Engineered Gadolinium-doped Carbon Dots For Magnetic Resonance Imaging-guided Radiotherapy Of Tumors

Objective:Radiotherapy is one of most efficient protocols used for solid tumors. Despite these advantages, but two critical radiotherapy-related challenges limit the clinical potentials of radiotherapy. Firstly, the inaccuracy in tumor localization could lead to inadequate dose coverage to the tumour target and overexposure to surrounding normal tissues, resulting in the occurrence of radiation-induced toxicity. Secondly, inherent radioresistance of tumors needs high irradiation dose for effective eradication of cancerous cells. In this study, we fabricated a bifunctional gadolinium-doped carbon dots(Gd-doped CDs) by simple hydrothermal carbonization for effective magnetic resonance(MR) imaging and radiosensitization of tumors to overcome the inaccuracy in tumor localization and inherent radioresistance of tumors. Methods:We prepared Gd-doped CDs by simple hydrothermal carbonization of gadopentetic acid(Gd-DTPA) as the gadolinium source material and glycine as the passivation agent. The physical and Chemical Properties of the Gd-doped CDs was characterized by transmission electron microscopy(TEM), X-ray diffraction(XRD), Fourier transform infrared spectrometry(FTIR), and X-ray photoelectron spectroscopy(XPS). The optical properties of the Gd-doped CDs were characterized with UV/Vis and photoluminescence(PL) spectroscopy. The magnetic properties of the Gd-doped CDs were characterized by Nuclear Magnetic Resonance Spectrometer. The biocompatibility characterization of Gd-doped CDs was characterized by CCK-8 assay, hemolysis assay, fluorescence microscopy images, histological analysis and ICP-MS. The MR imaging for tumor of Gd-doped CDs was demonstrated by BALB/c mice, the subcutaneous and in situ tumor models, and histopathological and immunohistochemical staining assay. The radiosensition of Gd-doped CDs was investigated by clonogenic assay in vitro and subcutaneous tumor models in vivo. Results:1. Synthesis and characterization of Gd-doped CDsWe successfully synthesized the Gd-doped CDs. As shown in the TEM images, the Gd-doped CDs obtained were discrete and quasi-spherical, with an average size of ~18 nm and relatively narrow size distribution. High resolution TEM found low diffraction and no discernible lattice fringes. X-ray diffraction(XRD) pattern further investigated the phase structure of Gd-doped CDs were between graphene-like and graphite oxide(GO)-like structure. FTIR spectroscopy showed the Gd-doped CDs composed of C=O and COO-. The XPS survey spectrum of the as-produced Gd-doped CDs showed Gd atoms incorporated into the nanoparticles and mainly composed of carbon, nitrogen, oxygen and gadolinium atoms. UV/Vis and photoluminescence(PL) spectroscopy illustrated that the prepared Gd-doped CDs exhibit favorable fluorescent properties and high dispersibility in an aqueous solution. The r1 value of Gd-doped CDs was measured to be 6.45 mM-1s-1.2. Biocompatibility characterization of Gd-doped CDsGd-doped CDs exhibited extremely low cytotoxicity in both cell lines, with cell viability at approximately 90% even at a Gd-doped CD concentration of 200μg/mL and a 72 h exposure time(p?0.05). No obvious hemolysis was observed in different concentrations of Gd-doped CDs groups(50-400μg/m L). Gd-doped CDs possess stable and bright green fluorescence emitted from UV. After post-injection, spleen and heart have uptake of the Gd-doped CDs, especially spleen and heart, with an uptake of 0.129 g/Kg in the spleen and 0.032 g/Kg in the heart. Histological assessment of the harvested susceptible organs(liver, heart, spleen, lung, and kidney) showed that the Gd-doped CDs did not cause obvious adverse effects, such as tissue damage or inflammation, when compared to the control group, showing good organ compatibility.3. Experimental study on the MR imaging for tumor using engineered gadolinium-doped carbon dotsT1-weighted images of mice were enhanced after intravenously injecting of Gd-DTPA and Gd-doped CDs, especially in brighter regions of kidney and bladder. The signal intensity of kidney and bladder after injecting of Gd-DTPA for 1h was a little decreased, with no contrast-enhancement for 6h, on the contrary, the signal intensity in the major organ of Gd-doped CDs was still higher for 6h with contrast-enhancement. The signal intensity of subcutaneous tumor region had no difference in the surrounding, but much higher in the normal tissues. The RES of the subcutaneous tumor dramatically reached 1.93 and 1.74 with coronal and transverse scanning, respectively. The signal intensity of in situ liver cancer models was lower than the surrounding tissues, where the RES of the tumor in situ decreased to 0.74 and 0.77 with the coronal and transverse scanning. Histopathological H&E staining and immunohistochemical staining analyses showed the presence of pathological tissue changes and hepatocellular carcinoma in both the subcutaneous tumor samples and the liver.4. Study on the radiosensition of Gd-doped CDs in vitro and in vivoNo significant difference in inhibition was found in Gd-doped CDs group, while the combination of X-rays and Gd-doped CDs significantly inhibited colony formation(P<0.05). The as-prepared Gd-doped CDs have radiosensitivity effect. With increasing doses of radiation and Gd-doped CDs, the survival fraction of HepG2 cells dramatically decreased to 64.9% during treatment with 8Gy radiation and 200μg/m L of Gd-doped CDs(P<0.05). The tumor growth curve showed tumor volume increased rapidly both in control and single Gd-doped CDs groups during the entire period of investigation, enlarging nearly 3-fold in 7 days. In the group treated with X-ray irradiation alone, tumor growth was inhibited during this process, as seen by a decrease in tumor volume of approximately 12%. In contrast, the Gd-doped CDs treatment combined with radiation group showed that the tumors shrank by a maximum volume percentage of 53%(P<0.05). Conclusion:1. We successfully synthesized the Gd-doped CDs by simple hydrothermal carbonization of gadopentetic acid(Gd-DTPA) as the gadolinium source material and glycine as the passivation agent.2. Gd-doped CDs were discrete with favorable solubility in water and relatively narrow size distribution, the phase structure were between graphene and graphite oxide(GO), composed of C=O and COO-, with Gd atoms incorporating into the nanoparticles and mainly composed of carbon, nitrogen, oxygen and gadolinium atoms. UV/Vis and photoluminescence(PL) spectroscopy illustrated that the prepared Gd-doped CDs exhibit favorable fluorescent properties and high dispersibility in an aqueous solution. Gd-doped CDs possess good biocompatibility.3. Gd-doped CDs with high r1 value have the potential to enhance MRI contrast on T1 weighted sequences and provide a great opportunity for tumor-targeting imaging through the long residence time an EPR effect.4. The as-prepared Gd-doped CDs have been demonstrated to be capable of enhancing irradiation dose deposition by a radiosensitizing effect.