Competitive Mg ²⁺ Regulation of Biomolecular Condensate Microenvironments Enables Diverse Macrophage Response

The intrinsic microenvironments of biomolecular condensates play decisive roles in applications spanning synthetic cell construction, targeted drug delivery systems, cell engineering, bioreactor development, and precision disease interventions. Recent studies highlight that divalent cations play a central role in modulating the internal condensate microenvironments. However, the complex multivalent interaction networks within condensates create significant challenges in unraveling the molecular mechanisms. This study employs model systems of cationic peptides (arginine decamer (R10), lysine decamer (K10)) and polyanionic polymers (polyadenylic acid (PolyA), polyinosinic acid (PolyI), polyglutamic acid (PolyE), polyaspartic acid (PolyD)) to systematically investigate Mg²⁺-mediated modulation of condensate properties. Mg²⁺ enrichment dynamically controls ionic microenvironments through competitive interactions with polyelectrolytes. When interpolyelectrolyte affinity dominates (e.g., R10/PolyA), weakly bound Mg²⁺ enhances the surface potential, promoting small-molecule enrichment and ribozyme catalytic efficiency. Conversely, when Mg²⁺-polyelectrolyte binding prevails (e.g., R10/PolyE), stable ion-polyelectrolyte complexes reduce the system polarity and amplify dye accumulation but compromise phase stability. Macrophage coculture experiments demonstrate that R10/PolyA@Mg condensates enable targeted magnesium delivery, significantly boosting TNF-α secretion and immune regulation. These findings establish a mechanistic framework for ion-mediated control of condensate microenvironments, offering theoretical insights into the intracellular ionic regulation of phase separation. This work suggests a Mg²⁺-responsive condensate design strategy for modulating macrophage responses, providing a foundation for the design of biomaterials with a tunable immunostimulatory potential.