Development and Validation of Novel Z-360-Based Macromolecules for the Active Targeting of CCK2-R

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-002_G-Rho, IP-002_L-Rho, and IP-002_M-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-002_H-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-002_x-Rho association with A431-CCK2-R⁺ cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002_M-Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002_G-Rho and IP-002_L-Rho, respectively. Unexpectedly, IP-002_H-Rho showed a similar cell association to that of IP-002_L-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-002_G-Rho, IP-002_L-Rho, and IP-002_M-Rho, respectively, proving IP-002_M-Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R⁺ cells incubated with IP-002_M-Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002_M-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.