胰腺癌LyP-1靶向性磁共振—荧光双模态分子探针实验

目的 构建胰腺癌LyP-1靶向性磁共振—荧光双模态分子探针,观察其表征并进行MR显像。方法 构建50 nm核壳结构的磁共振—荧光双模态介孔探针,表面经环形多肽LyP-1（Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys）修饰,通过荧光及MR T2WI联合成像,验证该分子探针能否特异性识别并结合胰腺癌细胞。结果 新型LyP-1靶向性磁共振—荧光双模态分子探针可用于小鼠原位胰腺癌的荧光显像和磁共振成像,可有效结合胰腺癌细胞并在体外使T2WI信号明显降低。体内实验表明,该分子探针可与荧光标记的胰腺癌组织靶向结合,并作为MRI对比剂显示C57BL/6小鼠原位胰腺癌。结论 LyP-1靶向性磁共振—荧光双模态分子探针可靶向性地高效结合原位移植的小鼠胰腺癌细胞,用于早期胰腺癌诊断的转化研究。     

携IR780碘化物液态氟碳纳米粒造影剂的制备及体外双模态成像

目的 制备携带IR780碘化物的液态氟碳纳米粒（IFNPs）,观察其体外超声/光声双模态成像效果。方法 以羟基端乳酸/羟基乙酸共聚物（PLGA-COOH）、IR780碘化物和全氟戊烷（PFP）为原料,采用双乳化法制备PLGA包裹的纳米粒IFNPs。以光学显微镜、共聚焦显微镜、透射电子显微镜及扫描电子显微镜检测其一般物理特性;以马尔文粒径分析仪分析其粒径大小、分布及表面电位;以紫外分光光度计测定IFNPs中IR780的包封率;观察IFNPs体外光声成像和超声成像的能力。结果 成功制备出IFNPs,其形态规则,大小均一,透射电子显微镜下表现为外壳黑色、内部灰白色的球形结构,扫描电子显微镜显示纳米粒表面光滑。IFNPs粒径为（241.87±3.47）nm,电位为（-0.766±0.096）mV,IR780包封率为（90.38±0.48）%。随IFNPs浓度增加,体外光声成像及超声成像效果均显著增强。结论 携带IR780碘化物的液态氟碳纳米粒造影剂制备成功,可用于体外超声/光声双模态成像。     

诊疗一体化胶质瘤靶向纳米粒增效声动力治疗及多模态显像：体外实验研究

目的 制备胶质瘤靶向诊疗一体化纳米粒,观察体外多模态显像及对U87细胞主动靶向能力和增效声动力治疗（SDT）效果。方法 以双乳化法制备载IR780和全氟戊烷（PFP）纳米粒（IR780-PFP@PLGA）,验证其体外光声（PA）/超声（US）/荧光（FL）多模态显像能力;以细胞计数试剂盒（CCK-8）检测细胞毒性,激光共聚焦显微镜观察其主动靶向能力,通过单线氧荧光探针（SOSG）检测法、JC-1染色法、2'',7''-二氯荧光黄双乙酸盐（DCFH-DA）染色法、钙黄绿素/碘化丙啶（Calcein-AM/PI）双染及CCK-8评价其增效SDT效果。结果 所制备IR780-PFP@PLGA呈圆形,形态规则,粒径均匀,平均（240.67±1.30） nm,表面电位（-9.50±0.06） mV,IR780包封率（92.03±0.42）%;经激光辐照后其PA和FL信号强度呈浓度依赖性,经低强度聚焦超声（LIFU）辐照后表现出良好US对比度成像能力和较强U87细胞靶向能力,并呈活性氧（ROS）浓度依赖。于U87细胞中检出大量ROS,线粒体膜电位明显降低,细胞毒作用明显。结论 成功构建的IR780-PFP@PLGA不仅可用于PA/US/FL多模态显像并具有良好体外寻靶能力,还可通过声致相变（ADV）增强SDT疗效,有望用于多模态成像指导下早期诊断和靶向精准治疗脑胶质瘤并评估疗效。     

超声响应性纳米粒子用于超声/光声成像引导下声动力/饥饿治疗小鼠结直肠癌

目的 制备载四苯基卟啉锌（TPZ）、葡萄糖氧化酶（GOD）与全氟戊烷的超声响应性纳米粒子（NP）TPZ-GOD NP,并评估其超声/光声双模态成像效果及介导声动力治疗（SDT）联合饥饿治疗小鼠结直肠癌的效果。方法 以薄膜水化法制备载TPZ及GOD的NP。观察TPZ-GOD NP基本特性,以紫外分光光度计检测其超声辐照下释放GOD量,以流式细胞仪检测结直肠癌CT-26细胞与TPZ-GOD NP共同孵育后细胞吞噬情况,于荧光显微镜下观察超声辐照后TPZ-GOD NP细胞毒性活性氧产生情况。检测TPZ-GOD NP增强体内外超声/光声双模态成像效果;观察TPZ-GOD NP介导SDT、饥饿治疗及二者联合对移植瘤小鼠模型的作用。结果 成功制备TPZ-GOD NP,颗粒呈球形,大小均一,平均粒径（262.10±62.92）nm。超声辐照后,GOD释放量随时间而增加。TPZ-GOD NP可被CT-26细胞吞噬,超声辐照后可产生大量细胞毒性活性氧;可发生液气相变,增强体内外超声造影效果。体外光声成像显示,TPZ-GOD NP光声信号值随浓度而呈线性增加。经尾静脉注射TPZ-GOD NP后,小鼠肿瘤区域内光声信号明显增强;SDT或饥饿治疗均可杀伤部分CT-26细胞,二者联合效果更佳。结论 制备TPZ-GOD NP具有超声响应性,可用于超声/光声双模态成像引导下声动力联合饥饿治疗小鼠结直肠癌。     

基于肝素-丙二醇酯的新型磁共振对比剂用于小鼠肿瘤成像

目的 观察基于肝素-丙二醇酯的新型磁共振对比剂（Gd-PPF-SS-CA）用于小鼠皮下瘤成像的价值。方法 首先对比Gd-PPF-SS-CA与钆喷替酸葡甲胺（DTPA-Gd）的体外纵向弛豫率。之后选取荷瘤BALB/c小鼠10只,随机分为实验组和对照组各5只;实验组以Gd-PPF-SS-CA、对照组以DTPA-Gd为对比剂,行小鼠肿瘤部位增强扫描,比较2组肿瘤体内增强效果、增强幅度及强化持续时间。将10只健康BALB/c小鼠随机分为Gd-PPF-SS-CA组与生理盐水组各5只,分别经静脉注射0.08 mmol Gd/kg体质量或同等量生理盐水,24小时后通过病理学检查观察Gd-PPF-SS-CA在体急性毒性作用。结果 Gd-PPF-SS-CA纵向弛豫率为12.87 L/（mmol·s）,为DTPA-Gd 的3.67倍。在体MR成像中,实验组肿瘤部位较对照组增强更明显,增强幅度更高、持续时间更长。组织病理学检查中,Gd-PPF-SS-CA组未见明显异常。结论 Gd-PPF-SS-CA体外纵向弛豫率高于 DTPA-Gd;用于小鼠体内肿瘤成像增强效果明显,强化持续时间长,且生物相容性良好,临床应用前景可期。     

基于七甲川菁构建并初步评估双模态PET/近红外荧光探针

目的 基于七甲川菁染料MHI148构建新型PET/近红外荧光(NIRF)双模态探针，并初步评估其用于肿瘤模型小鼠成像的能力。方法 采用双功能螯合剂DOTA修饰MHI148构建MHI148-DOTA,对其光物理、光热性能进行表征；以放射性核素⁶⁸Ga标记MHI148-DOTA,合成探针⁶⁸Ga-MHI148,测定其放射化学纯度及稳定性。建立小鼠乳腺癌4T1及人脑胶质母细胞瘤U-87MG皮下瘤模型，以⁶⁸Ga-MHI148探针对其进行小动物PET显像，观察成像效果及该探针在小鼠体内的生物分布。结果 MHI148-DOTA光物理及光热性能优异。以之构建的探针⁶⁸Ga-MHI148放射化学纯度>99%,稳定性良好；小动物PET成像可清晰显示肿瘤，肿瘤摄取随时间延长而逐渐升高，6 h时U-87MG及4T1肿瘤/肌肉摄取比值分别为4.55±0.20及8.08±2.26;⁶⁸Ga-MHI148小鼠体内主要分布于血液、肺、肝及肾脏。结论 成功构建的双模态⁶⁸Ga-M...     

多功能纳米液滴超声造影剂对胰腺癌分子靶向诊疗的实验研究

目的探讨自制的诊疗一体化相变纳米液滴"IR780/叶酸-纳米液滴-多西紫杉醇(IR780/FA-Nds-DTX)"作为分子靶向超声造影剂, 对胰腺癌荷瘤裸鼠的精准诊断和联合靶向治疗效果。方法体外培养胰腺癌细胞系, 建立异位皮下移植胰腺癌荷瘤裸鼠模型50只, 双模态靶向成像分实验组(IR780/FA-Nds-DTX)和4个对照组[生理盐水、Nds(FITC)、FA-Nds(FITC)、IR-780]。小动物活体成像验证IR780/FA-Nds-DTX对肿瘤的体内高效靶向探测能力及近红外荧光成像, 低强度聚焦超声诱导相变及进一步超声造影成像验证IR780/FA-Nds-DTX在肿瘤局部的高效聚集及对肿瘤轮廓的精准评估。治疗效果观察分实验组(IR780/FA-Nds-DTX)和4个对照组(FA-Nds-IR780、FA-Nds-DTX、FA-Nds和生理盐水)。低强度聚焦超声辐照30 s诱导各组相变后的微泡爆破后, 808 nm光热治疗仪分组辐照肿瘤局部, 治疗前及治疗后3 d、9 d、15 d分别用二维超声监测各组荷瘤裸鼠肿瘤体积变化并记录, 对比各组肿瘤体积变化率及抑制率。结果 IR78...     

前列腺干细胞抗原特异性分子探针的构建及其在体MR成像的实验研究

目的 应用纳米金磁微粒标记抗人前列腺干细胞抗原(PSCA)单抗7F5,构建PSCA特异性MR分子探针7F5@GoldMag,检测其与前列腺癌细胞结合的特异性,并探讨其用于前列腺癌体外及体内MR成像的可行性。方法 应用非共价键耦联方法将纳米金磁微粒标记7F5,构建PSCA特异性MR分子探针7F5@GoldMag,检测其与前列腺癌细胞结合的特异性;应用3.0T磁共振扫描仪,观察不同浓度金磁微粒MR成像情况,探讨MRI可分辨的最低浓度;培养人前列腺癌细胞PC-3和肝癌细胞SMMC-7721,将这两种细胞分别与7F5@GoldMag、无关抗体@GoldMag交叉配对,对各组细胞行T2WI并分别测量其信号强度。建立PC-3和SMMC-7721荷瘤裸鼠模型,对两组荷瘤裸鼠经尾静脉注射7F5@GoldMag和无关抗体@GoldMag,分别在注射前、注射后6、12和24 h行MR扫描,观察其对肿瘤T2WI信号的影响,分别测量其信号强度,探讨其用于MR体内成像的可行性,分析比较其信号强度的差异。结果 成功构建PSCA特异性MR分子探针7F5@GoldMag;流式细胞术检测其与PC-3细胞结合率为92.11％;激光共聚焦显微镜及透射电镜观察见PC-3细胞与7F5@GoldMag有特异性结合。纳米金磁微粒稀释至1 ∶ 640,与1％琼脂糖凝胶相比,T2WI信号强度差异有统计学意义;7F5@GoldMag可显著特异性降低PC-3细胞T2WI信号。PC-3荷瘤裸鼠注射7F5@GoldMag 6、12和24 h后肿瘤组织T2WI信号强度较平扫显著降低(F=43.675,P＜0.05)。而SMMC-7721荷瘤裸鼠在平扫和注射对比剂后6、12和24 h肿瘤信号强度差异无统计学意义。结论 PSCA特异性MR分子探针7F5@GoldMag具有良好的理化性质和免疫活性,可特异性降低PC-3细胞的T2WI信号,对活体前列腺癌组织具有靶向性的增强效果。     

一种产气超声/光声双模态纳米粒的制备及性质检测

目的 研制一种包碳酸氢铵溶液的脂质纳米粒,并观察其超声/光声成像效果。方法 采用薄膜水化法加挤出法制备脂质包裹碳酸氢铵溶液的纳米粒,光镜、电镜、激光粒径仪和电位检测仪检测纳米粒一般物理特性,并通过光声成像仪观察其超声/光声成像效果。结果 制备的纳米粒呈圆球形,形态规则,大小分布均匀,无明显聚集,平均粒径为(230.90±54.58)nm,电位为(-22.81±5.75)mV。碳酸氢铵纳米粒有超声/光声信号,双蒸水纳米粒无超声/光声信号。结论 成功制备包碳酸氢铵溶液脂质纳米粒,可用于超声及光声成像,为进一步体外、体内成像实验奠定了基础。     

MRT2加权成像与增强CT对原发直肠癌术前T分期诊断的比较

目的 比较MR T2WI和增强CT对原发直肠癌术前T分期的诊断效能。方法 51例经活检病理证实的原发直肠腺癌,术前其中27例接受常规MR检查,43例接受CT检查,19例接受MR和CT检查。用MR T2WI和增强CT图像对肿瘤浸润深度进行判断,并与术后病理结果对照。结果 MR T2WI对直肠癌术前T分期与术后病理的一致性优于增强CT(Kappa值0.72 vs 0.52,P<0.05),对原发直肠癌T1/T2和T3/T4分期准确率高于增强CT(88.89% vs 79.07%);MR T2WI鉴别T1/T2、T3/T4期肿瘤的特异度(94.74% vs 81.25%)、阳性预测值(85.71% vs 57.14%)均优于增强CT。MR T2WI对T1/T2和T3/T4期肿瘤的分期效能优于CT。结论 MR T2WI在原发直肠癌术前T分期评价中的效能优于增强CT。     

Tracking of Tumor Cell–Derived Extracellular Vesicles In Vivo Reveals a Specific Distribution Pattern with Consecutive Biological Effects on Target Sites of Metastasis

Purpose

Extracellular vesicles, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators of intercellular communication. The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis.

Procedures

Extracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell–derived versus extracellular vesicles from healthy serum.

Results

In contrast to control extracellular vesicles, DiR-labeled extracellular vesicles from tumor cells preferentially accumulated in lung, liver, and spine. Subsequent flow cytometry of the immune cell composition of lung tissue samples revealed an increase of cytotoxic CD8+ T cells and a decrease of CD4+ T-helper cells as well as an increase in mature macrophages in response to tumor cell EV.

Conclusions

In conclusion, distribution of tumor cell–derived extracellular vesicles follows a specific pattern and can be monitored, using dedicated imaging. Extracellular vesicles alter the immune cell composition in target organs of metastasis, using a specific proteome cargo.

     

Fluorescence lifetime imaging of activatable target specific molecular probes

In vivo optical imaging using fluorescently labeled self‐quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target‐to‐background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self‐quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2⁺ and BALB/3T3 (HER2^?) cell lines. Changes in fluorescence lifetime correlated with temperature‐ and time‐dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2⁺ and BALB/3T3 (HER2^?)] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target‐specific activatable antibody–fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

Magnetically targeted nanoparticles for imaging-guided photothermal therapy of cancer

Over the past several decades, nanocarriers have constituted a vital research area for accurate tumor therapy. Herein, magnetically targeted nanoparticles (IRFes) for photothermal therapy were generated by integrating IR780, a molecule with strong emission and absorption in the NIR spectrum and the ability to produce heat after laser irradiation, with Fe₃O₄ nanoparticles (NPs). These IRFes were guided to the tumor site by the application of an external magnetic field. In particular, the strong NIR absorption of IR780 was used for NIRF imaging, and we also demonstrated effective magnetic targeting for the photothermal ablation of tumors. In vitro cell viability and in vivo antitumor experiments showed that these IRFes can ablate 4T1 cells or transplanted 4T1 cell tumors when exposed to 808 nm laser irradiation and a magnetic field. In vivo experiments showed that IRFes only act on tumors, do not damage other organs and can be used to image tumors. These results demonstrate the enormous potential of local photothermal therapy for cancer under the guidance of external magnetic fields and reveal the prospect for the use of multifunctional nanoparticles in tumor therapy.

Magnetically targeted nanoparticles (IRFes) for photothermal therapy were generated by integrating IR780, a molecule with strong emission and absorption in the NIR spectrum and the ability to produce heat after laser irradiation, with Fe₃O₄ nanoparticles.     

Design and testing of biological scaffolds for delivering reparative cells to target sites in the lung

This study summarizes the development and testing of a scaffold to promote engraftment of cells in the distal lung. A fibrinogen–fibronectin–vitronectin hydrogel (FFVH) was developed and optimized with respect to its mechanical and biological properties for this application. In vitro, FFVH scaffolds promoted attachment, histiotypic growth and expression of basement membrane proteins by primary ovine lung mesenchymal cells derived from lung biopsies. In vivo testing was then performed to assess the ability of FFVHs to promote cell engraftment in the sheep lung. Treatment with autologous cells delivered using FFVH was clinically well tolerated. Cells labelled with a fluorescent dye (PKH‐26) were detected at treatment sites after 1 month. Tissue mass (assessed by CT imaging) and lung perfusion (assessed by nuclear scintigraphy) were increased at emphysema test sites. Post‐treatment histology demonstrated cell proliferation and increased elastin expression without scarring or collapse. No treatment‐related pathology was observed at healthy control sites. FFVH scaffolds promote cell attachment, spreading and extracellular matrix expression in vitro and apparent engraftment in vivo, with evidence of trophic effects on the surrounding tissue. Scaffolds of this type may contribute to the development of cell‐based therapies for patients with end‐stage pulmonary diseases. Copyright © 2009 John Wiley & Sons, Ltd.     

SPECT/CT融合显像术前定位诊断异位胃黏膜

目的 探讨SPECT/CT显像术前对异位胃黏膜病灶的定位诊断价值。方法 对25例异位胃黏膜患儿进行SPECT/CT检查,对动态平面显像20 min内腹部有固定异常放射性浓聚灶者,于20 min末行SPECT/CT断层扫描并进行同机断层融合,评价断层融合显像对病灶的定位结果与术中所见的一致性。结果 25例平面显像阳性,其中断层融合显像阳性17例,阴性8例。14例（14/17,82.35%）断层融合显像所示病灶位置与术中所见相同,一致性高（Kappa=0.746,P<0.05）。结论 异位胃黏膜SPECT/CT融合显像可准确定位病灶,有较高的应用价值。     

A Molecular Imaging Paradigm to Rapidly Profile Response to Angiogenesis-directed Therapy in Small Animals

Purpose  The development of novel angiogenesis-directed therapeutics is hampered by the lack of non-invasive imaging metrics capable of assessing treatment response. We report the development and validation of a novel molecular imaging paradigm to rapidly assess response to angiogenesis-directed therapeutics in preclinical animal models. Procedures  A monoclonal antibody-based optical imaging probe targeting vascular endothelial growth factor receptor-2 (VEGFR2) expression was synthesized and evaluated in vitro and in vivo via multispectral fluorescence imaging. Results  The optical imaging agent demonstrated specificity for the target receptor in cultured endothelial cells and in vivo. The agent exhibited significant accumulation within 4T1 xenograft tumors. Mice bearing 4T1 xenografts and treated with sunitinib exhibited both tumor growth arrest and decreased accumulation of NIR800-αVEGFR2ab compared to untreated cohorts (p = 0.0021). Conclusions  Molecular imaging of VEGFR2 expression is a promising non-invasive biomarker for assessing angiogenesis and evaluating the efficacy of angiogenesis-directed therapies. Electronic supplementary material    The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and Evaluation of Diindole-Based MRI Contrast Agent for In Vivo Visualization of Necrosis

Purpose

Noninvasive imaging of cell necrosis can provide an early evaluation of tumor response to treatments. Here, we aimed to design and synthesize a novel diindole-based magnetic resonance imaging (MRI) contrast agent (Gd-bis-DOTA-diindolylmethane, Gd-DIM) for assessment of tumor response to therapy at an early stage.

Procedures

The oil-water partition coefficient (Log P) and relaxivity of Gd-DIM were determined in vitro. Then, its necrosis avidity was examined in necrotic cells in vitro and in rat models with microwave ablation-induced muscle necrosis (MAMN) and ischemia reperfusion-induced liver necrosis (IRLN) by MRI. Visualization of tumor necrosis induced by combretastatin A-4 disodium phosphate (CA4P) was evaluated in rats bearing W256 orthotopic liver tumor by MRI. Finally, DNA binding assay was performed to explore the possible necrosis-avidity mechanism of Gd-DIM.

Results

The Log P value and T1 relaxivity of Gd-DIM is ??2.15?±?0.01 and 6.61 mM^?1 s^?1, respectively. Gd-DIM showed predominant necrosis avidity in vitro and in vivo. Clear visualization of the tumor necrosis induced by CA4P was achieved at 60 min after administration of Gd-DIM. DNA binding study indicated that the necrosis-avidity mechanism of Gd-DIM may be due to its binding to exposed DNA in necrotic cells.

Conclusion

Gd-DIM may serve as a promising necrosis-avid MRI contrast agent for early assessment of tumor response to therapy.

     

Direct tissue analysis by MALDI-TOF mass spectrometry in human hepatocellular carcinoma

BackgroundThe prognosis of hepatocellular carcinoma (HCC) relies mainly on histopathological imaging examinations after surgical removal of the tumor. However, the rate of tumor recurrence is still high. Defining molecular signatures comprised of a number of distinct peptide ions specific for various tumor regions may improve the classification and prognosis of HCC patients.MethodsMALDI imaging technology, cluster analysis and classification software were applied to investigate patients with hepatitis B virus (HBV)-related HCC to obtain differences in protein abundance and distribution from non-tumor to tumor regions.ResultsA number of mass spectra obtained from non-tumor and HCC tumor sections were readily distinguishable. Progressive change was found in a distance-dependent manner from non-tumor to tumor regions within the junction section of HCC. Fourteen of the peaks were determined from non-tumor and tumor sections as classifiers to classify various non-tumor and tumor regions of the junction section of HCC. The performance of the classification test for the tumor region with a coefficient of variation (CV) of 0.16 was better than the non-tumor region, which reached a CV of 0.53.ConclusionsOur findings provide peptide information pertaining to the classification of various tumor regions to supplement current histopathological analysis in tumor margins.