Targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides.
Targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides.
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DOI：
10.2967/jnumed.109.068007
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发表时间：
2010-03
期刊：
影响因子：
--
通讯作者：
Gambhir SS
中科院分区：
文献类型：
--
作者：
Willmann JK;Kimura RH;Deshpande N;Lutz AM;Cochran JR;Gambhir SS
靶向超声造影成像作为一种在分子水平上检测和定量肿瘤血管生成的有力成像工具，正日益被人们所认识。本研究的目的是开发和测试一类新的靶向配体，用于肿瘤血管生成的靶向对比增强超声成像，其具有可以偶联到超声造影剂表面的小的构象受限的肽。使用定向进化来工程化小的二硫键约束的胱氨酸结（knottin）肽，其以低纳摩尔亲和力结合αvβ3整联蛋白（KnottinIntegrin）。通过将KnottinIntegrin连接到全氟化碳填充的微泡（MB-KnottinIntegrin）的外壳来产生靶向对比增强的超声成像造影剂。使用具有乱序序列的结蛋白肽来产生对照微泡（MB-KnottinScrambled）。在细胞培养物结合实验中评估MB-结蛋白整合素和MB-结蛋白扰乱与αvβ3整合素阳性细胞和对照细胞的结合，并与偶联抗αvβ3整合素单克隆抗体（MBαvβ3）的微泡和偶联肽模拟剂c（RGDfK）（MBcRGD）的微泡进行比较。采用高分辨率40 MHz超声系统，对42只荷人卵巢腺癌异种移植瘤小鼠体内不同类型微泡超声造影成像信号进行定量分析。MB-结蛋白整合素与αvβ3整合素阳性细胞（每个细胞1.76 ± 0.49 [平均值± SD]个微泡）的粘附显著高于对照细胞（0.07 ± 0.006）。对照MB-KnottinScrambled与αvβ3整合素阳性细胞的粘附低于MB-KnottinIntegrin（0.15 ± 0.12）。阻断整合素后，MB-结蛋白整合素与αvβ3整合素阳性细胞的粘附能力明显降低。施用MB-结蛋白整合素后的体内超声成像信号显著高于施用MBαvβ3或对照MB-结蛋白混乱后的信号。在体内阻断整联蛋白受体后，施用MB-结蛋白整联蛋白后的成像信号显著降低（降低64%）。施用MB-结蛋白整合素后的成像信号在用MB-结蛋白整合素和用MBcRGD成像的荷瘤小鼠组中没有显著差异。体外免疫荧光证实整合素在人卵巢腺癌异种移植瘤的内皮细胞上表达。整合素结合结蛋白肽可以缀合到微泡表面，并用于肿瘤血管生成的体内靶向对比增强超声成像。我们的研究结果表明，结合到小肽靶向配体的微泡提供的成像信号高于由大抗体分子提供的信号。
Targeted contrast-enhanced ultrasound imaging is increasingly being recognized as a powerful imaging tool for the detection and quantification of tumor angiogenesis at the molecular level. The purpose of this study was to develop and test a new class of targeting ligands for targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with small, conformationally constrained peptides that can be coupled to the surface of ultrasound contrast agents. Directed evolution was used to engineer a small, disulfide-constrained cystine knot (knottin) peptide that bound to αvβ3 integrins with a low nanomolar affinity (KnottinIntegrin). A targeted contrast-enhanced ultrasound imaging contrast agent was created by attaching KnottinIntegrin to the shell of perfluorocarbon-filled microbubbles (MB-KnottinIntegrin). A knottin peptide with a scrambled sequence was used to create control microbubbles (MB-KnottinScrambled). The binding of MB-KnottinIntegrin and MB-KnottinScrambled to αvβ3 integrin-positive cells and control cells was assessed in cell culture binding experiments and compared with that of microbubbles coupled to an anti-αvβ3 integrin monoclonal anti-body (MBαvβ3) and microbubbles coupled to the peptidomimetic agent c(RGDfK) (MBcRGD). The in vivo imaging signals of contrast-enhanced ultrasound with the different types of microbubbles were quantified in 42 mice bearing human ovarian adenocarcinoma xenograft tumors by use of a high-resolution 40-MHz ultrasound system. MB-KnottinIntegrin attached significantly more to αvβ3 integrin-positive cells (1.76 ± 0.49 [mean ± SD] microbubbles per cell) than to control cells (0.07 ± 0.006). Control MB-KnottinScrambled adhered less to αvβ3 integrin-positive cells (0.15 ± 0.12) than MB-KnottinIntegrin. After blocking of integrins, the attachment of MB-KnottinIntegrin to αvβ3 integrin-positive cells decreased significantly. The in vivo ultrasound imaging signal was significantly higher after the administration of MB-KnottinIntegrin than after the administration of MBαvβ3 or control MB-KnottinScrambled. After in vivo blocking of integrin receptors, the imaging signal after the administration of MB-KnottinIntegrin decreased significantly (by 64%). The imaging signals after the administration of MB-KnottinIntegrin were not significantly different in the groups of tumor-bearing mice imaged with MB-KnottinIntegrin and with MBcRGD. Ex vivo immunofluorescence confirmed integrin expression on endothelial cells of human ovarian adenocarcinoma xenograft tumors. Integrin-binding knottin peptides can be conjugated to the surface of microbubbles and used for in vivo targeted contrast-enhanced ultrasound imaging of tumor angiogenesis. Our results demonstrate that microbubbles conjugated to small peptide-targeting ligands provide imaging signals higher than those provided by a large antibody molecule.
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