Spatiotemporal Organization of Nanoreactors in Artificial Cells by Liquid–Liquid Phase Separation for Regulation of Cascade Enzymatic Reactions

ABSTRACT Living cells regulate biochemical reactions through compartmentalization, achieving high efficiency by precisely controlling spatial and temporal factors. Inspired by this principle, we developed multicompartmentalized artificial cells using liquid–liquid phase separation (LLPS) to regulate cascade enzymatic reactions. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were separately encapsulated into PEGylated and non‐PEGylated liposomes, forming enzyme‐loaded nanoreactors. These nanoreactors were assembled within a PEG/dextran aqueous two‐phase system, allowing controlled spatial arrangement of nanoreactors. Depending on PEGylation, liposomes localized either at the microdroplet interface or inside the lumen, creating four distinct artificial cell models in one step. Among them, the organization with GOx‐loaded liposomes at the interface (Models 1 and 2) exhibited the highest catalytic efficiency. This enhancement arose from improved substrate accessibility, reduced diffusion barriers, and optimized nanoreactor separation. Other models with less favorable spatial arrangements showed slower reaction rates. Our results highlight how spatial organization within artificial cells can critically influence cascade reaction performance. This modular and biomimetic strategy offers a versatile platform for designing synthetic cells, programmable biocatalysts, and functional microreactors for applications in biosensing, metabolic engineering, and therapeutics.