pH-responsive self-assembly of natural saponin glycyrrhizic acid

Glycyrrhizic acid (GA), as a naturally occurring saponin biosurfactant, has been widely used to construct supramolecular functional materials, derived from its hierarchical self-assembly behavior and advantages like bioavailability, biocompatibility, and biodegradability. However, the underlying mechanisms of its stimuli-responsive self-assembly in solution, which is crucial for designing advanced structures, remains unclear. In this study, we investigated the self-assembly behavior of GA in aqueous solution across a pH range of 2-8, using the combination of a range of microscopy techniques, small-angle X-ray scattering, nuclear magnetic resonance, isothermal titration calorimetry, and molecular dynamics simulations. We propose a comprehensive model of GA assembly: (1) Around pH 4, where one carboxyl group is deprotonated, GA forms nanofibrils stabilized by lateral hydrophobic interactions between triterpene moieties, wherein diglucuronic units form the outer shell and provide electrostatic repulsion that contributes to fibril stabilization; (2) At pH below 4, complete protonation of carboxyl groups neutralizes the shell, leading to reduced electrostatic repulsion and enhanced hydrogen-bond aggregation of GA nanofibrils into thicker fibril bundles; (3) At pH above 5, as two or three carboxyl groups deprotonate, GA nanofibrils disintegrate and re-assemble into spherical aggregates. In this stage, electrostatic repulsion weakens lateral hydrophobic interactions, resulting in loosely packed triterpene cores with diglucuronic shells exposed to water. Especially at pH above 8, GA exists primarily as monomers. These findings indicate that GA self-assembly is controlled by a pH-tuned interplay of hydrogen bonding, hydrophobic and electrostatic interactions, modulated through the degree of its carboxyl group dissociation.