Secondary Site Ligand for Integrin <i>α</i> V <i>β</i> 3 Enables Targeted mRNA Delivery

ABSTRACT Poor perfusion and abnormal vasculature constrain direct drug delivery to solid tumors. Hence, targeting neighboring tumor endothelial cells via the upregulated marker integrin α V β 3 is a promising strategy. Orthosteric arginine–glycine–aspartate (RGD) ligands of α V β 3 achieve high affinities but suffer from cross‐reactivity. Alternatively, selective targeting of α V β 3 could potentially be achieved via low‐affinity ligands, displayed multivalently on nanoparticles to leverage avidity. To avoid orthosteric site competition, we performed a fragment screening under RGD saturation. Structure–activity relationship (SAR) analysis of the initial hit revealed its binding motif, a 4‐methylpyrimidine‐2‐amine core and a conjugation‐tolerant position for linker attachment. Multivalent display of the lead compound on liposomes and lipid nanoparticles (LNPs) led to time‐, dose‐, and valency‐dependent uptake in α V β 3‐expressing model cells and in primary human umbilical vein endothelial cells (pHUVECs). In contrast to RGD‐decorated NPs, fragment‐targeted NPs show superselective behavior ( α κ ≈3), that is, a sharp valency‐dependent threshold, enabling selective targeting of cells overexpressing α V β 3. Crucially, mRNA delivered by targeted LNPs translated into functional protein expression six‐fold over control. To our knowledge, these findings present the first non‐orthosteric, exogenous small‐molecule ligands for α V β 3 and outline their application as a multivalent, superselective delivery platform.