Bimodal Fluorescence andMagnetic Resonance Imaging Using Water-Soluble Hexagonal NaYF 4 : Ce , Tb , Gd Nanocrystals

1 Department of Physics, Key Laboratory of Artificial Microand Nano-Structures of Ministry of Education and School of Physics and Technology, Wuhan University, Wuhan 430072, China 2 Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital of Sichuan University, Chengdu 610041, China 3 State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China 4 Laboratory of Stem Cell Biology, State Key Laboratory of the Biotherapy, West China Hospital of Sichuan University, Chengdu 610041, China


Introduction
Multimodal bioimaging, which shows advantages over traditional single-imaging modality, has been regarded as a new research frontier in biological and medical sciences [1][2][3].Among various imaging techniques, fluorescence imaging provides the highest sensitivity and spatial resolution for in vitro bioimaging.However, fluorescence imaging suffers from lacking of detailed anatomical and physiological information in vivo.Magnetic resonance imaging (MRI), a noninvasive diagnostic method in clinic, offers excellent anatomical and functional information for in vivo bioimaging, but it is limited due to the poor sensitivity and resolution for cell imaging [3][4][5].Therefore, a probe with the combination of fluorescence and MR imaging could bridge gaps in resolution and depth of bioimaging and would provide a useful diagnostic tool for both in vitro and in vivo studies [4,[6][7][8][9].By virtue of this bimodal combination, some applica-tions have been exploited such as biological marking, photodynamic therapeutic intervention, tumors targeting, and drug delivering [10][11][12].Realization of such bimodal probes with high performance and good biocompatibility has aroused great research interests [13,14], though it is still a great challenge now.
In recent years, nanocrystals (NCs) of rare-earth (RE) compounds have been proposed to be a promising new class of biological probes due to their unique optical and chemical features [15][16][17][18].Ascribed to their special electron configuration, lanthanide ions (such as Tb 3+ , Eu 3+ , Er 3+ , and Nd 3+ ) exhibit sharp fluorescent emissions with long lifetime, superior photostability, and high resistance to photobleaching and photoblinking [19,20].In addition, other lanthanide ions including Gd 3+ possess a large number of unpaired electrons, which could become a paramagnetic relaxation agent with positive contrast enhancement in T 1 -weighted MRI [21].Among various RE compounds, hexagonal-phase NaYF 4 has attracted increasing attention as a matrix for lanthanide ions, because it is considered to be one of the most efficient host materials for supporting fluorescence of lanthanide ions [22][23][24].What is more, at present high-quality NaYF4 nanocrystals have been synthesized by a user-friendly method [25], which makes this material more attractive on bioimaging applications.On the road to realize bimodal imaging, incorporation of fluorescence RE 3+ ions and Gd 3+ ions in NaYF 4 nanocrystals have been demonstrated as an efficient route [4].Nevertheless, the combination of the fluorescence and MR bioimaging for both in vitro and in vivo studies has rarely been studied.
In the present work, a bimodal nanoprobe was synthesized by codoping Ce 3+ , Tb 3+ , and Gd 3+ ions in NaYF 4 .The synthesized hexagonal-phase NaYF 4 :Ce,Tb,Gd NCs exhibit good water solubility, low cytotoxicity and demonstrate both high fluorescence efficiency (26%) and high MR enhancement (58%).Using the NaYF 4 :Ce,Tb,Gd NCs as probes, we successfully demonstrate fluorescence imaging of C6 glioma cells in vitro and MR imaging for liver, spleen, and kidney of rat in vivo.These results provide an insight into this novel type of biological agent in multimodal bioimaging.

Synthesis.
In a typical procedure to synthesize PEI-coated NaYF 4 :10%Ce,5%Tb,10%Gd, 5 mL of PEI solution, 1.0 mL of NaCl, 0.1 mL of GdCl 3 , 0.1 mL of CeCl 3 and 0.05 mL of TbCl 3 , were added to 15 mL ethanol in sequence.After 5 min vigorous agitation, a stoichiometric amount of NH 4 F was charged.After another 15 min stirring, the resulting solution was transferred to a 50 mL Teflon-lined autoclave and eventually heated at 200 • C for 3 h.After naturally cooling down, the product was collected by centrifugation, washed with ethanol and ultrapure water for several times, then dissolved in 20 mL of GdCl 3 (0.1 M) and stirred for 3 h.Finally, the sample was obtained by centrifugation, washed with ethanol and ultrapure water several times, and dried in vacuum.

Characterization. The X-ray powder diffraction (XRD)
analysis was carried out on a Bruker D8 ADVANCE X-ray diffractometer with Cu Kα1 irradiation (λ = 1.5406Å).The transmission electron microscopy (TEM) measurements were performed with a JEOL 2010 HT microscope (operated at 200 kV).

In Vitro Cell
Imaging.Folic acid (75 mg) and EDC (180 mg) were dissolved in 30 mL of DMSO.After a halfhour mild agitation, 3 mL of DMSO containing 15 mg NaYF 4 :Ce,Tb,Gd NCs was slowly added.The mixture was stirred at room temperature for 4 h.The products were collected using centrifugation, washed with DMSO and ultrapure water several times, and then dispersed in 3 mL of ultrapure water.
Prior to cell fluorescence imaging, live rat C6 glioma cells (obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) were incubated with folic acid conjugated by NaYF 4 :Ce,Tb,Gd NCs at the concentration of 1000 μg/mL at 37 • C and 5% CO 2 for 12 h.The living cell fluorescence was observed by an inverted fluorescence microscope (Olympus IX 70), coupled with a digital camera (Nikon Coolpix 5400) and Image-pro Plus 5.0 image analysis software.

MRI Relaxation Measurements.
In order to evaluate the effectiveness of NaYF 4 :Ce,Tb,Gd NCs as an MRI contrast agent, the relaxation properties of NaYF 4 :Ce,Tb,Gd NCs solutions were examined on a 3T MR scanner (Siemens trio system, Huaxi MR Research Center, Chengdu, China) with a 12-channel phased array coil.Samples with concentration varying from 0.06 mg/mL to 1.67 mg/mL were prepared for T 1 -weighted MR imaging by diluting them in deionized water.Deionized water was used as the reference sample.All the samples were scanned using standard Spin-Echo (SE) sequence (TR = 500 ms; TE = 9.4 ms; FOV = 138 * 180; matrix = 170 * 384; slice thickness = 3 mm; number of signal averages = 2).
To further investigate the contrast effect of NaYF 4 :Ce,Tb, Gd NCs, specific relaxivity value r 1 was measured for each of the samples, respectively.Inversion recovery pulse sequence was used for the measurement of T 1 values (TR/TE = 15 ms/1.95ms; 10 different inversion-recovery waiting delay values (TI) between 23 ms and 3 s; FOV = 160 * 160; matrix = 768 * 768; slice thickness = 3 mm).The signal intensity of each tube on MRI was measured by placing a circle region of interest (ROI) with voxel size of 200 pixels in the center of the tube.T 1 values of each tube were deduced from the following formula: S (TI) = S0 × [1 − 2 exp(−TI/T 1 )] by performing a nonlinear least-squares fit.A plot of R 1 versus Gd 3+ concentration yielded a straight line with the slope defined as the NaYF 4 :Ce,Tb,Gd NCs T 1 relaxivity value r 1 .

In Vivo MRI Studies.
In vivo experiments were performed on white Sprague-Dawley (SD) rats (3-60 g) under authorization of the Animal and Human Ethics Committee of the West China Hospital at Sichuan University.MR scanning was performed on a Philips Achieva 3.0 T system with a phased array coil for rat (Shanghai Chenguang Medical Technologies Co., Ltd).After intraperitoneal injection of 10% chloral hydrate 0.4 mL, the rat was placed pronely in the MR system with its abdomen at the center of the coil.The NaYF 4 :Ce,Tb,Gd NCs solution was injected intravenously, and dynamic MRI 3D images (TR/TE = 18 ms/8 ms, matrix = 148 * 124, slices = 30) were obtained before and 0.67, 1.5, 3, 9, 24, and 48 h after the administration of contrast agent for each animal (maintained at normal body temperature).Signal intensity was measured at the each time point by using Siemens Syngo software, and the relative signal intensity changes were plotted against time.

Results and Discussion
The characterization of the PEI-coated NaYF 4 :Ce,Tb,Gd NCs (Y : Ce : Tb : Gd = 75 : 10 : 5 : 10) was demonstrated in Figure 1.As indicated in Figure 1(a), the products took on a rod-like shape with an average diameter of approximately 30 nm.The XRD pattern in Figure 1(b) exhibited that peak positions were in good accordance with the data in the JCPDS standard card (28-1192) for hexagonal NaYF 4 crystals.It was known that the NaYF 4 nanocrystal in hexagonalphase had been regarded as one of the most efficient host materials for RE fluorescence [22][23][24].
The surface properties of the products were exhibited by using FTIR spectrum.As shown in Figure 2, two strong bands (3428 and 1635 cm −1 ), originating from O-H stretching and H-O-H bending modes of vibration, render the NCs water soluble.Furthermore, the absorption peaks from internal vibration of amide bonds (1382 cm −1 ) and CH 2 stretching vibrations (2863 and 2927 cm −1 ) demonstrated the presence of PEI on the particle surface.The PEI-coated NCs could give a direct conjugation of biomolecules to the NCs. Figure 3 showed the excitation and emission spectra of the NaYF 4 :Ce,Tb,Gd NCs in water solution.In the NaYF 4 :Ce,Tb,Gd NCs, the Ce 3+ absorbed energy effectively from ultraviolet (UV) light and transferred it to the Tb 3+ that emitted green light.Under a 254 nm UV lamp irradiation, the particle water solution exhibited bright green emissions (Figure 2).Monitored with the emission wavelength of 542 nm, excitation spectrum consisted of a broad and strong band with a peak at 249 nm, which corresponded to the transitions from the ground state 2F 5/2 of Ce 3+ to different components of the excited Ce 3+ 5d stated split by the crystal field [26].The emission spectrum showed the characteristic and strong emission of Tb 3+ with the 5D 4 -7FJ (J = 6-3) transitions ranging from 475 to 650 nm.A broadband Ce 3+ emission (5d-4f transition) between 300 and 400 nm, as well as a sharp line Gd 3+ emission (6PJ−8S7/2 transition) at about 310 nm, could also be observed due to incomplete energy transfer [27].
The quantum yield of the NaYF 4 :Ce,Tb,Gd NCs was further determined by using quinine bisulfate in 0.5 M H 2 SO 4 as the standard samples [28].The quantum yield was calculated from the equation Y sam = Y sam (I sam A sta /I sta A sam ), in which A referred to the absorption intensity and I referred to the integral fluorescence intensity.The quantum yield of quinine bisulfate was 54.6% [29].By measuring the luminescence of both terbium and cerium, for the NaYF 4 :Ce, Tb,Gd NCs dispersed in water, a quantum yield of 26% was found, which was higher than that of the LaF 3 :Ce,Tb NCs reported previously [30,31].
In vitro bioimaging studies were conducted in live C6 glioma cells which were treated by folic acid conjugated with NaYF 4 :Ce,Tb,Gd NCs for 12 h.As shown in Figure 4, the comparison between fluorescence and bright field images suggested the signal distributions strongly correlated with the C6 glioma cells, proving the fine attachment of NCs on the surface of cells.No conspicuous cell death was observed, which further indicated that the NCs were not cytotoxic to the cells.The results demonstrated that the NaYF 4 :Ce,Tb,Gd NCs could be used as an efficient probe for fluorescence bioimaging.
In vitro, T 1 -weighted imaging for the NaYF 4 :Ce,Tb,Gd NCs was performed with the 4 different concentrations of Gd 3+ varying from 0.03 to 0.82 mM (pure water for the background signal) on a 3T MR scanner.A significant dose-dependent positive enhancement was observed on MRI with increasing of Gd 3+ (Figure 5(a)).The solution with maximum concentration (0.82 mM Gd 3+ ) presented the most effective positive MR signal enhancement which is about 200%.To further evaluate these properties, we calculated the MR relaxivity value r 1 of the NaYF 4 :Ce,Tb,Gd NCs by fitting a linear relationship between the R 1 relaxation rates and the Gd 3+ concentration (Figure 5(b)).The r 1 of NaYF 4 :Ce,Tb,Gd NCs, 2.87 mM −1 s −1 , was higher than those of 0.14 mM −1 s −1 of NaYF 4 :Yb,Er,Gd NCs [4] and 1.4 mM −1 s −1 of NaGdF 4 :Er,Yb/NaGdF 4 NCs [16].Furthermore, the r 1 of our products was only slightly lower than that of commonly used Gd-DTPA (3.7 mM −1 s −1 ) [32,33].The results suggested that the NaYF 4 :Ce,Tb,Gd NCs could be a decent contrast agent for MRI application.
In vivo, MRI experiments were carried out on a Philips Achieva 3.0 T MR system.Health SD rats were injected with NaYF 4 :Ce,Tb,Gd NCs (4.17 mg/kg) through tail veins.Three-dimensional T 1 -weighted images were acquired at the time points of 1.5, 3, and 48 hours after injection of the probes.Signal alteration was defined using "enhancement" (ENH) from the equation: ENH = (SI pre − SI post )/SI pre , according to previous studies [34].In order to quantitatively assess the enhanced effect of NCs, Siemens Syngo software was used to measure the signal intensity of the liver, spleen, and kidney of rats.As shown in Figure 6(c), obvious enhancement of the MR signal intensity was observed within 1.5 h after injection.The maximum relative enhancements were 58%, 36%, and 37% in the liver, spleen, and kidney, respectively.After 3 h after injection, the MR signal decreased gradually.After 48 h after injection, few probe MR signals were observed only in the intestinal tract, indicating the almost clearance of the injected probes [35].All the treated rats had survived for more than two months without any obvious toxicity response.Considering that the injected probe dosage was much lower than conventional clinical dosage and well tolerated by the animals, the results demonstrated that the probes were effective contrast agent for noninvasive MR imaging.In clinical applications, they would show good performance in indicating organ lesions, evaluating atherosclerosis, and labeling tumors [36,37].

Conclusions
In summary, a convenient and green route has been established for synthesizing water-soluble hexagonal-phase NaYF 4 :Ce,Tb,Gd NCs, which demonstrated high efficiency for both fluorescence imaging and T 1 -positive contrast-enhanced MRI.Compared with previously reported bioprobes, the NaYF 4 :Ce,Tb,Gd NCs provided the dual-modality bioimaging, in which the fluorescence imaging could provide cellular-or molecular-level information with near singlemolecule sensitivity, while MRI images could provide noninvasive in vivo imaging to clearly show organ lesions.Due to the combined presence of efficient optical and MR imaging capabilities, the proposed NaYF 4 :Ce,Tb,Gd nanoprobes held great promise for simultaneous bimodal fluorescence and MR bioimaging.

Figure 6 :
Figure 6: Series of in vivo 3D-T 1 MR images at a few representative time points of different organs in rats.On the top of arrays (a) were axial images of liver and at the bottom (b) were coronal images of spleen and kidney.(c) Mean values of relative signal intensity of different organs collected from before and after NaYF 4 :Ce,Tb,Gd NCs solution injection (rats No.-3).