Disposition of E-selectin-targeting liposomes in tumor spheroids with a perfusable vascular network.

We constructed tumor spheroids with a perfusable vascular network to assess drug delivery systems that target the tumor vasculature. A tricultured tumor spheroid containing human umbilical vein endothelial cells (HUVECs) was placed in the central compartment of a microfluidic device, and the HUVECs were seeded into the microslit channels on both sides. Angiogenic sprouts began to form within a few days, from both the tumor spheroids and microchannels, and became more abundant and branched, while attracting each other, over time. A continuous vascular network of HUVECs was fully formed on Day 7. The uptake of 3'-(1-carboxy)ethyl sialyl Lewis X mimic (3'-CE sLeX mimic) liposomes, which have previously been proven to recognize E-selectin, in vascular-perfusable tumor spheroids was assessed. 3'-CE sLeX mimic and pegylated liposomes were rarely taken up, but when the vascular network was pretreated with TNF-α and IL-1ß, 3'-CE sLeX mimic liposomes accumulated considerably more in endothelial cells and their vicinity. Taken together, along with the known in vivo expression of E-selectin in tumor angiogenic blood vessels, these results suggest that 3'-CE sLeX mimic liposomes are a promising carrier for targeting tumor vasculature. Furthermore, proinflammatory cytokine treatment may be appropriate for use with vascular-perfusable tumor spheroids in pharmacokinetic studies.

Analysis of biochemical features of ST8 α-N-acetyl-neuraminide α2,8-sialyltransferase (St8sia) 5 isoforms.

Gangliosides are important components of the membrane and are involved in many biological activities. St8sia5 is an α2,8-sialyltransferase involved in ganglioside synthesis, and has three isoforms. In this study, we analyzed the features of three isoforms, St8sia5-S, -M, and -L that had not been analyzed, and found that only St8sia5-L was localized in the Golgi, while the majority of St8sia5-M and -S were localized in the ER. The localization of Golgi of St8sia5 depended on the stem region. In addition, the incorporation of exogenous GD3 was upregulated only in St8sia5-L expressing cells. Taken together, the localization of St8sia5 is important for the activity of the enzyme.

Development of Fluorescent Ganglioside GD3 and GQ1b Analogs for Elucidation of Raft-Associated Interactions.

b-Series gangliosides are abundant in central nervous tissues and are involved in important nerve processes. However, their functions are complicated because of their properties of forming dynamic domains in cell plasma membranes (PMs), called lipid rafts. In this study, we aim to develop fluorescently labeled b-series gangliosides that are useful for single-molecule imaging. The chemical synthesis of fluorescent GD3 and GQ1b was achieved using sialylation and ganglioside synthetic methods previously developed by our group. Furthermore, biophysical evaluations demonstrated that synthesized fluorescent GD3 and GQ1b behaved as raft molecules on cell PMs, suggesting their applicability to the study of raft-associated interactions.

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

In this study, we developed novel E-selectin-targeting liposomes, i.e., 3'-(1-carboxy)ethyl sialyl LewisX (3'-CE sLeX) mimic liposomes, for targeted delivery of everolimus (EVE) in anti-angiogenic therapy. We investigated the uptake and efficacy of these E-selectin targeting liposomes in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs). The uptake of EVE in 3'-CE sLeX mimic liposomes increased steadily and almost caught up with the uptake of plain EVE at 3 h, which was higher than that in PEGylated liposomes (PEG-liposomes). Inhibition of uptake by anti-E-selectin antibody suggested involvement of E-selectin-mediated endocytotic processes. Migration in cells treated with EVE/3'-CE sLeX mimic liposomes was suppressed by more than half when compared to the control. This treatment was also seen to significantly inhibit the formation of capillary tubes and networks. In addition, Thr389 phosphorylation of pS6 kinase, as a marker of mTOR activity, was remarkably suppressed to less than endogenous levels by EVE/3'-CE sLeX mimic liposomes. In conclusion, the present study demonstrated that EVE/3'-CE sLeX mimic liposomes were intracellularly taken up by E-selectin and prompted anti-angiogenic effects of EVE involved in the mTOR signaling pathway. However, moderate retention of EVE in the liposomes might limit the targeting ability of 3'-CE sLeX mimic liposomes.

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods.

Syntheses of Fluorescent Gangliosides for the Studies of Raft Domains.

Gangliosides, glycosphingolipids containing one or more sialic acids in the glycan chain, are involved in various important biological processes in cell plasma membranes (PMs). However, the behaviors and functions of gangliosides are poorly understood, primarily because of the lack of fluorescent analogs that are equivalent to native gangliosides that can be used as chemical and physical probes. In this study, we developed entirely chemical methods to synthesize fluorescent gangliosides (GM3, GM2, GM1, and GD1b) in which the glycan components are site-specifically labeled with various fluorescent dyes. The functional evaluations of the synthesized fluorescent gangliosides demonstrated the great influence of fluorescent dye on the physical properties of gangliosides in PMs and revealed the fluorescent ganglioside analogs which show similar behaviors to the native gangliosides.

Raft-based interactions of gangliosides with a GPI-anchored receptor.

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.

Structure-Activity Relationship Study of the Neuritogenic Potential of the Glycan of Starfish Ganglioside LLG-3 (‡).

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically synthesized and evaluated in vitro. The methyl group at C8 of the terminal sialic acid residue was crucial for neuritogenic activity, and the terminal trisaccharide moiety was the minimum active motif. Furthermore, the trisaccharide also stimulated neuritogenesis in human neuroblastoma SH-SY5Y cells via mitogen-activated protein kinase (MAPK) signaling. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rapidly induced by adding 1 or 10 nM of the trisaccharide. The ratio of phosphorylated ERK to ERK reached a maximum 5 min after stimulation, and then decreased gradually. However, the trisaccharide did not induce significant Akt phosphorylation. These effects were abolished by pretreatment with the MAPK inhibitor U0126, which inhibits enzymes MEK1 and MEK2. In addition, U0126 inhibited the phosphorylation of ERK 1/2 in response to the trisaccharide dose-dependently. Therefore, we concluded that the trisaccharide promotes neurite extension in SH-SY5Y cells via MAPK/ERK signaling, not Akt signaling.

Extending the glucosyl ceramide cassette approach: application in the total synthesis of ganglioside GalNAc-GM1b.

The development of a novel cyclic glucosyl ceramide cassette acceptor for efficient glycolipid syntheses was investigated. p-Methoxybenzyl (PMB) groups were selected as protecting groups at C2 and C3 of the glucose residue with the aim of improving the functionality of the cassette acceptor. The choice of the PMB group resulted in a loss of ß-selectivity, which was corrected by using an appropriate tether to control the spatial arrangement and the nitrile solvent effect. To investigate the effect of linker structure on the ß-selectivity of intramolecular glycosylation, several linkers for tethering the glucose and ceramide moiety were designed and prepared, namely, succinyl, glutaryl, dimethylmalonyl, and phthaloyl esters. The succinyl ester linker was the best for accessing the cassette form. The newly designed glucosyl ceramide cassette acceptor was then applied in the total synthesis of ganglioside GalNAc-GM1b.

The first total synthesis of ganglioside GalNAc-GD1a, a target molecule for autoantibodies in Guillain-Barré syndrome.

The first synthesis of ganglioside GalNAc-GD1a, featuring efficient glycan assembly and a cyclic glucosyl ceramide as a versatile unit for ganglioside synthesis is described. Although ganglioside GalNAc-GD1a was first found as a brain ganglioside, IgG autoantibodies to GalNAc-GD1a were subsequently found to be closely related to a human peripheral-nerve disorder, Guillain-Barré syndrome, which is the commonest cause of acute flaccid paralysis worldwide. In this study, the characteristic hexasaccharide part carrying two sialic acid residues was synthesized efficiently by use of a readily accessible GM2-core unit as a common unit. The potentially difficult coupling of the oligosaccharide and ceramide moieties was carried out by using a cyclic glucosyl ceramide as a coupling partner for the hexasaccharide part, thereby successfully providing the framework of the target compound. Global deprotection delivered the homogenous ganglioside GalNAc-GD1a. An enzyme-linked immunosorbent assay showed that sera from patients with Guillain-Barré syndrome reacted both with natural and with synthetic GalNAc-GD1a.