Gd3+ Co-Doped Hydroxyapatite

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Luminescence Enhanced Eu3+/Gd3+ Co-Doped Hydroxyapatite Nanocrystals as Imaging Agents In Vitro and In Vivo Yunfei Xie, Wangmei He, Fang Li, Thalagalage Shalika Harshani Perera, Lin Gan, Yingchao Han, Xinyu Wang, Shipu Li, and Honglian Dai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01814 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 10, 2016

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ACS Applied Materials & Interfaces

Luminescence

Enhanced

Eu3+/Gd3+

Co-Doped

Hydroxyapatite Nanocrystals as Imaging Agents In Vitro and In Vivo Yunfei Xie, ‡,§ Wangmei He, ‡,§ Fang Li,‡,§ Thalagalage Shalika Harshani Perera,⊥,‡ Lin Gan,‡,§ Yingchao Han,*,‡,§ Xinyu Wang, ‡,§ Shipu Li, ‡,§ and Honglian Dai*, ‡,§ ‡

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan

University of Technology, Wuhan 430070, P.R. China §

Biomedical Materials and Engineering Research Center of Hubei Province, Wuhan University

of Technology, Wuhan 430070, P.R.China ⊥

Faculty of Applied Sciences, Sabaragamuwa University of Sri Lanka, 70140 Belihuloya, Sri

Lanka KEYWORDS: imaging, hydroxyapatite, Eu/Gd co-doping, nanocrystals, luminescence enhancement, intracellular degradation, tissue distribution ABSTRACT Biocompatible, biodegradable and luminescent nano material can be used as an alternative bioimaging agent for early cancer diagnosis, which is crucial to achieve successful treatment. Hydroxyapatite (HAP) nanocyrstals have good biocompatibility and biodegradability, and can be used as an excellent host for luminescent rare earth elements. In this study, based on the energy transfer from Gd3+ to Eu3+, the luminescence enhanced imaging agent of Eu/Gd co-

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doping HAP (HAP:Eu/Gd) nanocrystals are obtained via co-precipitation with plate-like shape and no change in crystal phase composition. The luminescence can be much elevated (up to about 120%) with nonlinearly increase versus Gd doping content, which is due to the energy transfer (6PJ of Gd3+→5HJ of Eu3+) under 273 nm and the possible combination effect of the cooperative upconversion and the successive energy transfer under 394 nm respectively. Results demonstrate that the biocompatible HAP:Eu/Gd nanocrystals can successfully perform the cell labeling and in vivo imaging. The intracellular HAP:Eu/Gd nanocrystals display good biodegradability with an cumulative degradation of about 65% after 72 h. This biocompatible, biodegradable and luminescence enhanced HAP:Eu/Gd nanocrystal has the potential to act as a fluorescent imaging agent in vitro and in vivo.

INTRODUCTION Early diagnosis of cancer is crucial to achieve successful treatment for improving the life quality of patients. With the development of nanotechnology, various novel imaging agents with single or multiple functions such as fluorescence, magnetism and radioactivity are showing great potential in cancer diagnosis.1-6 Recently, the research on improving the in vivo imaging ability attracts many attentions on luminescence and magnetic response properties.7-11 Hydroxyapatite (Ca10(PO4)6(OH)2, HAP) is the key inorganic component of bone and teeth of mammals with excellent biocompatibility and bioactivity.12-14 Therefore, synthesized HAP materials as bone substitutes have been widely investigated in the clinical application for repairing bone defects in the past decades.15 Recently, nano HAP has attracted more attentions in disease diagnosing16 and treating17-18 due to its good biocompatibility and biodegradability. The characteristic hexagonal structure and space group endow HAP with the ability that the Ca2+ in the crystal lattice can be easily substituted by rare earth (RE) ions and rare earth ions at Ca2+ site


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can emit specific luminescence.19-20 However, it is still required to further improve the luminescent signal of RE-HAP for bioimaging. Normally, the luminescence intensity of REHAP can be enhanced by increasing RE doping content. However, excess RE doping content may result in fluorescence quenching and cytotoxicity. Also, calcinations at high temperature can improve the luminescence intensity of RE-HAP by improving the HAP' crystallinity and enhancing the diffusion of rare earth ions into HAP' crystalline lattice. But calcinations may lead to the size increase and agglomeration of HAP crystals, which is not suitable for cellular uptake. Therefore, the method to enhance luminescence intensity should be explored avoiding excess RE doping content and calcination at high temperature. It is an effective way to enhance the luminescence intensity based on energy transfer and electron transfer process between rare earth elements.21-23 Herein, based on the luminescence enhancement effect of Eu3+ sensitized by Gd3+, Eu/Gd co-doped HAP (HAP:Eu/Gd) nanocrystals were synthesized by co-precipitation method. The effects of molar ratios of Eu/Gd and calcining temperature on the properties of HAP:Eu/Gd nanocrystals including phase composition, crystal size and luminescence were investigated. The energy transfer mechanism for the luminescence enhancement was discussed. Moreover, the hemolysis and cytotoxicity of HAP:Eu/Gd nanocrystals were evaluated respectively. The uptake, location and degradation of HAP:Eu/Gd in cancer cells as well as tissues distribution were studied. The application of HAP:Eu/Gd nanocrystals for fluorescent imaging were carried out in vivo and in vitro. RESULTS AND DISCUSSION Composition and Morphology. The XRD patterns and FTIR spectra of as prepared HAP:Eu/Gd powders were shown in Figure S1. The XRD patterns (Figure S1a) display the characteristic peaks of HAP (ICDD 01-074-0566), demonstrating that the dopants of Eu and Gd


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don’t lead to the change of crystalline phase composition in the experimental doping amount. The broadened diffraction peaks also reveal that the obtained HAP:Eu/Gd possesses nanosized nature. Moreover, the varied REu:Gd has no significant influence on the crystallinity degree and crystallite size. The crystallinity degree is about 40%-50%. And the crystallite sizes are 34-40 nm (d002), 10-16 nm (d310) and 20-26 nm (d210) (Table S1). The FT-IR spectra show the characteristic functional groups of HAP. As shown in Figure S1b, the bands centered at about 3430 cm-1 and 1636 cm-1 are due to the OH vibration of absorbed water. The bands at 3569 cm-1 and 631 cm-1 are attributed to the stretching and bending vibrations of OH in HAP. The peaks at 962 cm-1, 472 cm-1, 1034 cm-1 and 1107 cm-1, 564 and 602 cm-1 are assigned to the ν1, ν2, ν3, ν4 vibration modes of PO43-, respectively. Peak at 876 cm-1 is caused by the HPO42- group. And carbonate vibration is observed at 1424 cm-1 indicating samples are partially carbonate substituted HAP:Eu/Gd. SEM image shows that HAP:Eu/Gd was plate-like nanoparticle with a size of about 11.0±2.1 nm in thickness and 83.8±27.3 nm in the plane direction (Figure 1a). EDS result displays the elements of Ca, P, O, Gd, Eu and the calculated molar ratios of Eu/Gd, (Eu+Gd)/(Eu+Gd+Ca) and (Eu+Gd+Ca)/P are 1.500, 0.024 and 1.617, which are close to the setting values (Figure 1b). Also, TEM image shows lots of plate-like nanocrystals with a size of about 30-100 nm in plane direction (Figure 1c). As shown in Figure S2, after calcinations at 600 °C the size of HAP:Eu/Gd in the plane direction is increased a little to about 100-200 nm with the crystal growth and agglomeration. Although HAP:Eu/Gd crystals without calcinations also appear agglomeration in SEM and TEM which is mainly due to the sample preparation of SEM and TEM, they show a much smaller DLS size (137 nm) than the samples calcined at 600 °C (247 nm), indicating the observed agglomeration is attributed to soft agglomerate (Figure S3).


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Figure 1. SEM image (a), EDS analysis results (b) and TEM image (c) of HAP:Eu/Gd (2:1.5) with no calcinations.


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The XPS of HAP:Eu/Gd (2:1.5) shows the binding energy (calibrated using C (1s, 284.07 eV) as the reference) of Ca (2p, 346.71 eV), P (2p, 133,78 eV) and O (1s, 531.11 eV) (Figure S4). The Eu 3d and Gd 3d signals at 1138.27 eV and 1198.85 eV are assigned to the Eu ion and Gd ion state of +3, respectively. Compared to the XPS result of HAP:Eu (2%), Eu in HAP:Eu/Gd (2:1.5) shows a binding energy shift of -2.6 eV, which is accompanied with shifts of Ca (+0.15 eV), P (+1.08 eV) and O (+0.37 eV) (Figure S5). This can demonstrate that the doping of Gd leads to the change of Eu located environment in HAP:Eu/Gd crystal.

Figure 2. Emission spectra of HAP:Eu/Gd with no calcinations excited at 273 nm (a) and 394 nm (b) respectively. Luminescence Properties of HAP:Eu/Gd. Figure 2 shows the emission spectra of Eu/Gd codoped HAP (80 °C) excited at 273 and 394 nm, respectively. Results show that the emission spectra excited at 394 nm have same peak positions to those excited at 273 nm; however, the intensity of the former is much stronger than that of the latter. All the spectra display four main emission peaks located at 594, 617, 655 and 699 nm. The highest peak at 617 nm corresponds to the 5D0→7F2 transition of Eu3+ ions and the peaks at 594, 655 and 699 nm are attributed to 5

D0→7FJ (J=1, 3, 4) transitions of Eu3+ ions. The emission at 617 nm (electric dipole transition)


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indicates the location of Eu3+ in non inversion symmetry and the emission at 594 nm (magnetic dipole transition) implies the location of Eu3+ in inversion symmetry. Higher relative intensity of the 5D0 → 7F2 emission demonstrates that Eu3+ ions prefer to situate in crystallographic sites without an inversion center. Moreover, the luminescence intensity of HAP:Eu/Gd is changed while varying the REu:Gd value (Figure 3). Nevertheless, the luminescence is enhanced nonlinearly along with the increase of Gd3+ content. In our experiment, while the REu:Gd is 2:0.5 the luminescence intensity reaches the sub-high value and the highest intensity of emission peaks are observed at REu:Gd of 2:1.5. The luminescence enhancement ratios are about 60% and 120% respectively. It is demonstrated that the luminescence intensity of Eu3+ can be improved by codoping Gd3+. However, there exists concentration quenching effect due to Gd3+-Gd3+ interaction. So, a proper REu/Gd value is crucial to obtain enhanced luminescence.


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Figure 3. Relative ratio of luminescent intensity of HAP:Eu (2%) /Gd (x%, x=0.1-2) to HAP:Eu (2%) without calcinations (80 °C) and with calcinations (600 °C). To investigate the influence of annealing on the luminescence enhancement effect, HAP:Eu/Gd was calcined at 600 °C for 3h. Results show that the annealing generates influence on the luminescence intensity and enhancement effect (Figure S6). First, after calcination at 600 °C, the luminescence intensities of calcined samples are much enhanced. In addition to the emission bands at same wavelength (594, 617, 655, 699 nm), a new peak at 576 nm assigned to 5

D0→7F0 was detected. Second, the luminescence is still nonlinearly increased and the largest

luminescence enhancement effect appears at smaller REu:Gd (2:0.5) value corresponding to about 60% enhancement ratio. The luminescence intensity of HAP:Eu/Gd (2:0.5) at 600 °C is about 2.3 and 1.7 times of that of samples (80 °C) with REu:Gd of 2:0.5 and 2:1.5 respectively (Figure S7). After calcination at 600 °C, in addition to the three main peaks at 394 nm (7F0→5L6), 363 nm (7F0→5D4) and 382 nm (7F0→5G2), a weak and broad band from 200-300 nm with a maximum at about 280 nm is observed, which is due to the charge transfer band (CTB) O2-→Eu3+ (Figure S8). Especially, the band is easy to be distinguished when REu/Gd is 2:0.5. Because of the overlap between CTB and the 8S7/2-6I6/11 transition of Gd3+, it can be inferred that there is the energy transfer from Gd3+ to Eu3+ for the samples calcined at 600 °C under 273 nm excitation.24 However, for samples synthesized at 80 °C, CTB is not observed due to the lower crystalline degree as well as much more crystal defects. Therefore, at 80 °C, it can be concluded that the observed luminescent enhancement under 273 nm can’t be explained by this Gd3+ to Eu3+ energy transfer. An alternative energy transfer process from 6PJ (Gd3+) to 5HJ (Eu3+) transition23 can account for the luminescent enhancement of samples synthesized at 80 oC. It can be inferred by the emission (λex=273 nm) of HAP:Gd (2%) (Figure S9). One emission band at 313 nm is


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assigned to the 6PJ-8S7/2 transition of Gd3+. The other band at 626 nm (6GJ to 6PJ transition of Gd3+) may be achieved by the excited state absorption (ESA) process, in which a Gd3+ at a metastable level of 6P7/2 absorbs the next UV photon and reaches to the high-energy levels of 6

GJ. Briefly, under 273 nm excitation the observed luminescence enhancement is mainly based

on the energy transfer from 6PJ (Gd3+) to 5HJ (Eu3+). According to the Dieke energy level scheme, the unactivated Gd3+ can’t transmit energy to Eu3+ under 394 nm unlike the energy transfer under 273 nm. However, under the excitation at 394 nm, the luminescence enhancements were all observed for samples synthesized at 80 °C and 600 °C. Another interpretation combining the cooperative upconversion (CU) and the successive energy transfer (SET) is put forward to illustrate the intrinsic mechanism of luminescent enhancement. Normally, under 394 nm the excited Eu3+ (5L6) transits to

5

D0 by

nonradiationrelaxation and further 7FJ with luminescence emissions, which is accompanied with the energy loss. With the existence of Gd3+, it might be performed that two excited Eu3+ (5L6) ions could transmit energy to a Gd3+ ion via CU process. Consequently, the Gd3+ may be excited to 6GJ energy level from ground state. After the energy retransfer from Gd3+ to Eu3+ by SET process, Eu3+ ions are activated to the energy level that is matched with the 6GJ energy level of Gd3+. Subsequently, the Eu3+ ions generate gradual energy level transitions to 5HJ, 5D0 and 7FJ of Eu3+ by radiation transition. This process with lower loss of energy may play the key role on the luminescence enhancement. Biological Safety and Cell Labeling In Vitro. As biological cell labels, good biosafety and biocompatibility are vital for HAP:Eu/Gd. The hemolysis and cytotoxicity of HAP:Eu/Gd, two important factors, were evaluated firstly. All hemolysis rates of HAP:Eu/Gd (80 °C) at different concentrations (0.025-0.2 mg/mL) are much less than 5% (Figure S10a). The cytotoxicity


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experiments to L02 cells were conducted by the evaluation of cell viability. As shown in the results, after co-cultured with HAP:Eu/Gd (80 °C) for 1 day and 3 days, cell proliferation was not inhibited at concentrations of 0.0125-0.2 mg/mol (Figure S10b). Hemolysis and cytotoxicity results demonstrate that Eu/Gd co-doped HAP has good biocompatibility and biosafety. In order to evaluate the ability of HAP:Eu/Gd for bioimaging in vitro, the uptake, location, luminescent signal and amount of intracellular HAP:Eu/Gd nanocrystals in HepG2 cancer cells were further determined. After incubation with HepG2 cells in a 6-wells plate for a given time, the amounts of intracellular Eu and Gd are detected and the molar ratios of Eu/Gd were calculated (Figure 4a). Then the total amounts of HAP:Eu/Gd in cells and amount of HAP:Eu/Gd per cell were also calculated (Figure 4b). With the increasing of co-culture time, the detected amount of intracellular Eu and Gd is gradually increased, indicating the increasing amount of HAP:Eu/Gd in cells. However, the intracellular amount of HAP:Eu/Gd per cell almost reaches up to the maximum at 24 h. The calculated molar ratios of Eu/Gd are close to the setting value (about 1.333). As shown in Figure 5a, the biological TEM graph indicates that some nanoparticles are internalized into cells and located in intracellular vacuoles around the nucleus, and the further EDS determination shows the signals of Ca, P, O, Eu and Gd (Figure S11), indicating the observed nanoparticles should be HAP:Eu/Gd nanocrystals. Furthermore, under LSCM (405nm), the strong red luminescence is observed in cells (Figure 5b). So, it can be concluded that HAP:Eu/Gd nanocrystals are internalized into cells by the observations of TEM and LSCM.


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Figure 4. Intracellular Eu3+ and Gd3+ (a) and HAP:Eu/Gd (b) after co-culture of HepG2 cells with non-calcined HAP:Eu/Gd (2:1.5) nanocrystals at different co-culture time. HAP nanoparticles possess higher solubility in the intracellular acidic environment.25 It can be expected that HAP:Eu/Gd should have good biodegradability in cells and can be cleared after cell labeling.26 The results show that the extracellular amount of Eu3+ ion is increased along with the increase of co-culture time (Figure S12). This may be because the intracellular HAP:Eu/Gd is gradually dissolved in the acidic environment of cells and released out from cells.27

Figure 5. TEM image (a) and LSCM image (b) of HepG2 cells co-cultured with non-calcined HAP:Eu/Gd (2:1.5) nanocrystals for 4h.


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In Vivo Fluorescent Imaging and Distribution in Tissues. The HAP:Eu/Gd can perform the fluorescent bioimaging in vivo through enterocoelia and vein injection (Figure 6). After intraperitoneal injection for about 10 minutes, the distinguished fluorescent signal is observed in enterocoelia (Figure 6b). In the way of tail vein injection, the fluorescent signals are visible mainly in tail right after injection (5 minutes) and weak fluorescent signal is observed in liver (Figure 6c). After 1.5 h, the fluorescent signal in tail becomes weak accompanied with the enhancing signal in liver. After the fluorescent imaging observation via vein injection (about 3 h), the blood and organs were collected and the concentrations of Eu3+ ions were quantitatively measured for denoting the amount of HAP:Eu/Gd in tissues (Figure 7). Results show that the concentration of Eu3+ ions in blood is 17.58±0.88 µg/mL, indicating about 0.58 µmol/mL HAP:Eu/Gd in blood circulation. Liver tissue displays the most accumulation of HAP:Eu/Gd (about 160±37 nmol/g). The in vivo imaging results and detection data of Eu3+ ions in tissues are consistent and all display the high accumulation in liver. This is mainly due to the uptake of HAP:Eu/Gd nanocrystals by the reticuloendothelial system (RES).28 Additionally, the nanosize nature of HAP:Eu/Gd nanocrystals might generate the NanoEL effect and enter into organs.29-30 The RES accumulation will influence the tumor imaging of HAP:Eu/Gd in vivo. It can be expected that the accumulation in RES may be lowered by having the “stealth” property after surface modification of polyethylene glycol31. Also, the tumor passive and active targeting by controlling size and grafting targeting ligands on surface is helpful to increase the accumulation in tumor.4,32 These will be mainly investigated in the following experiments.


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Figure 6. In vivo fluorescent imaging of non-calcined HAP:Eu/Gd (2:1.5) nanocrystals in BALB/c-nu mice. (a) with no injection, (b) with injection in enterocoelia, (c) 5 minutes after vein injection (d) 1.5 h after vein injection.

Figure 7. Tissue biodistribution of non-calcined HAP:Eu/Gd (2:1.5) nanocrystals in BALB/c-nu mice via vein injection for about 3 h.

CONCLUSIONS


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We have presented a strategy for improving the luminescence of rare earth doped HAP nanoparticles by the energy transfer from Gd3+ to Eu3+. Furthermore, the HAP:Eu/Gd nanocrystals are demonstrated to have no hemolysis and cytotoxicity. The biocompatible HAP:Eu/Gd nanocrystals can successfully perform the cell labeling and in vivo imaging. They can exist in blood circulation for at least 3 h after vein injection, although their accumulation appears in liver due to the RES. Importantly, HAP:Eu/Gd nanocrystals have good biodegradability in intracellular acidic environment proved by the intracellular degradation experiment. The active surface of HAP can be easily modified using tumor targeting ligands. So, the HAP:Eu/Gd nanocrystals can be expected to possess tumor targeting function. In summary, the luminescence enhanced HAP:Eu/Gd nanocrystal is hopeful to become a biocompatible and biodegradable fluorescent imaging agent in vitro and in vivo. MATERIALS AND METHODS Materials. All raw materials including EuCl3·6H2O (Aladdin industrial Co., Ltd.), GdCl3·6H2O (Aladdin industrial Co., Ltd.), Ca(NO3)·4H2O (Sinopharm Chemical Reagent Co., Ltd.), (NH4)2HPO4 (Sinopharm Chemical Reagent Co., Ltd.) and NH3·H2O (Sinopharm Chemical ReagentCo., Ltd.) used in this study were analytically pure. Preparation of HAP:Eu/Gd. HAP:Eu/Gd (Eu = 2 mol%, Gd = 0, 0.5, 1, 1.5, 2 mol%) were prepared by a co-precipitation method. In the synthesis process, 100mL (NH4)2HPO4 (0.01 M) solution were introducedinto 100ml mixed solution of Ca(NO3)2, EuCl3 and GdCl3 (total concentrationof Ca2+, Eu3+ and Gd3+ is 0.0167 M) at 80 °C. The molar ratio of (Ca+Eu+Gd)/P was fixed at 1.67. Next, the pH value was adjusted to about 10 using NH3·H2O aqueous solution. After violent stirring for 1 h, the obtained precipitation was separated by centrifugation and washed thrice using de-ionized water, and finally dried in a freeze dryer.


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Characterization. The phase composition of sample was recorded by a Powder X-ray diffractometer (XRD, D8 Advance, Germany) with Cu-Kα radiation (1.54056 Å). And the crystallinity degree (XC) and crystallite size were calculated using Jade 6.0 software. The functional groups of sample were characterized using a fourier transform infrared spectroscopy (FT-IR, Thermo Nicolet 6700, USA) in the range of 400 cm-1 to 4000 cm-1. High resolution transmit electronic microscopy (HRTEM, JEM2100F, USA) and Scanning electronic microscopy (SEM, Zeiss Ultra Plus, Germany) equipped with an EDS were used to observe the morphology and determine elements content. The X-ray photoelectron spectra (XPS) were taken on an Al-K-Alpha (1486.68eV) X-ray Photoelectron Spectrometer (Thermo Scientific, USA). The emission spectrum was recorded between 550 nm and 750 nm excited at 396 nm by a fluorescence spectrophotometer (970CRT, China). Hemolysis Test. Venous blood samples (4 mL) were fleshly drawn from the ear edge of rabbitsand put into a centrifuge tube containing 1% (wt%) heparin sodium solution. Then, the blood was centrifuged at 2000 r/min for 5 minutes. The supernatantwas removed and 50 mL 0.9% (wt%) NaCl aqueous solution was added. Next, the red blood cells (RBCs) suspension was centrifuged at1000 r/min for 10 minutes to purify RBCs. This process was repeated 4 times. The obtained purified RBCs was re-suspended to 5% suspension using 0.9% (wt%) NaCl aqueous solution. Then, 1 mL suspension of RBCs was incubated (37 °C) with 1mL HAP:Eu/Gd suspension which has been pre-dispersed in 0.9% (wt%) NaCl aqueous solution at different concentrations (0.025, 0.05, 0.1, 0.2 mg/mL). The positive and negative controls were set by adding de-ionized water and 0.9% NaCl solution into RBC suspension respectively. After incubation of 1 h, the absorbance values (OD) were detected at 545 nm. The hemolysis% was calculated according to equation and recorded for three independent experiments (mean±SEM).33


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Hemolysis% = (ODsample-ODnegative)/(ODpositive-ODnegative) × 100% Cytotoxicity Experiments. The human normal hepatocyte (L02) was maintainedin humidified air (5% CO2) at 37 °C in RPMI-1640, supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin. HAP:Eu/Gd powders were ultrasonically dispersed in de-ionized water using heparin sodium (2 mg/mL) and then sterilized in an autoclave at 121 oC for 1 h. For cytotoxicity experiments, cells were seeded into 96-well culture plates at 5000 cells per wellwith 0.1 mL culture medium. After 1 day, the culture medium was removed and fresh culture mediumcontaining HAP:Eu/Gd was added. The concentration of HAP:Eu/Gd in culture medium was between 0.0125 mg/mL and 0.2 mg/mL. After co-cultured for 1 day and 3 days, remove the culture medium and wash cells thrice by phosphate buffer saline (PBS, pH 7.4). 100 µL fresh culture medium containing 1% (v/v) CCK-8 solution was placed into each well and cellswere incubated for1 h at 37 °C. The absorbance values were measured at 450 nm with reference wavelength of 650 nm using a microplate reader. The degree of inhibition (%) was calculated according to the equation: Inhibition% = [(ODcontrol-ODtreated)/ODcontrol] × 100%, where ODcontrol and ODtreated are the OD values of control and treated groups, respective. Intracellular Determination of HAP:Eu/Gd. HepG2 cells were seeded in a 6-well culture plate at a density of 100,000 per well and co-cultured with HAP:Eu/Gd (0.2 mg/mL, dispersed in culture medium). At a given interval time, the culture medium was removed and cells were washed thrice using PBS. Next, cells were dried in an oven at 80 °C and 3 mL nitric acid solution (pH 2) was added to dissolve the HAP:Eu/Gd. Then, the content of Eu and Gd was determined by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES, Optima 4300DV, USA). The amount of HAP:Eu/Gd was calculated according to the content of Eu and Gd in HAP:Eu/Gd.


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Biological TEM Observation. Human liver cancer cells (HepG2) were seeded in a culture flask and cultured in RPMI-1640 at 37 °C under 5% CO2. After 3 days, the culture medium was replaced by fresh culture medium containing HAP:Eu/Gd suspension (0.2 mg/mL) and the cells were co-cultured with HAP:Eu/Gd for 4 h. At the end of incubation, cells were washed 4 times by PBS and fixed using glutaradehyde (2.5%) in PBS for 4 h. Then, cells were dehydrated with a series of alcohols, embedded with resin. Finally the resin was sectioned for observation of TEM (H-7000FA, JAPAN) at an acceleration voltage of 75 kV. Laser Scanning Confocal Microscopy. HepG2 cells were seeded in a culture dish and cultured in RPMI-1640 at 37 °C under 5% CO2. After one day, the culture medium was replaced by fresh culture medium containing HAP:Eu/Gd (0.2 mg/mL) and the cells were co-cultured with HAP:Eu/Gd for 4 h. At the end of incubation, cells were washed 4 times by PBS and fixed using glutaradehyde (2.5%) in PBS for 4 h. Then, cells in culture dish were observed under LSCM (A1 MP STORM, JAPAN) (405 nm). Intracellular Degradation Evaluation of HAP:Eu/Gd via Determining Extracellular Eu3+ Ions. HepG2 cells (seeded in 6-wells plate, 1×106 per well) were co-cultured with HAP:Eu/Gd (0.2 mg/mL, dispersed in culture medium) for one day. Then, the medium was drawn out and cells were washed thrice by PBS. Next, fresh medium was added and cells were continued to be cultured. At a given interval time, the medium was replaced with the same volume fresh medium for continuing culture. The withdrawn medium was determined for the Eu3+ ions after addition of nitric acid solution (pH 2) and centrifugation referring to the Eu3+ fluorescent quantitative determination method.21 The supernatant was diluted by DELFIA Enhancement Solution and the emission intensity at 613 nm was recorded under an excitation of 340 nm. Finally, the Eu3+ ions concentration was calculated using the linear standard curve between the fluorescent intensity (y)


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and concentration (x) of Eu3+ ions (y=-0.1+15.8x, R2=0.99). The corresponding amount of HAP:Eu/Gd was calculated from Eu3+ ions concentration. In Vivo Fluorescent Imaging of HAP:Eu/Gd. The animal experimental procedures were carried out according to the institutional animal use and care regulations. The BALB/c-nu mice (about 20 g) were purchased from Animal Experiment Center of Wuhan University. 0.2 mL HAP:Eu/Gd (1 mg/mL, dispersed in water) were injected through intraperitoneal injection and tail vein injection, respectively. The mice were imaged with a Maestro In Vivo Imaging System (CRi, USA). Determination of HAP:Eu/Gd in Tissues. After the in vivo fluorescent imaging experiments (about 3 h), mice with tail vein injection were euthanized and the liver, spleen, heart, lung, kidney were collected, washed and weighted. Then, samples of organs were placed into 2 mL of 2 M nitric acid solution and homogenizedin a glass tissue homogenizer. After centrifugation, the supernatant was diluted by DELFIA Enhancement Solution and the emission intensity at 613 nm was recorded under an excitation of 340 nm. The Eu3+ ions concentration was determined and the amount of HAP:Eu/Gd was calculated using the same method to the above experiment part of intracellular degradation. The amount of HAP:Eu/Gd was normalized by the tissue weight denoted as HAP:Eu/Gd per tissue (nmol/g). ASSOCIATED CONTENT Supporting Information. XPS, Photoluminescence spectra, hemolysis and cytotoxicity, intracellular degradation, Xc values and crystallite sizes. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51002109, 81190133), the HongKong, Macro and Taiwan Science & Technology Cooperation Program of China (2015DFH30180), the Natural Science Foundation of Hubei Province (2015CFB551), the Wuhan International Science and Technology Cooperation Project, the Fundamental Research Funds for the Central Universities (2016-CL-A1-42), and the project supported by State Key Laboratory of Advanced Technology for Materials Synthesis and Processing (Wuhan University of Technology). REFERENCES (1) Kwon, O. S.; Song, H. S.; Conde, J.; Kim, H. I.; Artzi, N.; Kim, J. H. Dual-Color Emissive Upconversion Nanocapsules for Differential Cancer Bioimaging In Vivo. ACS Nano 2016, 10,1512-1521. (2) Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.; Zink, J. I. Multifunctional Inorganic Nanoparticles for Imaging, Targeting, and Drug Delivery. ACS Nano 2008, 2, 889−896. (3) Sun, C.; Pratx, G.; Carpenter, C. M.; Liu, H.; Cheng, Z.; Gambhir, S. S.; Xing, L. Synthesis and Radioluminescence of PEGylated Eu3+-doped Nanophosphors as Bioimaging Probes. Adv.


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Table of Contents Graphic


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Gd3+ Co-Doped Hydroxyapatite

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