Yan Fang1, Ruichen Yang2, Yi Hou3, Yabin Wang4, Ning Yang4, Mengqi Xu4, Sulei Li4, Shan Gao4, Min Jiang4, Jingyang Fan4, Yazhuo Hu5, Zhenzhen Xu6, Lei Gao7, Feng Cao4. 1. 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. 2. Department of Cardiology, The Affiliated Hospital of Jiangnan University, Wuxi No.4 People Hospital, Jiangsu, 21400, Wuxi, China. 3. College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China. houyi@iccas.ac.cn. 4. Medical School of Chinese PLA, Department of Cardiology in Second Medical Center &, National Research Center for Geriatric Diseases, Chinese PLA General Hospital, Beijing, 100853, China. 5. Institute of Gerontology institute, Second Medical Center, Chinese PLA General Hospital, Beijing, 100853, China. 6. National Research Center for Geriatric Diseases, Chinese PLA General Hospital, Beijing, 100853, China. 7. Medical School of Chinese PLA, Medical Department of Cardiovascular Disease in Sixth Medical Center, Chinese PLA General Hospital, Beijing, 100853, China. nkgaolei2010@126.com.
Abstract
AIM: 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). METHODS: 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 NaGdF4:Yb,Er@NaGdF4 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. RESULTS: 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. CONCLUSIONS: 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.
AIM: 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). METHODS: 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 NaGdF4:Yb,Er@NaGdF4 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. RESULTS: 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. CONCLUSIONS: 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.