Cooperative self-assembly of nanoparticle-encapsulating hybrid protein cages

Protein cages 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 vesicle-like protein cages 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, leucin zippers, and fluorescent proteins were employed to assemble vesicle-like protein cages via temperature-triggered liquid-liquid phase separation in the presence of fluorescent polystyrene nanoparticles. Analysis of nanoparticle encapsulation density and protein cage size indicates cooperative interactions between protein building blocks and nanoparticles that mediate the formation of protein-nanoparticle coacervate intermediates, which subsequently convert into core-shell hybrid protein cages, as further supported by kinetics studies. We demonstrate the self-assembly hybrid protein cages 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 development of advanced hybrid materials.