Atomically Precise Mn₁₂ Clusters For Cancer Radiosensitization And Radioprotection

Objective:Radiation therapy（radiotherapy）is one of the most widely used tumor treatment in recent decades.However,its efficacy is limited by the radiation dose and tumor tissues may not be completely eradicated and cause further relapse and metastasis.The effect of tumor radiotherapy is usually limited by the radiation resistance of hypoxic tumor cells,and excessive radiation dose may damage the healthy tissue around the lesion and produce serious toxic side effects.In this work,Mn₁₂ clusters with stable structure and accurate atomicity are selected,and their catalytic direction changes in different pH environments are used to achieve the dual functions of radiosensitization and radiation protection.Methods:1.Synthesis and characterization of Mn₁₂ clusters.Synthesizing high-yield Mn₁₂clusters by a two-step protocol.Transmission Electron Microscope（TEM）image and X-ray photoelectron spectroscopy（XPS）were used to analyze the appearance,structural composition and characteristics of the clusters.UV-vis spectra of Mn₁₂ clusters were obtained by ultraviolet spectraphotometer.The cyclic voltammetry was used to evaluate the catalytic ability of Mn₁₂ clusters at different pH environments.The salicylic acid method was used to determine the scavenging ability of the clusters to hydroxyl radicals under different pH conditions.2.In vitro behaviors of Mn₁₂ clusters.Mouse breast cancer cells 4T-1 and mouse ovarian cells CHO were used to evaluated in vitro radiation sensitization and radiation protection of Mn₁₂ clusters.The MTT method was used to detect the cytotoxicity of Mn₁₂clusters.DCFH-DA（2’,7’-dichlorofluorescent yellow diacetate）probe was used to detect intracellular reactive oxygen species（ROS）changes.3.In vivo tumor radiosensitization of Mn₁₂ clusters.To prepare the tumor model,murine breast cancer 4T1 cells were subcutaneously injected into the back of each Balb/c mouse.The tumor-bearing mice were randomly divided into three groups:radiation only group,Mn₁₂ cluster plus radiation group and control group.The mice in the administration plus irradiation group and the mice in the radiation only group were irradiated with gamma rays of 5 Gy,3 Gy,and 3 Gy on day 0,day 5,and day 10,respectively,after 12 hours post-injection of PBS or Mn₁₂ clusters.The weights and volume changes of the mice in each group were observed and recorded every two days.After the treatment,the main organs and tumor tissues of the mice were taken for staining analysis,and the oxygen content in the tumor tissues and the pathological changes of the main organs were analyzed.4.In vivo radiation protection experiment of Mn₁₂ clusters.Mice were treated with Mn₁₂clusters（200μl）at a concentration of 10 mg/ml 30 minutes before whole body irradiation.The survival fraction was observed for 30 days.Bone marrow nucleated cells,bone marrow DNA,and levels of superoxide dismutase（SOD）and malondialdehyde（MDA）in the liver and lungs were analysed after 30 days irradiation.Results:1.Synthesis and characterization of Mn₁₂ clusters.High-yield Mn₁₂ clusters were synthesized by a two-step method using insoluble Mn₁₂Ac as an intermediate product.Transmission Electron Microscope（TEM）image revealed that the synthesized Mn₁₂ clusters had a uniform particle size and good dispersibility.Characteristic absorption peaks at 297nm were obtained by UV-vis spectra analysis of Mn₁₂ clusters.X-ray photoelectron spectroscopy analysis found that Mn³⁺and Mn⁴⁺were coexisted in the Mn₁₂ clusters,and the content ratio was about 2:1.The clusters exhibited pH-dependent catalytic capacity by cyclic voltammetry.As the pH value decreased,the oxygen production was gradually increased.Subsequently,the salicylic acid method was used to determine the hydroxyl radicals scavenging ability of the clusters.It was found that the clusters owned higher scavenging ability of radicals in a neutral environment.The characterization analysis of Mn₁₂ clusters laid the foundation for further in vivo and in vitro radiosensitization and radiation protection research.2.In vitro behaviors of Mn₁₂ clusters.The results of in vitro experiments show that the clusters do not exhibit obvious cytotoxicity even at high drug concentration of 300μg/ml.As the dose of radiation increased,the survival rate of 4T1 cells cultured with the clusters decreased significantly.The detection of intracellular reactive oxygen species found that the production of radiation-induced ROS increased in the tumor cells after treated with Mn₁₂clusters.While the ROS levels in normal CHO cells reduced under the neutral pH environment after radiation.The different effects of this clusters in different cells provided great support for subsequent in vivo experiments.3.In vivo tumor radiosensitization of Mn₁₂ clusters.Compared with the radiation only group and the control group,the tumor growth of the Mn₁₂ cluster plus radiation group was significantly inhibited,indicating that the clusters could significantly improve the radiation therapy effect in vivo.In addition,positive signal of HIF-1αwas barely detected in the Mn₁₂clusters plus radiation group,confirming that the tumor hypoxia status was dramatically relieved by the catalytic oxidation process of Mn₁₂ clusters in acidic TME.It was proved by TUNEL staining and H&E staining that levels of apoptosis in the Mn₁₂ clusters plus radiation group was higher than the other control groups.4.In vivo radiation protection experiment of Mn₁₂ clusters.From the 30 days survival rate observation of the mice,the survival rate of the Mn₁₂ clusters plus radiation group was100%,while the survival rate of the radiation only group was only 50%.Through the research of radiation protection mechanism,it was found that bone marrow total DNA and bone marrow nucleated cells of the Mn₁₂ clusters plus radiation group backed to normal level30 days after radiation,while the total DNA content of the radiation alone group was still lower than that in the normal group,suggesting that Mn₁₂ clusters could effectively protect the hematopoietic system during the radiation process.Evaluation of the levels of oxidative stress revealed that there was no significant difference between the Mn₁₂ clusters plus radiation group and normal control group in the levels of SOD and MDA,indicating that the level of oxidative stress in the body could return to normal level after treated with the clusters.It was further confirmed that the clusters could achieve good radiation protection function for normal tissues.Conclusion:Atomically precise Mn₁₂ clusters possess highly catalytic selectivity ability in different pH environments.The clusters could achieve radiosensitization at the tumor site and protect normal tissues from radiation damage simultaneously.In the acidic tumor microenvironment,Mn₁₂ clusters could increase the generation of oxygen in the tumor site during radiotherapy.The killing ability of radiation therapy to tumor tissues was increased by accompanying with Mn₁₂ clusters.At the same time,in the environment where the pH of normal tissues is neutral,the Mn₁₂ clusters could scavenge excessive free radicals during radiation process and protect normal tissues from radiation damage.