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Dan Xu,1 Shu-Ting Lu,2 Yu-Shuang Li,2 Aju Baidya,2 Hao Mei,2 Yong He,1 Bo Wu2 1Department of Nuclear Medicine, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, PR China; 2Department of Radiology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, PR China Background: Gliomas are one of the most common types of primary brain tumors. It is usually evaluated by gadolinium(III)-based contrast agents by magnetic resonance imaging (MRI) in the clinic. Methotrexate (MTX), as a type of folate analog that inhibits the enzyme dihydrofolate reductase, is widely used as a chemotherapeutic agent to treat gliomas in the experiment.Purpose: In this study, a novel theranostic agent MTX-DOTA-Gd (MTX-Gd) was synthesized, which integrates magnetic resonance imaging (MRI) with anticancer treatment.Methods: MTX-Gd was synthesized by connecting MTX and Gd through 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The characterization of MTX-Gd was detected by ultraviolet (UV) and infrared spectroscopy (IR). To confirm the antitumor effect of MTX-Gd, the cytotoxicity of MTX-Gd was examined by the MTT assay. The contrast enhancement of the MTX-Gd was measured through MRI in vitro. Then, nude mice bearing C6 tumor xenografts were used to study in vivo imaging capabilities.Results: The ultraviolet-visible-near infrared radiation (UV-NIR) absorption curve indicated that MTX-Gd had a broad absorption in the region of 500-700 nm. The formation of MTX-Gd was confirmed from the characteristic bands of MTX-DOTA-Gd in the 1413 cm-1 (C-N), 1577 cm-1 (-NH2), and 3429 cm-1 (N-H), in the fourier-transform infrared (FTIR) spectra. MTX-Gd showed little difference in the cell viability compared with MTX, except for the highest concentration (270 μM). In vitro, the imaging of MTX-Gd was significantly brighter than Gd-DOTA at the same concentration, and the brightness and signal intensity of MRI were increased followed by the increased concentration of MTX-Gd. And it also showed that MTX was not visualized on MRI. The other images revealed that the concentration of 4 mM MTX-Gd had the same imaging effect with the concentration of 10 mM Gd-DOTA. Then, MTX-Gd was injected in nude mice bearing C6 tumor xenografts through the tail vein. Significant contrast enhancement was observed at the tumor site from 0.5 h to 3 h. The signal of tumor area was strongest at 3 h due to accumulation by size effect of macromolecules.Conclusion: A novel stable and unique theranostic agent(MTX-Gd) was successfully synthesized, and it has good stability, strong anticancer ability and excellent magnetic capacity. The methotrexate component of MTX-Gd, as a chemotherapeutic agent, played an important role in targeted therapies of cancer. The DOTA-Gd component of MTX-Gd performed as the MRI contrast agent. The superior MRI imaging performance and synergetic chemical antineoplastic ability of MTX-Gd was revealed, and it has great potential in the diagnosis and treatment of glioma and potentially other cancers, with prospects of clinical application in the near future. Keywords: methotrexate, gadolinium, glioma, theranostics

Bo Wu,1,* Bing Wan,2,* Shu-Ting Lu,1 Kai Deng,3 Xiao-Qi Li,1 Bao-Lin Wu,1 Yu-Shuang Li,1 Ru-Fang Liao,1 Shi-Wen Huang,3 Hai-Bo Xu1,2 1Department of Radiology, Zhongnan Hospital of Wuhan University, 2Department of Radiology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, 3Department of Chemistry, Key Laboratory of Biomedical Polymers, Ministry of Education, Wuhan University, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: The major challenge in current clinic contrast agents (CAs) and chemotherapy is the poor tumor selectivity and response. Based on the self-quench property of IR820 at high concentrations, and different contrast effect ability of Gd-DOTA between inner and outer of liposome, we developed “bomb-like” light-triggered CAs (LTCAs) for enhanced CT/MRI/FI multimodal imaging, which can improve the signal-to-noise ratio of tumor tissue specifically. IR820, Iohexol and Gd-chelates were firstly encapsulated into the thermal-sensitive nanocarrier with a high concentration. This will result in protection and fluorescence quenching. Then, the release of CAs was triggered by near-infrared (NIR) light laser irradiation, which will lead to fluorescence and MRI activation and enable imaging of inflammation. In vitro and in vivo experiments demonstrated that LTCAs with 808nm laser irradiation have shorter T1 relaxation time in MRI and stronger intensity in FI compared to those without irradiation. Additionally, due to the high photothermal conversion efficiency of IR820, the injection of LTCAs was demonstrated to completely inhibit C6 tumor growth in nude mice up to 17 days after NIR laser irradiation. The results indicate that the LTCAs can serve as a promising platform for NIR-activated multimodal imaging and photothermal therapy. Keywords: light triggered, near-infrared light, tumor-specific, multimodal imaging, photothermal therapy, contrast agents