Bioinspired Mineralizing Microenvironments Generated by Liquid–Liquid Phase Coexistence

Abstract

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 Ca 2+ /polyaspartate-rich coacervate phase and a second, adjacent phase hosting a carbonateproducing enzyme. CaCO 3 mineralization precursors, Ca 2+ and CO 3 2− , are thus spatially separated when the reaction is initiated. Diffusion of CO 3 2− into the Ca 2+ /polyaspartate-rich coacervates results in CaCO 3 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.

Liquid–Liquid Phase Separation of Short Histidine- and Tyrosine-Rich Peptides: Sequence Specificity and Molecular Topology

“…Second, the process of delivery and flow through the extracellular environment is further facilitated by the low interfacial energy of coacervate microdroplets with the dilute phase , as well as by their shear-thinning rheological characteristics. , Third, microdroplets can recruit both inorganic ions as well as larger macromolecules to compartmentalize, protect, and/or enhance localized chemical activity. For example, protein microdroplets can recruit inorganic ions such as Ca 2+ , CO 3 2– , and PO 4 3– and stabilize intermediate phases, thereby delaying premature precipitation which is important during biomineralization. In other cases, compartmentalization within the droplet is beneficial to regulate or delay chemical reactivity, such as oxidation of dihydroxylphenylalanine (Dopa) in mussel adhesive proteins , or in sandcastle worm glue, in turn allowing the organism to spatiotemporally control the chemical reactions required to form the final matured structure.…”

Precipitation of Calcium Carbonate Inside Giant Unilamellar Vesicles Composed of Fluid-Phase Lipids

“…These studies consistently demonstrated that confinement already strongly impacts the crystallization kinetics and CaCO 3 polymorphism. To assess the influence of a lipidic interface on CaCO 3 mineralization in confined space, Keating and co-workers , used aqueous emulsion droplets stabilized by lipid vesicles. However, these systems do not resemble a continuous lipid bilayer like it is found in biomineral-forming vesicles in nature.…”

Surface-Induced Coacervation Facilitates Localized Precipitation of Mineral Precursors from Dilute Solutions

“…Dense polymer–Ca 2+ phases, or coacervates, have been suggested to be functional precursors in the formation of Ca-based minerals. ,, The unique aspect of a mechanism involving polymer–Ca 2+ phases in comparison to other multistep pathways is that it separates temporally the precipitation of the cations from the addition of the anions. , Such a step-by-step mechanism can be advantageous in biomineralization processes, where calcium carbonate minerals form within the Ca 2+ -poor environment of the cell. Our results provide mechanistic insights into such processes, demonstrating how functional polymers can scavenge Ca 2+ from solutions with concentrations in the submillimolar range.…”

“…This dense CAP-Ca 2+ phase was shown to form in the absence of carbonate ions. , Nevertheless, if carbonate ions are added to the dense CAP-Ca 2+ phase after its formation, it transforms into a calcium carbonate phase that ultimately converts into calcite . These observations might be an indication that a dense polymer–ion complex, or coacervate, − is a functional precursor phase to mineral formation. − The dynamics of the CAP-Ca 2+ dense phase formation, however, have not been explored, and we have yet to understand the chemical conditions and interactions controlling this precipitation process.…”