Charge Density Mismatch Drives Demixing in Multicomponent Polyelectrolyte Complexes

Coexisting complex coacervate phases, or multiphase complex coacervates, have experienced a surge in popularity as simple models for biomolecular condensates and their potential as synthetic cells. However, given the vast structural and chemical diversity of commonly studied polycation/polyanion combinations, deeper insights into the fundamental physics governing the phase behavior of these interesting structures are needed. Here, we show that multicomponent mixtures of charge density-mismatched polyelectrolytes with high chemical and structural resemblance yield coexisting, nested complex coacervate phases. Using homologous polycations and polyanions with linear charge densities ranging from f = 0.30–1.0, 36 systems containing two polyanions and two dye-labeled polycations were examined by brightfield and fluorescence microscopy. Notably, at least two polycations and two polyanions were required for demixing into multiphase droplets, as a mixture of three polyelectrolytes remained miscible in single-phase droplets. Miscibility was found to increase in the presence of at least two strongly charged polyelectrolytes. Our results corroborate the prediction by the random phase approximation, which stipulates that systems containing more than two oppositely charged polyelectrolytes, theoretically identical except for a mismatch in their linear charge densities, undergo demixing to yield stable, coexisting liquid phases. The coexisting phases are more than just the sum of their parts, as a new equilibrium is established with the redistribution of polyelectrolytes across inner and outer droplets. The segregation of fluorescently labeled polycations between phases decreased as the difference between the critical salt concentrations of coacervates (Δcs*) decreased. Our findings suggest that while Δcs* is necessary to drive demixing, it is not a sufficient parameter to govern it, as, in addition, a significant chemical dissimilarity of macromolecules is also required for their multiphase separation.