Engineering Protein Cage Nanoparticles as Tunable Multifunctional Nanoplatforms for Effective Diagnosis and Therapy

Abstract

Protein cage nanoparticles are biomolecule-based supramolecular biopolymers and attractive candidates for nano-scale cargo delivery vehicles. While the interior surfaces of the protein cages have been used for encapsulation, attachment and synthesis of organic and inorganic materials, their exterior surfaces have been used for multivalent presentations of molecules, including affinity tags, antibodies, fluorophores, carbohydrates, nucleic acids, and peptides, for molecular targeting and hierarchical structure formation. Multiple displays of two or more targeting ligands on a polyvalent single template would make it possible to construct versatile multiplex probes that can visualize two or more target cells individually without the need for a set of individual probes. A lumazine synthase protein cage nanoparticle isolated from Aquifex aeolicus (AaLS) was used as a multiple ligand-displaying nanoplatform to display polyvalently both TRAIL and EGFR binding affibody molecules (EGFRAfb) to form AaLS/TRAIL/EGFRAfb. The dual-ligand-displaying AaLS/TRAIL/EGFRAfb exhibited a dramatically enhanced cytotoxicity on TRAIL-resistant and EGFR-overexpressing A431 cancer cells both in vitro and in vivo. For full application, we genetically engineered protein cage nanoparticles as modular nanoplatforms and applied two different post-translational ligation methods, i.e., split intein and SpyTag/SpyCatcher, for the selective functionalization of both the interior space and the exterior surface of the engineered Encap individually to form a multifunctional nanoparticle with the desired functionalities. Modular functionalization of the interior space and the exterior surface of a protein cage nanoparticle may offer the opportunity to develop new protein-based nanomedical tools.