骨桥蛋白靶向磁性纳米探针对动脉斑块的分子成像

Molecular imaging of atherosclerotic plaques via osteopontin targeted Fe3O4 nanoparticles

目的通过构建新型骨桥蛋白（OPN）靶向的四氧化三铁（Fe2O4）纳米颗粒，实现对动脉粥样硬化斑块中平滑肌增殖的荧光成像。方法通过-COOH和-NH2之间的脱水缩合反应将OPN抗体偶联至DMSA修饰的Fe3O4纳米颗粒表面，进而将荧光染料NHS-Cy5．5连接获得纳米成像探针。通过噻唑蓝（MTT）和TUNEL方法检测探针对巨噬细胞的毒性。动脉粥样硬化动物模型通过高脂喂养ApoE^-/-小鼠20周构建成功，继而将探针（5mgFe／kg）经小鼠尾静脉注射，24h后用小动物活体成像仪系统进行光学三维成像，离体血管的冰冻切片做Cy5．5的激光共聚焦检测。结果MTT结果显示，加不同浓度探针孵育：0，5，10，15，20，25，30mg／L相较于对照组来说，细胞活性并未表现出差异阻490nm：（1．10±0．03），（1．05±0．03），（1．03±0．02），（0．96±0．02），（0．96±0．03），（0．93±0．03）vs（1．11±0．05），P〉0．05]；TUNEL结果显示，加不同浓度的探针孵育：0，10，20，25，30mg／L与对照组比较来说，细胞凋亡率差异无统计学意义[（21．2±1．5）％，（21．8±1．1）％，（21．5±1．2）％，（22．3±1．2）％vs（20．5±1．0）％，P〉0．05]，证实探针在我们所运用浓度范围内对细胞几乎是无毒性的。探针经尾静脉注射到动脉粥样硬化模型小鼠体内后，24h光学成像可见颈部有明显的信号，且组织切片的激光共聚焦结果显示，探针主要集中在斑块内，HE染色结果进一步显示斑块的性质。结论本实验所构建的探针在所使用浓度范围内基本无任何明显毒性，且对动物颈部动脉粥样硬化斑块有较好的识别效果。

Objective To present atherosclerotic plaques via fluorescence imaging by designing and constructing a probe Cy5.5-anti-OPN-DMSA-MNPs （OPN： osteopontin, DMSA-MNPs： meso-2,3-dimercaptosuccinic acid-Fe3O4 magnetic nanoparticles）. Methods OPN antibody and Cy5.5 dye were conjugated to DMSA-MNPs by amide reaction between carboxylic group and amine group. The cytotoxicity of probes for mouse macrophages was evaluated by MTT and TUNEL assays. ApoE-/- mice were fed with high fat diet for 20 weeks to build atherosclerosis models, and then were given an injection of the probes （5mg Fe/kg） via tail vein. Fluorescence imaging was performed in vivo by IVIS Kinetic System 24h later. Then, the carotid arteries were collected for frozen section examination by confocal fluorescent microscopy. Results The MTT results showed that there was no significant discrepancy in cell viability between the macrophages treated by 5, 10, 15, 20, 25 and 30mg/L probe respectively and the control cells （A490nm： 1.10 ± 0.03, 1.05 ±0.03, 1.03 ±0.02, 0.96 ±0.02, 0.96 ±0.03, 0.93 ±0.03 vs 1.11 ±0.05, P〉 0.05）. The TUNEL indicated that no significant difference was found in the number of positive nuclei in the treated cells （10, 20, 25 and 30mg/L probe） and control cells [（21.2 ±1.5）%, （21.8 ±1.1）%, （21.5 ±1.2）%, （22.3 ±1.2）% vs （20.5 ±1.0）%, P 〉 0.05]. These results exhibited that the probe had no influence on cell survival at above-used concentrations. After the probes were injected into the atherosclerosis models, thefluorescence imaging clearly displayed the signal of atherosclerotic plaques in carotid artery, and confocal fluorescent microscopy showed that the probes were mainly located in the plaque which was further verified by HE staining. Conclusion Our self-made probes have barely no cytotoxicity on cells within the concentrations we used, and are effective in detection of atherosclerotic plaques in carotid artery in vivo.