Molecular imaging with molecularly targeted probes is a powerful tool for studying the spatio-temporal interactions between complex biological processes. The pivotal role of the receptor for advanced glycation end products (RAGE), and its involvement in numerous pathological processes, aroused the demand for RAGE-targeted imaging in various diseases. In the present study, we evaluated the use of a diagnostic imaging agent for RAGE quantification in an animal model of peripheral artery disease, a multimodal dual-labeled probe targeted at RAGE (MMIA-CML). PAMAM dendrimer was conjugated with Nε-carboxymethyl-lysine (CML) modified albumin to synthesize the RAGE-targeted probe. A control untargeted agent carried native non-modified human albumin (HSA). Bifunctional p-SCN-Bn-NOTA was used to conjugate the 64Cu radioisotope. Surgical right femoral artery ligation was performed on C57BL/6 male mice. One week after femoral artery ligation, mice were injected with MMIA-CML or MMIA-HSA labeled with 64Cu radioisotope and 60 min later in vivo microPET-CT imaging was performed. Immediately after PET imaging studies, the murine hindlimb muscle tissues were excised and prepared for gene and protein expression analysis. RAGE gene and protein expression was assessed using real-time qPCR and Western blot technique respectively. To visualize RAGE expression in excised tissues, microscopic fluorescence imaging was performed using RAGE-specific antibodies and RAGE-targeted and -control MMIA. Animals subjected to PET imaging exhibited greater MMIA-CML uptake in ischemic hindlimbs than non-ischemic hindlimbs. We observed a high correlation between fluorescent signal detection and radioactivity measurement. Significant RAGE gene and protein overexpression were observed in ischemic hindlimbs compared to non-ischemic hindlimbs at one week after surgical ligation. Fluorescence microscopic staining revealed significantly increased uptake of RAGE-targeted nanoparticles in both ischemic and non-ischemic muscle tissues compared to the control probe but at a higher level in ischemic hindlimbs. Ischemic tissue exhibited explicit RAGE dyeing following anti-RAGE antibody and high colocalization with the MMIA-CML targeted at RAGE. The present results indicate increased expression of RAGE in the ischemic hindlimb and enable the use of multimodal nanoparticles in both in vitro and in vivo experimental models, creating the possibility for imaging structural and functional changes with a RAGE-targeted tracer.

中文翻译：

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分子靶向纳米颗粒：一种用于评估外周动脉疾病小鼠模型中晚期糖基化终产物受体表达的新兴工具

分子靶向探针的分子成像是研究复杂生物过程之间时空相互作用的有力工具。晚期糖基化终产物 (RAGE) 受体的关键作用及其参与众多病理过程，引起了对各种疾病中 RAGE 靶向成像的需求。在本研究中，我们评估了在外周动脉疾病动物模型中使用诊断成像剂进行 RAGE 定量，这是一种针对 RAGE (MMIA-CML) 的多模式双标记探针。PAMAM 树枝状聚合物与 Nε-羧甲基-赖氨酸 (CML) 修饰的白蛋白结合以合成 RAGE 靶向探针。对照非靶向试剂携带天然未修饰的人白蛋白 (HSA)。双功能 p-SCN-Bn-NOTA 用于共轭 64Cu 放射性同位素。对 C57BL/6 雄性小鼠进行手术右股动脉结扎。股动脉结扎后一周，给小鼠注射用 64Cu 放射性同位素标记的 MMIA-CML 或 MMIA-HSA，60 分钟后进行体内 microPET-CT 成像。PET 成像研究后，立即切除小鼠后肢肌肉组织并准备进行基因和蛋白质表达分析。分别使用实时 qPCR 和蛋白质印迹技术评估 RAGE 基因和蛋白质表达。为了使切除组织中的 RAGE 表达可视化，使用 RAGE 特异性抗体和 RAGE 靶向和对照 MMIA 进行显微荧光成像。接受 PET 成像的动物在缺血性后肢中表现出比非缺血性后肢更高的 MMIA-CML 摄取。我们观察到荧光信号检测和放射性测量之间的高度相关性。在手术结扎后一周，与非缺血后肢相比，在缺血后肢中观察到显着的 RAGE 基因和蛋白质过度表达。荧光显微镜染色显示，与对照探针相比，缺血和非缺血肌肉组织中 RAGE 靶向纳米颗粒的吸收显着增加，但在缺血后肢中的吸收水平更高。缺血组织在抗 RAGE 抗体后表现出明确的 RAGE 染色，并与针对 RAGE 的 MMIA-CML 高度共定位。目前的结果表明，缺血后肢中 RAGE 的表达增加，并且能够在体外和体内实验模型中使用多模式纳米粒子，