Designing Peptide Bunches on Nanocage for Bispecific or Superaffinity Targeting

Ferritin cage nanoparticles are promising platforms for targeted delivery of imaging and therapeutic agents because their cage structure can accommodate small molecules and their surfaces can be decorated with multiple functionalities. However, selective targeting is still a challenge for translating ferritin-based nanomedicines into the clinic, especially for heterogeneous diseases such as cancer. Targeting peptides can be genetically fused onto the surface of a ferritin cage, forming peptide bunches on nanocages (PBNCs) that offer synergistic increases in binding avidity. Here, we utilized two sites of the ferritin monomer, the N-terminus and the loop between the fourth and fifth helices, which are exposed on the surface of the assembled 24-subunit ferritin cage, to ligate one or two types of peptides to achieve "super affinity"? and bispecificity, respectively. PBNCs formed by ligation of the IL-4 receptor-targeting peptide, AP1, to both sites (48AP1-PBNCs) tethered IL-4R, expressing tumor cells with greater affinity than did PBNCs with AP1 ligated to a single site (24AP1-PBNCs). Moreover, bispecific PBNCs containing 24 RGD peptides and 24 AP1 peptides (24RGD/24AP1-PBNCs) were capable of independently targeting cells expressing the corresponding receptors. Bispecific and superaffinity PBNCs could be useful for efficient targeting of ferritin-based therapeutic/diagnostic agents in a clinical setting.