Synthesis and Functional Characterization of Novel Sialyl LewisX Mimic-Decorated Liposomes for E‑selectin-Mediated Targeting to Inflamed Endothelial Cells

Sialyl LewisX (sLeX) is a natural ligand of E-selectin that is overexpressed by inflamed and tumor endothelium. Although sLeX is a potential ligand for drug targeting, synthesis of the tetrasaccharide is complicated with many reaction steps. In this study, structurally simplified novel sLeX analogues were designed and linked with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG) for E-selectin-mediated liposomal delivery. The sLeX structural simplification strategies include (1) replacement of the Gal-GlcNAc disaccharide unit with lactose to reduce many initial steps and (2) substitution of neuraminic acid with a negatively charged group, i.e., 3′-sulfo, 3′-carboxymethyl (3′-CM), or 3′-(1-carboxy)­ethyl (3′-CE). While all the liposomes developed were similar in particle size and charge, the 3′-CE sLeX mimic liposome demonstrated the highest uptake in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs), being even more potent than native sLeX-decorated liposomes. Inhibition studies using antiselectin antibodies revealed that their uptake was mediated primarily by overexpressed E-selectin on inflamed HUVECs. Molecular dynamics simulations were performed to gain mechanistic insight into the E-selectin binding differences among native and mimic sLeX. The terminally branched methyl group of the 3′-CE sLeX mimic oriented and faced the bulk hydrophilic solution during E-selectin binding. Since this state is entropically unfavorable, the 3′-CE sLeX mimic molecule might be pushed toward the binding pocket of E-selectin by a hydrophobic effect, leading to a higher probability of hydrogen-bond formation than native sLeX and the 3′-CM sLeX mimic. This corresponded with the fact that the 3′-CE sLeX mimic liposome exhibited much greater uptake than the 3′-CM sLeX mimic liposome.