载Fe_3O_4高分子造影剂多模态显像及增效HIFU治疗实验研究
|
摘要
第一部分载Fe_3O_4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（Fe_3O_4）的聚乳酸-羟基乙酸（PLGA）多功能造影剂（Fe_3O_4/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Fe_3O_4/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Fe_3O_4/PLGA（DiI-Fe_3O_4/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Fe_3O_4/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Fe_3O_4/PLGA溶于双蒸水后呈棕色乳液，光镜及扫描电镜观察，Fe_3O_4/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Fe_3O_4颗粒较均匀分布于外壳结构中。Malvern激光粒径仪检测出Fe_3O_4/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Fe_3O_4/PLGA吸收光谱位于590nm和650nm附近。磁滞回线显示，Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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Fe_3O_4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Fe_3O_4高分子造影剂增强超声、磁共振及光声显像实验
目的观察Fe_3O_4/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Fe_3O_4/PLGA对兔肝癌磁共振成像效果的影响，了解Fe_3O_4/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Fe_3O_4/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Fe_3O_4/PLGA微球的光声信号，及被MDA细胞吞噬入胞的Fe_3O_4/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Fe_3O_4/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，Fe_3O_4/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，Fe_3O_4/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，Fe_3O_4/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Fe_3O_4/PLGA光声信号也较强。兔肝癌磁共振成像显示，注射Fe_3O_4/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，Fe_3O_4/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Fe_3O_4/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，Fe_3O_4/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Fe_3O_4高分子造影剂增效HIFU治疗实验
目的观察Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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、Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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。
PART PREPARATION AND CHARACTERIZATION OFIRON OXIDE-LOADED POLYMER CONTRAST AGENTS
Objective To prepare the superparamagnetic Fe_3O_4-loaded polymermicrocapsules as a novel multi-functional contrast agents (Fe_3O_4/PLGA),and investigate their properties of physics, optics, magnetism and biosafety.
Methods Fe_3O_4/PLGA microcapsules were fabricated by a typicaldouble emulsion evaporation method. The surface morphology andstructure, the mean size and zeta potential distribution, the concentration ofFe_3O_4loading in PLGA microcapsules, the absorption spectrum and themagnetic properties of Fe_3O_4/PLGA were assessed using several analyticalmethods. The DiI labeled Fe_3O_4/PLGA (DiI-Fe_3O_4/PLGA) microcapsules,which were prepared using the same process as Fe_3O_4/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 Fe_3O_4/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 Fe_3O_4/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 Fe_3O_4particlesdistributed in the shell structure were observed under transmission electronmicroscope (TEM). The diameter of Fe_3O_4/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 Fe_3O_4loading in PLGAmicrocapsules was46.58μg/ml detected by an atomic absorptionspectrophotometer (AAS). The Fe_3O_4/PLGA absorption spectrum wasaround590nm and650nm measured by a UV-Vis spectrophotometry. TheM-H hysteresis loop behavior showed the Fe_3O_4/PLGA typicalsuperparamagnetic properties. A time-dependent Fe_3O_4/PLGA uptake byMDA cells was observed under the light microscope. The amount ofFe_3O_4/PLGA uptake increased with the increasing incubation time. MTTresults showed the cells proliferation activity was not affected afterFe_3O_4/PLGA engulfed by MDA cells. And the Fe_3O_4/PLGA had nosignificant effect on rabbit liver, kidney and heart after injection into the rabbits.
Conclusion Fe_3O_4/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 IMAGING
Objective To observe the effect of Fe_3O_4/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 Fe_3O_4/PLGA in MR imaging.
Methods The prepared Fe_3O_4/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 Fe_3O_4/PLGA microcapsule andFe_3O_4/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 Fe_3O_4/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 Fe_3O_4/PLGA showed hyperechogenicity in USimaging, the echo intensity (EI) decreased with the concentration andmechanical index decreasing. The Fe_3O_4/PLGA exhibited MRI negativelyenhanced effect in T2*-weighted images, the signal intensity (SI) decreasedwith the increasing Fe concentration in PLGA. On the other hand,Fe_3O_4/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 Fe_3O_4/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 Fe_3O_4/PLGA group, this phenomenon was notoccurred in liver tumor tissue and groups with injection of saline andPLGA.
Conclusion The Fe_3O_4/PLGA microcapsules are successfully acted asmulti-modality biological imaging contrast agents for US, MR and PAimaging in vitro. And The Fe_3O_4/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 ABLATION
Objective To investigate the efficacy of Fe_3O_4/PLGA combined withHIFU in bovine liver tissue ablation, and to investigate the influence ofFe_3O_4/PLGA combined with HIFU in rabbits bearing breast tumor.
Methods The degassed bovine liver in vitro was directly injected into200μl Fe_3O_4/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 Fe_3O_4/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 ofFe_3O_4/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 Fe_3O_4/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, Fe_3O_4nanoparticles were seen depositing in residual tumor cells under TEM.
Conclusion The Fe_3O_4/PLGA microspheres can effectively enhanced thebiomedical effect of HIFU ablation both on bovine liver tissue and onrabbits bearing VX2breast tumor, The Fe_3O_4/PLGA was a potentialsynergistic agent for HIFU cancer surgery.
目的制备一种载超顺磁性氧化铁纳米颗粒（Fe_3O_4）的聚乳酸-羟基乙酸（PLGA）多功能造影剂（Fe_3O_4/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Fe_3O_4/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Fe_3O_4/PLGA（DiI-Fe_3O_4/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Fe_3O_4/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Fe_3O_4/PLGA溶于双蒸水后呈棕色乳液，光镜及扫描电镜观察，Fe_3O_4/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Fe_3O_4颗粒较均匀分布于外壳结构中。Malvern激光粒径仪检测出Fe_3O_4/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Fe_3O_4/PLGA吸收光谱位于590nm和650nm附近。磁滞回线显示，Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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Fe_3O_4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Fe_3O_4高分子造影剂增强超声、磁共振及光声显像实验
目的观察Fe_3O_4/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Fe_3O_4/PLGA对兔肝癌磁共振成像效果的影响，了解Fe_3O_4/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Fe_3O_4/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Fe_3O_4/PLGA微球的光声信号，及被MDA细胞吞噬入胞的Fe_3O_4/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Fe_3O_4/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，Fe_3O_4/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，Fe_3O_4/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，Fe_3O_4/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Fe_3O_4/PLGA光声信号也较强。兔肝癌磁共振成像显示，注射Fe_3O_4/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，Fe_3O_4/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Fe_3O_4/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，Fe_3O_4/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Fe_3O_4高分子造影剂增效HIFU治疗实验
目的观察Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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、Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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Fe_3O_4/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。
PART PREPARATION AND CHARACTERIZATION OFIRON OXIDE-LOADED POLYMER CONTRAST AGENTS
Objective To prepare the superparamagnetic Fe_3O_4-loaded polymermicrocapsules as a novel multi-functional contrast agents (Fe_3O_4/PLGA),and investigate their properties of physics, optics, magnetism and biosafety.
Methods Fe_3O_4/PLGA microcapsules were fabricated by a typicaldouble emulsion evaporation method. The surface morphology andstructure, the mean size and zeta potential distribution, the concentration ofFe_3O_4loading in PLGA microcapsules, the absorption spectrum and themagnetic properties of Fe_3O_4/PLGA were assessed using several analyticalmethods. The DiI labeled Fe_3O_4/PLGA (DiI-Fe_3O_4/PLGA) microcapsules,which were prepared using the same process as Fe_3O_4/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 Fe_3O_4/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 Fe_3O_4/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 Fe_3O_4particlesdistributed in the shell structure were observed under transmission electronmicroscope (TEM). The diameter of Fe_3O_4/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 Fe_3O_4loading in PLGAmicrocapsules was46.58μg/ml detected by an atomic absorptionspectrophotometer (AAS). The Fe_3O_4/PLGA absorption spectrum wasaround590nm and650nm measured by a UV-Vis spectrophotometry. TheM-H hysteresis loop behavior showed the Fe_3O_4/PLGA typicalsuperparamagnetic properties. A time-dependent Fe_3O_4/PLGA uptake byMDA cells was observed under the light microscope. The amount ofFe_3O_4/PLGA uptake increased with the increasing incubation time. MTTresults showed the cells proliferation activity was not affected afterFe_3O_4/PLGA engulfed by MDA cells. And the Fe_3O_4/PLGA had nosignificant effect on rabbit liver, kidney and heart after injection into the rabbits.
Conclusion Fe_3O_4/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 IMAGING
Objective To observe the effect of Fe_3O_4/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 Fe_3O_4/PLGA in MR imaging.
Methods The prepared Fe_3O_4/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 Fe_3O_4/PLGA microcapsule andFe_3O_4/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 Fe_3O_4/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 Fe_3O_4/PLGA showed hyperechogenicity in USimaging, the echo intensity (EI) decreased with the concentration andmechanical index decreasing. The Fe_3O_4/PLGA exhibited MRI negativelyenhanced effect in T2*-weighted images, the signal intensity (SI) decreasedwith the increasing Fe concentration in PLGA. On the other hand,Fe_3O_4/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 Fe_3O_4/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 Fe_3O_4/PLGA group, this phenomenon was notoccurred in liver tumor tissue and groups with injection of saline andPLGA.
Conclusion The Fe_3O_4/PLGA microcapsules are successfully acted asmulti-modality biological imaging contrast agents for US, MR and PAimaging in vitro. And The Fe_3O_4/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 ABLATION
Objective To investigate the efficacy of Fe_3O_4/PLGA combined withHIFU in bovine liver tissue ablation, and to investigate the influence ofFe_3O_4/PLGA combined with HIFU in rabbits bearing breast tumor.
Methods The degassed bovine liver in vitro was directly injected into200μl Fe_3O_4/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 Fe_3O_4/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 ofFe_3O_4/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 Fe_3O_4/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, Fe_3O_4nanoparticles were seen depositing in residual tumor cells under TEM.
Conclusion The Fe_3O_4/PLGA microspheres can effectively enhanced thebiomedical effect of HIFU ablation both on bovine liver tissue and onrabbits bearing VX2breast tumor, The Fe_3O_4/PLGA was a potentialsynergistic agent for HIFU cancer surgery.
引文
[1]王志刚.超声分子影像学研究进展[J].中国医学影像技术.2009,25(6):84-86
[2] Wang Zhigang, Ling Zhiyu, Ran Haitao, et al. Ultrasound-mediated microbubbledestruction enhances VEGF gene delivery to the infarcted myocardium in rats[J].Clinical Imaging.2004,28(6):395-398
[3] Tural B, Ozenbas M, Atalay S, et al. Rapid synthesis and characterization ofmaghemite nanoparticles[J]. J Nanosci Nanotechnol.2008,8(2):861-866
[4] Sun C, Lee JS, Zhang M. Magnetic nanoparticles in MR imaging and drugdelivery[J]. Adv Drug Deliv Rev.2008,60(11):1252-1265
[5] Ling Y, Wei K, Luo Y, et al. Dual docetaxel/superparamagnetic iron oxide loadednanoparticles for both targeting magnetic resonance imaging and cancer therapy[J].Biomaterials.2011,32(29):7139-7150
[6] Liu ZY, Wang Y, Liang CH, et al. In vitro labeling of mesenchymal stem cells withsuperparamagnetic iron oxide by means of microbubble-enhanced US exposure:initial experience[J]. Radiology.2009,253(1):153-159
[7] Degen CL, Poggio M, Mamin HJ, et al. Nanoscale magnetic resonance imaging[J].Proc Natl Acad Sci USA.2009,106(5):1313-1317
[8] Weissleder R, Pittet MJ. Imaging in the era of molecular oncology[J]. Nature.2008,452(7187):580-589
[9] Jun YW, Lee JH, Cheon J. Chemical design of nanoparticle probes for high-performance magnetic resonance imaging[J]. Angew Chem Int Ed.2008,47(28):5122-5135
[10] Qiao RR, Yang CH, Gao MY. Superparamagnetic iron oxide nanoparticles: Frompreparations to in vivo MRI applications[J]. J Mater Chem.2009,19(35):6274-6293
[11] Norton SJ, Vo-Dinh T. Imaging the distribution of magnetic nanoparticles withultrasound[J]. IEEE Trans Med Imaging.2007,26(5):660-665
[12] Liu Z, Lammers T, Ehling J, et al. Iron oxide nanoparticle-containing microbubblecomposites as contrast agents for MR and ultrasound dual-modality imaging[J].Biomaterials.2011,32(26):6155-6163
[13] Kruskal JB. Can USPIO-enhanced spinal MR imaging help distinguish acuteinfectious osteomyelitis from chronic infectious and inflammatory processes[J]?Radiology.2008,248(1):1-3
[14]李奥，王志刚，吴伟，等.载Fe3O4液态氟碳纳米粒增强磁共振成像的实验研究[J].中国医学影像技术.2011,27(3):453-456
[15]乔瑞瑞，贾巧娟，曾剑峰，等.磁性氧化铁纳米颗粒及其磁共振成像应用[J].生物物理学报.2011,27(4):272-288
[16] Lihong V. Wang. Prospects of photoacoustic tomography[J]. Med Phys.2008,35(12):5758-5767
[17] Chen Z, Yang S, Xing D. In vivo detection of hemoglobin oxygen saturation andcarboxyhemoglobin saturation with multiwavelength photoacoustic microscopy[J].Opt Lett.2012,37(16):3414-3416
[18] Andreev VG, Karabutov AA, Oraevsky AA. Detection of ultrawide-bandultrasound pulses in optoacoustic tomography. IEEE Trans Ultrason Ferroele-ctr Freq Control.2003,50(10):1383-1390
[19] Ermilov SA, Khamapirad T, Conjusteau A, et al. Laser optoacoustic imagingsystem for detection of breast cancer. J Biomed Opt.2009,14(2):024007
[20] Li C, Wang LV. Photoacoustic tomography and sensing in biomedicine. Phys. Med.Biol.2009,54(19):R59-97
[21] Grootendorst DJ, Jose J, Fratila RM, et al. Evaluation of superparamagnetic ironoxide nanoparticles (Endorem) as a photoacoustic contrast agent forintra-operative nodal staging[J]. Contrast Media Imaging.2013,8(1):83-91
[22] Eric Strohm, Min Rui, Ivan Gorelikov, et al. Vaporization of perfluorocarbondroplets using optical irradiation[J].Biomed Opt Express.2011,2(6):1432-1442
[23] Orsi F, Arnone P, Chen W, et al. High intensity focused ultrasound ablation: a newtherapeutic option for solid tumors[J]. J Cancer Res Ther.2010,6(4):414-420
[24] Lynn JG, Zwemer RL, Chick AJ, et al. A new method for the generation and use offocused ultrasound in experimental biology[J]. J Gen Physiol.1942,26(2):179-193
[25] Gelet A, Chapelon JY, Poissonnier L, et al. Local recurrence of prostate cancerafter external beam radiotherapy: early experience of salvage therapy usinghigh-intensity focused ultrasonography[J]. Urology.2004,63(4):625-629
[26] Chan AH, Fujimoto VY, Moore DE, et al. In vivo feasibility of image-guidedtransvaginal focused ultrasound therapy for the treatment of intracavitaryfibroids[J]. Fertil Steril.2004,82(3):723-730
[27] Esnault O, Franc B, Menegaux F, et al. High-intensity focused ultrasound ablationof thyroid nodules: first human feasibility study[J]. Thyroid.2011,21(9):965-973
[28] Kennedy JE. High-intensity focused ultrasound in the treatment of solid tumours[J].Nat Rev Cancer.2005,5(4):321-7
[29] ter Haar G. Ultrasound focal beam surgery[J]. Ultrasound Med Biol.1995,21(9):1089-1000
[30]王公正，莫润阳，刘亚红.微米磁性复合材料超声波吸收检测研究[J].西北大学学报.2007,37(1):67-72
[31] Ao M, Wang Z, Ran H, et al. Gd-DTPA-loaded PLGA microbubbles as bothultrasound contrast agent and MRI contrast agent-a feasibility research[J]. JBiomed Mater Res B Appl Biomater.2010,93(2):551-556
[32]张辉，冉海涛，张亚萍，等.结合链霉亲和素的高分子造影剂的制备及其初步应用[J].中国医学影像技术.2010,26(6):997-1000
[33]冉海涛，任红，王志刚，等.包裹阿霉素的高分子材料微泡声学造影剂制备及显影效果实验研究[J].临床超声医学杂志.2005,7(4):217-220
[34]冉海涛，任红，王志刚，等.一种新型高分子聚合材料微泡超声造影剂的制备与体外显影实验[J].中华超声影像学杂志.2005,14(10):774-776
[35]郑元义,王志刚，冉海涛，等.自制高分子材料超声造影剂及初步实验研究[J].中国超声医学杂志.2004,20(12):888-890
[36]沈红霞，郑元义，周洋，等.包裹液态氟碳的高分子超声造影剂的制备及体外显像实验[J].中国介入影像学.2012,9(4):291-294
[37] Gullapalli RR, Demirel MC, Butler PJ. Molecular dynamics simulations ofDiI-C18(3) in a DPPC lipid bilayer[J]. Phys Chem Chem Phys.2008,10(24):3548-3560
[38] Kikkawa YS, Pawlowski KS. Cochlear neuronal tracing for frequency mappingwith DiI, NeuroVue, and Golgi method[J]. Acta Otolaryngol Suppl.2007,559:19-23
[39] Acharya S, Sahoo SK. PLGA nanoparticles containing various anticancer agentsand tumour delivery by EPR effect[J]. Adv Drug Deliv Rev.2011,63(3):170-183
[40] Wheatley MA, Forsberg F, Oum K, et al. Comparison of in vitro and in vivoacoustic response of a novel50:50PLGA contrast agent[J]. Ultrasonics.2006,44(4):360-367
[41] Xu JS, Huang J, Qin R, et al. Synthesizing and binding dual-mode poly(lactic-co-glycolic acid)(PLGA) nanobubbles for cancer targeting and imaging[J].Biomaterials.2010,31(7):1716-1722
[42] Schweiger C, Pietzonka C, Heverhagen J, et al. Novel magnetic iron oxidenanoparticles coated with poly(ethylene imine)-g-poly(ethylene glycol) forpotential biomedical application: synthesis, stability, cytotoxicity and MRimaging[J]. Int J Pharm.2011,408(1-2):130-137
[43] Lu BQ, Zhu YJ, Ao HY, et al. Synthesis and characterization of magnetic ironoxide/calcium silicate mesoporous nanocomposites as a promising vehicle for drugdelivery[J].2012,4(12):6969-6974
[44]刘岘.超顺磁性氧化铁类对比剂及其在肝病变的应用[J].国外医学临床放射学分册.2000,23(6):325-327
[45] Yilmaz A, Dengler MA, van der Kuip H, et al. Imaging of myocardial infarctionusing ultrasmall superparamagnetic iron oxide nanoparticles: a human study usinga multi-parametric cardiovascular magnetic resonance imaging approach[J].EurHeart J.2013,34(6):462-475
[46] Yang D, Zeng L, Pan C, et al. Noninvasive photoacoustic detecting intraocularforeign bodies with an annular transducer array[J]. Opt Express.2013,21(1):984-991
[47] Kircher MF, Willmann JK. Molecular body imaging:MR imaging, CT, and US[J].Part1. principles.2012,263(3):633-643
[48] Jun YW, Seo JW, Cheon J. Nanoscaling laws of magnetic nanoparticles and theirapplicabilities in biomedical sciences[J]. Acc Chem Res.2008,41(2):179-189
[49] Jun YW, Lee JH, Cheon J. Chemical design of nanoparticle probes forhigh-performance magnetic resonance imaging[J]. Angew Chem Int Ed Engl.2008,47(28):5122-5135.
[50] Corot C, Robert P, Idée JM, et al. Recent advances in iron oxide nanocrystaltechnology for medical imaging[J]. Adv Drug Deliv Rev.2006,58(14):1471-1504
[51] Ling Y, Wei K, Gao X, et al. Dual docetaxel/superparamagnetic iron oxidenanoparticles for both targeting magnetic resonance imaging and cancer therapy[J].Biomaterials.2011,32(29):7139-7150
[52] Ma HL, Xu YF, Qi XR, et al. Superparamagnetic iron oxide nanoparticles stabilizeby alginate, pharmacokinetics, tissue distribution, and applications in detectingliver cancers[J]. Int J Pharm.2008,354(1-2):217-226
[53] Smirnov P, Laverqne E, Gazeau F, et al. In vivo cellular imaging of lymphocytetrafficking by MRI: a tumor model approach to cell-based anticancer therapy[J].Magn Reson Med.2006,56(3):498-508
[54] Brahler M, Georgieva R, Buske N, et al. Magnetic-loaded carrier erythrocytes ascontrast agents for magnetic resonance imaging[J]. Nano Lett.2006,6(11):2505-2509
[55] Brindle K. New approaches for imaging tumor responses to treatment[J]. Nat RevCancer.2008,8(2):94-107
[56] Han Z, Fu A, Wang H, et al. Noninvasive assessment of cancer response totherapy[J]. Nat Med.2008,14(3):343-349
[57] Jun YW, Seo JW, Cheon A. Nanoscaling laws of magnetic nanoparticles and theirapplicabilities in biomedical sciences[J]. Acc Chem Res.2008,41(2):179-189
[58] Yang L, Peng XH, Wang YA, et al. Receptor-targeted nanoparticles for in vivoimaging of breast cancer[J]. Clin Cancer Res.2009,15(14):4722-4732
[59]李和，周莉.免疫化学与免疫组织化学检测[M].北京：人民卫生出版社，2008.59
[60] Li YY, Sha WH, Zhou YJ, et al. Short and long term efficacy of high intensityfocused ultrasound therapy for advanced hepatocellular carcinoma[J]. JGastroenterol Hepatol.2007,22(12):2148-2154
[61] Li JJ, Xu GL, Gu MF, et al. Complications of high intensity focused ultrasound inpatients with recurrent and metastatic abdominal tumors[J]. World J Gastroenterol.2007,13(19):2747-2151
[62]邓凤莲，姜振东，邹建中，等.高强度聚焦超声治疗子宫肌瘤临床并发症分析[J].临床超声医学杂志.2010,12(5):326-328
[63] Wen Luo, Xiaodong Zhou, Xiaolong Ren, et al. Enhancing effects of SonoVue, amicrobubble sonographic contrast agent, on high-intensity focused ultrasoundablation in rabbit livers in vivo[J]. Ultrasound Med.2007,26(4):469-476
[64] Wen Luo, Zhou X, Qian Y, et al. Analysis of apoptosis and cell proliferation afterhigh intensity-focused ultrasound ablation combined with microbubbles in rabbitlivers[J]. Eur J Gastroenterol Hepatol.2007,19(11):962-968
[65] Chung DJ, Cho SH, Lee JM, et al. Effect of microbubble contrast agent during highintensity focused ultrasound ablation on rabbit liver in vivo[J]. Eur J Radiol.2012,81(4):519-523
[1]李奥,王志刚.液态氟碳纳米粒——一种多功能影像学造影剂[J].临床超声医学杂志.2008,10(12):830-832
[2]李奥,王志刚,余进洪,等.液态氟碳纳米粒增强CT成像在兔VX2肝癌模型中的应用[J].中国医学影像技术.2010,26(5):809-811
[3] Rapoport N, Nam KH, Gupta R, et al. Ultrasound-mediated tumor imaging andnanotherapy using drug loaded, block copolymer stabilizedperfluorocarbonna-noemulsions[J]. J Control Release.2011,153(1):4-15
[4] Hu G, Lijowski M, Zhang H, et al. Imaging of VX2rabbit tumors alpha(nu)beta3-integrin-targeted111In nanoparticles[J]. Int J Cancer.2007,120(9):1951-1957
[5] Liu Z, Lammers T, Ehling J, et al. Iron oxide nanoparticle-containing micro-bubblecomposites as contrast agents for MR and ultrasound dual-modality imaging[J].Biomaterials.2011,32(26):6155-6163
[6] Fan C, Gao W, Chen Z, et al. Tumor selectivity of stealth multi-functionalizedsuperparamagnetic iron oxide nanoparticles[J]. Int J Pharm.2011,404(1-2):180-190
[7] Arifin DR, Long CM, Gilad AA, et al. Trimodal Gadolinium-gold microcapsulescontaining pancreatic islet cells restore normoglycemia in diabetic mice and can betracked by using US, CT, and positive-contrast MR imaging[J]. Radiology.2011,260(3):790-798
[8] Kim D, Jeong YY, Jon S. A drug-loaded aptamer-gold nanoparticle bioconjugatefor combined CT imaging and therapy of prostate cancer[J]. ACS Nano.2010,4(7):3689-3696
[9]柯亨特，邢占文，岳磊，等.新型荧光-超声双功能复合成像剂的制备和表征[J].中国组织工程研究与临床康复.2009,13(29):5798-5800
[10] Ren JL, Xu CS, Wang ZG, et al. A novel ultrasound microbubble carrying gene andtat peptide: preparation and characterization[J]. Acad Radiol.2009,16(12):1457–1465
[11] Zhou ZX, Zhang BG, Zhang H, et al. Drug packaging and delivery usingperfuorocarbon nanoparticles for targeted inhibition of vascular smoothmuscle cells[J]. Acta Pharmacol Sin.2009,30(11):1577-1584
[12] Winter PM, Schmieder AH, Caruthers SD, et al. Minute dosages of alpha(nu)beta3-targeted fumagillin nanoparticles impair VX-2tumor angiogenesisand development in rabbits[J]. FASEB J.2008,22(8):2758-2767
[13] Maeng JH, Lee DH, Jung KH, et al. Multifunctional doxorubicin loadedsuperparamagnetic iron oxide nanoparticles for chemotherapy and magneticresonance imaging in liver cancer[J]. Biomaterials.2010,31(18):4995-5006
[14] Dilnawaz F, Singh A, Mohanty C, et al. Dual drug loaded superparamagnetic ironoxide nanoparticles for targeted cancer therapy[J]. Biomaterials.2010,31(13):3694-3706
[15] Ke H, Wang J, Dai Z, et al. Gold-nanoshelled microcapsules: a theranostic agentfor ultrasound contrast imaging and photothermal therapy. Anqew Chem Int EdEnql[J].2011,50(13):3017-3021
[16] Jing Y, Zhu Y, Yang X, et al. Ultrasound-triggered smart drug release frommultifunctional core-shell capsules one-step fabricated by coaxial electrospraymethod[J]. Langmuir.2010,27(3):1175-1180
[17] Famy CH, Glynn Holt R, Roy RA.The correlation between bubble-enhancedHIFU heating and cavitation power[J]. IEEE Trans Biomed Eng.2010,57(1):175-184
[18] Yu T, Wang G, Hu K, et al. A microbubble agent improves the therapeuticefficiency of high intensity focused ultrasound: a rabbit kidney study[J]. Urol Res.2004,32(1):14-19
[19] Kennedy JE, Ter Haar GR, Wu F, et al.Contrast-enhanced ultrasound assessment oftissue response to high-intensity focused ultrasound[J].Ultrasound Med Biol.2004,30(6):851-854
[20]张闻，陈建科，刘学明，等.超声造影在评价高强度聚焦超声治疗子宫平滑肌瘤疗效中的价值[J].中国肿瘤.2007,16(11):956-958
[2] Wang Zhigang, Ling Zhiyu, Ran Haitao, et al. Ultrasound-mediated microbubbledestruction enhances VEGF gene delivery to the infarcted myocardium in rats[J].Clinical Imaging.2004,28(6):395-398
[3] Tural B, Ozenbas M, Atalay S, et al. Rapid synthesis and characterization ofmaghemite nanoparticles[J]. J Nanosci Nanotechnol.2008,8(2):861-866
[4] Sun C, Lee JS, Zhang M. Magnetic nanoparticles in MR imaging and drugdelivery[J]. Adv Drug Deliv Rev.2008,60(11):1252-1265
[5] Ling Y, Wei K, Luo Y, et al. Dual docetaxel/superparamagnetic iron oxide loadednanoparticles for both targeting magnetic resonance imaging and cancer therapy[J].Biomaterials.2011,32(29):7139-7150
[6] Liu ZY, Wang Y, Liang CH, et al. In vitro labeling of mesenchymal stem cells withsuperparamagnetic iron oxide by means of microbubble-enhanced US exposure:initial experience[J]. Radiology.2009,253(1):153-159
[7] Degen CL, Poggio M, Mamin HJ, et al. Nanoscale magnetic resonance imaging[J].Proc Natl Acad Sci USA.2009,106(5):1313-1317
[8] Weissleder R, Pittet MJ. Imaging in the era of molecular oncology[J]. Nature.2008,452(7187):580-589
[9] Jun YW, Lee JH, Cheon J. Chemical design of nanoparticle probes for high-performance magnetic resonance imaging[J]. Angew Chem Int Ed.2008,47(28):5122-5135
[10] Qiao RR, Yang CH, Gao MY. Superparamagnetic iron oxide nanoparticles: Frompreparations to in vivo MRI applications[J]. J Mater Chem.2009,19(35):6274-6293
[11] Norton SJ, Vo-Dinh T. Imaging the distribution of magnetic nanoparticles withultrasound[J]. IEEE Trans Med Imaging.2007,26(5):660-665
[12] Liu Z, Lammers T, Ehling J, et al. Iron oxide nanoparticle-containing microbubblecomposites as contrast agents for MR and ultrasound dual-modality imaging[J].Biomaterials.2011,32(26):6155-6163
[13] Kruskal JB. Can USPIO-enhanced spinal MR imaging help distinguish acuteinfectious osteomyelitis from chronic infectious and inflammatory processes[J]?Radiology.2008,248(1):1-3
[14]李奥，王志刚，吴伟，等.载Fe3O4液态氟碳纳米粒增强磁共振成像的实验研究[J].中国医学影像技术.2011,27(3):453-456
[15]乔瑞瑞，贾巧娟，曾剑峰，等.磁性氧化铁纳米颗粒及其磁共振成像应用[J].生物物理学报.2011,27(4):272-288
[16] Lihong V. Wang. Prospects of photoacoustic tomography[J]. Med Phys.2008,35(12):5758-5767
[17] Chen Z, Yang S, Xing D. In vivo detection of hemoglobin oxygen saturation andcarboxyhemoglobin saturation with multiwavelength photoacoustic microscopy[J].Opt Lett.2012,37(16):3414-3416
[18] Andreev VG, Karabutov AA, Oraevsky AA. Detection of ultrawide-bandultrasound pulses in optoacoustic tomography. IEEE Trans Ultrason Ferroele-ctr Freq Control.2003,50(10):1383-1390
[19] Ermilov SA, Khamapirad T, Conjusteau A, et al. Laser optoacoustic imagingsystem for detection of breast cancer. J Biomed Opt.2009,14(2):024007
[20] Li C, Wang LV. Photoacoustic tomography and sensing in biomedicine. Phys. Med.Biol.2009,54(19):R59-97
[21] Grootendorst DJ, Jose J, Fratila RM, et al. Evaluation of superparamagnetic ironoxide nanoparticles (Endorem) as a photoacoustic contrast agent forintra-operative nodal staging[J]. Contrast Media Imaging.2013,8(1):83-91
[22] Eric Strohm, Min Rui, Ivan Gorelikov, et al. Vaporization of perfluorocarbondroplets using optical irradiation[J].Biomed Opt Express.2011,2(6):1432-1442
[23] Orsi F, Arnone P, Chen W, et al. High intensity focused ultrasound ablation: a newtherapeutic option for solid tumors[J]. J Cancer Res Ther.2010,6(4):414-420
[24] Lynn JG, Zwemer RL, Chick AJ, et al. A new method for the generation and use offocused ultrasound in experimental biology[J]. J Gen Physiol.1942,26(2):179-193
[25] Gelet A, Chapelon JY, Poissonnier L, et al. Local recurrence of prostate cancerafter external beam radiotherapy: early experience of salvage therapy usinghigh-intensity focused ultrasonography[J]. Urology.2004,63(4):625-629
[26] Chan AH, Fujimoto VY, Moore DE, et al. In vivo feasibility of image-guidedtransvaginal focused ultrasound therapy for the treatment of intracavitaryfibroids[J]. Fertil Steril.2004,82(3):723-730
[27] Esnault O, Franc B, Menegaux F, et al. High-intensity focused ultrasound ablationof thyroid nodules: first human feasibility study[J]. Thyroid.2011,21(9):965-973
[28] Kennedy JE. High-intensity focused ultrasound in the treatment of solid tumours[J].Nat Rev Cancer.2005,5(4):321-7
[29] ter Haar G. Ultrasound focal beam surgery[J]. Ultrasound Med Biol.1995,21(9):1089-1000
[30]王公正，莫润阳，刘亚红.微米磁性复合材料超声波吸收检测研究[J].西北大学学报.2007,37(1):67-72
[31] Ao M, Wang Z, Ran H, et al. Gd-DTPA-loaded PLGA microbubbles as bothultrasound contrast agent and MRI contrast agent-a feasibility research[J]. JBiomed Mater Res B Appl Biomater.2010,93(2):551-556
[32]张辉，冉海涛，张亚萍，等.结合链霉亲和素的高分子造影剂的制备及其初步应用[J].中国医学影像技术.2010,26(6):997-1000
[33]冉海涛，任红，王志刚，等.包裹阿霉素的高分子材料微泡声学造影剂制备及显影效果实验研究[J].临床超声医学杂志.2005,7(4):217-220
[34]冉海涛，任红，王志刚，等.一种新型高分子聚合材料微泡超声造影剂的制备与体外显影实验[J].中华超声影像学杂志.2005,14(10):774-776
[35]郑元义,王志刚，冉海涛，等.自制高分子材料超声造影剂及初步实验研究[J].中国超声医学杂志.2004,20(12):888-890
[36]沈红霞，郑元义，周洋，等.包裹液态氟碳的高分子超声造影剂的制备及体外显像实验[J].中国介入影像学.2012,9(4):291-294
[37] Gullapalli RR, Demirel MC, Butler PJ. Molecular dynamics simulations ofDiI-C18(3) in a DPPC lipid bilayer[J]. Phys Chem Chem Phys.2008,10(24):3548-3560
[38] Kikkawa YS, Pawlowski KS. Cochlear neuronal tracing for frequency mappingwith DiI, NeuroVue, and Golgi method[J]. Acta Otolaryngol Suppl.2007,559:19-23
[39] Acharya S, Sahoo SK. PLGA nanoparticles containing various anticancer agentsand tumour delivery by EPR effect[J]. Adv Drug Deliv Rev.2011,63(3):170-183
[40] Wheatley MA, Forsberg F, Oum K, et al. Comparison of in vitro and in vivoacoustic response of a novel50:50PLGA contrast agent[J]. Ultrasonics.2006,44(4):360-367
[41] Xu JS, Huang J, Qin R, et al. Synthesizing and binding dual-mode poly(lactic-co-glycolic acid)(PLGA) nanobubbles for cancer targeting and imaging[J].Biomaterials.2010,31(7):1716-1722
[42] Schweiger C, Pietzonka C, Heverhagen J, et al. Novel magnetic iron oxidenanoparticles coated with poly(ethylene imine)-g-poly(ethylene glycol) forpotential biomedical application: synthesis, stability, cytotoxicity and MRimaging[J]. Int J Pharm.2011,408(1-2):130-137
[43] Lu BQ, Zhu YJ, Ao HY, et al. Synthesis and characterization of magnetic ironoxide/calcium silicate mesoporous nanocomposites as a promising vehicle for drugdelivery[J].2012,4(12):6969-6974
[44]刘岘.超顺磁性氧化铁类对比剂及其在肝病变的应用[J].国外医学临床放射学分册.2000,23(6):325-327
[45] Yilmaz A, Dengler MA, van der Kuip H, et al. Imaging of myocardial infarctionusing ultrasmall superparamagnetic iron oxide nanoparticles: a human study usinga multi-parametric cardiovascular magnetic resonance imaging approach[J].EurHeart J.2013,34(6):462-475
[46] Yang D, Zeng L, Pan C, et al. Noninvasive photoacoustic detecting intraocularforeign bodies with an annular transducer array[J]. Opt Express.2013,21(1):984-991
[47] Kircher MF, Willmann JK. Molecular body imaging:MR imaging, CT, and US[J].Part1. principles.2012,263(3):633-643
[48] Jun YW, Seo JW, Cheon J. Nanoscaling laws of magnetic nanoparticles and theirapplicabilities in biomedical sciences[J]. Acc Chem Res.2008,41(2):179-189
[49] Jun YW, Lee JH, Cheon J. Chemical design of nanoparticle probes forhigh-performance magnetic resonance imaging[J]. Angew Chem Int Ed Engl.2008,47(28):5122-5135.
[50] Corot C, Robert P, Idée JM, et al. Recent advances in iron oxide nanocrystaltechnology for medical imaging[J]. Adv Drug Deliv Rev.2006,58(14):1471-1504
[51] Ling Y, Wei K, Gao X, et al. Dual docetaxel/superparamagnetic iron oxidenanoparticles for both targeting magnetic resonance imaging and cancer therapy[J].Biomaterials.2011,32(29):7139-7150
[52] Ma HL, Xu YF, Qi XR, et al. Superparamagnetic iron oxide nanoparticles stabilizeby alginate, pharmacokinetics, tissue distribution, and applications in detectingliver cancers[J]. Int J Pharm.2008,354(1-2):217-226
[53] Smirnov P, Laverqne E, Gazeau F, et al. In vivo cellular imaging of lymphocytetrafficking by MRI: a tumor model approach to cell-based anticancer therapy[J].Magn Reson Med.2006,56(3):498-508
[54] Brahler M, Georgieva R, Buske N, et al. Magnetic-loaded carrier erythrocytes ascontrast agents for magnetic resonance imaging[J]. Nano Lett.2006,6(11):2505-2509
[55] Brindle K. New approaches for imaging tumor responses to treatment[J]. Nat RevCancer.2008,8(2):94-107
[56] Han Z, Fu A, Wang H, et al. Noninvasive assessment of cancer response totherapy[J]. Nat Med.2008,14(3):343-349
[57] Jun YW, Seo JW, Cheon A. Nanoscaling laws of magnetic nanoparticles and theirapplicabilities in biomedical sciences[J]. Acc Chem Res.2008,41(2):179-189
[58] Yang L, Peng XH, Wang YA, et al. Receptor-targeted nanoparticles for in vivoimaging of breast cancer[J]. Clin Cancer Res.2009,15(14):4722-4732
[59]李和，周莉.免疫化学与免疫组织化学检测[M].北京：人民卫生出版社，2008.59
[60] Li YY, Sha WH, Zhou YJ, et al. Short and long term efficacy of high intensityfocused ultrasound therapy for advanced hepatocellular carcinoma[J]. JGastroenterol Hepatol.2007,22(12):2148-2154
[61] Li JJ, Xu GL, Gu MF, et al. Complications of high intensity focused ultrasound inpatients with recurrent and metastatic abdominal tumors[J]. World J Gastroenterol.2007,13(19):2747-2151
[62]邓凤莲，姜振东，邹建中，等.高强度聚焦超声治疗子宫肌瘤临床并发症分析[J].临床超声医学杂志.2010,12(5):326-328
[63] Wen Luo, Xiaodong Zhou, Xiaolong Ren, et al. Enhancing effects of SonoVue, amicrobubble sonographic contrast agent, on high-intensity focused ultrasoundablation in rabbit livers in vivo[J]. Ultrasound Med.2007,26(4):469-476
[64] Wen Luo, Zhou X, Qian Y, et al. Analysis of apoptosis and cell proliferation afterhigh intensity-focused ultrasound ablation combined with microbubbles in rabbitlivers[J]. Eur J Gastroenterol Hepatol.2007,19(11):962-968
[65] Chung DJ, Cho SH, Lee JM, et al. Effect of microbubble contrast agent during highintensity focused ultrasound ablation on rabbit liver in vivo[J]. Eur J Radiol.2012,81(4):519-523
[1]李奥,王志刚.液态氟碳纳米粒——一种多功能影像学造影剂[J].临床超声医学杂志.2008,10(12):830-832
[2]李奥,王志刚,余进洪,等.液态氟碳纳米粒增强CT成像在兔VX2肝癌模型中的应用[J].中国医学影像技术.2010,26(5):809-811
[3] Rapoport N, Nam KH, Gupta R, et al. Ultrasound-mediated tumor imaging andnanotherapy using drug loaded, block copolymer stabilizedperfluorocarbonna-noemulsions[J]. J Control Release.2011,153(1):4-15
[4] Hu G, Lijowski M, Zhang H, et al. Imaging of VX2rabbit tumors alpha(nu)beta3-integrin-targeted111In nanoparticles[J]. Int J Cancer.2007,120(9):1951-1957
[5] Liu Z, Lammers T, Ehling J, et al. Iron oxide nanoparticle-containing micro-bubblecomposites as contrast agents for MR and ultrasound dual-modality imaging[J].Biomaterials.2011,32(26):6155-6163
[6] Fan C, Gao W, Chen Z, et al. Tumor selectivity of stealth multi-functionalizedsuperparamagnetic iron oxide nanoparticles[J]. Int J Pharm.2011,404(1-2):180-190
[7] Arifin DR, Long CM, Gilad AA, et al. Trimodal Gadolinium-gold microcapsulescontaining pancreatic islet cells restore normoglycemia in diabetic mice and can betracked by using US, CT, and positive-contrast MR imaging[J]. Radiology.2011,260(3):790-798
[8] Kim D, Jeong YY, Jon S. A drug-loaded aptamer-gold nanoparticle bioconjugatefor combined CT imaging and therapy of prostate cancer[J]. ACS Nano.2010,4(7):3689-3696
[9]柯亨特，邢占文，岳磊，等.新型荧光-超声双功能复合成像剂的制备和表征[J].中国组织工程研究与临床康复.2009,13(29):5798-5800
[10] Ren JL, Xu CS, Wang ZG, et al. A novel ultrasound microbubble carrying gene andtat peptide: preparation and characterization[J]. Acad Radiol.2009,16(12):1457–1465
[11] Zhou ZX, Zhang BG, Zhang H, et al. Drug packaging and delivery usingperfuorocarbon nanoparticles for targeted inhibition of vascular smoothmuscle cells[J]. Acta Pharmacol Sin.2009,30(11):1577-1584
[12] Winter PM, Schmieder AH, Caruthers SD, et al. Minute dosages of alpha(nu)beta3-targeted fumagillin nanoparticles impair VX-2tumor angiogenesisand development in rabbits[J]. FASEB J.2008,22(8):2758-2767
[13] Maeng JH, Lee DH, Jung KH, et al. Multifunctional doxorubicin loadedsuperparamagnetic iron oxide nanoparticles for chemotherapy and magneticresonance imaging in liver cancer[J]. Biomaterials.2010,31(18):4995-5006
[14] Dilnawaz F, Singh A, Mohanty C, et al. Dual drug loaded superparamagnetic ironoxide nanoparticles for targeted cancer therapy[J]. Biomaterials.2010,31(13):3694-3706
[15] Ke H, Wang J, Dai Z, et al. Gold-nanoshelled microcapsules: a theranostic agentfor ultrasound contrast imaging and photothermal therapy. Anqew Chem Int EdEnql[J].2011,50(13):3017-3021
[16] Jing Y, Zhu Y, Yang X, et al. Ultrasound-triggered smart drug release frommultifunctional core-shell capsules one-step fabricated by coaxial electrospraymethod[J]. Langmuir.2010,27(3):1175-1180
[17] Famy CH, Glynn Holt R, Roy RA.The correlation between bubble-enhancedHIFU heating and cavitation power[J]. IEEE Trans Biomed Eng.2010,57(1):175-184
[18] Yu T, Wang G, Hu K, et al. A microbubble agent improves the therapeuticefficiency of high intensity focused ultrasound: a rabbit kidney study[J]. Urol Res.2004,32(1):14-19
[19] Kennedy JE, Ter Haar GR, Wu F, et al.Contrast-enhanced ultrasound assessment oftissue response to high-intensity focused ultrasound[J].Ultrasound Med Biol.2004,30(6):851-854
[20]张闻，陈建科，刘学明，等.超声造影在评价高强度聚焦超声治疗子宫平滑肌瘤疗效中的价值[J].中国肿瘤.2007,16(11):956-958