Role of Surface RGD Patterns on Protein Nanocages in Tumor Targeting Revealed Using Precise Discrete Models

Abstract The effectiveness of active targeting in cancer nanomedicine is becoming increasingly more debatable. Here, the role of the ligand functionalization patterns (number and distribution) on nanoparticle surfaces in tumor targeting is investigated using a 9 nm sized miniferritin protein nanocage, Dps modified with Arg‐Gly‐Asp (RGD) ligands whose functionalization patterns are precisely controlled. In vitro and in vivo experiments show that RGD modification endows Dps with tumor targeting capacity no matter what the surface pattern is. The tumor targeting of 2‐ligand Dps, which is better than that of 1‐ligand Dps, rivals or surpasses that of the 12‐ or 24‐ligand Dps. The 12‐ligand Dps with clustered RGD distribution shows 2.3 times the in vivo targeting efficiency of that with even distribution. The surface ligand pattern effects are correlated at least to receptor clustering and opsonization. This study provides insights into the understanding of the controversial findings on active tumor targeting in the literature and highlights the necessity of precise functionalization to achieve optimal active targeting in developing cancer nanomedicine.