Probing Cell-Surface Carbohydrate Binding Proteins with Dual-Modal Glycan-Conjugated NanoparticlesJ. Am. Chem. Soc.


Sungjin Park, Gun-Hee Kim, Seong-Hyun Park, Jaeyoung Pai, Dominea Rathwell, Jin-Yeon Park, Young-Sun Kang, Injae Shin
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Probing Cell-Surface Carbohydrate Binding Proteins with DualModal Glycan-Conjugated Nanoparticles

Sungjin Park,†,§ Gun-Hee Kim,†,§ Seong-Hyun Park,† Jaeyoung Pai,† Dominea Rathwell,† Jin-Yeon Park,‡

Young-Sun Kang,‡ and Injae Shin*,† †Department of Chemistry, Yonsei University, Seoul 120-749 Korea ‡Department of Veterinary Medicine, Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701 Korea *S Supporting Information

ABSTRACT: Dual-modal fluorescent magnetic glyconanoparticles have been prepared and shown to be powerful in probing lectins displayed on pathogenic and mammalian cell surfaces.

Blood group H1- and Leb-conjugated nanoparticles were found to bind to BabA displaying Helicobacter pylori, and Lea- and Lebmodified nanoparticles are both recognized by and internalized into DC-SIGN and SIGN-R1 expressing mammalian cells via lectin-mediated endocytosis. In addition, glyconanoparticles block adhesion of H. pylori to mammalian cells, suggesting that they can serve as inhibitors of infection of host cells by this pathogen. It has been also shown that owing to their magnetic properties, glyconanoparticles are useful tools to enrich lectin expressing cells. The combined results indicate that dual-modal glyconanoparticles are biocompatible and that they can be employed in lectin-associated biological studies and biomedical applications. ■ INTRODUCTION

Multiple earlier studies have shown that interactions between glycans and carbohydrate binding proteins (lectins) displayed on the cell surface are involved in a variety of physiological and pathological processes.1 For example, cell surface lectins in the immune system recognize glycans expressed on the exterior of pathogens, and these interactions lead to stimulation of immune responses to pathogens.2 In addition, infections caused by pathogens, including Helicobacter pylori and influenza virus, takes place by initial adhesion of the pathogens to host cells through interactions of the pathogenic lectins (adhesins) with the host cell surface glycans.3 Consequently, detection of cell surface lectins and an understanding of recognition events that take place between lectins and glycans are of great importance in both basic research and development of more efficacious drugs and diagnostic tools.

H. pyroli, which colonizes the human stomach, is one of the most widespread infectious pathogens affecting nearly one-third of the world’s population. H. pyroli causes chronic gastritis, which may lead to peptic ulcer disease and gastric cancer.4 The cell surface of this pathogen frequently contains adhesins that recognize specific host cell glycans,5 as exemplified by the wellcharacterized blood group antigen-binding adhesin (BabA).6 In this case, infection of hosts by H. pyroli is triggered by binding of BabA to Leb present in the gastric mucosa. Because of their unique roles, H. pylori adhesins have been actively investigated.

Because H. pyroli strains express carbohydrate binding proteins on their surfaces, glycan-based detection methods have been exploited as part of methods to diagnose diseases associated with this pathogen,7 and materials that block associations of H. pylori with host glycans have been developed to prevent infections caused by this pathogen.8

Mammalian cells also express lectins on their surfaces. For instance, human DC-SIGN (dendritic cell-specific ICAM-3grabbing nonintegrin) is a C-type lectin displayed on the surface of dendritic cells and macrophages.9 Mouse SIGN-R1 (SIGN-related 1) is a homologue of human DC-SIGN that is expressed largely on macrophages in the splenic marginal zone and the medullar lymph nodes.10 Two lectins recognize mannose-rich or fucosylated glycans in a Ca2+-dependent manner with subtle differences in their glycan binding properties.11,12 When DC-SIGN and SIGN-R1 interact with glycans on host cells, bacteria or viruses, glycan antigens are internalized into cells via lectin-mediated endocytosis to induce immune activation. Because of their biological significance, mammalian cell surface lectins have been widely studied for an understanding of lectin-associated cellular processes.2,13

It is well-known that multivalent interactions of glycans with lectins enhance otherwise weak binding affinities of monomeric sugars to proteins.14 Over a decade, glycan-conjugated nanoparticles have been constructed in biological and biomedical research efforts because they have high surface area-to-volume ratios in comparison to other glycoclusters, such

Received: January 19, 2015

Published: May 5, 2015

Article © 2015 American Chemical Society 5961 DOI: 10.1021/jacs.5b00592

J. Am. Chem. Soc. 2015, 137, 5961−5968 as neoglycopeptides, glycolipid micelles and glycoproteins, and thus provide higher lectin binding capacity.15 Gold nanoparticles were first used as metal-based multivalent scaffolds to present glycan ligands.16 Later, glycan-conjugated single-modal fluorescent17 or magnetic nanoparticles7,18 were prepared for detection and capture of lectin displaying cells as well as noninvasive imaging of glycan-protein recognition events in animals. Although single-modal glyconanoparticles have been prepared for glycan-associated biomedical and basic research, dual-modal glyconanoparticles that integrate the advantageous features of both fluorescent and magnetic properties have rarely been employed for these purposes.19 In the study described below, we prepared oligosaccharide-conjugated dual-modal fluorescent magnetic nanoparticles (FMNPs) to probe mammalian and pathogenic cell surface lectins. The results of this effort show that dual-modal glyconanoparticles are useful tools in studies aimed at gaining an understanding of the biological roles of cell surface lectins, diagnosing pathogenassociated diseases and preventing pathogen infection. ■ RESULTS AND DISCUSSION

Preparation and Characterization of Glycan-Conjugated FMNPs. FMNPs conjugated with multiple copies of three types of fucose-bearing oligosaccharides [Lewis a (Lea) and Lewis b (Leb) antigens and a blood group H type 1 (H1)] were constructed to target pathogenic and mammalian cell surface lectins (Figure 1). The required oligosaccharides were synthesized in β-aminoethylated forms for conjugation to NPs as delineated in Scheme S1−S3.20 The oligosaccharides were then coupled to carboxylic acid moieties on the FMNPs (a molar ratio of FMNPs to glycan = 1:30), which were composed of cobalt ferrite magnetic nanoparticles coated with a shell of amorphous silica containing rhodamine B isothiocyanate,21 under amide coupling conditions to afford Lea-, Leb- and H1FMNPs. Unreacted carboxylic acids on the NPs were then capped with 2-aminoethanol to suppress nonspecific adsorption during detection of cell-surface lectins.