Aqueous Emulsion Droplets Stabilized by Lipid Vesicles as Microcompartments for Biomimetic Mineralization.

Mineral deposition within living cells relies on control over the distribution and availability of precursors as well as the location and rates of nucleation and growth. This control is provided in large part by biomolecular chelators, which bind precursors and regulate their availability, and compartmentalization within specialized mineralizing vesicles. Biomimetic mineralization in self-assembled lipid vesicles is an attractive means of studying the mineralization process, but has proven challenging due to vesicle heterogeneity in lamellarity, contents, and size across a population, difficulties encapsulating high and uniform precursor concentrations, and the need to transport reagents across an intact lipid bilayer membrane. Here, we report the use of liposome-stabilized all-aqueous emulsion droplets as simple artificial mineralizing vesicles (AMVs). These biomimetic microreactors allow the entry of precursors while retaining a protein catalyst by equilibrium partitioning between internal and external polymer-rich phases. Small molecule chelators with intermediate binding affinity were employed to control Ca(2+) availability during CaCO3 mineralization, providing protection against liposome aggregation while allowing CaCO3 formation. Mineral deposition was limited to the AMV interior, due to localized production of CO3(2-) by compartmentalized urease. Particle formation was uniform across the entire population of AMVs, with multiple submicrometer amorphous CaCO3 particles produced in each one. The all-aqueous emulsion-based approach to biomimetic giant mineral deposition vesicles introduced here should be adaptable for enzyme-catalyzed synthesis of a wide variety of materials, by varying the metal ion, enzyme, and/or chelator.

Bioreactor droplets from liposome-stabilized all-aqueous emulsions.

Artificial bioreactors are desirable for in vitro biochemical studies and as protocells. A key challenge is maintaining a favourable internal environment while allowing substrate entry and product departure. We show that semipermeable, size-controlled bioreactors with aqueous, macromolecularly crowded interiors can be assembled by liposome stabilization of an all-aqueous emulsion. Dextran-rich aqueous droplets are dispersed in a continuous polyethylene glycol (PEG)-rich aqueous phase, with coalescence inhibited by adsorbed ~130-nm diameter liposomes. Fluorescence recovery after photobleaching and dynamic light scattering data indicate that the liposomes, which are PEGylated and negatively charged, remain intact at the interface for extended time. Inter-droplet repulsion provides electrostatic stabilization of the emulsion, with droplet coalescence prevented even for submonolayer interfacial coatings. RNA and DNA can enter and exit aqueous droplets by diffusion, with final concentrations dictated by partitioning. The capacity to serve as microscale bioreactors is established by demonstrating a ribozyme cleavage reaction within the liposome-coated droplets.

Biocatalyzed mineralization in an aqueous two-phase system: effect of background polymers and enzyme partitioning.

The formation of minerals in living organisms occurs in crowded microenvironments generated by the organization of soft matter. Here, we used a biphasic aqueous polymer medium to mimic the macromolecular crowding and compartmentalization of intracellular environments. Mineralization was performed in an aqueous two-phase system (ATPS) containing two nonionic polymers, poly(ethylene glycol) (PEG, 8 kDa) and dextran (Dx, 10 kDa). The enzyme urease was used to catalyze CaCO3 formation by hydrolyzing urea to produce CO3 2-, which reacted with Ca2+ already present in solution. Urease partitioning into the Dx-rich phase provided a mechanism for localizing the hydrolysis reaction, which consequently restricted mineral formation to this phase, despite the initially equal concentration of Ca2+ in both phases. Spatially confined mineralization was quantified by sampling the phases during bulk reactions and also directly observed in microscale systems by optical microscopy. Decreasing the volume of the Dx-rich phase relative to that of the PEG-rich phase significantly enhanced the local urease concentration in the Dx-rich phase, increasing local reaction rates. The PEG and Dx polymers, though present at up to 30 wt% in the ATPS, did not strongly influence the morphology of CaCO3(s) observed. However, addition of ovalbumin (1.5 wt%) caused marked changes in crystal morphology. The PEG/dextran ATPS reaction medium captured several key aspects of the biological environment including macromolecular crowding, localized reagent production via enzymatic activity, and reaction compartmentalization while not precluding the use of structure-directing additives such as proteins.