Self-Assembling Synthetic Trehalose Glycolipids for Mincle Activation: Biophysical Properties, Molecular Dynamics...

Synthetic trehalose glycolipids (TGLs) that modulate the Macrophage Inducible C-Type Lectin receptor (Mincle) are attractive targets for adjuvant development. However, natural ligands such as trehalose dimycolate (TDM), as well as existing synthetic mimetics, are limited by poor physicochemical properties and reactogenicity, motivating the development of rationally designed synthetic TGLs. Herein, we investigated the biophysical properties of amphiphilic TGLs featuring hydrophobic, aromatic moieties attached to the polar trehalose headgroup via ester, amide, or triazole linkages. The formation of homogeneous and colloidally stable aqueous nanodispersions was readily achieved through focused ultrasonication, eliminating the need for cosolvents and/or surfactants that might interfere with the self-assembling and biological properties of these compounds. These self-assembled particles were characterized using DLS, cryo-TEM, and synchrotron small-angle X-ray scattering to gain insight into their particle morphology and liquid crystalline phase behavior. Additionally, molecular dynamics (MD) simulations were conducted to evaluate the critical packing parameter (CPP), a predictive descriptor of the supramolecular structures formed by self-assembling amphiphiles, under conditions that closely mimic experimental environments. The synthetic Mincle ligands formed lyotropic liquid crystals, including cubosomal or hexagonal phases as well as lamellar phases. In vitro and in vivo testing of these self-assemblies revealed that all tested TGLs exhibited measurable immunological activity. Analogues forming cubic phases were associated with comparatively stronger cytokine production and enhanced antigen-specific humoral and cell-mediated immune responses within this compound set. Taken together, the data support a model in which both molecular structure and supramolecular organization contribute to immunostimulatory activity, although these effects cannot be fully decoupled in the present study. Additionally, we developed an in silico model capable of predicting supramolecular assembly, providing insight into the self-assembly tendencies of trehalose-based amphiphiles, and offering broader relevance to other biologically active, self-assembling systems.