In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)4
In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)4
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DOI：
10.2310/7290.2007.00002
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发表时间：
2007-01-01
影响因子：
2.8
通讯作者：
Coll, Jean-Luc
中科院分区：
文献类型：
--
作者：
Jin, Zhao-Hui;Razkin, Jesus;Coll, Jean-Luc
我们报道了区域选择性可寻址功能化模板（RAFT）-c（-RGDfK-）（4）呈递四个环状（Arg-Gly-Asp）（cRGD）肽，与单体cRGD相比，其以改善的特异性靶向整合素α（v）β（3）。在这项研究中，我们通过创建一个“隐形”分子来改进这种载体，其中荧光猝灭剂（Q）通过二硫键（-SS-）与Cy 5连接。RAFT-c（RGDfK-）（4）-Cy 5-SS-Q荧光被淬灭，除非在内化过程中被还原激活。与对照相比，RAFT-c（-RGDfK-）（4）-Cy 5-SS-Q荧光可忽略不计，但在二硫桥断裂后完全恢复。共聚焦显微镜显示，使用RAFT-c（-RGDfK-）（4）-Cy 5-SS-Q只能检测到细胞内Cy 5信号，证实未切割的细胞外分子不可见。静脉注射RAFT-c（-RGDfK-）（4）-Cy 5-SS-Q的皮下肿瘤小鼠的全身成像显示，与未淬灭的分子相比，肿瘤中的荧光对比度非常显著地增强。肿瘤的组织学证实了Cy 5的细胞内积累。这些结果表明，在靶向载体和药物模拟物之间存在不稳定的二硫桥是细胞内递送染料或药物的有效方式。此外，这种淬灭的RAFT-c（-RGDfK-）（4）-Cy 5-SS-Q探针是用于成像肿瘤块和研究体内RGD介导的内化的非常强大的载体。
We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)(4) presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin alpha(v)beta(3) with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a "stealth" molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(RGDfK-)(4)-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)(4)-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)(4)-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)(4)-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.