Gadolinium-Functionalized Aggregation-Induced Emission Dots as Dual-Modality Probes for Cancer Metastasis Study

Understanding the localization and engraftment of tumor cells at postintravasation stage of metastasis is of high importance in cancer diagnosis and treat-ment. Advanced ﬂ uorescent probes and facile methodologies for cell tracing play a key role in metastasis studies. In this work, we design and synthesize a dual-modality imaging dots with both optical and magnetic contrast through integration of a magnetic resonance imaging reagent, gadolinium(III), into a novel long-term cell tracing probe with aggregation-induced emission (AIE) in far-red/near-infrared region. The obtained ﬂ uorescent-magnetic AIE dots have both high ﬂ uorescence quantum yield (25%) and T 1 relaxivity (7.91 m M − 1 s − 1 ) in aqueous suspension. After further conjugation with a cell membrane penetrating peptide, the dual-modality dots can be efﬁ ciently internalized into living cells. The gadolinium(III) allows accurate quantiﬁ cation of biodistribution of cancer cells via intraveneous injection, while the high ﬂ uorescence provides engraftment information of cells at single cellular level. The dual-modality AIE dots show obvious synergistic advantages over either single imaging modality and hold great promises in advanced biomedical studies. as metastatic tumor cells from the primary neoplasm to a distant in aggregates due to the restriction of intramolecular rotations

Understanding the localization and engraftment of tumor cells at postintravasation stage of metastasis is of high importance in cancer diagnosis and treatment. Advanced fl uorescent probes and facile methodologies for cell tracing play a key role in metastasis studies. In this work, we design and synthesize a dual-modality imaging dots with both optical and magnetic contrast through integration of a magnetic resonance imaging reagent, gadolinium(III), into a novel long-term cell tracing probe with aggregation-induced emission (AIE) in far-red/near-infrared region. The obtained fl uorescent-magnetic AIE dots have both high fl uorescence quantum yield (25%) and T 1 relaxivity (7.91 mM − 1

s − 1 ) in aqueous suspension. After further conjugation with a cell membrane penetrating peptide, the dual-modality dots can be effi ciently internalized into living cells. The gadolinium(III) allows accurate quantifi cation of biodistribution of cancer cells via intraveneous injection, while the high fl uorescence provides engraftment information of cells at single cellular level. The dualmodality AIE dots show obvious synergistic advantages over either single imaging modality and hold great promises in advanced biomedical studies.
diseases always lead to poor prognosis as metastatic tumor cells can move from the primary neoplasm to a distant location in a living body. [ 1 ] In modern biomedical research, it has become a major concern to understand the cancer metastasis mechanism and process, which will dramatically benefi t therapeutic treatments and improve patient outcomes. [ 2 ] Although mimic complete metastatic process can be achieved through xenograft studies with tumor cell lines known to metastasize in,vivo, such assays always suffer from poor quantifi cation and slow completion. [ 3 ] As such, simple and direct experimental metastasis assays are ideal models to simulate the postintravasation stage of metastasis, in which tumor cells are injected into the bloodstream to monitor the location, distribution, and engraftment of administrated cells. [ 4 ] To understand the behavior of transplanted cells, different direct celllabeling strategies involving optical, magnetic resonance (MR) and radionuclide imaging techniques have been reported. [5][6][7] However, each imaging approach has its own advantages and disadvantages. For instance, fl uorescence imaging has high sensitivity but cannot provide quantitative evaluation of cell distribution upon transplantation, whereas MRI and radionuclide imaging provide desired tissue penetration depth but suffer from low sensitivity and resolution under single cellular level. [ 8 ] As compared with single-modal imaging probes, integration of two or more imaging modalities into a single probe will afford multimodal imaging probe with synergistic benefi ts, which is highly desirable in modern imaging. [9][10][11] To date, fl uorescent nanomaterials have been actively used as a platform to design multimodal imaging probes. Although inorganic quantum dots (QDs) have shown promising merits in fl uorescence imaging (e.g., high fl uorescence effi ciency and remarkable resistance to photobleaching), the potential toxicity and fl uorescence quenching phenomenon at low pH greatly impede their in vivo applications in real-world. [ 12 , 13 ] In previous studies, we have demonstrated that a novel class of photostable and biocompatible organic dots with aggregation-induced emission (AIE) in far-red/near-infrared (FR/NIR) region can greatly outperform its commercial inorganic QD counterpart in both in vitro and in vivo long-term cell tracing. [ 14 ] The AIE fl uorogen is nonemissive in solution but becomes highly fl uorescent in aggregates due to the restriction of intramolecular rotations that opens the radiative decay channel. [ 15 ] The AIE phenomenon is opposite to the notorious aggregation-caused quenching effect of organic fl uorophores, [ 16 , 17 ] making AIE fl uorogens ideal materials for highly fl uorescent nanoparticles. In addition, the versatile encapsulation matrices with different surface functional groups, e.g., -maleimide (-Mal), -NH 2 , and -COOH terminated distearoylsn -glycero-3-phosphoethanolaminepoly(ethylene glycol) (DSPE-PEG) derivatives, facilitate further functionalization to achieve the goal of multimodal imaging. As DSPE-PEG derivatives with different terminal groups have been reported to form heterogeneous patches on nanoparticle and microparticle surfaces, [ 18 ] this property is benefi cial to sequential surface functionalization with minimal steric interference.
In this contribution, we fabricated fl uorescent-magnetic dualmodality AIE dots for in vivo tumor cell metastasis studies. A mixture of amine-and Mal-terminated DSPE-PEG (DSPE-PEG-NH 2 and DSPE-PEG-Mal) was used as the encapsulation matrix to synthesize biocompatible AIE dots with different surface functionalities. Diethylenetriaminepentaacetic (DTPA) dianhydride was employed to conjugate with amine groups on AIE dot surface for further chelation of gadolinium(III) to endow magnetic contrast, whereas the maleimide groups were used to immobilize the cell penetrating peptide via maleimide-thiol coupling reaction to enhance the living cell labeling effi ciency. The obtained Tat-Gd-AIE dots were further used to label C6 glioma cells before being injected into mice through tail vein to evaluate the cell distribution and engraftment at the postintravasation stage of metastasis. Ex vivo analysis reveals that the incorporation of Gd(III) allows accurate quantifi cation of transplanted cell biodistribution and the high fl uorescence of AIE dots ensures excellent single cell imaging to provide valuable information of cancer cell engraftment in organ tissues. The developed dualmodality AIE dots not only provide mutual advantages in metastasis analysis but also inspire further exploration of multimodal imaging dots for in vivo monitoring of transplanted cells with both high temporal and spatial resolution.

Synthesis and Characterization of Dual-Modality AIE Dots
The AIE fl uorogen, TPETPAFN, was synthesized according to literature. [ 14 ] The AIE dots were synthesized through a nanoprecipitation approach reported by our group. [ 19 ]  The THF solution was rapidly mixed with water, followed by sonication to yield AIE dots with abundant surface amine and maleimide groups. After THF evaporation, the dots were fi ltered through a 0.2 μ m syringe fi lter. High-resolution transmission electron microscopy (HR-TEM) image reveals that the AIE dots are in spherical shape ( Figure 1 ), with an average particle size of 31.7 ± 2.1 nm. The water suspension of AIE dots shows maximum absorption and emission at 512 and 670 nm, respectively, with an intense emission tail till 900 nm. The quantum yield of AIE dots in water was measured to be 25% using 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4Hpyran in methanol as a standard ( Φ F = 43%). [ 20 ] To integrate the MR contrast with the fl uorescent AIE dots, DTPA dianhydride was used to conjugate with surface amine groups for chelation of Gd(III). The obtained Gd(III)-chelated dots were then modifi ed with HIV-1 Tat peptide (RKKR-RQRRRC) through conjugation between maleimide groups on dot surface and thiol groups at C-terminus of Tat peptide. The Gd(III) content in the fi nally obtained Tat-Gd-AIE dots was quantifi ed using ion coupled plasma-mass spectrometry (ICP-MS). The results reveal that the number of successfully chelated Gd(III) ions on each dot is estimated to be ≈ 136. To investigate the stability of chelate Gd(III) on Tat-Gd-AIE dots, 1 mL of the Tat-Gd-AIE dot suspension was dialyzed against water for 48 h at 37 ° C. ICP-MS results suggest that the Gd(III) concentration in the dot suspension keeps consistent before and after dialysis, indicating that the chelate Gd(III) elements are stable on Tat-Gd-AIE dot surface. High-performance liquid chromatography (HPLC) was employed to determine the number of Tat peptide conjugated on the dot surface, suggesting that there are ≈ 850 peptide molecules on each dot.

Cell Imaging and Cytotoxicity
The performance of Tat-Gd-AIE dots for direct cell labeling was fi rst examined using C6 glioma cells and the fl uorescence images were acquired by confocal as shown in Figure 2 . Upon 12 h incubation with 2 × 10 − 9 M Tat-Gd-AIE dots at 37 ° C, intense fl uorescence signal can be detected from C6 glioma cells upon excitation at 514 nm (1 mW). The 3D confocal image of Tat-Gd-AIE dotlabeled C6 cells was also obtained, indicating that the dots were internalized into the cells to emit intense fl uorescence signal ( Figure S1, Supporting Information). It is noteworthy that no autofl uorescence can be detected from the cells without incubation with Tat-Gd-AIE dots (Figure 2 c), which further proves that the intense fl uorescence in Figure 2 a is from the dots internalized into C6 cells. Gd-AIE dots without Tat peptide functionalization were also used as a control group to highlight the direct cell labeling ability of Tat-Gd-AIE dots. As shown in Figure 2 b, under the same experimental conditions, only very weak fl uorescence signal can be detected from the Gd-AIE dot-treated cells, indicating that at the same dot concentration, much fewer Gd-AIE dots were internalized into C6 cells as compared with that for Tat-Gd-AIE dots. As a result, the surface conjugation of Tat peptide on fl uorescent dots plays a crucial role in enhancing living cell internalization effi ciency, which is desirable for cell tracing.
A major concern in fl uorescence imaging of biotargets is the biocompatibility of the probes. To obtain reliable experimental results, fl uorescent probes should possess very low toxicity to target cells during cell tracing studies. To evaluate the cytotoxicity of pure AIE dots without surface modifi cation and Tat-Gd-AIE dots, methylthiazolyldiphenyltetrazolium bromide (MTT) assays were employed to determine the metabolic viability of C6 glioma cells after incubation with 1, 2, and 4 × 10 − 9 M dots for 24 and 48 h. As shown in Figure S2 (Supporting Information), the cell viability remains above 90% after 48 h incubation with the dual-modality dots, indicating their low cytotoxicity under the studied conditions.

Cell Tracing in Mice
The performance of Tat-Gd-AIE dots in in vivo cell tracing was then evaluated using an animal model. After incubation with 2 × 10 − 9 M Tat-Gd-AIE dots in culture medium at 37 ° C for 12 h, living C6 glioma cells were trypsinalized and suspended in freshly prepared culture medium, followed by immediate intravenous injection into the mice through tail vain (1.5 × 10 6 cells in 0.2 mL of medium for one mouse). After designated time intervals, the mice (Four groups, n = 3 for each group) were sacrifi ced and various organs including heart, liver, spleen, lung, and kidney were isolated and imaged using an IVIS Spectrum Imaging System ( Figure 3 a). At each time point post injection, intense fl uorescence is observed from the lung and liver while the spleen also emits visible fl uorescence signal upon excitation at 560 nm, indicating the preferential accumulation of injected cells in these organs. On the contrary, fl uorescence signal from the kidney and heart is negligible at all time points. It is noteworthy that the obvious accumulation of injected cells in the lung is consistent with the results from previously reported cell transplantation studies, suggesting that intravenously delivered cells are likely to be trapped inside the pulmonary system due to the pulmonary microvascular barrier. [ 21 , 22 ] To further study the biodistribution of transplanted cells in living animal bodies, the collected organs were decomposed by concentrated nitric acid and analyzed using ICP-MS to measure the Gd(III) contents. The percentage of cells engrafted in each organ from the total amount of cells localized in all the fi ve organs was fi rst calculated, suggesting that ≈ 35% and 48% of the cells are engrafted in the lung and liver at 1 h post injection, respectively. On the other hand, only ≈ 17% of the cells are in heart, spleen, and kidney. The Gd(III) concentrations in organs at all the time points were further studied to investigate the time course of accumulation of the C6 cells. As shown in Figure 3 b, the concentrations of Gd(III) in lung, spleen and  liver tissues are determined to be 21.5, 10.9, and 2.2 × 10 − 3 μ g g − 1 tissue at 1 h post injection of the cells, respectively. It should be noted that the concentration of Gd(III) in each organ is normalized to per gram tissue and the concentration of Gd(III) in liver is much lower than that in spleen (the ratio of Mass liver to Mass spleen is ≈ 100:1). Moreover, the concentration of cells accumulated in lung decreased at 6 h post injection, whereas that in other organs are relatively consistent as time elapses. This agrees with the previous reports that injected cells were fi rst captured by lung or liver and then released from the organs to enter circulation for further migration. [ 23 , 24 ] As compared with semiquantitative evaluation using fl uorescence techniques, the incorporation of Gd(III) in AIE dots allows accurate quantitation of the biodistribution of transplanted cells.

Localization of Cells in Organ Tissues
To investigate the localization of injected cells in organs, one mouse was sacrifi ced at 24 h post injection and the lung, spleen, liver, heart, and kidney tissues were collected for study using confocal laser scanning microscopy. As shown in Figure S3 (Supporting Information), intense red fl uorescent spots are clearly observed in the collected organ tissues, indicating the engraftment of injected cells in the animal body. In the enlarged confocal image of lung tissue ( Figure S3f, Supporting Information), three individual cells labeled by Tat-Gd-AIE dots in the view can be seen to engraft in the tissue while the surrounding tissue cells are not fl uorescent. These results clearly demonstrate the ability of Tat-Gd-AIE dots to monitor cell engraftment after transplantation with high resolution and sensitivity under single cellular level, which is of high importance in cancer metastasis and cell therapy studies.
Furthermore, the deep tissue imaging was also performed upon exciting the lung at 560 nm using one-photon excited fl uorescence microscope. To study the effi cient penetration depth of Tat-Gd-AIE dots in deep tissue, fl uorescence signal from the lung was collected layer-by-layer at 3 μ m interval. As shown in Figure S3g (Supporting Information), the 3D color-coded projection of deep tissue image indicates that the fl uorescence of labeled cells can be detected up to 150 μ m depth. The eminent performance of Tat-Gd-AIE dots in one-photon excited deep tissue imaging can be attributed to the high molar extinction coeffi cient at long wavelength (3.5 × 10 7 M − 1 cm − 1 at 560 nm).

Magnetic Resonance Imaging
The magnetic property of Tat-Gd-AIE dots was investigated by measuring longitudinal relaxation time ( T 1 ) of water protons as a function of Gd(III) concentration in aqueous solutions using a 7-Tesla Bruker ClinScan MR scanner. Figure 4 a shows the T 1weighted MR images of Tat-Gd-AIE dots in water suspensions at various Gd(III) concentrations, indicating that the signal intensity gradually increases upon increasing the Tat-Gd-AIE dot concentration. The T 1 relaxivity ( r 1 ) value that refers to the amount in 1/ T 1 per Gd(III) unit concentration of agent is calculated to be 7.91 m M − 1 s − 1 (Figure 4 b). The spin-spin relaxation time ( T 2 ) of the dual-modality dots was also calculated, giving a T 2 relaxivity ( r 2 ) value of 10.9 m M − 1 s − 1 . As a result, the r 2 / r 1 value is below 2, indicating that the probe belongs to positive contrast reagent. [ 25 ] Under the same experimental conditions, Dotarem ® , a commercial Gd(III)-complex of T 1 contrast reagent, shows a r 1 value of 3.761 m M − 1 s − 1 . Overall, the small r 2 / r 1 value of Tat-Gd-AIE dots and higher r 1 value as compared with Dotarem ® endow the dual-modality probes great potential to serve as a novel T 1 contrast reagent in bioimaging tasks.
In vivo MRI experiments were further conducted on an animal model and the C6 cells labeled by Tat-Gd-AIE dots (3 × 10 6 cells) were injected into the mice following the same experimental protocol as described in the fl uorescence imaging. The cross-sectional T 1 -weighted MR images of mice were recorded at 7.0 T before and after injection of Tat-Gd-AIE dot-labeled cells. As shown in Figure S4 (Supporting Information), the liver is not lightened at 1 and 24 h post injection and the brightness of these organs remains similar to that of the muscle tissues. The negative MRI results could be attributed to the fact that the Gd(III) content injected into the mice is lower than the detection threshold [ 26 ] (ICP-MS result suggests that there are ≈ 1.2 × 10 − 4 μ mol Gd(III) in total 3 × 10 6 cells injected into one mouse). These results clearly indicate that MRI suffers from low sensitivity in direct cell tracing. [ 27 ] However, the incorporation of Gd(III) can provide precise information of the biodistribution of transplanted cells.

Conclusions
In this work, we report a general strategy to synthesize dualmodality dots through attaching DTPA-Gd(III) ligands to the Adv. Healthcare Mater. 2013, 2, 1600-1605 www.MaterialsViews.com

FULL PAPER
www.advhealthmat.de surface of fl uorescent AIE dots and demonstrate their great potential in cell tracing studies using an animal model to mimic postintravasation stage of metastasis. Upon surface conjugation with Tat peptides, the obtained Tat-Gd-AIE dots have shown greatly enhanced living cell internalization effi ciency. The incorporation of Gd(III) on dual-modality imaging dots allows accurate quantifi cation of the biodistribution of injected cancer cells. On the other hand, the high brightness of the dots facilitates ex vivo imaging of transplanted cells at single cell level, which is of high importance to obtain information of the engrafted metastatic cancer cells in the organs. Although the Tat-Gd-AIE dots have been proven to be an effi cient T 1 contrast reagent with desired longitudinal relaxation time, the labeled cells can not be detected through MRI upon intravenous injection into the mice, due to the relatively low sensitivity of MRI and the insuffi cient amount of Gd(III) in the injected cells. To enhance the capability for Gd(III) chelation to realize in vivo cell tracing through MRI, optimization of the AIE dot formulation (e.g., attachment of hyperbranched polyglycerols to AIE dot surface to afford more active sites for conjugation with DTPA-Gd) will be highly desired. Taking advantage of the versatile surface functionalities of AIE dots, integration of highly sensitive imaging modalities (e.g., radioactive reagent) into the AIE dots is also expected to provide both high spatial and temporal resolution to benefi t in vivo cell tracing.
Characterization : The UV-vis spectra were recorded on a Shimadzu UV-1700 spectrometer. The fl uorescence spectra were measured using a fl uorometer (LS-55, PerkinElmer, USA). Fluorescence quantum yield of the AIE dots was determined using 4-(dicyanomethylene)-2-methyl-6-( p -dimethylaminostyryl)-4H-pyran in methanol (43%) as a standard. Average particle sizes were determined by laser light scattering with a particle size analyzer (90 Plus, Brookhaven Instruments Co., USA) at a fi xed angle of 90 ° at room temperature. HR-TEM images were recorded using a JEM-2010F microscopy (JEOL, Japan) with an accelerating voltage of 200 kV.
Synthesis of Surface-Functionalized AIE Dots : TPETPAFN, DSPE-PEG 2000 , and DSPE-PEG 2000 -NH 2 (1 mg for each) were dissolved in 1 mL of THF to form homogeneous solution. The solution was added into 9 mL of water and the mixture was sonicated using a microtip probe sonicator (XL2000, Misonix Incorporated, NY) for 60 s at 12 W output. The obtained dot suspension was vigorously stirred at room temperature overnight in fumehood at 800 rpm to evaporate the organic solvent to yield TPETPAFN dots (AIE dots) with surface amine and maleimide groups in 8 mL of water. The AIE dots were fi ltered using a 0.2 μ m syringe driven fi lter to purify. 1 mL of borate buffer (pH = 8.5) and 200 μ L of DTPA dianhydride (2 mg) solution in DMSO was added into 4 mL of AIE dots water suspension and the mixture was stirred at room temperature for 24 h, followed by dialysis against MilliQ water using a 3.5 kDa molecular weight cutoff dialysis membrane for 2 d. Gadolinium(III) chloride hexahydrate (4 mg) was further added to the DTPA-AIE dots suspension for 24 h at room temperature. The free Gd(III) was removed by dialysis against MilliQ water for 2 d using a 3.5 kDa molecular weight cutoff dialysis membrane. To attach HIV-1 Tat peptides on the dots, Gd-AIE dots were mixed with HIV1-Tat peptide (0.1 μ mol) and stirred overnight. After dialysis using a 3.5 kDa molecular weight cutoff dialysis membrane, the fi nal product, Tat-Gd-AIE dots were obtained and concentrated through freeze-drying. The water after each run of dialysis was collected and concentrated for HPLC measurements to determine the number of peptides conjugated on dot surface. The Gd(III) content in Tat-Gd-AIE dots was determined by ICP-MS.
In Vitro Cell Imaging : C6 glioma cells were precultured in four-well confocal chamber (Costar, IL, USA) to achieve 80% confl uence. The medium was then removed and the cells were washed twice with 1 × PBS buffer, followed by addition of 2 × 10 − 9 M Tat-Gd-AIE dots in DMEM for incubation at 37 ° C. After 12 h, the cell monolayer was washed with 1 × PBS buffer and fi xed by 75% ethanol for 20 minutes. The confocal images were obtained using Leica TCS SP 5X upon excitation at 514 nm (1 mW) and the fl uorescence signal was collected using a 550-800 nm bandpass fi lter.
Cytotoxicity Studies : Cytotoxicity of Tat-Gd-AIE dots was evaluated using C6 glioma cells through MTT assay. In brief, C6 glioma cells were seeded in 96-well plates (Costar, IL, USA) at a density of 4 × 10 4 cells mL − 1 . After 24 h incubation, the medium was discarded and Tat-Gd-AIE dots in DMEM with various concentrations were added into each sample wells for further incubation at 37 ° C. To eliminate the UV absorption interference of TPETPAFN at 570 nm, the cells incubated with a series of Tat-Gd-AIE dots at the same doses but not post treated by MTT were used as the control. After the designated time intervals, the sample wells were washed twice with 1 × PBS buffer and 100 μ L of freshly prepared MTT (0.5 mg mL − 1 ) solution in culture medium was added into each well. After 3 h incubation in the incubator, the MTT medium solution was carefully removed, followed by addition of 100 μ L of DMSO into each well and the plate was gently shaken for 10 min at room temperature to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan). Cell viability was expressed by the ratio of the absorbance of the cells incubated with Tat-AIE dot suspension to that of the cells incubated with culture medium only.
In Vivo Cell Tracing : All animal experiments were performed in compliance with guidlines set by the Institutional Animal Care and Use Committee (IACUC), Sigapore General Hospital. C6 glioma cells were cultured in 6-well plates (Costar, IL, USA) to achieve 80% confl uence. After medium removal and washing with 1 × PBS buffer, 2 × 10 − 9 M Tat-Gd-AIE dots in DMEM medium were then added to the wells for incubation at 37 ° C. After 12 h, the cells were washed twice with 1 × PBS buffer and detached by 1 × trypsin. Trypsin was discarded through centrifugation at 2000 rpm for 5 min and the labeled cells were resuspended in culture medium. The labeled C6 glioma cells (0.1 mL, 1 × 10 6 cells) were then intravenously injected into each mouse. Three mice were used for each group. After designated time intervals post injection, the mice were imaged using an IVISSpectrum imaging system (Caliper Life Sciences) while under anesthesia. The fl uorescence images were recorded with 1 s exposure using a fi lter 660/20 nm upon excitation at 535 nm. Scans were carried out on 1, 3, 6, and 24 h, respectively. The autofl uorescence was removed using the software of IVIS Spectrum imaging system.
Ex Vivo One-Photon Excited Fluorescence Imaging : After in vivo imaging at designated time intervals upon injection of labeled C6 glioma cells, the mice were euthanized with CO 2 to harvest the organs (heart, kidney, spleen, lung, and liver) for isolated tissue imaging using the IVIS ® Spectrum imaging system. The organs collected at 24 h post injection of labeled C6 glioma cells were then fi xed in 4% paraformaldehyde and imaged using confocal microscope (Leica TCS SP 5X) upon excitation at 560 nm with a 600-800 nm bandpass fi lter. The one-photon excited fl uorescence images of consecutive layers with approximately 3 μ m interval were recorded to generate 3D colored projection to demonstrate the penetration depth of fl uorescence signal from Tat-Gd-AIE dotslabeled cells in the lung.
In Vivo MRI Studies : The labeled Tat-Gd-AIE dot-labeled C6 glioma cells (0.2 mL, 3 × 10 6 cells) were intravenously injected into the mice. After designated time intervals post injection, the MR images of mice under anesthesia were conducted on a 7Tesla MRI Bruker ClinScan. T 1weighted images were acquired at 1 and 24 h.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.