Molecular MRI and fluorescence imaging of atherosclerosis using annexin A5-functionalized bimodal nanoparticles
Molecular MRI and fluorescence imaging of atherosclerosis using annexin A5-functionalized bimodal nanoparticles
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使用膜联蛋白 A5 功能化双峰纳米颗粒进行动脉粥样硬化的分子 MRI 和荧光成像
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发表时间：
2009
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通讯作者：
K. Nicolay
中科院分区：
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作者：
G. V. Tilborg;Esad Vucic;G. Strijkers;D. Cormode;T. Skajaa;C. Reutelingsperger;Z. Fayad;W. Mulder;K. Nicolay
双峰胶束由Gd-DTPA-BSA（50%）、PEG 2000-DSPE（39%）、马来酰亚胺PEG 2000-DSPE（10%）和近红外（NIR）Cy5.5-PEG-DSPE（1%）组成，流体动力学直径约为15 nm。蛋白膜联蛋白A5（anxA 5）共价结合，以获得对PS的特异性。体外：将凋亡Jurkat细胞与非靶向对照胶束或anxA 5胶束（1 mM总脂质，30分钟）一起孵育，固定、核染色（DAPI）并用共聚焦激光扫描显微镜（CLSM）研究。体内试验：将10只雄性apoE -/-小鼠（60-68周龄，高胆固醇饮食）用于体内MRI实验和另外的离体荧光成像或显微镜检查。小鼠接受2.5 μmol对照胶束（n=4）或anxA 5胶束（n=6）。使用脂肪抑制的黑血自旋回波序列（TR/TE= 800/8.6 ms，NEX = 16，101.6 x 101.6 μm 2，0.5 mm层厚），在9.4T下，在造影前和造影后24小时采集腹主动脉的T1加权MR图像。MRI检查后，解剖主动脉，并在两组和对照主动脉的相同曝光时间下获得整个主动脉的近红外荧光反射图像。接下来，将动脉瘤冷冻切片，对凋亡细胞（TUNEL）或巨噬细胞（CD 68）进行染色，并用CLSM进行研究。结果通过对凋亡Jurkat细胞的体外结合测定证实了anxA 5-胶束的靶特异性（比较A和G）。apoE -/-小鼠腹主动脉在注射anxA 5-胶束后24小时的体内T1加权MRI显示，与造影前水平相比，主动脉壁中的平均信号强度适度增加（10.7 ± 1.7%;比较H和I）。在注射对照胶束后24小时，信号强度增加较少（6.7 ± 3.4%;比较B和C）。离体近红外荧光成像显示，切除的整个髂动脉对膜联蛋白A5-胶束的摄取最明显（比较D和J），并且主要在富含动脉粥样硬化斑块的区域[3]，例如主动脉分叉进入髂动脉。此外，共聚焦激光扫描显微镜（CLSM）显示，靶向药物与巨噬细胞（K）和凋亡细胞（L）相关，而非特异性对照药物未显示出被此类细胞明确摄取（E，F）。结论本研究中提出的膜联蛋白A5功能化造影剂可能允许对被认为显著促进斑块不稳定性的细胞类型进行无创评估，因此可能对动脉粥样硬化病变表型的诊断有价值。
Bimodal micelles, with a hydrodynamic diameter of ~15 nm, were composed of Gd-DTPA-BSA (50%), PEG2000-DSPE (39%), maleimidePEG2000-DSPE (10 %), and near-infrared (NIR) Cy5.5-PEG-DSPE (1%). The protein annexin A5 (anxA5) was covalently conjugated to obtain specificity for PS. In vitro: Apoptotic Jurkat cells were incubated with untargeted control-micelles or anxA5-micelles (1mM total lipid, 30 min.), fixed, stained for nuclei (DAPI) and studied with confocal laser scanning microscopy (CLSM). In vivo: 10 male apoE -/- mice (60-68 weeks of age, high cholesterol diet) were used for in vivo MRI experiments and additional ex vivo fluorescence imaging or microscopy. Mice received 2.5 μmol control-micelles (n=4) or anxA5-micelles (n=6). T1-weighted MR images of the abdominal aorta were acquired pre-contrast and 24 hrs post-contrast at 9.4T, using a fat-suppressed black blood spin echo sequence (TR/TE= 800/8.6 ms, NEX = 16, 101.6 x 101.6 μm 2 , 0.5 mm slice thickness). Following MRI, aortas were dissected and near-infrared fluorescence reflectance images of whole aortas were acquired with identical exposure times for both groups and a control aorta. Next, aortas were cryo-sectioned, stained for apoptotic cells (TUNEL) or macrophages (CD68) and studied with CLSM. Results Target-specificity of the anxA5-micelles was confirmed with in vitro binding assays to apoptotic Jurkat cells (compare A and G). In vivo T1weighted MRI of the abdominal aorta in apoE -/- mice at 24 hours post-injection of the anxA5-micelles revealed a modest increase of the mean signal intensity in the aortic wall compared to pre-contrast levels (10.7 ± 1.7%; compare H and I). The signal intensity was less increased at 24 hours after injection of control-micelles (6.7 ± 3.4%; compare B and C). Ex vivo near-infrared fluorescence imaging of excised whole aortas demonstrated most pronounced uptake of the annexin A5-micelles (compare D and J), and predominantly in areas that were rich of atherosclerotic plaque [3], such as the aortic bifurcation into the iliac arteries. Furthermore, confocal laser scanning microscopy (CLSM) revealed that the targeted agent was associated with macrophages (K) and apoptotic cells (L), whereas the non-specific control agent showed no clear uptake by such cells (E, F). Conclusion The annexin A5-functionalized contrast agent presented in this study potentially allows non-invasive assessment of cell types that are considered to significantly contribute to plaque instability, and therefore may be valuable for the diagnostics of atherosclerotic lesion phenotype.