Bioinspired Mineralizing Microenvironments Generated by Liquid-Liquid Phase Coexistence

Living organisms direct the location, structure, and properties of biominerals by tightly controlling reactant concentration profiles, biopolymer identity and availability, and other aspects of reaction microenvironments. Such control at the microscale is difficult to exert in synthetic systems. Inspired by the scalability of emulsions and the effectiveness of liquid-liquid phase separation in organizing subcellular biochemistry, we introduce mineralizing microreactors based on vesicle-coated multiphase droplets of a semistabilized, all-aqueous Pickering emulsion. All phases are macromolecularly crowded, which mimics biological milieu. Each droplet contains both a Ca²⁺/polyaspartate-rich coacervate phase and a second, adjacent phase hosting a carbonate-producing enzyme. CaCO₃ mineralization precursors, Ca²⁺ and CO₃ ^2-, are thus spatially separated when the reaction is initiated. Diffusion of CO₃ ^2- into the Ca²⁺/polyaspartate-rich coacervates results in CaCO₃ formation and release of polyaspartate, such that the composition of the mineralizing microenvironment evolves during the reaction. The resulting amorphous calcium carbonate (ACC) microspheres have smooth surfaces and are rich in polyaspartic acid, with nearly 30 wt % organics and a dense shell/porous core morphology. Although ACC normally converts over time to calcite, these particles are stable against crystallization for at least one year, which we attribute to their high organic loading. The crowded, compartmentalized nature of the reaction medium dictated the amorphous structure, spherical core-shell morphology, and organic-rich composition of these particles. Preorganization of mineralizing media by phase coexistence is a powerful way to shape reaction microenvironments. The approach introduced here should be broadly generalizable to the synthesis of other materials; we demonstrate a straightforward adaptation for calcium phosphate production.