Medical School of Chinese PLA, Medical Department of Cardiovascular Disease in Sixth Medical Center & National Research Center for Geriatric Diseases, Chinese PLA General Hospital Chinese PLA General Hospital, Beijing, 100853, China.
Department of Cardiology, The Affiliated Hospital of Jiangnan University, Wuxi No.4 People Hospital, Jiangsu, 21400, Wuxi, China.
Mol Imaging Biol. 2022 Oct;24(5):721-731. doi: 10.1007/s11307-022-01721-5. Epub 2022 May 23.
Angiogenesis plays a major role in atherosclerotic plaque development and instability. Our study aims to develop a novel optical and magnetic resonance (MR) dual-modality molecular imaging probe to early detect unstable plaques in vivo by targeting biomarkers of angiogenesis in murine models of atherosclerosis (AS).
Immunofluorescence and western blot were used to detect the expression of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in activated Human Umbilical Vein Endothelial Cells (HUVECs). After synthesis and identification of novel short peptide VRBP1-targeted VEGFR2, HUVECs were co-cultured with FITC-VRBP1 to test specific affinity of VRBP1. Then VRBP1-UCNPstargeting VEGFR2 were constructed by conjugating VRBP1 to the surface of NaGdF:Yb,Er@NaGdF nanoparticles. The characterization of the nanoparticles was performed by transmission electron microscopy (TEM), distribution of size, hydrodynamic size, zeta potential, absorption spectra, emission spectra, imaging intensity of different concentrations, binding affinity and cytotoxicity of nanoprobes in vitro. The upconversion luminescence (UCL) and MR imaging were performed to identify unstable atherosclerotic plaque in ApoE mice in vivo and ex vivo. Morphological staining was used to verify AS model and angiogenesis, and Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) was used to confirm accumulation of the nanoparticles after imaging.
After induced by hypoxia and ox-LDL, the expression of VEGFR2 in activated HUVECs was enhanced. FITC-VRBP1 can specifically bind to the HUVECs. Characterization of the nanoparticles showed that particles size is uniform with a stable structure, specific optical and MR signal, good binding affinity to VEGFR2 and low cytotoxicity. In vivo and ex vivo UCL imaging and quantitative analysis revealed that distinctive optical signal was observed in the regions of left carotid common arteries (LCCAs) of AS group after injection of VRBP1-UCNPs. Higher signal intensity on T1-weighted MR imaging appeared in the LCCA wall of AS group after injection. The results of morphological staining demonstrated angiogenesis in the atherosclerotic plaques, Gd ions in LCCAs, aortic arch and renal arteries bifurcations detected by ICP-AES confirmed accumulation of the nanoparticles in plaque.
We successfully design and synthesize a novel UCNPs using peptide VRBP1 targeting to VEGFR2. In vivo imaging demonstrates that VRBP1-UCNPs can be used to perform optical/MR dual-modality imaging targeting angiogenesis in plaques, which is a promising technique to early detect unstable atherosclerosis.
血管生成在动脉粥样硬化斑块的发展和不稳定中起着重要作用。我们的研究旨在通过靶向动脉粥样硬化（AS）小鼠模型中血管生成的生物标志物，开发一种新型的光学和磁共振（MR）双重模态分子成像探针，以早期体内检测不稳定斑块。
免疫荧光和蛋白质印迹用于检测激活的人脐静脉内皮细胞（HUVEC）中血管内皮生长因子受体 2（VEGFR2）的表达。在合成并鉴定新型短肽 VRBP1 靶向 VEGFR2 后，将 FITC-VRBP1 与 HUVEC 共培养，以测试 VRBP1 的特异性亲和力。然后通过将 VRBP1 连接到 NaGdF:Yb，Er@NaGdF 纳米粒子的表面来构建 VRBP1-UCNP 靶向 VEGFR2。通过透射电子显微镜（TEM）、粒径分布、水动力粒径、zeta 电位、吸收光谱、发射光谱、不同浓度的成像强度、纳米探针的体外结合亲和力和细胞毒性对纳米粒子进行表征。在体内和体外进行上转换发光（UCL）和磁共振成像（MRI）以识别 ApoE 小鼠的不稳定动脉粥样硬化斑块。形态学染色用于验证 AS 模型和血管生成，电感耦合等离子体原子发射光谱法（ICP-AES）用于在成像后确认纳米粒子的积累。
在缺氧和 ox-LDL 诱导后，激活的 HUVECs 中 VEGFR2 的表达增强。FITC-VRBP1 可以特异性地与 HUVECs 结合。纳米粒子的特性表明，颗粒大小均匀，具有稳定的结构、特定的光学和磁共振信号、良好的与 VEGFR2 的结合亲和力和低细胞毒性。体内和体外 UCL 成像和定量分析显示，注射 VRBP1-UCNPs 后，AS 组左颈总动脉（LCCA）区域可见明显的光学信号。注射后，AS 组 LCCA 壁上 T1 加权 MR 成像的信号强度增加。形态学染色的结果表明斑块中有血管生成，ICP-AES 检测到 LCCA、主动脉弓和肾动脉分叉处的 Gd 离子证实了斑块中纳米粒子的积累。
我们成功设计并合成了一种新型的使用肽 VRBP1 靶向 VEGFR2 的 UCNPs。体内成像表明，VRBP1-UCNPs 可用于进行针对斑块血管生成的光学/MR 双重模态成像，这是一种早期检测不稳定动脉粥样硬化的有前途的技术。
