胆囊收缩素 2 型受体 (CCK2-R) 是癌症治疗的理想靶点，因为它在多种肿瘤中过度表达，并且与不良预后相关。 Nastorazepide (Z-360) 是一种选择性 CCK2-R 拮抗剂，作为 CCK2-R 配体用于靶向治疗已被广泛研究；然而，其高疏水性可能限制了细胞选择性和最佳体内生物分布。在这里，我们提出了三种新的荧光 Z-360 衍生物（IP-002G-Rho、IP-002L-Rho 和 IP-002M-Rho），其中纳斯托拉西肽通过带有不同糖类（葡萄糖 (G)、乳糖 (G) 的间隔基连接）。 L) 和麦芽三糖 (M)) 转化为磺基罗丹明 B。还合成了不含侧糖的第四种化合物 (IP-002H-Rho) 作为对照。通过二维 (2D) 和三维 (3D) 体外研究，我们评估了化合物与 CCK2-R 过表达细胞 (A431-CCK2-R ) 与 CCK2-R 表达不足细胞 (A431 WT ）。 2D 体外研究强调，IP-002x-Rho 与 A431-CCK2-R 细胞的关联性根据接头亲水性逐渐增加，即麦芽三糖 > 乳糖 > 葡萄糖 > 氢，IP-002M-Rho 显示出 2.4- 和吸收率分别比 IP-002G-Rho 和 IP-002L-Rho 高 1.36 倍。出乎意料的是，IP-002H-Rho 显示出与 IP-002L-Rho 相似的细胞关联，但两种测试的细胞系之间没有差异。相反，与 A431 WT 相比，IP-002G-Rho、IP-002L-Rho 和 IP-002M 与 A431-CCK2-R 细胞的关联性高出 1.08、1.14 和 1.37 倍-Rho，分别证明 IP-002M-Rho 是性能最佳的化合物，这也得到了竞争研究的证实。对与 IP-002M-Rho 一起孵育的 A431-CCK2-R 细胞进行的贩运研究表明，受体介导的内吞作用和简单扩散并存。相反，仅在嵌入细胞的 3D 支架上观察到 A431-CCK2-R 细胞对 IP-002M-Rho 的高选择性摄取，强调了 3D 模型在体外初步评估中的重要性。

The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives (IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound (IP-002H-Rho) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R+) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002x-Rho association with A431-CCK2-R+ cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002M-Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002G-Rho and IP-002L-Rho, respectively. Unexpectedly, IP-002H-Rho showed a similar cell association to that of IP-002L-Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R+ cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho, respectively, proving IP-002M-Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R+ cells incubated with IP-002M-Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002M-Rho by A431-CCK2-R+ cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.