Macromolecular Fingerprints and Thermodynamic–Kinetic Regulation of Liquid–Liquid Phase Separation in Polysaccharide-Derived Biocondensates

The relationship between chemical functionalization and the thermoresponsive phase separation of polysaccharide-derived biocondensates remains insufficiently investigated. In this study, we systematically examine how the substitution site, polymer concentration, solvent environment, and thermal history influence the phase behavior of methacrylated dextran derivatives using multidimensional nuclear magnetic resonance (NMR) spectroscopy, diffusion-ordered spectroscopy (DOSY), temperature-dependent turbidimetry, isothermal titration calorimetry (ITC), and live time-lapse microscopy. NMR analysis revealed preferential methacrylation of C2 and C3 hydroxyl positions at low degrees of substitution, with increasing functionalization at the C4 position as the substitution degree increases. DOSY measurements further demonstrated distinct solvent- and substitution-dependent changes in polymer mobility under aqueous-phase-separating conditions. The lower critical solution temperature decreased as the Dex-MA concentration increased and shifted to higher temperatures with an increasing heating rate. Repeated thermal cycling progressively altered the transition temperature and reduced thermal hysteresis, particularly at higher concentrations, demonstrating a pronounced thermal-history dependence. ITC characterizations showed concentration- and temperature-dependent exothermic responses associated with polymer–solution interactions, while live imaging directly captured the progressive nucleation, growth, and coalescence of polymer-rich domains upon heating. Together, these findings establish molecular-to-mesoscale relationships linking chemical functionalization, polymer–solvent interactions, and thermodynamic–kinetic factors to the liquid–liquid phase separation of methacrylated polysaccharides, providing insights for the rational design of thermoresponsive polysaccharide-based biomaterials.