Protein Cages Engineered for Interaction-Driven Selective Encapsulation of Biomolecules

Hollow protein cages have become attractive drug delivery vehicles with high biocompatibility and precise functional/structural manipulability. However, difficulties in effective cargo loading inside the cages have been limiting further development of protein cage-based drug carriers. Here, we developed a specific interaction-driven encapsulation and cellular delivery strategy for various biomolecules by engineering a porous protein cage. The computationally designed hyperstable mi3 protein cage was circularly permuted to fuse the cancer targeting RGD tripeptide to the cage surface and SpyTag (ST), which forms a covalent bond with SpyCatcher (SC), to the cage inner cavity. SC-fused proteins with different sizes and charges could be stably and actively encapsulated in the engineered nanocage via the ST/SC reaction. Cargo protein encapsulation inside the cage was directly confirmed by cryo-electron microscopy (EM) structure determination. In addition, SC-fused monomeric avidin was added to the nanocage to encapsulate various biotinylated (nonprotein) cargos such as oligonucleotides and the anticancer drug doxorubicin. All cargo molecules loaded onto the engineered mi3 were effectively delivered to cells. This work introduces a highly versatile cargo loading/delivery strategy, where loading/delivery interactions, cargo molecules, and cell targeting moieties can be further varied for optimal cellular drug delivery.