SpyTag-Embedded Protein Nanoparticles Enable pH-Dependent Protein Loading

Functional protein particles are emerging as versatile biomaterials with applications spanning nano-designs, drug delivery and biocatalysis. Their inherent biocompatibility, structural stability, and genetic modularity, when combined with nanoscale engineering, allow for the design of customizable systems with diverse functionalities. However, the reliable assembly of such nanostructures remains challenging due to the complexity of intermolecular interactions and surface charge variability. In this study, we present a modular strategy to assemble bioactive protein nanoparticles (NPs) using spontaneously crystallizing scaffolds derived from Bacillus thuringiensis. We generated stable, bioactive protein particles by genetically fusing the Cry1Ac scaffold to either monomeric red fluorescent protein or SpyTag002. These NPs maintained their structural integrity and fluorescence over extended period, highlighting their potential for sustained release applications. Modular recruitment on the NPs was achieved via the SpyTag/SpyCatcher system, a highly specific and covalent protein conjugation strategy, enabling targeted loading of functional proteins to the particle surface. Interestingly, SpyTag-embedded NPs exhibited pH-dependent binding to SpyCatcher, confirming the platform's pH-responsive functionality. The pH sensitivity and stability of these NPs position them as promising candidates for therapeutic delivery in acidic microenvironments, such as tumor tissues.