Cell-compatible, integrin-targeted cryptophane-(129)Xe NMR biosensors.
Cell-compatible, integrin-targeted cryptophane-(129)Xe NMR biosensors.
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DOI：
10.1039/c1sc00041a
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发表时间：
2011-06
期刊：
影响因子：
8.4
通讯作者：
Dmochowski IJ
中科院分区：
文献类型：
--
作者：
Seward GK;Bai Y;Khan NS;Dmochowski IJ
肽修饰的 Cryptophane 能够使用超极化 129Xe NMR 光谱灵敏地检测蛋白质分析物。在这里，我们报告了改进的 Cryptophane 向表达 αvβ3 整合素受体的细胞的靶向和递送，该受体在许多人类癌症中过度表达。 Cryptophane 使用环状 RGDyK 肽和 Alexa Fluor 488 染料进行功能化，并通过共焦激光扫描显微镜监测细胞内化。竞争性阻断试验证实了 Cryptophane 内吞作用是通过 αvβ3 整合素受体介导的途径实现的。通过 MTT 测定，在通常用于超极化 129Xe NMR 生物传感实验的微摩尔隐蛋白浓度下，肽-隐蛋白缀合物被确定对正常人肺成纤维细胞无毒。流式细胞术显示，与正常细胞相比，过度表达整合素受体的癌细胞的细胞内化程度高出 4 倍。测量了 Cryptophane 生物传感器对玻连蛋白与 αvβ3 整合素结合以及纤维蛋白原与 αIIbβ3 整合素结合的纳摩尔抑制浓度（IC50 = 20–30 nM）。具有两个丙酸基团的缀合物的官能化提高了超极化 129Xe NMR 光谱研究的水溶性，这揭示了 129Xe-cryptophane-环状 RGDyK 生物传感器在 67 ppm 处的单共振。在洗涤剂溶液中引入 αIIbβ3 整联蛋白受体产生了新的“结合”129Xe 生物传感器峰，该峰从“游离”129Xe 生物传感器向低场移动 4 ppm。
Peptide-modified cryptophane enables sensitive detection of protein analytes using hyperpolarized 129Xe NMR spectroscopy. Here we report improved targeting and delivery of cryptophane to cells expressing αvβ3 integrin receptor, which is overexpressed in many human cancers. Cryptophane was functionalized with cyclic RGDyK peptide and Alexa Fluor 488 dye, and cellular internalization was monitored by confocal laser scanning microscopy. Competitive blocking assays confirmed cryptophane endocytosis through an αvβ3 integrin receptor-mediated pathway. The peptide–cryptophane conjugate was determined to be nontoxic in normal human lung fibroblasts by MTT assay at the micromolar cryptophane concentrations typically used for hyperpolarized 129Xe NMR biosensing experiments. Flow cytometry revealed 4-fold higher cellular internalization in cancer cells overexpressing the integrin receptor compared to normal cells. Nanomolar inhibitory concentrations (IC50 = 20–30 nM) were measured for cryptophane biosensors against vitronectin binding to αvβ3 integrin and fibrinogen binding to αIIbβ3 integrin. Functionalization of the conjugate with two propionic acid groups improved water solubility for hyperpolarized 129Xe NMR spectroscopic studies, which revealed a single resonance at 67 ppm for the 129Xe-cryptophane–cyclic RGDyK biosensor. Introduction of αIIbβ3 integrin receptor in detergent solution generated a new “bound” 129Xe biosensor peak that was shifted 4 ppm downfield from the “free” 129Xe biosensor.