scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis.
scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis.
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DOI：
10.1097/rli.0b013e3181efd581
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发表时间：
2010-10
影响因子：
6.7
通讯作者：
Klibanov AL
中科院分区：
文献类型：
--
作者：
Anderson CR;Rychak JJ;Backer M;Backer J;Ley K;Klibanov AL
开发一种新型的微泡（MB）超声造影剂共价偶联到一个重组单链血管内皮生长因子构建体（scVEGF），通过均匀的位点特异性结合肿瘤血管生成的超声成像。首先用荧光团（BODIPY-胱氨酸）验证了通过硫醚键合的配体与带有马来酰亚胺的MB的缀合，并通过荧光测定法和流式细胞术检测共价结合的染料。随后将MB位点特异性缀合至scVEGF中含有半胱氨酸的Cys标签，并通过酶联免疫吸附测定定量结合的scVEGF。使用重组鼠VEGFR-2底物和表达人VEGFR-2的猪内皮细胞（PAE/KDR），通过体外平行板流动室试验研究scVEGF-MB的靶向粘附。使用无壁超声流动体模（其流动通道涂覆有固定的VEGFR-2）通过超声造影成像检测scVEGF-MB的粘附。使用结肠腺癌的鼠模型来评估在体内肿瘤血管生成期间scVEGF-MB的保留与造影超声成像。用BODIPY-半胱氨酸荧光团证明了配体与带有马来酰亚胺的MB缀合的原理证明。BODIPY与MB的缀合相对于MB表面上的马来酰亚胺基团以10倍摩尔过量BODIPY饱和。MB与scVEGF反应，导致每个MB结合1.2 × 105个scVEGF分子。在平行板流动室测定中显示sc-VEGF-MB的功能性粘附。在1.0 dynes/cm 2的剪切应力下，与非靶向对照MB相比，scVEGF-MB对重组VEGFR-2底物和VEGFR-2表达内皮细胞的粘附性高5倍。此外，scVEGF-MB靶向固定的VEGFR-2在超声流动体模显示了8倍的平均声信号增加相对于酪蛋白包被的控制通道。在肿瘤血管生成的体内模型中，与非靶向对照MB（1.58 ± 0.83 dB）相比，scVEGF MB在肿瘤中显示出显著更高的超声造影信号增强（8.46 ± 1.61 dB）。这些结果证明了一种新型的携带scVEGF的MB造影剂的功能，其可用于基础科学和药物发现研究中VEGFR-2的分子成像。
To develop a novel microbubble (MB) ultrasound contrast agent covalently coupled to a recombinant single-chain vascular endothelial growth factor construct (scVEGF) through uniform site-specific conjugation for ultrasound imaging of tumor angiogenesis. Ligand conjugation to maleimide-bearing MB by thioether bonding was first validated with a fluorophore (BODIPY-cystine), and covalently bound dye was detected by fluorometry and flow cytometry. MBs were subsequently site-specifically conjugated to cysteine-containing Cys-tag in scVEGF, and bound scVEGF was quantified by enzyme-linked immunosorbent assay. Targeted adhesion of scVEGF-MB was investigated with in vitro parallel plate flow chamber assays with recombinant murine VEGFR-2 substrates and human VEGFR-2-expressing porcine endothelial cells (PAE/KDR). A wall-less ultrasound flow phantom, with flow channels coated with immobilized VEGFR-2, was used to detect adhesion of scVEGF-MB with contrast ultrasound imaging. A murine model of colon adenocarcinoma was used to assess retention of scVEGF-MB with contrast ultrasound imaging during tumor angiogenesis in vivo. Proof-of-principle of ligand conjugation to maleimide-bearing MB was demonstrated with a BODIPY-cysteine fluorophore. Conjugation of BODIPY to MB saturated at 10-fold molar excess BODIPY relative to maleimide groups on MB surfaces. MB reacted with scVEGF and led to the conjugation of 1.2 × 105 molecules scVEGF per MB. Functional adhesion of sc-VEGF-MB was shown in parallel plate flow chamber assays. At a shear stress of 1.0 dynes/cm2, scVEGF-MB exhibited 5-fold higher adhesion to both recombinant VEGFR-2 substrates and VEGFR-2-expressing endothelial cells compared with nontargeted control MB. Additionally, scVEGF-MB targeted to immobilized VEGFR-2 in an ultrasound flow phantom showed an 8-fold increase in mean acoustic signal relative to casein-coated control channels. In an in vivo model of tumor angiogenesis, scVEGF MB showed significantly higher ultrasound contrast signal enhancement in tumors (8.46 ± 1.61 dB) compared with nontargeted control MB (1.58 ± 0.83 dB). These results demonstrate the functionality of a novel scVEGF-bearing MB contrast agent, which could be useful for molecular imaging of VEGFR-2 in basic science and drug discovery research.