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Dec 31, 2016 - Carcinoembryonic antigen (CEA) antibody is conjugated onto the surface of UCNPs to achieve the targeted imaging of early CC tumors, which ...
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Yingying Jin, Dalong Ni, Jiawen Zhang,* Fang Han, Jing Wang, Lu Gao, Hua Zhang, Yanyan Liu, Zhaowen Cui, Zhenwei Yao, Xiaoyuan Feng, and Wenbo Bu* Therefore, early diagnosis of CC is crucial for patients’ prognosis. At present, the imaging techniques adopted for the diagnosis of CC include computerized tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET).[5] Among these imaging modalities, MRI has been proven to be inherently superior.[6] MRI can depict anatomical structures clearly and accurately owing to its character­ istics of high tissue resolution, multi­ parameter imaging, and nonradiological injury.[7] To improve the resolution of MRI, contrast agents (CAs), such as clini­ cally used gadolinium diethylenetriamine penta-acetic acid (Gd-DTPA), are used to brighten MRI signals.[8] The application of Gd-DTPA, however, in the early diagnosis of CC is seriously compromised by its low aggregation in colon tissue,[9] poor tumor-targeting ability,[10] and rapid renal clearance.[11] There­ fore, novel CC-targeting MR CAs are required to realize early diagnosis. Besides the modality of CT, MRI, and PET, fluorescence imaging is another powerful tool for cancer diagnosis. Upcon­ version nanoparticles (UCNPs) can be excited by near-infrared (NIR) light and emit strong visible-to-NIR luminescence signal.[12] Compared with the existing fluorescent probes, UCNPs have more narrow emission peak, longer lifetimes, higher photostability, and lower toxicity, which enable UCNPs to be ideal nanoprobes for bioimaging[13] and biodetection.[14] In addition, fluorescence nanoparticles are also of great impor­ tance in cancer destruction.[15] Several approaches destructing cancer with fluorescence nanoprobes have been reported, such as supplementing conventional radiation therapy with photo­ dynamic therapy for cancer treatment.[16] Therefore, UCNPs have been widely used in multimodal imaging, drug delivery, cancer treatment, etc. As the origin of the magnetism of CAs is attributed to Gd3+ ions, Gd3+ ions could be doped to improve T1 relaxivity. On the basis of UCNPs, MR/fluorescence bimodal imaging can be realized with doping of rare-earth ions and Gd3+ ions. Gd3+ based NPs have plenty of advantages over Gd-DTPA, including longer blood circulation half-lives,[17] higher T1 relaxivity, and suitable surface modifications.[18] Meanwhile, rare-earth UCNPs can acquire favorable luminescent proper­ ties with high photostability, high tissue penetration, etc.[19] In addition, considering that carcinoembryonic (CEA) antigens

Colon cancer (CC) is one of the most common intestinal malignancies and is difficult to detect in its early stage by magnetic resonance imaging (MRI) with currently used contrast agents (CAs). The development of targeted CAs contributes to the early diagnosis of CC and thereby enables early intervention and timely therapy. Considering the outstanding performance of upconversion nanoprobes (UCNPs) in high-performance MR and fluorescence imaging, a new type of nanoprobes with considerably enhanced imaging performance is developed herein. Carcinoembryonic antigen (CEA) antibody is conjugated onto the surface of UCNPs to achieve the targeted imaging of early CC tumors, which overexpress CEA. Both toxicity tests and histological/hematological examinations demonstrate the excellent biocompatibility of these CC-targeting nanoprobes, which possess great potential for clinical application in the early diagnosis of CC.

1. Introduction Colon cancer (CC) is one of the most aggressive and common intestinal malignancies, and causes millions of deaths every year.[1] As there is no obvious clinical feature such as bleeding and abdominal pain at the early stage, people do not notice it until the tumor is too late to cure.[2] Known as a “silent disease,” early colon cancer is of great difficulty to diagnose. It has been reported that the survival rate of CC patients depends on the tumor stage at diagnosis.[3] The five-year survival rate for stage I cancer is 93.2%, while that for stage IV cancer is only 8.1%.[4] Dr. Y. Jin, Prof. J. Zhang, Dr. F. Han, Dr. J. Wang, Dr. L. Gao, Dr. H. Zhang, Prof. Z. Yao, Prof. X. Feng Department of Radiology Huashan Hospital Fudan University 12 Middle Urumqi Road, Shanghai 200040, China E-mail: [email protected] Dr. D. Ni, Dr. Y. Liu, Dr. Z. Cui, Prof. W. Bu State Key Laboratory of High Performance Ceramics and Superfine Microstructures Shanghai Institute of Ceramics Chinese Academy of Sciences 1295 Dingxi Road, Shanghai 200050, China E-mail: [email protected] Prof. W. Bu Shanghai Key Laboratory of Green Chemistry and Chemical Processes School of Chemistry and Molecular Engineering East China Normal University Shanghai 200062, P. R. China

DOI: 10.1002/ppsc.201600393

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Targeting Upconversion Nanoprobes for Magnetic Resonance Imaging of Early Colon Cancer

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2. Results and Discussion 2.1. Preparation of Core@Shell-Structured NaYF4:Yb/Er@NaGdF4 Nanoprobes

Scheme 1. A) Synthesis of carcinoembryonic antigen (CEA)-upconversion nanoprobes (UCNPs). B) Schematic diagram of CEA-UCNPs as contrast agents to target in situ early colon cancer (CC) for magnetic resonance imaging (MRI).

are overexpressed on the surface of CC cells,[20] specific mono­ clonal CEA antibodies can be used as targeting ligand by being conjugated onto the surface of UCNPs.[21] Herein, we design a novel kind of CC-targeting CAs with CEA decoration, CEA-UCNPs, to realize diagnosis of in situ early colon cancer for both MR and fluorescence imaging. To verify the targeting and imaging capabilities, both in vitro and in vivo investiga­ tions were carried out, and all the results demonstrated that CEA-UCNPs hold a great potential in future MR diagnosis of early CC.

The synthetic process involves three typical steps, as illus­ trated in Scheme 1A. First, monodispersed core@shell-struc­ tured NaYF4:Yb/Er@NaGdF4 nanoparticles were prepared by growing a NaGdF4 shell on NaYF4:Yb/Er core according to our previous work.[19,22] Yb3+ and Er3+ were doped into the core to emit visible optical signal under the excitation of 980 nm NIR light.[23] The coated ultrathin NaGdF4 shell can significantly enhance MRI contrast performance as well as upconversion luminescence intensity. Then, to address the hydrophobicity of UCNPs, hydrochloric acid was used to transfer hydrophobic UCNPs into an aqueous phase by removing oleic acid (OA).[24] Biocompatible SH–polyethylene glycol (PEG)–NH2 was conju­ gated onto the surface of the UCNPs based on the interaction between the thiol groups and lanthanide ions.[25] Finally, the PEGylated UCNPs were covalently decorated with CEA via a “click” reaction between the amino groups on the probe surface and the carboxyl group of CEA,[26] yielding CC-specific nano­ probes (CEA-UCNPs). Transmission electron microscopy and scanning electron microscopy (TEM and SEM, respectively), shown in Figure 1, revealed that the core and core@shell structured nanoprobes were synthesized successfully with uniform spherical shape and well-defined size distributions. The average particle sizes were 30.24 ± 1.5 and 31.35 ± 1.2 nm, respectively. Thus, the thick­ ness of the NaGdF4 shell was ≈1 nm. The pure hexagonal phase structure of the nanoprobes was clearly seen in powder X-ray diffraction spectrum (Figure S1A,B, Supporting Information),

Figure 1. Transmission electron microscopy images of A) core (NaYF4:Yb/Er), B) upconversion nanoprobes (UCNPs; NaYF4:Yb/Er@NaGdF4), C) carcinoembryonic antigen (CEA)-UCNPs. D) Scanning electron microscopy image of CEA-UCNPs. E–K) Element mappings (Na, F, Y, Gd, Yb, and Er) of CEA-UCNPs.

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2.2. Cellular Uptake of CEA-UCNPs The specific targeting ability of CEA-UCNPs was verified in vitro by confocal microscopic imaging. LoVo (a human CC strain) with CEA overexpression and bovine caruncular epi­ thelial cells (BCECs; endothelial cells) without CEA expression were cocultured with CEA-UCNPs and PEG-UCNPs under the same conditions. In the LoVo cell strain incubated with CEA-UCNPs (Figure 2A), the maximum upconversion fluores­ cence under 980 nm laser excitation was observed around the nucleus, demonstrating that the CEA-UCNPs had been effec­ tively taken into the cytoplasm of CC cells. In contrast, there were only faint fluorescence signals in those LoVo cells which cocultured with PEG-UCNPs (Figure 2B). Given the fact that CEA was overexpressed on the surface of LoVo cells,[30] the notably higher uptake of CEA-UCNPs than PEG-UCNPs by

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LoVo cells should be attributable to the targeting interaction between antibody and antigen.[31] This was further proven by a typical blocking assay. The fluorescence observed around the nucleus was quite weak when LoVo cells were incubated with CEA-UCNPs under high-dose CEA blocking (Figure 2C). In BCEC strain incubated with either CEA-UCNPs or PEG-UCNPs (Figure 2D–F), no significant fluorescence signal was observed around the nucleus, demonstrating that CEA-UCNPs have no specific targeting ability toward cells without CEA antigen. Therefore, CEA-UCNPs can be significantly taken in by LoVo cells owing to their specific targeting ligand. We also carried out a typical 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay to test the in vitro cytotoxicity against LoVo cells and BCECs. The results showed that the nanoprobes had low toxicity (85% survival rate) even at an extremely high concentration (1000 µg mL−1) for 24 h (Figure S4A, Supporting Information).

2.3. MR and Fluorescence Imaging of CC CC-bearing mice were used as model to verify the in vivo CCtargeting capability of CEA-UCNPs. To confirm the tumor loca­ tion, we performed plain T1-weighted MRI before the injection of CAs. Then, contrast T1-weighted MRI was performed at 0.5, 3, 6, and 9 h after the intravenous injection of Gd3+ based CEAUCNPs, Gd3+ based PEG-UCNPs, or Gd-DTPA (control). Under the same scanning conditions, the tumors were more obviously enhanced and the tumor boundary was more clearly deline­ ated in the targeting group (Gd3+ based CEA-UCNPs) than in the nontargeting group (Gd3+ based PEG-UCNPs) and the control group (Gd-DTPA) (Figure 3A–C). Moreover, at 9 h after the injection, the tumor in the nontargeting group was slightly enhanced and could be barely differentiated from surrounding normal intestine (Figure 3B, arrows). As a complication of colon cancer, intestinal obstruction presented as the stasis of CAs and dilatation of intestine adjacent to tumor, and indi­ rectly displayed the location of colon cancer (Figure 3B, 9 h). The boundary of the tumor in Gd-DTPA group cannot been seen clearly as shown in Figure 3C. Quantitative analysis of the signal intensity (SI) of the tumor area further confirmed the tumor-targeting capability of Gd3+ based CEA-UCNPs (Figure S8, Supporting Information). During 24 h after injection, SI of Gd3+ based CEA-UCNPs increased significantly to twofold at 3 h after injection and decreased to 1.2-fold at 9 h. Then, there was a slight drop in SI after that. Owing to the enhanced per­ meability and retention effect,[32] the SI in the Gd3+ based PEGUCNP group experienced a rapid increase and reached a peak to about 1.3-fold at 1–2 h after the injection. In the Gd-DTPA group, the SI rose slightly over ≈30 min and declined due to rapid renal clearance from the blood pool. In the whole period, the SI of Gd3+ based CEA-UCNPs remained consistently higher than that of Gd3+ based PEG-UCNPs and Gd-DTPA, due to the highly efficient CC-targeting capability of CEA antibodies. From the MRI and corresponding SI enhancement data, we can conclude that Gd3+ based CEA-UCNPs indeed possessed many advantages over Gd-DTPA in CC-targeting MRI, such as higher imaging performance and longer imaging time. These advantages should be ascribed to CEA antibodies decorated

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and all the expected basic chemical elements (Na, Y, Yb, Er, Y, Gd) were displayed in the energy-dispersive X-ray (EDX) spec­ trum (Figures S2B and S3B, Supporting Information). During the entire synthesis procedure, there were no obvious changes in the composition and morphology of the UCNPs after sur­ face modification, as verified by TEM (Figure 1A–C), SEM (Figure 1D), and Scanning Transmission Electron Microscopy (STEM) (Figure 1E–K). In the dynamic light scattering meas­ urements (Figure S4, Supporting Information), the growing size of the UCNPs after each step of surface modification con­ firmed the presence of hydrated layers, PEG chains, and CEA modifications, while the zeta-potential (Figure S4, Supporting Information) measurement showed a negative shift trend. Moreover, the changes of functional groups were verified by Fourier transform infrared spectra (Figure S5, Supporting Information). The existence of S and O elements in the EDX spectra of the CEA-UCNPs further demonstrated the successful conjugation of PEG and CEA (Figure 3B). The surface-associated Gd ions offered the nanoprobes a concentration-dependent MRI contrast effect. The MRI con­ trast enhancement effect of Gd3+ based NPs was evaluated on a 3.0 T MRI instrument. By linear regression fitting of the R1 values at different Gd ion concentrations, the relaxivity (r1) of the nanoprobes was tested to be 9.672 × 10−3 m−1 s−1 (Figure S6, Supporting Information). According to the reported “negativelattice shield effect,” the shortened T1-weighted relaxation time of water protons was mainly attributed to the surface Gd in the nanoprobes.[27] Consequently, core@shell structured Gd3+ based NPs with higher surface Gd ion concentrations possessed a dramatically increased r1 value compared with the core structured nanoparticles. Moreover, the PEGylation endowed Gd3+ based NPs with good biocompatibility as well as long in vivo blood circulation half-life.[28] Compared with GdDTPA, which undergoes rapid renal metabolization,[29] Gd3+ based NPs can avoid rapid uptake by the reticuloendothelial system, resulting in a long circulation half-life. In addition, as the Upconversion Luminescence (UCL) spectra of nanoprobes showed, there were two weak bands (510–550 nm) and a strong NIR band (650 nm) under 980 nm excitation (Figure S7, Sup­ porting Information). Therefore, Gd3+ based NPs may improve MRI contrast imaging quality effectively.

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Figure 2. Confocal electron microscopic images of LoVo cells and BCECs incubated with A,D) carcinoembryonic antigen (CEA)-upconversion nanoprobes (UCNPs), B,E) polyethylene glycol (PEG)-UCNPs, and C,F) CEA-UCNPs under CEA blocking. The concentration of all nanoprobes is 200 µg mL−1.

on the Gd3+ based NPs which can target CEA antigens over­ expressed by CC cells. By anchoring on tumors, Gd3+ based CEA-UCNPs may not be washed away by blood flow immedi­ ately and provide a better contrast imaging possibility. Moreover, upconversion luminescence imagings of tumor sections excised from CC-bearing mice were acquired at 3 h after injection of CEA-UCNPs (Figure S9, Supporting Informa­ tion). In the bright field image, the shape of tumor could be seen directly. The UCL image demonstrated the distribution of CEA-UCNPs. After merging with 4′,6-diamidino-2-phenylin­ dole (DAPI) staining imaging, we could see that nanoprobes were taken in by CC cells effectively. In Figure S9B (Supporting Information), it was obvious that the luminescent signal was very strong under the excitation of 980 nm light, displaying significant fluorescence property. The consistent distribution of nanoprobes (green) and nuclear (blue) in Figure S9C (Sup­ 1600393  (4 of 7)

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porting Information) showed high targeting uptake rate of CEA-UCNPs by CC cells. The hematoxylin and eosin (H&E) staining further confirmed the existence of tumor in the colon tissue (Figure S10, Supporting Information).

2.4. Toxicity Studies To enable the clinical application of nanoprobes, it is crucial that the in vivo biocompatibility is meticulously tested. Herein, we used body weight monitoring, blood biochemistry, and hematology tests, and H&E staining analysis to evaluate the in vivo toxicity of CEA-UCNPs. Fluctuation in body weight is considered to be a direct indicator of in vivo biocompatibility. The body weights of mice that were or were not injected with CEA-UCNPs were recorded for one month, and the results are

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FULL PAPER Figure 3.  In vivo MRI of in situ colon cancer-bearing mice before and after the injection of A) carcinoembryonic antigen (CEA)-upconversion nanoprobes (UCNPs), B) polyethylene glycol (PEG)-UCNPs, and C) gadolinium diethylenetriaminepenta-acetic acid (Gd-DTPA).

displayed in Figure S11 (Supporting Information). As there was no obvious difference in the variation in body weights between the injected and noninjected mice, we can conclude that CEA-UCNPs have no overall toxicity. In order to assess the organ-specific toxicity, tissue sections from the heart, liver, spleen, lung, and kidney were stained with H&E after the injection of CEA-UCNPs for 3, 15, and 30 d. No visible tissue damage or any other side effect was observed in these organs compared with the control group (Figure S12, Supporting Infor­ mation), demonstrating good biocompatibility of CEA-UCNPs. Finally, we examined the effects of CEA-UCNPs on serological and hematological indices. Analysis of various liver-function markers (alanine transaminase [ALT], aspartate transaminase [AST], and alkaline phosphatase [ALP]) and kidney-function markers (blood urea nitrogen [BUN] and creatinine) indicated that CEA-UCNPs had negligible hepatic and renal toxicity (Figure 4A–C). Furthermore, hematological analysis revealed that the blood parameters in the test group tended to be within normal limits. Collectively, the above results demonstrate that CEA-UCNPs have low toxicity, good biocompatibility, and great potential as new CAs for CC-targeted MRI.

3. Conclusion In summary, we successfully synthesized a novel type of CCtargeting nanoprobes with greatly enhanced MRI contrast per­ formance and significant fluorescence properties. Owing to the tumor-targeting specificity, CEA-UCNPs exhibited dramatically increased capability for detecting early CC, as demonstrated

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by both cellular and animal experiments. Compared with GdDTPA, CEA-UCNPs showed better MRI performance and longer blood circulation half-life. Moreover, there were no obvious side effects observed in the toxicity tests, implying good biocompatibility of CEA-UCNPs. Therefore, it can be concluded that CEA-UCNPs have the great potential to serve as markers for the early diagnosis of CC in the future.

4. Experimental Section Materials: YCl3·6H2O, YbCl3·6H2O, ErCl3·6H2O, GdCl3·6H2O, 1-octacene, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and ammonium fluoride were acquired from Sigma-Aldrich. OA was purchased from Shanghai Lingfeng Chemical Reagent Co. Ltd. Sodium hydroxide and hydrochloric acid were obtained from Sinopharm Chemical Reagent Co. Ltd. Gd-DTPA was given by Huashan Hospital. CEA antibody (ab105364) was acquired from Abcam. Enhanced Cellular Uptake In Vitro: The LoVo strain and BCEC strain were cultured in an incubator (Thermo) at 37 °C under 5% CO2. CEAUCNPs and PEG-UCNPs, with a concentration of 200 µg mL−1, were dispersed in F12K and RPMI 1640 media, respectively. These media were added into culture flask 0.5 h before DAPI staining. Then, phosphatebuffered saline was used to wash away the free nanoprobes, and methanal was applied for fixation. Subsequently, confocal fluorescence microscopy was carried out with a CW NIR (Continuous Wave Near Infred) laser at 980 nm to measure the photoluminescence of the nanoprobes and at 358 nm to assess DAPI staining. Images were acquired with a 60× oil immersion objective lens. In the blocking groups, cells were pretreated with a high concentration of CEA (3 mg mL−1) for 20 min. Cytotoxicity Test: LoVo cells were cultured in F12K medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under

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Figure 4.  Toxicity studies of nanoprobes in vivo. A) Liver-function indicators. B,C) Kidney-function indicators. D–P) Blood routine examinations. All of the tested blood samples were obtained from mice intravenously injected with CEA-UCNPs (n = 4, dose = 15 mg Y per kg) at various time points or from the control group mice, which were not injected with anything (according to homogeneity test of variance, p value > 0.05, there is no difference among these four groups). BPC, blood platelet count; BUN, blood urea nitrogen; CEA, carcinoembryonic antigen; CREA, creatinine; HCT, hematocrit; HGB, hemoglobin; LYM, lymphocytes; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MONO, monocytes; NEUT, neutrophils; PDW, platelet distribution width; RBC, red blood cell; RDW-SD, red cell distribution width-standard deviation; UCNP, upconversion nanoprobe; WBC, white blood cell. 5% CO2, while human capillary endothelial cells (BCECs) were incubated in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS and 1% penicillin/streptomycin under the same conditions. Typical MTT assay was performed to examine the in vitro cell cytotoxicity. Cells were seeded into 96-well cell-culture plates with graded concentrations (7.81, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 µg mL−1) of nanoprobes dispersed in the medium. The cells were incubated with the nanoprobes for 24 h, and then, their survival ratio was tested by Enzyme Linked Immunosorbent Assay (ELISA). In Vivo MRI of CC: All animal experiments complied with the guidelines of the Institutional Animal Care and Use Committee. The tumor-bearing mouse model was established by Silaike Laboratory Animal Co. Ltd. (Nanyang No. 1, Songjiang District). A piece of tumor tissue was sutured onto the surface of the colon to create the tumorbearing model. In vivo MRI was performed using a 3.0 T clinical MRI instrument (GE Discovery 3.0 T) with the turbo spin echo (TSE) sequence: repetition time, 400 ms; excitation time, 12 ms; Time decay (TD), 0.0 ms; slice thickness, 1.0 mm; field of view read, 8 mm; fat suppression, none; and water suppression, none. Before the performance of MRI, CEA-UCNPs, PEG-UCNPs, and Gd-DTPA were intravenously injected at a dose of 100 µL (6 mg Gd per kg, n = 3). Images were collected at 0, 0.5, 3, 6, and 9 h after the

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injection. The SIs of regions of interest on contrast-enhanced MRI were also measured at postprocessing work station. Fluorescence Imaging of CC: CEA-UCNPs were intravenously injected at a dose of 100 µL (6 mg Gd per kg, n = 3). The mice were sacrificed 3 h after injection to get the DAPI staining of cancer tissue. Then, confocal fluorescence microscopy was carried out with the same parameters as cellular uptake in vitro. Images were acquired with a 60× oil immersion objective lens. Toxicity Studies: Kunming mice (average weight, 25 g) were purchased from Laboratory Animal Center, Shanghai Medical College, Fudan University for toxicity studies. CEA-UCNPs were intravenously injected in the mice. Then, blood samples and tissues were harvested after 3, 15, and 30 d. A group of mice that received no injections served as the control group. Three important hepatic-function indicators (ALT, AST, and ALP) and two kidney-function indicators (creatinine and BUN) were tested in each group. In addition, routine blood analysis was performed. After the collection of blood samples, the mice were sacrificed for tissue section. H&E staining of the heart, liver, spleen, lung, and kidney specimens was proceeded to examine the tissue damage caused by the injected nanoprobes. All the above hematology tests and H&E staining analysis were carried out at Huashan Hospital.

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

Acknowledgements Y.J. and D.N. contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant Nos. 51372260, 81271632), the Shanghai Excellent Academic Leaders Program (Grant No.16XD1404000), and the Shanghai Health and Family Planning Commission Scientific Research Foundation (Grant No. 20114186). The authors thank Prof. Lixin Chen (Shanghai Silaike Laboratory Animal Technology Co.) for his help with building the in situ CC mice model. The authors also thank Zunguo Du (Huashan Hospital) for his help with H&E staining and pathologic analysis. The authors are grateful to Heliang Yao and Linlin Zhang (Shanghai Institute of Ceramics, Chinese Academy of Science) for their useful discussions. Received: December 5, 2016 Revised: December 31, 2016 Published online:

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