New fluorescent molecular probes, which can selectively target specific cell surface receptors, are needed for microscopy, in vivo imaging, and image guided surgery. The preparation of multivalent probes using standard synthetic chemistry can be a laborious process due to low reaction yields caused by steric effects. In this study, fluorescent molecular probes were prepared by a programmed non‐covalent pre‐assembly process that used a near‐infrared fluorescent squaraine dye to thread a macrocycle bearing a cyclic arginine‐glycine‐aspartate peptide antagonist (cRGDfK) as a cancer targeting unit. Cell microscopy studies using OVCAR‐4 (ovarian cancer) and A549 (lung cancer) cells that express high levels of the integrin αvβ3 or αvβ5 receptors, respectively, revealed a multivalent cell targeting effect. That is, there was comparatively more cell uptake of a pre‐assembled probe equipped with two copies of the cRGDfK antagonist than a pre‐assembled probe with only one appended cRGDfK antagonist. The remarkably high photostability and low phototoxicity of these near‐infrared probes allowed for acquisition of long‐term fluorescence movies showing endosome trafficking in living cells. In vivo near‐infrared fluorescence imaging experiments compared the biodistribution of a targeted and untargeted probe in a xenograft mouse tumor model. The average tumor‐to‐muscle ratio for the pre‐assembled targeted probe was 3.6 which matches the tumor targeting performance reported for analogous cRGDfK‐based probes that were prepared entirely by covalent synthesis. The capability to excite these pre‐assembled near‐infrared fluorescent probes with blue or deep‐red excitation light makes it possible to determine if a target site is located superficially or buried in tissue, a probe performance feature that is likely to be very helpful for eventual applications such as fluorescence guided surgery.

中文翻译：

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非共价预组装的高性能近红外荧光分子探针，用于癌症成像

显微镜，体内成像和图像引导手术需要新型的能够选择性靶向特定细胞表面受体的荧光分子探针。由于空间效应引起的低反应产率，使用标准的合成化学方法制备多价探针可能是一个费力的过程。在这项研究中，荧光分子探针是通过程序化的非共价预组装过程制备的，该过程使用近红外荧光方酸染料将带有环状精氨酸-甘氨酸-天冬氨酸肽拮抗剂（cRGDfK）作为癌症靶向单元的大环穿入。使用分别表达高水平整联蛋白αvβ3或αvβ5受体的OVCAR-4（卵巢癌）和A549（肺癌）细胞进行的细胞显微镜研究显示了多价细胞靶向作用。那是，与仅附有一个cRGDfK拮抗剂的预组装探针相比，配备有两个拷贝的cRGDfK拮抗剂的预组装探针具有更高的细胞摄取率。这些近红外探针具有极高的光稳定性和低光毒性，因此可以获取显示活体内体运输的长期荧光电影。体内近红外荧光成像实验比较了异种移植小鼠肿瘤模型中靶向和非靶向探针的生物分布。预先组装的靶向探针的平均肿瘤与肌肉之比为3.6，与完全通过共价合成制备的类似基于cRGDfK的探针报道的肿瘤靶向性能相匹配。