Acidity-Triggered “Sticky Spotlight”: CCK2R-Targeted TME-Sensitive NIR Fluorescent Probes for Tumor Imaging In Vivo,Bioconjugate Chemistry

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Acidity-Triggered “Sticky Spotlight”: CCK2R-Targeted TME-Sensitive NIR Fluorescent Probes for Tumor Imaging In Vivo
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2024-03-21 , DOI: 10.1021/acs.bioconjchem.4c00040
Ruiqi Sun ₁ , Yuxin Wang ₁ , Wenhui Shi ₁ , Hongfu Zhang ₁ , Jianhua Liu ₁ , Weina He ₁

Affiliation

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：

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 NIR 荧光探针

癌症在全球范围内导致高死亡率，严重威胁公共卫生。迫切需要开发肿瘤特异性近红外（NIR）显像剂，以实现精确诊断并指导有效治疗。近年来，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，导致诊断中的信噪比较差，因此这些 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 敏感的七甲嘧啶花青与 CCK2R 配体、微型胃泌素类似物 11 （MG11）偶联合成胆囊收缩素-2 受体（CCK2R）靶向 TME 敏感的 NIR 荧光探针 R2SM 用于体内成像，其中 MG11 将靶向胃肠道间质瘤（GIST）中过表达的 CCK2R。细胞摄取实验显示，R2SM 对 CCK2R 表现出高亲和力，导致受体介导的内化，使探针最终在肿瘤细胞的溶酶体中积累，这表明在肿瘤组织中，探针分布在细胞外酸性 TME 和细胞内溶酶体中。当 pK_a 为 6.83 时，R2SM 可以在酸性 TME （pH = 6.5–6.8）和溶酶体（pH = 4.5–5.0）处被激活，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的荧光。体内成像显示，MG11 与 pH 敏感的 NIR 探针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。在 0 以内的肿瘤区域观察到高信号。注射后 5 小时，表明其在术中成像中的潜在应用。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 敏感性的协同作用在 NIR 成像探针的高信噪比中起着至关重要的作用。此外，不同种类的肿瘤靶向载体可以与 NIR 染料同时偶联，这将进一步提高受体亲和力和靶向效率。

更新日期：2024-03-21

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