Direct evidence of the impact of aqueous self-assembly on biological behavior of amphiphilic molecules: The case study of molecular immunomodulators Sulfavants

Sulfavant A (1) and Sulfavant R (2) are amphiphilic sulfoquinovoside-glycerol lipids under study as molecular adjuvants of vaccines. Their structures differ only for the configuration of the oxymethine carbon of glycerol that is R/S in 1 and R in 2. The in vitro activity of both products follows a bell-shaped dose-response curve but 1 gave the best response between 10 and 30 µM, while 2 was active at 10 nM. Characterization of aqueous self-assembly of these molecules by a multi-technique approach based on surface tensiometry, dynamic light scattering (DLS), nuclear magnetic resonance (NMR), fluorescence light phase contrast, and cryo-electron microscopy has finally clarified this divergent and controversial biological outcome. The study proved the occurrence of supramolecular structures at concentrations much lower than the critical aggregation concentration (CAC) calculated by surface tensiometry for both products. The shape and size of these aggregates varied as a function of the concentrations differently for 1 and 2. At nanomolar concentration, Sulfavant A (1) formed cohesive vesicular structures, while Sulfavant R (2) arranged in small spherical micellar particles whose reduced stability was responsible for an increase of the monomer concentration that well agreed with the immunomodulatory activity. On the other side, micromolar concentrations led to an increase of particle sizes of both compounds and a transition from micellar to the vesicular state of Sulfavant R (2). The thermodynamic stability of these aggregates correlated with the activity of 1 above 10 µM and the complete loss of efficacy of 2 at micromolar concentrations. The study of Sulfavants provides clear evidence of how self-aggregation and thus the equilibria between the monomers and the supramolecular forms of lipophilic products in aqueous media determine the overall response of these substances in cell-based assays.