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

Abstract

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.

Sialyl LewisX mimic-decorated liposomes for anti-angiogenic everolimus delivery to E-selectin expressing endothelial cells

“…32 The uorescent labelled 3 0 -CE sLeX mimic liposomes were prepared by the same method as reported previously. 24 Regardless of lm hydration or ethanol injection methods, 3 0 -CE sLeX mimic liposomes prepared possessed basically the same characteristics.…”

“…1) were synthesized by the method reported previously. 24 EVE was purchased from Chem Scene (South Brunswick Township, NJ, USA). Phosphate-buffered saline (PBS) was purchased from Nissui Pharmaceutical (Tokyo, Japan).…”

“…Cy5.5-labeled 3 0 -CE sLeX mimic liposomes were prepared according to the hydration method reported previously. 24 Briey, DSPC, cholesterol, 3 0 -CE sLeX mimic-linked DSPE-PEG, and DSPE-PEG-Cy5.5 were mixed in chloroform-methanol (1 : 1) at the molar ratio of 55 : 39 : 5 : 1 and then dried under reduced pressure using a rotary evaporator and vacuum desiccator. The lipid lm was swollen and was suspended in PBS at 65 C. The suspension was sonicated in a bath-type sonicator at 65 C for 10 min and with a tip-type sonicator for 3 min, and then extruded through a 100 nm pore membrane equipped in an extruder (Avanti Mini-Extruder, Avanti Polar Lipids, Alabaster, AL, USA) maintained at 65 C. The liposome solution was puried using a PD-10 gel ltration column.…”

“…Despite being a structurally simplied trisaccharide analog, the 3 0 -(1-carboxy) ethyl sLeX (3 0 -CE sLeX) mimic exhibited higher potency than native sLeX in terms of enhancement of liposome uptake in Eselectin-expressing endothelial cells. 24 The 3 0 -CE sLeX mimic liposomes appeared promising for the delivery of antiangiogenic drugs to tumor endothelial cells.…”

Glycopolymers Mimicking GM1 Gangliosides: Cooperativity of Galactose and Neuraminic Acid for Cholera Toxin Recognition

“…The general method to obtain oligo‐ and polysaccharides is by total synthesis . The synthesis of glycomimetics, that is, ligands mimicking carbohydrates, especially physiologically important carbohydrates, such as gangliosides, sialyloligosaccharides, and glycosaminoglycans has been reported. However, in most cases, multiple synthetic steps were required, and large‐scale synthesis is difficult because natural carbohydrates have different carbohydrate branch patterns and regioselective protection is required .…”