ASSEMBLY OF VACUOLATED COACERVATES USING FOLIC-ACID-CONJUGATED POLYESTER NANOPARTICLES: A TUNABLE STRATEGY FOR BIOMIMETIC COMPARTMENTALIZATION

Compartmentalization is fundamental to cellular organization, enabling the regulation of biochemical processes within specific organelles. This principle inspired synthetic biology to develop protocells with compartmentalized architectures, particularly membraneless organelles formed via liquid-liquid phase separation (LLPS). Complex coacervates, a class of phase-separated structures, mimic the dynamic and electrolyte-rich environment of the cytoplasm, playing a key role in intracellular organization and enzymatic regulation. Recent evidence suggests that many membraneless organelles exhibit multiphase architectures, including core-shell substructures and vacuolated germ granules, which enhance biomacromolecular segregation and selective permeability without relying on lipid membranes. Despite progress in assembling synthetic coacervates, challenges remain in achieving controlled permeability and stability. To address these challenges, we developed an innovative approach for assembling vacuolated coacervates using a polyelectrolyte pair of polysaccharides—DEAD-dextran and carboxymethyl amylose (AMC)—stabilized with negatively charged polyester-folic acid nanoparticles (PGlPDL-FA). By precisely modulating the amount of nanoparticle dispersion during phase separation, we controlled the transition from homogeneous coacervates to single- and multi-vacuolated architectures. These structures exhibit dynamic disassembly in response to cytosolic stimuli, such as changes in pH and salt concentration, and act as macromolecular reservoirs with selective permeability to fluorescent probes. This strategy provides a robust platform for spatiotemporal regulation of biomolecular interactions, with potential applications in drug delivery, synthetic organelles, and biomimetic systems.