well-dispersed THA nanoparticles were synthesized by the silica-template approach. First, silica spheres were synthesized via a solution method.41 Subsequently, the Si OH groups on the surface of SiO2 induced the nucleation of hydroxyapatite when Na2HPO4 3 12H2O and CaCl2 were added. To obtain the THA nanoparticles, the composites were suspended in NaOH (5 M) for 2 days, and SiO2 cores were chemically etched yielding well-dispersed THA nanoparticles, as shown in Scheme 1. For comparison, HA nanoparticles were also synthesized without using SiO2 templates. Time-dependent colloidal stability of HA and THA in PBS was studied. It can be seen that HA becomes

Figure 1. SEM images of HA (a), SiO2 HA (b), and THA (c) nanoparticles. TEM images of HA (d) and THA (e) nanoparticles. SAED patterns from the samples of THA (f). 18540

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Figure 3. Excitation (red) and emission (green) spectra of a dilute colloidal dispersion of THA-FITC nanoparticles in ethanol. The inset is the image of THA-FITC nanoparticles under daylight and UV lamp.

Figure 2. XRD patterns (a) and FTIR (b) spectra for THA nanoparticles.

aggregated after 3 min, while THA colloidal solutions exhibit good dispersion stability within the times used in this study (Figure S1, Supporting Information). The morphology and microstructure of the HA and THA nanoparticles were investigated by SEM and TEM. Figure1a shows the SEM images of HA nanoparticles, where serious particle aggregation was present; this is a great handicap for their biological applications. Figure 1b and Figure 1c show the SEM images of HA with SiO2 templates (SiO2 HA) before and after NaOH dissolution. HA nanoparticles were coated on the SiO2 templates well-proportioned, which can be also seen from the TEM image of SiO2 HA (Figure S2, Supporting Information). After SiO2 cores were etched, porous THA nanoparticles were obtained. Figure 1d and Figure 1e show the TEM image of HA and THA nanoparticles. It can be clearly seen that the resulting THA structures are needlelike nanocrystals and 50 100 nm in length. No aggregation of THA nanoparticles was observed by electron microscopy. This can be ascribed to the smaller size of THA. HA particles seem to be distributed in much larger aggregates. This indicated that THA is more suitable for biological applications. The selected area electron diffraction (SAED) patterns from the samples (Figure 1f) confirmed the THA nanocrystals to be multicrystalline. The phase structure and purity of the obtained products are investigated by powder XRD and FTIR. Figure 2a displays the XRD patterns of THA nanoparticles, and all the diffraction peaks can be well assigned to a hexagonal phase known from the bulk HA hexagonal phase (JCPDS card, no. 09-0432). The broad XRD peaks are attributed to the very small particle size, which is also demonstrated by TEM observation. Typically, the FTIR spectrum of the as-synthesized sample (Figure 2b) shows absorption bands at 3435, 1636, 1465, 1415, 1036, 860, 603, and 560 cm 1. The broad band at 3435 cm 1 is due to the presence of absorbed water.44 The band at 1636 cm 1 is assigned

to the ν2 bending mode of the H2O molecules.45 The bands at 1036, 603, and 560 cm 1 are assigned to vibrations of the phosphate group, PO43 . The bands at 860, 1415, and 1465 cm 1 are attributed to vibration modes of CO32 . This is understandable, as carbonate ions are a common impurity in HA.46 The surface area of THA nanoparticles measured from Brunauer Emmett Teller (BET) is 99.3 m2 g 1, which is larger than that of HA prepared without the template (51.4 m2 g 1). So the increased bovine serum albumin (BSA) adsorption was probably produced, which is beneficial for drug delivery.47 The preparation of THA-FITC nanoparticles takes two steps (Scheme 1). Firstly, HA was functionalized by APTES. The presence or absence of primary amine groups on the surface of modified materials can be confirmed by using a Kaiser test according to the literature.40,48 Samples untreated or treated with APTES were suspended in ethanol. After two drops of each Kaiser test reagent (phenol, potassium cyanide, and ninhydrin) were added, the suspensions were heated in a boiling water bath for a few minutes. The THA-APTES suspensions turned deep blue, whereas the untreated samples remain slightly pale yellow. The results suggest the successful APTES silylation of the THA nanoparticles. Second, the amino terminal groups on the surface or in the “channel” of THA reacted with FITC molecule. The solid products were obtained by centrifuge and dried overnight, followed by dialysis for 5 days to remove the free dyes, with fresh deionized water exchanged every 12 h until the dialysis solution is colorless. The fluorescence intensity of the THA-FITC nanoparticles before and after dialysis was examined by flow cytometry (Figure S3, Supporting Information). The very slight decrease of the fluorescence intensity indicates the fluorescent nanoparticles are very stable and that the FITC absorbed on the THA surface was removed. In contrast, the fluorescent HA nanoparticles prepared by adsorption of FITC exhibit no fluorescence after 48 h dialysis. As shown in Figure 3, the excitation and emission spectra of the THA-FITC nanoparticles are similar to those of the parent dye molecule, green for FITC at 500 nm, and THA nanoparticles in the aqueous solution produce a very bright green luminescence (inset in Figure 3). Since these dye molecules are chemically bound to the THA matrix, they cannot be easily removed by extracting with a large amount of solvent or applying sonication, and furthermore they are not easily photobleached. These properties make fluorescent THA nanoparticles very useful for biomarking and drug delivery. Toxicity is a common characteristic of materials typically used for intracellular delivery because of their ability to alter or disrupt 18541

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Figure 4. MTT assay of L929 cells cultured for 24, 48, and 72 h in media containing THA-FITC nanoparticles.

Figure 5. Flow cytometry analysis of HeLa cells incubated with THA-FITC nanoparticles (500 μg mL 1) for 2 h (a, b) and the control cells (c, d).

cell membranes.49 Since we attempted to develop particles to be used with cells, we quantified the effects of THA-FITC nanoparticles on the viability of L929 fibroblast cells in vitro. Cell viability was evaluated using the modified MTT assay.42,43 Figure 4 shows the viability of cells treated with THA-FITC over a range of concentrations for 24, 48, and 72 h. It can be seen that THA-FITC nanoparticles do not negatively affect cell viability over the full range of concentrations measured, indicating that they exhibit no cytotoxicity and would reasonably be used for labeling or intracellular detection. The increased measured viability seen with increasing concentrations of particles seems to be caused by particles activating cells. The bright field microscopy images of cells grown in the presence and absence of THA-FITC nanoparticles confirmed biocompatibility of this material (Figure S4, Supporting Information). These results are consistent with the interactions of cells with HA tubes.50 Cellular uptake of THA-FITC nanoparticles was first determined using flow cytometry (Figure 5). HeLa cells after being

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Figure 6. Confocal fluorescent images of HeLa cells incubated with THA-FITC nanoparticles with concentrations of (a) 100 μg mL 1, (b) 300 μg mL 1, and (c) 500 μg mL 1 for 2 h. Multilabeling laser scanning confocal microscope of HeLa cells incubated with THA-FITC nanoparticles with concentration of 300 μg mL 1 for 2 h: (d) fluorescence image showing THA-FITC color only; (e) fluorescence image of cytoplasm illuminated with red; (e) overlaid image of HeLa cells. Incubation was performed at 37 °C under a humidified atmosphere containing 5% CO2.

exposed with THA-FITC nanoparticles were treated with PBS to wash away the surface-bound nanoparticles and ensure that data from flow cytometry were due to the engulfed nanoparticles.51 Median fluorescence levels in the FL1 histograms (Figure 5a and 5b) indicate that THA-FITC nanoparticles were probably taken up by HeLa cells compared to the controlled cells (Figure 5c and 5d), suggesting that THA-FITC nanoparticles would be useful for high-sensitivity detection and characterization of cells by flow cytometry. Confocal microscopy was used to verify the location of the particles relative to the cells since flow cytometry cannot distinguish between surface-bound and intracellular nanoparticles. THA-FITC nanoparticles were subsequently allowed to interact with HeLa cells for 2 h at 37 °C at increasing concentrations, namely, 100, 300, and 500 μg mL 1, in the medium. The green fluorescence of FITC can be clearly seen in confocal images with an excitation wavelength of 495 nm (Figure 6a c). The green FITC appears localized as discrete dots and exhibited the morphology of cells, which indicate the nanoparticles probably have crossed the cell membrane and been internalized into cells. The detected green signal increased in proportion to the concentrations of THA-FITC added to the cells. This confirmed that the fluorescence signal detected follows a THA-FITC dose response. For the cell retention examination, cells incubated with THAFITC for 24 h were also evaluated (Figure S5, Supporting Information). The green FITC signal was detected inside the cells, indicating the photostability of THA-FITC nanoparticles. To confirm that the observed THA-FITC luminescence signal (green) was located intracellularly instead of adsorbing onto the cell surface and identify the exact intracellular location following their internalization, multilabeling laser scanning confocal microscope studies were performed. After incubation of THA-FITC nanoparticles with HeLa cells for 2 h at 37 °C (5% CO2), the cytoplasm of cells was stained red by rhodamine 6G. The confocal microscopy optical setup was in multichannel mode. The fluorescence intensity was evaluated under two sets of conditions: the first set of conditions was the same as that used to evaluate the fluorescence intensity of the THA nanoparticles 18542

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Figure 7. Z-stack image of interiors of HeLa cells incubated with THA-FITC nanoparticles with a concentration of 300 μg mL for 2 h: (a) fluorescence image showing THA-FITC color only and (b) overlaid image of cells.

containing FITC (excitation and emission wavelengths were 495 and 530 nm, respectively); the second set was the optimum condition for rhodamine (excitation and emission wavelengths were 520 and 580 nm, respectively). Figure 6d,f depicts confocal images of cells and THA-FITC nanoparticles. Red regions represent cytoplasm, and green indicates THA-FITC fluorescence. Yellow regions represent the colocalization of THA-FITC nanoparticles and cytoplasm. Figure 6f shows the uptake of fluorescent particles into cells (yellow). The particles are well distributed throughout the cell and appear to be adjacent to the nucleus, most likely through endocytosis. It could be found that there are also some particles attached on the cell surface, which is probably due to the large dose we used (300 μg mL 1). Some particles aggregated and attached on the cell surface. However, from the images, it could be considered that the cells ingested a relatively large amount of particles and that the cells retained cellular morphology without damage. Thus, we confirm that the incubation and the cellular internalization of THA-FITC particles have not caused any damage at the doses used in this study. These results are in complete agreement with the MTT results. The internalization of THA-FITC nanoparticles was further confirmed with Z-stack images, which is a kind of technology based on acquiring signals of the focal plane at different levels in cell or tissue samples, corresponding to the layer cross-sectional images.52 A series of cell cross sections at various z-axis values showed the intracellular distribution of nanoparticles (Figure 7). It can be clearly seen that THA-FITC nanoparticles were distributed on the surface of the cells and inside the cells and rarely particles inside the nuclei (Figure 7a). In addition, the fluorescence intensity of the nanoparticles is increased from the top slice to the bottom slice, so the imaging area turns from confuse to clear (Figure 7b). This verified that the THA luminescence signal was indeed localized within the cell. Since the HeLa cells are adherent cells, we can only get the Z-stack images from the top to the middle of the cell lines. Furthermore, at a dose of up to 500 μg mL 1 of THA-FITC nanoparticles used in confocal microscopy studies, no cell detachment or cell death was observed microscopically. As a result, it is indicated that THA-FITC nanoparticles exhibit superior performance as cellular fluorescence markers and promising application.

’ CONCLUSIONS In summary, we have synthesized dye-functionalized HA nanoparticles by using SiO2 nanoparticles as templates. The structure of the obtained composite was characterized by FITR,

XRD, and morphology analyses (SEM and TEM). For the first time, well-dispersed, FITC functionalized hydroxyapatite nanoparticles were demonstrated to be internalized into HeLa cells and exhibited good cell labeling capability. Also, the luminescent HA nanoparticles appeared to have no apparent cytotoxic effects on fibroblast cells. These findings indicated that the as-synthesized luminescent HA nanoparticles are applicable for intracellular distribution analysis or targeted therapeutics applications. Our study opens the way to the synthesis of well-controlled HA nanoparticles. The covalent cross-link of organic dye (fluorescein) with inorganic nanoparticles (HA) can provide a general method to fabricate luminescent materials in the future.

’ ASSOCIATED CONTENT

bS

Supporting Information. Time-dependent colloidal stability of HA and THA, TEM images of SiO2, SiO2-HA, and THA, flow cytometry analysis of THA-FITC before and after dialysis, bright field microscopy images of L929 cells in the presence of THA-FITC, and confocal fluorescent images of cells incubated with THA-FITC after 24 h. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: +86 931 8610877. Fax: +86 931 8912582. Zhengzhi Zeng, e-mail: [email protected]; [email protected]. Decheng Bai, e-mail: [email protected].

’ ACKNOWLEDGMENT This study was supported by the Foundation of Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and the NSFC (20171019). We thank Prof. Decheng Bai for his help with the confocal microscope examinations. ’ REFERENCES (1) Fukumori, Y.; Ichikawa, H. Adv. Powder Technol. 2006, 17, 1–28. (2) Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.; Zink, J. I. ACS Nano 2008, 2, 889–896. (3) Alivisatos, A. P.; Gu, W. W.; Larabell, C. Annu. Rev. Biomed. Eng. 2005, 7, 55–76. (4) Lee, S.; Xie, J.; Chen, X. Y. Biochemistry 2010, 49, 1364–1376. 18543

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