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

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 remain limited, due to incomplete understanding of the structural and biophysical mechanisms of some key MLGIs which has hampered the design of spatial matched multivalent therapeutics. Moreover, the overlapping glycan specificities of various lectins makes it difficult to target specific MLGIs with high potency and selectivity. To address this challenge, we have recently developed polyvalent glycan-nanoparticles (glycan-NPs) as new biophysical probes for MLGIs: the NPs’ unique, size-dependent optical properties are exploited as readouts for quantifying MLGI affinities and thermodynamics, while their nanoscale size and high electron microscopy contrast are employed for probing binding modes and site orientation. Despite success, how design features such as glycan type, density and linker flexibility govern glycan-NP MLGI properties remain underexplored. Herein, we coat gold nanoparticles (GNPs) with varying densities of lipoic acid-oligo(ethylene glycol)--manno--1,2-biose or -fucose 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 GNP surface greatly enhances their DC-SIGN affinity, with low nM Kds, ~300,000 fold tighter than the corresponding monovalent binding. Their bindings are enthalpy driven, with favorable enthalpic terms 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. Finally, we demonstrate GNP-glycans potently block DC-SIGN augmented viral entry to host cells with sub-nM IC50s, with IC50 values being positively linked to their DC-SIGN MLGI affinity.