【作者】 张伟；

【导师】 冉海涛；

【作者基本信息】 重庆医科大学 ， 影像医学与核医学， 2022， 博士

【摘要】 第一部分纳米材料的制备、表征及基本性能研究目的制备一种卟啉基金属有机框架PCN-224,并在其内装载MTH1抑制剂TH588,随后在金属有机框架表面用聚多巴胺(PDA)进行修饰,检测和表征纳米粒的基本理化性质、细胞毒性等。方法采用马尔文激光粒径及电位仪检测纳米粒的粒径分布和Zeta电位分布;采用透射电镜及扫描电镜检测纳米粒的微观结构和形貌,采用能谱仪分析纳米粒中元素成分分布。采用全自动比表面积和孔径分析仪检测纳米粒的比表面积和孔径。采用紫外-可见分光光度计检测纳米粒的紫外-可见吸收光谱。采用1,3-二苯基异苯丙呋喃(DPBF)探针检测纳米粒的声敏化单线态氧产生能力。采用CCK8法检测纳米粒PCN-224@PDA的细胞毒性,应用激光共聚焦显微镜、流式细胞术以及透射电镜评估乳腺癌细胞对纳米粒的吞噬和摄取情况。结果首先制备了金属有机框架PCN-224,其电镜下呈球形,形貌规则,比表面积为394.25 m2/g,平均孔径为4.54 nm,粒径为(105±16.86)nm,电位为(21.69±4.39)m V。进一步检测发现PCN-224在低强度聚焦超声的辐照下,能够显著产生单线态氧,且产生单线态氧的能力呈超声辐照时间的依赖性的特征。随后,金属有机框架经聚多巴胺修饰后,CCK-8法测得PCN-224@PDA纳米粒对细胞的毒性较小,当纳米粒的浓度高达200μg/m L时细胞存活率仍有85%以上。接着对制备的载药纳米粒PCN-224@PDA@TH588进行了表征,其电镜下呈形貌规则的球形,大小分布均匀,分散性较好,能谱分析发现该纳米粒主要由C、N、O、Zr这四种化学元素组成,平均粒径约为(118±18.75)nm,电位为(-33.67±3.88)m V;PCN-224@PDA与PCN-224@PDA@TH588的紫外吸收光谱最大吸收峰值均在413 nm附近。通过激光共聚焦显微镜观察、流式细胞术定量分析以及TEM观察发现在与纳米粒共孵育4小时后,小鼠乳腺癌细胞能够对其进行有效的吞噬和摄取。结论成功制备了形貌规则、具备均一粒径和较好分散性、生物相容性好的载MTH1抑制剂TH588的多功能纳米粒PCN-224@PDA@TH588,该纳米粒在可以作为声敏剂的同时,也是一种良好的药物载体,可实现将不溶性小分子或毒性化疗药物通过EPR效应靶向递送至肿瘤区域,为下一步进行体内外研究奠定了基础。第二部分纳米材料的光声成像、生物安全性及生物分布的研究目的探究PCN-224@PDA@TH588纳米粒在体内、体外光声的成像能力,评估纳米粒在小鼠体内的生物分布以及生物安全性。方法首先在体外使用小动物光声成像仪扫描不同浓度的PCN-224@PDA@TH588纳米材料,采集其光声图像并测量其信号值。分析尾静脉注射纳米粒后不同时间点(1、7、14天)的健康小鼠的血液常规指标、血生化指标以及主要脏器的病理切片,系统性检测纳米粒的在体内应用的生物安全性。构建小鼠乳腺癌原位移植瘤模型,观察在注射纳米材料之前以及注射后不同时间点小鼠肿瘤区域的光声信号,并测量信号强度。构建小鼠乳腺癌原位移植瘤模型,采用DIR染料标记的纳米粒注射到小鼠体内评估其在小鼠体内的生物分布。结果体外光声成像结果显示,纳米材料的光声信号随浓度的增加呈线性增强;体内光声成像结果显示小鼠肿瘤部位的光声信号随时间延长而逐渐增强,在注射纳米粒后24 h达到峰值,此后开始逐渐下降。与对照组相比,在注射纳米粒后小鼠的血常规、肝肾功能等血液指标以及小鼠主要器官的H&E染色切片均未见明显的异常,差异没有统计学意义。小动物活体成像结果表明在注射后24小时肿瘤区域荧光强度达到最大,并且在注射后48小时纳米粒主要积累在肝脏和脾脏。结论制备的PCN-224@PDA@TH588纳米材料在体内、体外均具有较好的光声成像效果,具备作为光声成像造影剂潜能。PCN-224@PDA@TH588能够通过EPR效应有效的聚集在小鼠肿瘤区域并且具有良好的体内生物相容性和安全性,为后期的动物体内治疗实验应用奠定了基础。第三部分负载TH588的纳米材料联合超声治疗乳腺癌效应及相关机制的研究目的评估PCN-224@PDA@TH588纳米粒联合低强度聚焦超声对小鼠乳腺癌细胞活性的影响及MTH1抑制剂TH588增效SDT的相关机制。方法4T1细胞被分为以下组:空白对照组、LIFU组、PCN-224@PDA@TH588组、PCN-224@PDA+LIFU组、PCN-224@PDA@TH588+LIFU组,采用CCK-8法、活/死细胞染色法、流式细胞术等方法检测经上述不同分组处理的纳米粒在细胞水平对小鼠乳腺癌细胞活性的影响及相关机制。建立小鼠乳腺原位移植瘤模型,随后将荷瘤小鼠随机分为以下组:对照组(PBS)、LIFU组、PCN-224@PDA@TH588组、PCN-224@PDA+LIFU组、PCN-224@PDA@TH588+LIFU组,评估载药纳米粒PCN-224@PDA@TH588联合低强度聚焦超声在体水平抗小鼠乳腺癌的效应。结果与其它治疗组相比较,携载MTH1抑制剂TH588的金属有机框架的载药纳米粒PCN-224@PDA@TH588联合超声辐照显示出更强的细胞杀伤效果,差异具有统计学意义(P<0.05);研究发现在超声作用下乳腺癌4T1细胞产生了大量的活性氧,并且随着纳米粒内负载TH588的逐步释放,抑制了MTH1活性,增加了8-羟基脱氧鸟苷(8-oxo-d G)的含量,导致癌细胞ROS防御系统被破坏,显著提高了细胞内活性氧的杀伤效果,产生了增效声动力治疗的作用。在小鼠原位移植瘤模型中,载药纳米粒PCN-224@PDA@TH588联合低强度聚焦超声辐照能够显著抑制小鼠乳腺移植瘤的生长,并且在治疗终末时小鼠肿瘤体积、肿瘤质量、肿瘤抑制率等与其它各组相比均有显著的统计学差异(P<0.05)。结论体内外治疗结果表明,载药纳米粒PCN-224@PDA@TH588联合超声辐照的抗瘤效果显著优于单纯的TH588以及单纯的声动力治疗,优异的细胞杀伤效率归因于纳米粒释放的MTH1抑制剂TH588诱导的MTH1表达的降低可以阻止细胞内DNA损伤的自我修复过程,导致癌细胞ROS防御系统被破坏,起到了显著增强SDT引起的氧化损伤的作用,从而协同并增效对肿瘤细胞杀伤,这为临床肿瘤的治疗提供了一种新的方法和策略。更多还原

【Abstract】 PART I PREPARATION AND CHARACTERIZATION OF NANOMATERIALSObjective To prepare the metal-organic framework PCN-224,which was loaded with MTH1 inhibitor TH588,and then modified with polydopamine on the surface of metal-organic framework to detect and characterize the basic physical and chemical properties and cytotoxicity of nanoparticles.Methods The particle size distribution and Zeta potential distribution of nanoparticles were detected by Malvern laser particle size and potentiometer.The microstructure and morphology of the nanoparticles were detected by transmission electron microscopy and scanning electron microscopy,and the elemental distribution in the nanoparticles was analyzed by energy dispersive spectroscopy.The specific surface area and pore size of nanoparticles were measured by automatic specific surface area pore-able analyzer.The UV-vis absorption spectra of nanoparticles were measured by UV-vis spectrophotometer.1,3-diphenylisophenylfuran(DPBF)probe was used to detect the singlet oxygen production efficiency of nanoparticles.CCK8 method was used to detect the cytotoxicity of PCN-224@PDA nanoparticles.Confocal laser microscopy,flow cytometry and transmission electron microscopy were used to evaluate the phagocytosis and uptake of nanoparticles by breast cancer cells.Results Firstly,the metal-organic framework PCN-224 was prepared.Under electron microscope,it was spherical with regular morphology,specific surface area of 394.25 m2/g,average pore size of 4.54 nm,particle size of(105 ± 16.86)nm,potential of(21.69 ± 2.85)m V.Further detection shows that PCN-224 can significantly produce singlet oxygen under lowintensity focused ultrasound irradiation,and the singlet oxygen generation ability is dependent on ultrasonic irradiation time.After the metal-organic framework was modified with polydopamine,CCK-8 method showed that PCN-224@PDA nanoparticles had less toxicity to cells,and the cell survival rate was still more than 85% when the concentration of nanoparticles was up to 200 μg/m L.The nanoparticle PCN-224@PDA@TH588 was characterized by electron microscope,which showed regular spherical shape,uniform size distribution and good dispersion.Energy spectrum analysis showed that the nanoparticle was mainly composed of four chemical elements C,N,O and Zr,with an average particle size of(118 ± 18.75)nm.The potential was(-30.35 ± 0.46)m V.The maximum absorption peaks of PCN-224@PDA and PCN-224@PDA@TH588 are around 413 nm.Confocal laser observation,flow cytometry quantitative analysis and TEM showed that breast cancer cells could effectively phagocytosis and uptake of nanoparticles after 4 hours of co-incubation with them.Conclusion Multifunctional nanoparticles PCN-224@PDA@TH588 containing MTH1 inhibitor TH588 with regular morphology,uniform particle size,good dispersity and good biocompatibility were successfully prepared.The nanoparticles can be used as a sound sensitizer and a good drug carrier.It can realize the targeted delivery of insoluble small molecules or toxic chemotherapy drugs to the tumor region through EPR effect,laying a foundation for the next in vivo and in vitro research.PART II STUDY ON PHOTOACOUSTIC IMAGING,BIOSAFETY AND BIODISTRIBUTION OF NANOMATERIALSObjective To investigate the photoacoustic imaging ability,biodistribution and biosafety of PCN-224@PDA@TH588 nanoparticles in vivo and in vitro.Methods PCN-224@PDA@TH588 nanoparticles with different concentrations were scanned by small animal photoacoustic imager in vitro,and their photoacoustic images and signal values were collected and measured.Blood routine indexes,blood biochemical indexes and pathological sections of major organs of healthy mice were analyzed at different time points(1,7 and 14 days)after tail vein injection of nanoparticles,and the biosafety of nanoparticles in vivo was systematically detected.The photoacoustic signals in the tumor region of mice were observed before and after the injection of nanoparticles,and the signal intensity was measured.The model of orthotopic transplanted tumor of breast cancer in mice was established,and the biological distribution was evaluated by injecting DIR dye labeled nanoparticles into mice.Results In vitro photoacoustic imaging results showed that the photoacoustic signal of nanoparticles increased linearly with the increase of concentration.The results of photoacoustic imaging in vivo showed that the photoacoustic signal of the tumor site in mice increased gradually with time, and reached the peak at 24 h after the injection of nanoparticles,then gradually decreased.Compared with the control group,there were no significant abnormalities in blood routine,liver and kidney function and H&E staining sections of main organs of mice after injection of nanoparticles,and the differences were not statistically significant.In vivo imaging results of small animals showed that the fluorescence intensity in tumor region reached the maximum 24 h after injection,and the nanoparticles mainly accumulated in liver and spleen 48 h after injection.Conclusion The prepared PCN-224@PDA@TH588 nanomaterial has good photoacoustic imaging effect both in vitro and in vivo,and has potential as a photoacoustic imaging contrast agent.PCN-224@PDA @TH588 has good biocompatibility and safety in vivo,which lays a foundation for the later experimental application of therapy.PART III TO EVALUATE THE EFFICACY AND RELATED MECHANISM OF TH588 LOADED NANOMATERIALS COMBINED WITH ULTRASOUND IN THE TREATMENT OF BREAST CANCERObjective To evaluate the therapeutic effect of PCN-224@PDA @Th588 nanoparticles combined with low intensity focused ultrasound on mouse breast cancer cells and the synergistic mechanism.Methods 4T1 cells were divided into the following groups: Control group,LIFU group,PCN-224@PDA@TH588 group,PCN-224@PDA + LIFU group,PCN-224@PDA@TH588 + LIFU group,CCK-8 assay,live/dead cell staining and flow cytometry were used to detect the effects of nanoparticles treated by different groups on the activity of breast cancer cells at the cellular level and the related mechanisms.A mouse mammary gland orthotopic tumor model was established,and the tumor-bearing mice were randomly divided into the following groups: Control group(PBS),LIFU group,PCN-224@PDA@TH588 group,PCN-224@PDA + LIFU group,PCN-224@PDA@TH588 + LIFU group.To evaluate the effect of low intensity focused ultrasound combined with PCN-224@PDA@TH588 nanoparticles on mouse breast cancer in vivo.Results Compared with other treatment groups,PCN-224@PDA@TH588 nanoparticles combined with ultrasonic irradiation showed stronger cell killing effect,and the difference was statistically significant(P<0.05);The study found that breast cancer 4T1 cells produced a large amount of reactive oxygen species under the irradiation of ultrasound,and with the gradual release of nano-particle loading agent TH588,the activity of MTH1 was inhibited,the content of 8-oxo-d G was increased,and the ROS defense system of cancer cells was destroyed,and the killing effect of intracellular reactive oxygen species was significantly improved.It has the effect of synergistic sonodynamic therapy.In the 4T1 tumor-bearing mice,PCN-224@PDA@TH588 nanoparticles combined with low-intensity focused ultrasound irradiation can significantly inhibit the growth of tumor,and at the end of treatment,tumor volume and tumor weight in mice were statistically significant compared with other groups(P< 0.05).Conclusion The results showed that the anti-tumor effect of PCN-224@PDA@TH588 nanoparticles combined with ultrasound irradiation is significantly better than TH588 alone and sonodynamic therapy alone both in vitro and in vivo.The excellent cell killing efficiency is attributed to the reduction of MTH1 expression induced by TH588,the MTH1 inhibitor released by nanoparticles,which prevents the intracellular self-repair process and leads to the destruction of cancer cells’ ROS defense system,thus significantly enhancing the oxidative damage caused by SDT,thus synergistically killing tumor cells.This provides a new method and strategy for the treatment of tumors.更多还原