【作者】 孙阳；

【导师】 王志刚；

【作者基本信息】 重庆医科大学 ， 影像医学与核医学， 2013， 博士

【摘要】 第一部分载Fe₃O₄高分子造影剂的制备及性能检测目的制备一种载超顺磁性氧化铁纳米颗粒（Fe₃O₄）的聚乳酸-羟基乙酸（PLGA）多功能造影剂（Fe₃O₄/PLGA），检测其物理学、光学、磁学及生物安全性能。方法采用双乳化法制备出Fe₃O₄/PLGA,对其表面形貌、结构，粒径、电位，载Fe量，吸收光谱及磁学性质进行检测；制备出DiI标记的Fe₃O₄/PLGA（DiI-Fe₃O₄/PLGA），与MDA细胞孵育，观察不同时间内（4h，12h，24h）造影剂被细胞吞噬情况及对细胞活性的影响；于健康兔耳缘静脉注射不同浓度的Fe₃O₄/PLGA，观察不同时间点（1d，3d，7d）该造影剂对兔肝、肾、心功能的影响。结果Fe₃O₄/PLGA溶于双蒸水后呈棕色乳液，光镜及扫描电镜观察，Fe₃O₄/PLGA呈球形，形态规则，大小尚均匀，分散度好，表面欠光滑。透射电镜下可见大量Fe₃O₄颗粒较均匀分布于外壳结构中。Malvern激光粒径仪检测出Fe₃O₄/PLGA平均直径为（885.6133.2）nm，Zeta电位为（-2.323.84）mV。原子吸收光谱法测得样品中铁含量为46.58μg/ml。紫外-可见分光光度计检测出Fe₃O₄/PLGA吸收光谱位于590nm和650nm附近。磁滞回线显示，Fe₃O₄/PLGA无磁滞现象，呈超顺磁性。细胞吞噬实验发现，MDA细胞于体外能够吞噬Fe₃O₄/PLGA，吞噬数量具有时间依赖性，MTT法测得吞噬后的造影剂对细胞活性无明显影响。在选定观察时间范围内，所用浓度的Fe₃O₄/PLGA对兔肝、肾及心功能指标无明显影响。结论成功制备出形态规则、呈球形，大小均匀，性质稳定，包封率较高，生物安全性好的载Fe₃O₄高分子造影剂。该造影剂具有良好的光学吸收性能及磁性能，具备增强光声显像及磁共振显像的潜能，是一种新型的多功能造影剂。第二部分载Fe₃O₄高分子造影剂增强超声、磁共振及光声显像实验目的观察Fe₃O₄/PLGA体外增强超声、磁共振及光声成像的效果，探讨其作为多模态显像剂的可行性；于体内实验观察Fe₃O₄/PLGA对兔肝癌磁共振成像效果的影响，了解Fe₃O₄/PLGA增强磁共振显像的能力及原理。方法制备体外成像模型，应用超声诊断仪及磁共振扫描仪对不同浓度Fe₃O₄/PLGA分别行超声及磁共振成像；利用光声成像仪，以波长为532nm的脉冲激光激发，分别检测单个Fe₃O₄/PLGA微球的光声信号，及被MDA细胞吞噬入胞的Fe₃O₄/PLGA微球光声信号。建立兔VX2肝癌模型12只，于肿瘤种植后21d，随机分为3组，分别经兔耳缘静脉注入生理盐水、PLGA以及Fe₃O₄/PLGA1ml/kg体质量，注射前后行MR平扫及增强扫描，分别测量肝实质和肿瘤的信号强度，计算肿瘤/肝实质信号强度比。磁共振扫描结束后，行HE染色及普鲁士蓝染色观察。结果体外超声显像，Fe₃O₄/PLGA呈高回声，回声强度随浓度及机械指数减小而降低；磁共振T2*加权像上，Fe₃O₄/PLGA呈负增强显像，即随Fe含量增高，MR信号强度显著降低；在532nm激光激发下，Fe₃O₄/PLGA能够产生较强的光声信号，且光声信号随激光能量的增高呈增强趋势。即使被MDA细胞吞噬后，于细胞内检测到的Fe₃O₄/PLGA光声信号也较强。兔肝癌磁共振成像显示，注射Fe₃O₄/PLGA后，肝实质信号强度显著下降，肿瘤信号无明显变化呈相对高信号，肿瘤/肝实质信号强度比明显高于生理盐水组及PLGA组（P <0.05）。普鲁士蓝染色显示，Fe₃O₄/PLGA组兔肝实质内分布有较多蓝染颗粒，而肿瘤组织，及生理盐水组、PLGA组肝实质中未见蓝染颗粒。结论Fe₃O₄/PLGA具有体外增强超声、磁共振及光声学多模态复合显像的能力，具备成为多模态影像学对比剂的潜能。体内实验中，Fe₃O₄/PLGA能有效增强兔肝脏肿瘤与正常组织间信号强度对比，对肝癌诊断具有一定的应用价值。第三部分载Fe₃O₄高分子造影剂增效HIFU治疗实验目的观察Fe₃O₄/PLGA联合HIFU消融离体牛肝效果，探讨其用于体内增效实验的最佳浓度和最优辐照参数;观察Fe₃O₄/PLGA联合HIFU对兔VX2乳腺移植瘤消融效果，探讨其作为HIFU增效剂的应用价值。方法取新鲜离体牛肝，局部注射不同浓度Fe₃O₄/PLGA（0.5mg/ml，1mg/ml，2mg/ml）200μl后，给予不同输出声功率（120W，150W，180W）、不同时间（2s，5s，10s）的HIFU辐照，通过计算辐照区灰度变化值及凝固性坏死体积评价Fe₃O₄/PLGA增强HIFU消融效果，以PBS液、PLGA对照。建立兔VX2乳腺癌模型32只，于肿瘤接种后14d，随机分为4组，第1组实验兔给予单纯HIFU辐照，第2、3、4组在HIFU辐照前，距肿瘤中心约3mm处，经肿瘤3、6、9、12点钟方向，分别注入生理盐水、PLGA、Fe₃O₄/PLGA共0.5ml，局部按摩1min后行HIFU定点辐照，辐照声功率150W，辐照时间5s。HIFU辐照结束后，观测靶区灰度变化值及凝固性坏死体积，采集标本行HE染色，增殖细胞核抗原（PCNA）检测及透射电镜观察。结果离体牛肝组织局部注射Fe₃O₄/PLGA后经HIFU辐照，辐照区灰度变化值及凝固性坏死体积较注射PBS液、PLGA组明显增大（P<0.05）。HIFU消融兔VX2乳腺癌实验中，联合Fe₃O₄/PLGA组，消融区灰度变化值及组织凝固性坏死体积明显增加（P<0.05），肿瘤细胞PCNA阳性表达率明显降低（P<0.05）。HE染色后光镜下观察，辐照区细胞结构被破坏，坏死区与周围组织分界清楚。电镜下观察，癌细胞结构欠清晰，细胞膜和核膜失去连续性，染色质固缩、碎裂呈团块状，残存细胞中可见Fe颗粒沉积于胞浆。结论Fe₃O₄/PLGA能有效增强HIFU消融离体牛肝及兔乳腺VX2肿瘤的生物学效应，是一种良好的HIFU增效剂。更多还原

【Abstract】 PART PREPARATION AND CHARACTERIZATION OFIRON OXIDE-LOADED POLYMER CONTRAST AGENTSObjective To prepare the superparamagnetic Fe₃O₄-loaded polymermicrocapsules as a novel multi-functional contrast agents （Fe₃O₄/PLGA）,and investigate their properties of physics, optics, magnetism and biosafety.Methods Fe₃O₄/PLGA microcapsules were fabricated by a typicaldouble emulsion evaporation method. The surface morphology andstructure, the mean size and zeta potential distribution, the concentration ofFe₃O₄loading in PLGA microcapsules, the absorption spectrum and themagnetic properties of Fe₃O₄/PLGA were assessed using several analyticalmethods. The DiI labeled Fe₃O₄/PLGA （DiI-Fe₃O₄/PLGA） microcapsules,which were prepared using the same process as Fe₃O₄/PLGA wereincubated with MDA cells for different hours （4h,12h,24h）, then thephagotrophic effect of these microcapsules in MDA cells was observed by light microscope, MTT assay was used to detect the viability of the cells.The Fe₃O₄/PLGA microcapsules were injected into normal rabbits by veinto investigate the effect of these microcapsules on rabbit liver, kidney andheart at different time （1d,3d, and7d）.Results Fe₃O₄/PLGA microcapsules became brown emulsion whendissolved in double distilled water. The microcapsules exhibited a perfectspherical morphology with not very smooth surface under light microscope（LM） and scanning electron microscope （SEM）. A lot of Fe₃O₄particlesdistributed in the shell structure were observed under transmission electronmicroscope （TEM）. The diameter of Fe₃O₄/PLGA was （885.6±133.2） nm,and zeta potential was （-2.32±3.84） mV measured by a Laser Particle SizeAnalyzer System. The concentration of Fe₃O₄loading in PLGAmicrocapsules was46.58μg/ml detected by an atomic absorptionspectrophotometer （AAS）. The Fe₃O₄/PLGA absorption spectrum wasaround590nm and650nm measured by a UV-Vis spectrophotometry. TheM-H hysteresis loop behavior showed the Fe₃O₄/PLGA typicalsuperparamagnetic properties. A time-dependent Fe₃O₄/PLGA uptake byMDA cells was observed under the light microscope. The amount ofFe₃O₄/PLGA uptake increased with the increasing incubation time. MTTresults showed the cells proliferation activity was not affected afterFe₃O₄/PLGA engulfed by MDA cells. And the Fe₃O₄/PLGA had nosignificant effect on rabbit liver, kidney and heart after injection into the rabbits.Conclusion Fe₃O₄/PLGA microcapsules have been successfullysynthesized; these microcapsules have good spherical morphology, uniformsize, high encapsulation efficiency and good biosafety. They have excellentoptical absorption and magnetic properties, which make them a potentialcandidate for photoacoustic （PA） and magnetic resonance （MR） imaging. PART Ⅱ IRON OXIDE-LOADED POLYMERCONTRAST AGENTS FOR ENHANCED US, MR ANDPA IMAGINGObjective To observe the effect of Fe₃O₄/PLGA for enhanced US, MRand PA imaging in vitro, to investigate the feasibility of thesemicrocapsules as a multi-modality imaging agents; to observe the influenceof these agents on rabbits liver tumor MR imaging in vivo, to investigatethe enhanced capacity and the principle of Fe₃O₄/PLGA in MR imaging.Methods The prepared Fe₃O₄/PLGA microcapsules with differentconcentrations were applied to perform US and MR imaging in vitro. Awavelength of532nm pulsed laser excitation was used to carry outphotoacoustic imaging. A single Fe₃O₄/PLGA microcapsule andFe₃O₄/PLGA microcapsules that were phagocytized by MDA cells wereselected to detect the PA signal.21days after implantation of VX2tumorsin liver,12rabbits were randomly divided into three equal groups with theinjection of saline, PLGA, and Fe₃O₄/PLGA1ml/kg via ear veinrespectively. MR imaging was performed before and after injection of theabove three agents, the signal intensity （SI） of liver parenchyma and livertumor were measured, then, the signal ratio of liver tumor to liverparenchyma was calculated before and after enhancement. After MR scanning, Prussian blue and HE staining were done.Results In vitro, the Fe₃O₄/PLGA showed hyperechogenicity in USimaging, the echo intensity （EI） decreased with the concentration andmechanical index decreasing. The Fe₃O₄/PLGA exhibited MRI negativelyenhanced effect in T2*-weighted images, the signal intensity （SI） decreasedwith the increasing Fe concentration in PLGA. On the other hand,Fe₃O₄/PLGA could produce strong PA signal even were phagocytized byMDA cells under532nm laser. The PA signal increased with increasinglaser energy. In vivo MR imaging, after injection of Fe₃O₄/PLGA, the SI ofliver parenchyma reduced markedly, however, no significant changes of SIwere found in liver tumor, the signal ratio of liver tumor to liverparenchyma increased significantly compared with the other two groups （P<0.05）. Prussian blue stain showed accumulation of the blue particleswithin liver parenchyma in Fe₃O₄/PLGA group, this phenomenon was notoccurred in liver tumor tissue and groups with injection of saline andPLGA.Conclusion The Fe₃O₄/PLGA microcapsules are successfully acted asmulti-modality biological imaging contrast agents for US, MR and PAimaging in vitro. And The Fe₃O₄/PLGA microcapsules can effectivelyenhanced the signal intensity contrast between the liver tumor and normaltissue in rabbits, which have a potential application value in the detection of liver carcinoma. PART Ⅲ IRON OXIDE-LOADED POLYMERCONTRAST AGENTS FOR HIGH INTENSITY FOCUSEDULTRASOUND CANCER ABLATIONObjective To investigate the efficacy of Fe₃O₄/PLGA combined withHIFU in bovine liver tissue ablation, and to investigate the influence ofFe₃O₄/PLGA combined with HIFU in rabbits bearing breast tumor.Methods The degassed bovine liver in vitro was directly injected into200μl Fe₃O₄/PLGA in PBS with different Fe concentrations just beforeHIFU ablation, the PBS （200μl） and pure PLGA （200μl） were employedfor control. After HIFU ablation, the gray-scale value change and thevolume of coagulated tissue irradiated by HIFU was calculated.14daysafter implantation of VX2tumor in breast,32rabbits were randomlydivided into four equal groups. In group Ⅱ, Ⅲ, and Ⅳ, the rabbits receiveda percutaneous injection of0.5ml saline, PLGA, and Fe₃O₄/PLGA solutioninto breast tumor at3,6,9,12dots, about3mm around the tumorrespectively, and massaged for1min before receiving HIFU ablation, whileHIFU group received HIFU ablation only. HIFU ablation parameters werethe same:“ablated-dot” mode, acoustic power was150W, exposureduration was5s. After HIFU ablation, the gray-scale value change and thevolume of coagulative necrosis was calculated, HE staining, TEM andimmunohistochemical examination of PCNA were performed to detect the structure changes of the targeted tissue caused by HIFU ablation.Results The gray-scale value change and the volume of coagulatedtissue in bovine liver tissue irradiated by HIFU after local injection ofFe₃O₄/PLGA was larger than that of PBS and pure PLGA （P<0.05）. Inrabbits with breast cancer, the gray-scale value change and the volume ofcoagulative necrosis caused by HIFU ablation was substantially larger andthe positive index （PI） of PCNA was lower in the necrotic region after theinjection of Fe₃O₄/PLGA microcapsules compared to the group withoutinjection agents, the group of saline and the group of pure PLGAmicrospheres under the same irritation （P<0.05）. Pathological examinationdemonstrated the targeted tissue was destroyed severely, Fe₃O₄nanoparticles were seen depositing in residual tumor cells under TEM.Conclusion The Fe₃O₄/PLGA microspheres can effectively enhanced thebiomedical effect of HIFU ablation both on bovine liver tissue and onrabbits bearing VX2breast tumor, The Fe₃O₄/PLGA was a potentialsynergistic agent for HIFU cancer surgery.更多还原