Developing Glycan-gold Nanoparticles for Targeting Specific Lectin-carbohydrate Interactions and Modulating Dendritic Cell Immune Functions

Multivalent lectin-glycan interactions (MLGIs) are fundamental to viral infection and immune defence, particularly those mediated by the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) receptor on Dendritic Cells (DCs). While DC-SIGN bridges innate and adaptive immunity by recognising pathogen-associated glycan patterns to initiate T-cell responses, this pathway is frequently subverted by pathogens, such as HIV and Ebola, to facilitate viral entry. Because these interactions rely on multivalent avidity driven by precise nanoscale topology rather than simple monovalent affinity, developing effective therapeutics requires biomimetic tools capable of replicating viral surface structures. This thesis employs gold nanoparticles (GNPs) as tuneable, biocompatible scaffolds to construct topological antigen models. By employing the unique physicochemical properties of GNPs, specifically their strong fluorescence quenching ability, this research further bridges the study of innate immunity with humoral immunity, investigating how spatial arrangement governs both lectin recognition and antibody clustering.

To investigate the MLGIs with DC-SIGN, Chapter 3 details the display of a synthetic glycomimetic, psDiMan, onto GNP scaffolds of varying sizes (~5 and ~13 nm). Fluorescence quenching assays revealed that these conjugates bind strongly to DC-SIGN, with sub-nM apparent Kds but minimal binding to DC-SIGNR, a closely related, almost identical tetrameric lectin to DC-SIGN; and thermodynamic analysis confirmed their interactions are enthalpy-driven, involving the all four Carbohydrate Recognition Domains (CRDs) in each DC-SIGN engaging in binding to one psDiMan-GNP conjugate. Importantly, the distinct MLGI properties were positively corelated to their biological functions: the psDiMan-GNP conjugates robustly blocked DC-SIGN-mediated cellular entry of Ebola-pseudotyped viruses (EBOV-GPpp) while showing negligible effect for the related DC-SIGNR receptor.

Chapter 4 shifts from individual protein interactions to intracellular processing by introducing G5-DiMan-OVA, a multifunctional nanoprobe for targeting DC-SIGN and tracking antigen (OVA peptide) presentation via a new Förster resonance energy transfer (FRET) readout method. Fluorescence microscopy confirmed robust DC-SIGN-mediated uptake into endo-lysosomal pathways by dendritic cells, leading to preferential MHC-II presentation (confirmed by flow cytometry), partial MHC-I cross-presentation (revealed by fluorescence microscopy), and distinct cytokine modulation profiles (revealed by ELISA). Furthermore, integration with a single-cell droplet microfluidics technique enables the high-throughput assessment of uptake dynamics and dissection of cellular heterogeneity of nanoprobe-treated dendritic cells.

To address the topological limitations of simple spherical glycosylated-GNP models, Chapter 5 presents the construction of hierarchical core-satellite GNP assemblies to mimic viral topography: a large central core represents the viral capsid surrounded by multiple small satellites representing glycoprotein spikes. Four different linkage strategies were explored: copper-catalysed click chemistry, strain-promoted copper-free click chemistry, covalent amide coupling, and DNA hybridisation, which gave different degrees of success, with the DNA hybridisation strategy being the most promising. Future research will focus on refining core-satellite assemblies by optimising satellite density and functionalising the satellite GNPs with di-mannose to accurately mimic viral spike presentation, enabling their application in DC-SIGN interaction studies.

Finally, Chapter 6 extends the investigation of topology to humoral immunity. Using a 13 nm GNP scaffold functionalised with His6 peptide as model epitopes at varying densities, this study examines the relationship between antigen presenting geometry and their antibody binding avidity. By using dynamic light scattering (DLS) and fluorescence quenching assays, the epitope density and deflection angle are found to strongly impact their antibody binding. A geometric analysis reveals that within the typical inter-distance spanned by the two Fabs in each antibody, strong bivalent binding is also influenced by the inter-epitope deflection angles.

Collectively, the findings presented in this thesis establish GNP scaffolds as powerful biophysical tools for probing MLGIs. By elucidating the critical roles of scaffold size, glycan display, and spatial topology, this work provides some useful insights into the rational design of polyvalent glycan displays for more potent viral entry inhibition and effective modulation of dendritic cell immune functions.