Construction of robust protein nanocage by designed disulfide bonds for active cargo molecules protection in the gastric environment

Notwithstanding the progress made, cargo molecules encapsulated within ferritin via oral administration in the gastric environment remains a persistent challenge. This study focuses on the strategic enhancement of ferritin stability in harsh gastric environment. By taking advantagie of computational-assisted design, we strategically introduced up to 96 disulfide bonds along three key inter-subunit interfaces to one single ferritin molecule with human H-chain ferritin and shrimp (Marsupenaeus japonicus) ferritin as starting materials, producing two kinds of robust ferritin nanocages with markedly enhanced acid and protease (pepsin and rennin) resistance. The crystal structure of ferritin nanocage confirmed our design at an atomic level. Encapsulation experiments demonstrated successful loading of bioactive cargo molecules (e.g., doxorubicin) into the engineered ferritin nanocages, with pronouncedly improved protection against leakage under acidic condition and the presence of pepsin and rennin as compared to their native counterparts. This study presents a potential approach for the design and engineering of protein nanocages for oral administration.