Probing Glycan-Gold Nanoparticle Architectures: Glycan Type, Density, and Linker Length, Governing Multivalent Lectin...

Multivalent lectin-glycan interactions (MLGIs) are widespread and vital for pathogen infection, cell-cell communication, and immune regulation, making them attractive therapeutic targets. Despite significant efforts, research progress in MLGI targeting therapeutics remains limited, due to our incomplete understanding of the structural and biophysical mechanisms of some key MLGIs, which has hampered the design of spatially matched multivalent therapeutics. Moreover, the overlapping glycan specificities of various lectins make it difficult to target MLGIs with high potency and selectivity. To address this challenge, we have recently developed polyvalent glycan nanoparticles (glycan-NPs) as biophysical probes for MLGIs. The NPs' unique, size-dependent optical properties are exploited as sensitive readouts for quantifying MLGI affinities and thermodynamics, while their nanoscale size and high electron microscopy contrast are exploited for probing binding modes and binding site orientation. Despite this success, how design features such as glycan type, density, and linker flexibility govern glycan-NP MLGI properties remains underexplored. In this work, we coated gold nanoparticles (GNPs) with varying densities of a lipoic acid-oligo(ethylene glycol)-α-manno-α-1,2-biose (DiMan) or fucose (Fuc) ligand of varying linker lengths and studied their MLGIs with DC-SIGN, an important tetrameric lectin viral receptor found on dendritic cells. Using our recently established GNP fluorescence quenching assay, we reveal that displaying DiMan or Fuc polyvalently on a GNP surface greatly enhances their DC-SIGN affinity, with low nanomolar apparent K_ds, ∼480 000-fold tighter than the corresponding monovalent binding. Their binding is driven by enthalpy, with favorable enthalpic but unfavorable entropic terms, and their absolute values depend on linker flexibility and glycan density. At high glycan densities, a short and less flexible linker is favored by maximizing enthalpic gains while minimizing entropic penalties, whereas at low glycan densities, a long and flexible linker is favored by increasing the reach and adaptivity of terminal glycans to maximize favorable enthalpic gains. These results reveal a delicate balance between glycan density and flexibility in controlling glycan-NP MLGI properties and their underlying thermodynamic mechanisms. Finally, we demonstrate that GNP-glycans potently block DC-SIGN-augmented viral entry into host cells with subnanomolar IC₅₀s, which are positively linked to their DC-SIGN MLGI affinity.