【作者】 张涛；

【导师】 张学文；

【作者基本信息】 吉林大学 ， 外科学， 2017， 博士

【摘要】 肝癌是我国常见的恶性肿瘤之一,严重威胁着人民群众的健康。当前,虽然随着诊疗技术手段的变革,肝癌患者总体生存期有了一定的提高,但其仍具有较高的癌症相关性死亡率,肝癌防治形式仍然十分严峻。计算机断层扫描技术(CT)因其高空间、高密度分辨率和深层组织穿透强的特点已经成为临床肝癌诊断一种重要工具。然而由于临近组织器官密度的相似使单纯CT成像难以清晰区分正常组织和病变组织。造影剂的使用成为一种必然需要。传统CT造影剂为小分子碘化芳香族药物,其快速肾清除率、非特异性分布以及毒副作用等缺陷严重限制其临床应用。采用纳米有机金属框架载体作为碘化物的载体,可以有效增加CT成像的靶向性及成像效果,并可降低药物用量及药物的毒副作用。纳米有机金属框架造影剂为恶性肿瘤的靶向诊断提供了新的机遇与思路。目的:本研究应用溶剂辅助配体交换技术制备含BODIPY纳米有机金属框架颗粒(UiO-PDT)。通过人源Hep G2细胞MTT实验、血液相容性实验以及昆明小鼠体内毒性试验验证UiO-PDT纳米颗粒生物相容性。应用鼠源肝癌细胞Walk 256建立SD大鼠原位肝癌模型,通过药物体外CT成像、体内肝癌CT成像以及与传统小分子CT造影剂泛影葡胺肝癌成像对比分析探讨含BODIPY纳米有机金属框架颗粒(UiO-PDT)用于肝癌CT成像对比剂的可行性及优良的成像效果。方法:(1)通过溶剂辅助配体交换技术将二碘代氟硼二甲川吡咯(I2-BDP)交换入锆基纳米配位聚合物体(UiO-66)得到含BODIPY纳米金属有机框架颗粒(UiO-PDT)。通过1H NMR、TEM、SEM、PXRD、UV-vis光谱和Micro CT测定纳米金属有机框架颗粒的理化性质并对其在体外环境中CT成像效果进行检测;(2)以人源肝癌细胞Hep G2为细胞实验对象,通过MTT法检测纳米药物在体外环境中的细胞相容性;通过血液相容性实验以及动物体内生物相容性相关实验验证UiO-PDT的生物安全性;(3)建立SD大鼠原位肝癌模型,通过尾静脉注射途径,总体评估UiO-PDT纳米晶体在肝癌增强成像效果以及成像图像变化规律。(4)对比小分子CT造影剂泛影葡胺在SD大鼠原位肝癌模型体内CT成像效果,分析UiO-PDT纳米颗粒用于肝癌CT造影剂的优势结果:(1)本研究成功合成制备了锆基纳米配位聚合物体(UiO-66),并成功将I2-BDP装载其中后得到UiO-PDT,其合成方法溶剂辅助配体交换反应,较其它纳米合成方法简便且易放大。在TEM和SEM下观察UiO-PDT均为正八面体结构,其粒径大小约为70nm。PXRD和UV-vis光谱结果间接显示晶体结构稳定性以及组成部分的化学成分及其自身结构相对完整性。在Micro CT下UiO-PDT纳米颗粒呈现出良好的浓度-CT值线性关系,为其用于CT造影剂的进一步研究奠定基础。(2)在本研究中,通过细胞学实验(细胞MTT毒性试验)、血液相容性实验(溶血实验和血浆凝血实验)、血液学分析(血常规和血生化指标)、体重变化曲线、组织病理学分析等结果证实UiO-PDT具有良好的生物相容性,为进一步临床转化应用提供良好生物学基础。(3)在本研究中,成功建立SD大鼠原位肝癌模型,UiO-PDT在SD大鼠原位肝癌模型体内CT成像结果UiO-PDT除了显著延长了其在体内的循环时间外,其在肝癌组织出的靶向聚集以及成像清晰度也获得令人满意的结果,证实其作为肝癌CT造影剂的设想是可行的,且具有广阔的开发应用前景。(4)通过对比小分子CT造影剂泛影葡胺在SD大鼠原位肝癌模型体内CT成像效果,UiO-PDT在体内的循环时间以及肿瘤部位显像时间显著增加;在相同碘含量的前提下,UiO-PDT的肝癌部位增强显像效果更好。结论:(1)在本研究中,成功设计并合成一种新型的纳米CT造影剂:含BODIPY纳米金属有机框架颗粒UiO-PDT。(2)UiO-PDT纳米颗粒具有良好的生物相容性,为其进一步临床应用研究奠定了坚实的基础。(3)与传统小分子造影剂相比,UiO-PDT具有更长的体内循环时间和肝癌CT增强显像时间以及更佳的肝癌CT增强显像效果,表明UiO-PDT作为肝癌CT造影剂的可行性以及具有良好的应用前景。更多还原

【Abstract】 Hepatocarcinoma is one of the common malignant tumor in our country,which seriously threatens people’s health.At present,although the liver cancer patients’ overall survival has been highly improved with the development of treatment,but there is still a high mortality of cancer,and the prevention form is still grim.High mortality of liver cancer is mainly attributed to the low early diagnosis rate and poor prognosis of advanced cancer treatment.The early diagnosis of tumor can greatly improve the prognosis of tumor patients after treatment.Computed Tomography(CT)has become an indispensable tool in clinical diagnosis for its unique characteristics of high spatial,density resolution,and deep tissue penetration.However,the attenuation values of different soft living tissues and organs usually display relatively high similarity,making it difficult to distinguish normal tissues and diseased tissues.Therefore,some of contrast agents contain heavy elements with high X-ray attenuation ability are necessary to obtain CT images with desired contrast enhancements.Conventional CT contrast agents in clinical practice are those small iodinated aromatic molecules.However,rapid pharmacokinetics,non-specific distribution and side effects have limited these materials in clinical diagnostic applications.The use of nanoscale metal-Organic frameworks as nanocarrier for iodine drugs could improve the targeted property of CT imaging and imaging effect,and also can reduce the drug dosage and decrease the side effects.Therefore,nanoscale metal-organic frameworks provide a new thought and opportunity for targeting diagnosis of malignant tumor.Objective:This study intends to synthesis BODIPY-containing Nanoscale Metal-OrganicFrameworks by solvent-assisted ligandexchange method(SALE).The biocompatibility of UiO-PDT nanoparticles is verified by MTT experiment on Hep G2 cells,blood compatibility test and toxicity test on kunming mice.SD rats bearing Walk 256 orthotopic hepatoma are intended to be prepared and the feasibility and excellent imaging effect of UiO-PDT used as CT contrast agent are intended to be vilified through the following aspects: contrast analysis with traditional small-molecule contrast agents meglumine diatrizoate,CT imaging in vitro and vivo.Method:(1)The carboxyl-functionalized diiodo-substituted BODIPYs(I2-BDP)is incorporated into the nanoscale UiO-MOFs(UiO-66)to exert the PDT into the final product of UiO-PDT by solvent-assisted ligand exchange method(SALE).The physicochemical properties are tested by 1H Proton nuclear magnetic resonance(1H NMR),transmission electron microscopy(TEM)、 scanning electron microscope(SEM)、 phase analysis of X ray diffraction(PXRD)and ultraviolet–visible spectroscopy(UV-vis).The CT imaging effect in vitro environment is tested by a Micro CT.(2)Anthropogenic cells Hep G2 are used as experiment cells.The cytotoxicity of UiO-PDT nanoparticales was detected by MTT method.The biological safety of UiO-PDT is verified through blood compatibility test and animal biocompatibility related experiments in vivo.(3)SD rats bearing Walk 256 orthotopic hepatoma are intended to be prepared.The overall evaluation of UiO-PDT nanocrystalline imaging effect and imaging change rule in vivo are carried out by the analysis of CT images and data post injection via tail vein.(4)By comparing the CT images of small molecule CT contrast agents meglumine diatrizoate in vivo orthotopic hepatoma SD rats,the advantages of UiO-PDT nanoparticles as contrast agents in hepatocarcinoma computed tomography are revealed.Result:(1)During the research,our team successfully synthesis nanoscale metal-organic frameworks(UiO-PDT).Octahedral structure of UiO-PDT is observed under TEMand SEM,and its particle size is about 70 nm.PXRD and UV-vis spectrum results suggest crystal stability of structure and the component of chemical composition.The Micro CT results of UiO-PDT in vitro shows that the CT values of nanocrystalline in vitro environment present a good linear relationship with concentration.(2)In the aspects of biocompatibility characterization,the superior biocompatibility characterization of UiO-PDT nanocrystalline is confirmed by MTT test on Hep G2 cells,blood compatibility tests in vitro(hemolysis experiment,plasma coagulation experiment)and kunming mice biocompatibility examinations in vivo(blood biochemical examination,histologic examination,weight change curve),and provided the theoretical basis for its clinical application.(3)In this study,SD rats bearing Walk 256 orthotopic hepatoma are successfully prepared.The CT imaging results of UiO-PDT in SD rats bearing Walk 256 orthotopic hepatoma shows UiO-PDT nanoparticles not only significantly prolong the circulation time in body,and also receive satisfactory results of the nanoparticles targeted gathering in hepatoma site,confirming that the idea for UiO-PDT as tumor CT contrast agenst was feasible,and had wide application prospects.(4)Compared with the small molecule CT contrast agents meglumine diatrizoate in vivo,UiO-PDT has a longer cycle time and a longer enhanced CT imaging time in the tumor sit.In the case of the same iodine content,the imaging contrast effect of UiO-PDT in hepatocarcinoma is better than the traditional small molecule contrast agents.Conclusion:(1)In this study,we successfully design and synthesize a new type of nanoscale CT contrast agent: BODIPY-containing nanoscale metal-organic frameworks,UiO-PDT.(2)UiO-PDT nanoparticles have a good biocompatibility and lays a solid foundation for further clinical application.(3)Compared with the traditional small molecule contrast agents,UiO-PDT has a longer cycle time in the body,CT enhanced imaging time and better effect of enhanced CT imaging in hepatocarcinoma,suggesting UiO-PDT as a feasible CT contrast agent in hepatocarcinoma and has a good application prospect.更多还原