Two different sized GNPs, with diameters of ~5 and ~13 nm (denoted as G5 and G13), respectively, were prepared by citrate reduction of H[AuCl4] in the absence (for G13) or presence of a small amount of tannic acid (for G5) by following the literature methods, and their core sizes were confirmed by TEM (SI, Fig. ). They were then incubated with the LA-EG4-psDiMan ligand in an aqueous solution under a total ligand: GNP molar ratio of 1000 and 3000 for G5 and G13, respectively, to prepare the desired Gx-psDiMan conjugates (x = 5 or 13). We have found previously that treating G5 with 1000 molar equivalent of LA-EGn-glycan ligand produced highly stable and densely glycosylated G5glycans. Here, a higher ligand: GNP molar ratio of 3000:1, about 2.4 times the ligand ratio required to coat G13 with a self-assembled monolayer of LA-EGn-ligand was used, ensuring that the G13 was fully coated with the desired glycan ligands (see SI, section 2.3). To investigate how GNP surface glycan density affect their MLGI properties with DC-DIGN, the GNPs were further incubated with mixed the LA-EG4-psDiMan and LA-EG2-EG2-OH ligands of varying ratios (but under a fixed total ligand: GNP ratio as above). In this way, a serial of Gx-psDiMan conjugates with the psDiMan content being systematically varied from 0, 6.3, 12.5, 25, 50, 75 to 100% were prepared (see Table ). Each LA-based ligand can form 2 Au-S bonds on binding onto the GNP surface, with a total bond enthalpy comparable to a typical covalent C-C single bond (~90 vs. ~83 kcal.mol -1 ). As a result, the LA-based ligands are expected to be non-mobile after self-assembly on the GNP surface. Thus, the LA-glycan and LA-spacer ligands should be randomly distributed on GNP surface without phase separation. Scheme 1. Synthetic route to the LA-EG4-psDiMan and LA-EG2-EG2-OH ligands.