Cooperative Self-Assembly of Nanoparticle-Encapsulating Hybrid Protein Cages

Protein cages (PCs) are versatile platforms capable of encapsulating a wide range of nanoparticle cargo within biocompatible protein shells while providing tunable functionalities. Here, we investigated a self-assembly system that forms PCs while simultaneously encapsulating nanoparticles at high density, yielding pomegranate-like protein-nanoparticle hybrid materials. Amphiphilic recombinant fusion protein building blocks based on elastin-like polypeptides, leucine zippers, and fluorescent proteins were employed to assemble PCs via temperature-triggered liquid-liquid phase separation in the presence of fluorescent polystyrene nanoparticles. Analysis of nanoparticle encapsulation density and PC size suggests the possibility of cooperative interactions between protein building blocks and nanoparticles that mediate the formation of protein-nanoparticle coacervate intermediates, which may subsequently convert into core-shell hybrid PCs, a proposed pathway that is consistent with the results of kinetics studies. We demonstrate the self-assembly of hybrid PCs incorporating a fluorescent calcium sensor protein and titanium oxide nanoparticles, which exhibit a drastic enhancement in their calcium-sensing capability as a result of nanoparticle encapsulation. This platform offers a broadly applicable strategy that integrates protein biofunctionality with diverse nanoparticle properties for the development of advanced hybrid materials.