Cancer which causes high mortality globally threatens public health seriously. There is an urgent need to develop tumor-specific near-infrared (NIR) imaging agents to achieve precise diagnosis and guide effective treatment. In recent years, imaging probes that respond to acidic environments such as endosomes, lysosomes, or acidic tumor microenvironments (TMEs) are being developed. However, because of their nonspecific internalization by both normal and tumor cells, resulting in a poor signal-to-noise ratio in diagnosis, these pH-sensitive probes fail to be applied to in vivo tumor imaging. To address this issue, a cholecystokinin-2 receptor (CCK2R)-targeted TME-sensitive NIR fluorescent probe R2SM was synthesized by coupling pH-sensitive heptamethine cyanine with a CCK2R ligand, minigastrin analogue 11 (MG11) for in vivo imaging, in which MG11 would target overexpressed CCK2Rs in gastrointestinal stromal tumors (GISTs). Cell uptake assay demonstrated that R2SM exhibited a high affinity for CCK2R, leading to receptor-mediated internalization and making probes finally accumulated in the lysosomes of tumor cells, which suggested in the tumor tissues, the probes were distributed in the extracellular acidic TME and intracellular lysosomes. With a pK_a of 6.83, R2SM can be activated at the acidic TME (pH = 6.5–6.8) and lysosomes (pH = 4.5–5.0), exhibiting an apparent pH-dependent behavior and generating more intense fluorescence in these acidic environments. In vivo imaging showed that coupling of MG11 with a pH-sensitive NIR probe facilitated the accumulation of probe and enhanced the fluorescence in CCK2R-overexpressed HT-29 tumor cells. A high signal was observed in the tumor region within 0.5 h postinjection, indicating its potential application in intraoperative imaging. Fluorescence imaging of R2SM exhibited higher tumor-to-liver and tumor-to-kidney ratios (2.1:1 and 2.3:1, respectively), compared separately with the probes that are lipophilic, pH-insensitive, or MG11-free. In vitro and in vivo studies demonstrated that the synergistic effect of tumor targeting with pH sensitivity plays a vital role in the high signal-to-noise ratio of the NIR imaging probe. Moreover, different kinds of tumor-targeting vectors could be conjugated simultaneously with the NIR dye, which would further improve the receptor affinity and targeting efficiency.

中文翻译：

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酸度触发的“粘性聚光灯”：用于体内肿瘤成像的 CCK2R 靶向 TME 敏感近红外荧光探针

导致全球高死亡率的癌症严重威胁着公众健康。迫切需要开发肿瘤特异性近红外（NIR）成像剂以实现精准诊断并指导有效治疗。近年来，人们正在开发对酸性环境（如内体、溶酶体或酸性肿瘤微环境（TME））做出反应的成像探针。然而，由于它们被正常细胞和肿瘤细胞非特异性内化，导致诊断信噪比较差，这些pH敏感探针无法应用于体内肿瘤成像。为了解决这个问题，通过将pH敏感的七次甲基花青与CCK2R配体小胃泌素类似物11（MG11）偶联，合成了一种针对胆囊收缩素2受体（CCK2R）的TME敏感的NIR荧光探针R2SM，用于体内成像，其中MG11将针对胃肠道间质瘤 (GIST) 中过度表达的 CCK2R。细胞摄取实验表明，R2SM对CCK2R表现出高亲和力，导致受体介导的内化，使探针最终积聚在肿瘤细胞的溶酶体中，这表明在肿瘤组织中，探针分布在细胞外的酸性TME和细胞内的溶酶体中。 。 R2SM 的ap Ka_值为6.83，可以在酸性 TME（pH = 6.5–6.8）和溶酶体（pH = 4.5–5.0）中被激活，表现出明显的 pH 依赖性行为，并在这些酸性环境中产生更强烈的荧光。体内成像表明，MG11 与 pH 敏感的 NIR 探针的偶联促进了探针的积累，并增强了 CCK2R 过表达的 HT-29 肿瘤细胞中的荧光。注射后0.5小时内在肿瘤区域观察到高信号，表明其在术中成像中的潜在应用。分别与亲脂性、pH 不敏感或不含 MG11 的探针相比，R2SM 的荧光成像表现出更高的肿瘤与肝脏和肿瘤与肾脏的比率（分别为 2.1:1 和 2.3:1）。体外和体内研究表明，肿瘤靶向与pH敏感性的协同效应对于近红外成像探针的高信噪比起着至关重要的作用。此外，不同种类的肿瘤靶向载体可以同时与近红外染料缀合，这将进一步提高受体亲和力和靶向效率。