Nanostructure Empowers Active Tumor Targeting in Ligand‐Based Molecular Delivery

Cell‐selective targeting is expected to enhance effectiveness and minimize side effects of cytotoxic agents. Functionalization of drugs or drug nanoconjugates with specific cell ligands allows receptor‐mediated selective cell delivery. However, it is unclear whether the incorporation of an efficient ligand into a drug vehicle is sufficient to ensure proper biodistribution upon systemic administration, and also at which extent biophysical properties of the vehicle may contribute to the accumulation in target tissues during active targeting. To approach this issue, structural robustness of self‐assembling, protein‐only nanoparticles targeted to the tumoral marker CXCR4 is compromised by reducing the number of histidine residues (from six to five) in a histidine‐based architectonic tag. Thus, the structure of the resulting nanoparticles, but not of building blocks, is weakened. Upon intravenous injection in animal models of human CXCR4+ colorectal cancer, the administered material loses the ability to accumulate in tumor tissue, where it is only transiently found. It instead deposits in kidney and liver. Therefore, precise cell‐targeted delivery requires not only the incorporation of a proper ligand that promotes receptor‐mediated internalization, but also, unexpectedly, its maintenance of a stable multimeric nanostructure that ensures high ligand exposure and long residence time in tumor tissue. Tumor‐homing peptides are not sufficient to mediate tumor accumulation of protein materials, as demonstrated by CXCR4‐targeted modular proteins forming nanoparticles. Destabilizing their nanoscale structure by subtle protein engineering abolishes the tumor‐targeting of the protein that accumulates in off‐target organs. The nanoscale organization of proteins is an unexpected but critical determinant of proper targeting and biodistribution, even in ligand‐empowered active targeting.