Secondary Site Ligand for Integrin αVβ3 Enables Targeted mRNA Delivery

Poor perfusion and abnormal vasculature constrain direct drug delivery to solid tumors. Hence, targeting neighboring tumor endothelial cells via the upregulated marker integrin αVβ3 is a promising strategy. Orthosteric arginine-glycine-aspartate (RGD) ligands of αVβ3 achieve high affinities but suffer from cross-reactivity. Alternatively, selective targeting of αVβ3 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 αVβ3-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 αVβ3. 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 αVβ3 and outline their application as a multivalent, superselective delivery platform.