Advances in multifunctional glycosylated nanomaterials: preparation and applications in glycoscience

Carbohydrate Research xxx (2014) xxx–xxx

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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres

Advances in multifunctional glycosylated nanomaterials: preparation and applications in glycoscience Avijit K. Adak, Ben-Yuan Li, Chun-Cheng Lin ⇑ Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan

a r t i c l e

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Article history: Received 8 May 2014 Received in revised form 30 July 2014 Accepted 31 July 2014 Available online xxxx Keywords: Glyconanoparticles Multivalent interaction Biolabels Cellular imaging Target-specific delivery Glycosyltransferase activity

a b s t r a c t Applications of glycosylated nanomaterials have gained considerable attention in recent years due to their unique structural properties and compatibility in biological systems. In this review, glyco-nanoparticles (glyco-NPs) are defined as compounds that contain a nano-sized metallic core, are composed of noble metals, magnetic elements, or binary inorganic nanoparticles, and that exhibit carbohydrate ligands on the surface in three dimensional polyvalent displays similar to the glycocalyx structures on cell membranes. Nanomaterials decorated with suitable biological recognition ligands have yielded novel hybrid nanobiomaterials with synergistic functions, especially in biomedical applications. This review focuses on strategies for building various types of glyco-NPs and highlights their potential in targeted drug delivery and molecular imaging as well as their uses in bioassays and biosensors. The most recent examples of glyco-NPs as vaccine candidates and probes for assaying enzymes with bond-forming activities are also discussed. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Carbohydrates are ubiquitous in all organisms and are commonly found on cell surfaces in the form of glycoproteins, glycolipids, and the glycocalyx. Because (oligo)saccharides of the glycocalyx are easily accessible, they are involved in carbohydrate–protein interactions that play essential roles in a range of pathological and physiological cell functions.1–3 Such interactions are also considered as a prerequisite step in the cycle of host infection by several microbes, including viruses, bacterial pathogens, and their toxins.4 The interactions between an individual carbohydrate ligand and a carbohydrate-binding protein (lectin) are relatively weak and nonspecific. Consequently, these interactions often (but not always) exhibit a low association constant (Ka) in the range of 103 M 1 and are difficult to quantify.5 To overcome this limitation, simultaneous multi-point contacts between clusters of carbohydrates and a corresponding multimeric protein, or a protein with multiple binding sites, exploit multivalent binding to achieve high avidity as a result of the cluster glycosidic effect.6 Such interactions are ubiquitous in biology, and the binding affinities are orders of magnitude higher than that of a monovalent binding event.7 As a result, both the clustering of carbohydrate binding sites in the protein partner and the presentation of the car-

⇑ Corresponding author. Tel.: +886 3 5753147; fax: +886 3 5711082. E-mail address: [email protected] (C.-C. Lin).

bohydrate at the cell surface in a polyvalent, oriented fashion have been utilized to overcome the typical low Ka of carbohydrate–protein interactions in living systems.8 Therefore, understanding the central role that glycans (free carbohydrates or carbohydrate fragments of glycoproteins, glycolipids, and proteoglycans) play in a wide variety of biological recognition events is crucial for developing efficacious drugs and diagnostic tools. In light of the above factors, numerous well-defined synthetic multivalent glycomimetics with variable valency, topology, and modes of ligand presentation have been introduced. These glycomaterials include peptides,9 dendrimers,10 polymers,11 liposomes,12 cyclodextrins,13 fullerenes,14 calixarenes,15 and nanoparticles (NPs).16 Despite many studies on multivalent glycosylated nanoscale systems, the precise requirement for the best scaffold relies heavily on a specific type of glycan binding event. When conjugated to affinity ligands such as glycans, nanomaterials provide excellent opportunities for studying carbohydrate-mediated biological interactions at the molecular level (Fig. 1). Because of their inherent high surface area-to-volume ratio compared to other traditional micrometer-sized counterparts, NPs provide a greater contact surface area capable of producing higher capacity receptor binding. In addition, multiple and different forms of carbohydrate ligands can be incorporated simultaneously on the surface of an NP, thus mimicking the polyvalent glycolipid structure on the cell surface. Such features facilitated the self-assembly of carbohydrate monolayers on the surface of colloidal gold nanoparticles (AuNPs), termed glyco-AuNPs, and were first employed as metal-based multivalent scaffolds to

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Figure 1. Examples and applications of different multivalent and multifunctional glyco-NPs.

present carbohydrate ligands.17,18 In this review, glyco-nanoparticles (glyco-NPs) are defined as compounds containing a nano-sized metallic core that is composed of noble metals, magnetic elements, or binary inorganic NPs and that exhibit biologically relevant carbohydrate ligands on the surface in three dimensional polyvalent displays. Earlier contributions have surveyed the methodologies involving the synthesis and characterization of gold, magnetic iron oxide, and binary inorganic (e.g., semiconductor quantum dots (QDs)) NPs and their applications in specific biomolecular recognition events.16,19 Metal-based NPs functionalized with natural polysaccharides, such as chitosan, heparin, and dextran, have been reviewed elsewhere20 and are therefore not covered in this review. Here, we highlight multivalent glycosylated nanomaterial systems based on gold, iron oxide, and QDs that have demonstrated enhanced binding affinity for glycans and proteins. Specifically, we focus on glyco-NP-based binding assays and biosensors for protein detection and present their potential in targeted drug delivery and molecular imaging. Finally, we discuss the advances in vaccine development and briefly outline the prospective applications of glyco-AuNPs in enzyme activity assays (Fig. 1). 2. A brief overview of the preparation of glyco-NPs Glyco-NPs consist of an inorganic core of nanoscale dimensions (less than 100 nm in diameter (d)) and an outer surface covered with a flexible organic layer (linker or polymer) connected either covalently or non-covalently to glycans. The chemical stability of gold colloids is advantageous in synthesis for controlling the size and shape of the NPs. The traditional method of fabricating AuNP uses the reduction of metal salts such as chloroauric acid (HAuCl4) in aqueous media to produce neutral Au atoms by modification of the Turkevich or Brust method.21,22 While the gold-colloid method uses the mild reducing agent sodium citrate in hot aqueous solution to produce colloids of a relatively broad size range (12– 100 nm in d), the Brust method utilizes a strong reducing agent, such as NaBH4, to yield AuNPs with a d of 2–5 nm. The covalent binding between AuNPs and biomolecules is easily achieved with a ligand-exchange process using thiolated molecules, that is, the generation of a self-assembled monolayer (SAM) on the colloidal

gold surface.23 The ligand density on the metal surface can be controlled by dilution with non-functionalized thiols. Thus, by changing the reaction conditions, the size, shape, and chemical composition of the surface of the AuNPs can be selectively adjusted. In addition, AuNPs have a unique surface plasmon band in the visible region (400–700 nm), making them a suitable tool for studying or monitoring biological recognition processes. Penadés and co-workers were the first to use carbohydrate-functionalized AuNPs, which contained lactose (Lac) and LewisX (LeX), for studying carbohydrate–carbohydrate interactions.17 Magnetic cores such as iron oxide (Fe3O4/Fe2O3) can also be used to functionalize desired glycans. The common methods for synthesizing magnetic NPs (MNPs) are co-precipitation, thermal decomposition, and microemulsion. However, the conjugation chemistries used in the surface modification are critical for ligand assembly and have been reviewed extensively.24 To attach glycans onto MNPs, carboxymethyldextran-coated MNPs or amine-functionalized dextran-coated MNPs were employed, thus achieving glycofunctionalization. For example, amine-functionalized dextran-coated MNPs have been used as a platform for the incorporation of multiple copies of sialyl LeX (sLeX) onto the MNP surface.25 Methods for the covalent functionalization of pre-fabricated MNPs through peptidic coupling26,27 or Cu(I)-catalyzed alkyne–azide [2+3] cycloaddition26,28 have also been reported with a high loading yield of the glycan. With the latter method, azido-functionalized NPs provide improved conjugation efficiency with alkynated carbohydrates compared to alkynated NPs with azide-bearing molecules.28 The typical size of glyco-MNPs (d 10–100 nm) is an average of two orders of magnitude smaller than that of a bacterium and allows multiple attachments of NPs onto the surface of a cell. In addition, MNPs exhibit high magnetization due to their superparamagnetic properties. These features make them useful in bioseparation and as a contrast agent for Magnetic Resonance Imaging (MRI). QDs are nano-sized fluorescent semiconductors and are characterized by a narrow emission bandwidth, high quantum yield, and long-term photostability compared to common organic dyes and fluorescent proteins.29 The most common procedure used to fabricate stable and water soluble QDs is the attachment of a hydro-

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philic surfactant onto the surface of semiconductor nanocrystals. Rosenzweig and co-workers successfully appended negatively charged carboxymethyldextran and positively charged polylysine residues onto succinate-modified CdSe/ZnS QDs using electrostatic interactions to form glyconanospheres (d 190 nm).30 Trioctylphosphine oxide (TOPO) is also widely used as a surfactant to prepare water soluble nanohybrids. The surface TOPOs are then exchanged with carbohydrates to yield glyco-QDs (d 5 nm).31 Of the several semiconductors studied, including InP, GaAs, and Si, core shell Cd-based QDs (CdSe, CdTe, CdS, and CdSe/ZnS) are the most commonly employed semiconductors in glycofunctionalization. Direct and one-step synthesis of glyco-QDs using thiolated glycans is also reported. For example, Penadés and co-workers prepared maltose- and tumor-associated carbohydrate antigen (TACA)- and LeX-encapsulated glyco-CdS QDs (d 2–5 nm) using Cd(NO3)2 and Na2S in the presence of thiolated carbohydrates.32 Since then, various glyco-QDs have been prepared to serve as useful multivalent fluorescent nanoprobes, especially in biolabeling.33 Recently, a continuous-flow microreactor system was developed to prepare ZnS coated with CdSe or CdTe QDs using a relatively low temperature (150 °C instead of 250–300 °C), and the surface of the NPs was further assembled using glycans.34

3. Multivalent glyco-NPs as probes in binding assays for lectins and protein toxins Taking advantage of the surface plasmon absorption of AuNPs, an initial investigation observing the lectin-mediated aggregation of glyco-AuNPs was completed with the model lectin Ricinus communis Agglutinin 120 (RCA120) and Lac-functionalized AuNPs (LacAuNPs) using UV–Vis absorption spectroscopy.18 RCA120 can be used as a surrogate for the bioterrorism agent ricin (RCA60), and it specifically recognizes terminal Lac and galactose (Gal) residues of glycoconjugates. Lac-stabilized AuNPs (d 8.9 nm) were prepared and selectively interacted with RCA120 to induce NP aggregation, which was observed as a broadening and red shift of UV absorbance as a function of lectin concentration. A nanomolar lectin concentration could be detected using this colorimetric bioassay. Russell and co-workers used Gal-AuNPs with different Gal densities for interaction with RCA120.35 The surface carbohydrate density was reduced by the addition of a non-functionalized thiol, the short-chain thiolated tri(ethylene glycol) linker (30% coverage). The bioassay results indicated that 16 nm glyco-AuNPs with the optimal 70% coverage of Gal could detect RCA120 concentrations as low as 9 nM. More recently, a design strategy similar to that of Russell et al. was employed to detect human influenza virus X31 (H3N2) (Fig. 2).36 The AuNPs were self-assembled with a trivalent a-2,6thio-linked sialyllactose and a short tetra(ethylene glycol) linker with an optimized surface ratio of 75:25. The colorimetric assay showed that glyco-AuNPs (d 16 nm) avidly bind to the H3N2 virus but not to the avian RG14 (H5N1) influenza virus due to the binding specificity of carbohydrate ligands. Thus, selective detection was achieved. A similar colorimetric assay based on the aggregation of AuNPs to induce a color change was used to detect cholera toxin at the low concentration of 54 nM within 10 min of incubation using Lac-AuNPs (d 16 nm).37 Although lectins and toxins have been selectively detected using multivalent glyco-AuNPs, quantitative measurements of the binding affinity of glyco-NPs have been investigated to a lesser extent, and only a few protocols have been reported that evaluate glyco-NP affinity to a corresponding binding partner. The specific binding between the lectin Con A and the a-D-linked Man residues is well documented and therefore constitutes a good model system for investigating the multivalent carbohydrate–lectin interactions.

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In this context, surface plasmon resonance (SPR) was used to quantitatively determine the binding affinities of glyco-AuNPs to Con A (Fig. 3).38 Different sizes of AuNPs were coated with various monosaccharides (glucose (Glc), Gal, and Man) containing different spacer lengths. The SPR studies indicated that the dissociation constant (Kd) of Man-AuNP with Con A is 2.3 nM, which is a binding affinity over six orders of magnitude higher than that of the corresponding monovalent methyl-a-D-mannopyranoside (Me-a-DMan). The inhibitory activity of the glyco-AuNPs (d 6 nm) follows the order of Man > Glc >> Gal, as expected from the known Con A specificity for these monosaccharides. Furthermore, the combination of a larger AuNP (d 20 nm) with a relatively short 5-carbon spacer length and with 680 Man per NP provides the highest (128-fold) affinity enhancement per Man on the surface. In addition to the SPR study, dynamic light scattering (DLS),39 quartz crystal microbalance (QCM),40 isothermal microcalorimetry (ITC),41 and a fluorescence-based competition assay42 were also reported for measuring the affinity between a number of different Man-AuNPs and the Con A lectin. The apparent Kd values of glycoNP–lectin interactions are, in most cases, determined to be in the nanomolar range based on the energetics of these interactions. Quantification of the inhibitory potency of glyco-AuNPs was completed by investigating the binding of pentameric B-subunits of Shiga-like toxin I (Slt-I) as a function of particle size and spacer length.43 AuNPs of different sizes (d 4, 13, and 20 nm) were functionalized with 100% globotriose (Gb3; Gala1,4Galb1,4Glcb or Pk-blood group antigen), linked by a short aliphatic or long hydrophilic spacer, and contained Gb3 ligands totaling 60–1970 saccharides on the surface. The Gb3-AuNPs were determined to be very potent inhibitors of Slt-I. The SPR results showed that the potency per Gb3 ligand increased from 1300 for the d 4 nm particle with a short spacer to 228,000 for the d 20 nm particle with a long spacer. By taking advantage of the strong affinity between Gb3AuNP and Slt-1, these glyco-AuNPs were further employed in a chip-based biosensing assay of the toxin on a randomly immobilized antibody microarray (Fig. 4). In this method, the antibodymediated capture of the toxin was coupled with a signal amplification technique based on AuNP-promoted reduction of silver ions to metallic silver (precipitation) for visualization by the naked eye with a detection sensitivity of 70 nM. Glyco-AuNPs (d 4 nm) coated with Gb3 and a Gb3 analog (Gb3-trisaccharide containing terminal GalNAc) with varying glycan densities were also used for the selective inhibition of different variants of pathogenic Shiga toxins (Stxs).44 A luciferase assay was used to measure the toxin-mediated inhibition of protein synthesis, and the results revealed that the Gb3-AuNPs were nontoxic to Vero monkey kidney cells and protected the Vero cells from Stx-mediated toxicity in a dose-dependent manner. These studies indicated that inhibition is dependent on the glycan density on the NP surface, and selective inhibition of Stx1 and the more clinically relevant Stx2 was achieved using terminal GalNAc (NHAcGb3-AuNPs) rather than Gal (Gb3-AuNPs). The inhibition of dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binding to human immunodeficiency virus (HIV-1) envelope glycoprotein gp120 was studied by a SPR assay using several (oligo)Man-AuNPs.45 The AuNPs were coated with various densities (10%, 50%, and 100%) of partial structures of the N-linked high-Man-type glycans of viral gp120, such as di-, tri-, tetra-, penta-, and heptamannosides. A 20,000-fold enhancement in inhibition was observed using multivalent Man-AuNPs composed of the disaccharide Mana1-2Man compared to the monomeric mannoside. Furthermore, the same design principle was applied to the synthesis of fluorescein-isothiocyanate (FITC)-conjugated (oligo)mannosylated-AuNPs and the evaluation of their uptake by DC-SIGN-transfected Burkitt lymphoma cells (Raji DC-SIGN cell line).46 Flow

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Figure 2. Schematic representation of the aggregation of glyco-AuNPs in the presence of the influenza virus. A trivalent a-2,6-thio-linked sialyllactose on the surface of the AuNPs binds to the hemagglutinin (HA) on the surface of the virus and induces aggregation. Reproduced from Ref. 36 with permission from The Royal Society of Chemistry.

Figure 3. An SPR-based competition binding assay. (A) A schematic representation of the interaction between glyco-AuNPs and Con A lectins on a biosensor chip used in the competition assays. (B) The inhibition of Con A (0.5 lM) binding to the chip by Man-AuNPs. A set of inhibition curves for 0, 0.175, 0.5, and 1.0 lM Man-AuNPs is shown (from top to bottom). Reproduced from Ref. 38 with permission from The Royal Society of Chemistry.

Figure 4. Schematic presentation of the chip-based assay for the detection of Slt-I. The antibody that recognizes A-Slt is immobilized on a glass slide. After being incubated with Slt-I, the presence of Slt-I on the chip is visualized by incubating the chip with the high-affinity Pk-AuNPs, followed by silver enhancement. Reproduced with permission from Ref. 43. Copyright (2008) John Wiley & Sons.

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cytometry and confocal laser scanning microscopy revealed that the glyco-AuNP of d 1.8 nm decorated with the tetramannoside (Mana1,2Mana1,2Mana1,3Mana) ligand was preferentially taken up and endocytosed following both DC-SIGN-dependent and independent mechanisms. These examples demonstrate that parameters such as NP size and spacer length as well as surface coverage (ligand presentation density) have profound effects on the multivalent interactions between glyco-NPs and their specific receptors. Taking advantage of synthetic flexibilities, several high affinity glyco-NPs have been reported based on rational design and have been discussed in a number of excellent reviews and conference proceedings.47,48 In addition to the use of glyco-NPs in bioassays and biosensors, glyco-NPs can also be designed as nanocarriers for the detection of microbial pathogens. In the following section, we will illustrate examples for the detection and capture of bacterial pathogens using multivalent glyco-NPs. 4. Glyco-NP mediated bacterial pathogen detection, capture, and decontamination The adhesion protein FimH mediates the attachment of uropathogenic Escherichia coli strains to the host cell glycocalyx and specifically recognizes structures containing terminal Man residues. In 2002, we demonstrated that glyco-AuNPs can be used to label bacterial pathogens (Fig. 5).49 Two E. coli strains, ORN178 (with FimH-expressing pili) and ORN208 (mutant without the fimH gene), were employed to evaluate the specific binding of ManAuNPs (d 6 nm) to FimH. The transmission electron microscope (TEM) image revealed that the Man-AuNP binds solely to the ORN178 strain, indicating that the glyco-AuNPs provide a simple and direct method for the specific detection of this bacterial strain. The Man-specific binding in FimH adhesion is well characterized. Therefore, many studies have focused on the isolation of type 1 fimbriated uropathogenic E. coli. For instance, silica-coated MNPs (d 10 nm) decorated with Man residues (Man-MNPs) have been used for adhesion-based detection of E. coli (ORN178) at a concentration of 104 cells/mL (Fig. 6).26 The Man-MNPs were able to remove 65% and 88% of E. coli from the media after 5 and 45 min, respectively, using an external magnetic field. The capture efficiency was found to be higher than those of other biomoleculefunctionalized MNPs, such as antibodies and lectins. Recently, perfluorophenyl azide groups on hematite-ironoxide-NPs (d 250 nm) have been utilized for conjugation with unmodified Man residues upon UV irradiation.50 The resulting Man-MNP was found to retain the expected recognition of the E. coli strain ORN178 pili and the lectin Con A. While the immobi-

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lization by photoirradiation is very flexible and may alter the glycan structures, this method may be especially suitable for the glycoconjugation of unmodified polysaccharides. The adhesionbased capture of the Gram-positive zoonotic pathogen Streptococcus suis using magnetic glycoparticles has also been reported; in this case, the glycoparticles specifically recognize the terminal galabiose (Gala1,4Gal) residues.51 Streptavidin-coated magnetic particles with submicrometer diameters were conjugated with both mono- and tetravalent biotinylated galabiose derivatives through biotin–streptavidin interactions. A standard luminescence-based adenosine triphosphate (ATP) detection assay was performed to evaluate S. suis growth after incubation and magnetic separation. The result indicated that smaller magnetic glycoparticles (d 250 nm) efficiently bind to bacterial cells at a concentration of 104 cfu/mL but larger particles (d 10 lm) failed to bind, providing additional evidence that the larger surface area of NPs facilitates higher binding affinity. The use of the magnetic separation technique significantly reduced the long operational time and contamination of the samples, which are commonly encountered in almost all conventional separation methods. For example, a microfluidic system consisting of Man-MNPs was developed to isolate target bacterial cells from a mixture.52 The integrated MNP-based cell sorting system can effectively separate up to 103 E. coli ORN178 cells in as little as 1 min, with an efficiency level of more than 70%. Glycosylated nanomaterials have been used in the isolation and removal of bacteria from contaminated solutions. For example, Lindhorst and co-workers studied the carbohydrate-mediated precipitation of bacteria using glyco-nanodiamonds (glyco-NDs) for water decontamination (Fig. 7).53 The NDs were readily functionalized with Man-derivatives by thiol-ene reactions and used for the aggregation of E. coli cells through specific binding with FimH. The precipitates were found to be mechanically stable, and more than 90% of the aggregated bacteria could be filtered off with a conventional filter with a 10 lm pore size. In a related study, mannosylated magnetic porous microparticles (MaPoS) were found to be 3-fold more effective for removing E. coli ORN178 from a contaminated solution compared to nonporous particles.54 The higher efficiency of the former technique is attributed to the increased binding efficacy with a larger surface area. TEM imaging was used to visualize the binding of the influenza virus to sialic acid (Sia)-functionalized-AuNPs (Sia-AuNPs).55 SiaAuNPs (d 2 and 14 nm) were prepared, and the interaction with the viral fusion protein hemagglutinin (HA) of the influenza A virus was investigated. The TEM images of the virion complexes with the large (d 14 nm) AuNPs depicted the binding of multiple individual AuNPs to viral HAs. Flow cytometry results confirmed that

Figure 5. Glyco-AuNPs mediated biolabeling of microbial pathogen. Selective binding of wild-type E. coli ORN178 by Man-AuNP. The mutant ORN208 showed no detectable binding. Reprinted with permission from Ref. 49. Copyright (2002) American Chemical Society.

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Figure 6. A schematic representation of glyco-MNP-based pathogen detection. Reprinted with permission from Ref. 26. Copyright (2007) American Chemical Society.

Figure 7. Glyco-ND based removal of bacterial cells for water decontamination. Reproduced with permission from Ref. 53. Copyright (2008) John Wiley & Sons.

the 14 nm Sia-AuNP is an effective inhibitor of the influenza virus due to a strong affinity toward viral HA. More recently, AuNPs bearing various densities of Gal (17%, 33%, 80%, 90%, and 100%) were synthesized, and their interactions with the lectin PA-IL from Pseudomonas aeruginosa were determined using a hemagglutinin inhibition assay (HIA).56 The results showed that the most effective gold glycocluster with 100% Gal density on the surface exhibited a Kd of 50 nM, which is a 3000-fold enhancement in ligand affinity compared to monovalent Gal (Ka = 3.4  104 M 1). Taking advantage of the high avidity between the lectin and glyco-AuNP, Galand Gb3-trisaccharide functionalized AuNPs were prepared for separation/purification of a specific protein, PA-IL lectin, from a protein mixture.57 The target protein was simultaneously captured and enriched at a low femtomole level due to the high selectivity of the glyco-AuNPs. This work also established that the whole nanocomplex could be directly analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDITOF MS) with minimum sample handling. Furthermore, affinitybased purification of the B-subunit of Slt-I (B-Slt) from the crude cell lysate has been developed using MNPs functionalized with Gb3.58 A single protein band that corresponds to the B-Slt monomer, as characterized by MALDI-TOF MS, was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), indicating the specific binding of Gb3-MNPs. The MNP-based purification system was determined to be highly efficient: ca. 31 lg of B-Slt was extracted by 1 mg of Gb3-MNP. These examples demonstrate the applications of metallic glyco-NPs as specific nanoprobes in detection, separation, and labeling of target microorganisms. In the next section, we discuss the use of glyco-NPs as multifunctional nanoprobes for in vitro and in vivo cellular imaging and target-specific drug delivery. 5. Glyco-NPs as nanoprobes for targeting and labeling cells MRI was demonstrated to be one of the most important noninvasive imaging techniques both in vitro and in vivo.59 Gadolin-

ium (Gd)-based contrast agents and superparamagnetic iron oxide (Fe3O4) NPs have shown great potential as imaging probes in clinical diagnostics. Penadés and co-workers reported a one-step synthesis of paramagnetic Gd(III)-based glyco-AuNPs (d 2–4 nm) functionalized with different sugar derivatives, such as Glc, Gal, and Lac.60 Tetraazacyclododecane triacetic acid (DO3A) ligands were selected to effectively chelate the Gd(III)-cation. In vivo imaging of gliomas in a mouse brain indicated that paramagnetic Gd-glyco-NPs carrying monosaccharides (Gal and Glc) and a disaccharide (Lac) showed ca. 2–5 times greater longitudinal relaxivities (r1) in the tumoral zones than the clinical contrast agent DotaremÒ. The super paramagnetic nature of iron oxide NPs was used for mono- and triantennary Gal (T-Gal) derivatives with different spacer lengths that were incorporated into fluorophore (Cy3)labeled silica-coated MNPs using a highly efficient peptidic coupling reaction (Fig. 8).61 These dual fluorescent-magnetic glyco-NPs specifically targeted the asialoglycoprotein receptor (ASGP-R) on the surface of hepatocellular carcinoma HepG2 cancer cells and triggered receptor-mediated endocytosis. In this study, the proper orientation and spacer length of the T-Gal residues on the NP surface were determined to be crucial for the avidity of ASGP-R. The silica-coated outer shells were believed to impart stability and protection to the inner magnetic core.62 CD62E (E-selectin) and CD62P (P-selectin) are sLeX-binding endothelial transmembrane proteins and play an essential role in the initial adherence of leukocytes to inflammation sites.63 MRI allowed direct detection of these endothelial transmembrane inflammatory protein markers (E- and P-selectin) in acute inflammation.25,64 In the first case, sLeX-functionalized super paramagnetic NPs (sLeX-MNPs) were used for in vivo imaging of an inflamed brain.25 In this study, the authors introduced a ‘masked’ S-cyanomethyl group as the linker to chemoselectively attach unprotected glycans to the amine-functionalized dextran-coated MNPs through the formation of an amidine linkage to afford high glycan loading (105–107 copies of sLex per particle, ca. 20 nmol/ mg). The glyco-MNPs were used for direct detection of the

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Figure 8. Synthesis of Gal-functionalized dual fluorescent magnetic glyco-NPs used for specific targeting of HepG2 cells. Reproduced with permission from Ref. 61. Copyright (2010) John Wiley & Sons.

activated endothelial marker, E-selectin, by MRI in an acute inflammation model. In the second case, amino-functionalized MNPs (d 18 nm) were used for glycoconjugation through peptidic coupling to yield sLeX-functionalized super paramagnetic silica MNPs (sLeX-MNPs or SX@MNPs) with a glycan load of ca. 10.5 nmol/mg (Fig. 9).64 The use of MNPs in a common stroke animal model (mice) revealed that sLeX-MNPs accumulated to a greater extent in the brain vasculature compared to the non-functionalized control particles (LeX-MNP), as determined by MRI. The specific accumulation of the targeted (sLex) NPs is due to the up-regulation of selectin expression over the entire brain as a result of inflammation. Recently, five different types of glyco-MNPs (d 6 nm), Man-, Gal-, fucose (Fuc)-, Sia-, and N-acetyl glucosamine (GlcNAc)-MNPs, were prepared as MRI agents via amide bond formation and Cu(I)catalyzed azide–alkyne cycloaddition (Fig. 10).65 The glyco-MNPs were used to detect and quantify cancer cells using MRI. Based on the different response patterns (percentage change (DT2) of the transverse relaxation time, T2) of the MRI signatures of the cells

Figure 9. sLeX-functionalized super paramagnetic silica MNPs (sLeX-MNPs) for targeting E-selectin in a clinically relevant animal model of stroke. Reprinted with permission from Ref. 64. Copyright (2014) American Chemical Society.

to the five glyco-MNPs, nine different types of cancer cells and one normal cell line were differentiated by linear discrimination analysis (LDA). In addition to differentiation between normal and cancer cells, incubation of the mouse melanoma B16F10 cells with the Gal-MNPs substantially reduced the cell adhesion to the surface (up to 50%), while unfunctionalized MNPs had no effect, suggesting that this glyco-MNP could be used as an anti-adhesive agent for diseased tissue such as tumor tissue. For targeted anticancer nanotherapy, AuNP-based binary drug nanocarriers comprising of a tumor-targeting ligand have been constructed as tools for active and controlled drug delivery.66 Advantages and disadvantages exist for each drug conjugate. When linked with tumor-targeting ligands such as monoclonal antibodies (mAbs) or Ab fragments, these NPs confer a high degree of specificity and a wide range of binding affinities for the cancer-specific receptors or tumors. However, several limitations such as being expensive to produce, large size, and difficulty in conjugation to NP surface while maintaining immune recognition function have hampered their uses. Conversely, smaller-sized non-Ab ligands such as carbohydrates are often easily available, inexpensive to prepare, and easy to handle, and exploit highly specific interactions with endogenous carbohydrate binding proteins which are either uniquely expressed or over expressed on the malignant cells relative to normal tissues.67 More specifically, the ASGP-R in the liver is a particularly promising target because of its typical high density on hepatocyte surfaces (50,000–500,000 per cell) with specific affinity to Gal residues.68 However, lectin receptors those are not regularly involved in the endocytosis may also be targeted. To specifically deliver a drug using functionalized nanocarriers, fluorescent silicon oxide NPs decorated with a biofunctional ligand that targets a specific glycan binding protein and a chemotherapeutic agent were developed.69 The targeting ligand, T-Gal (see above for its structure), and a therapeutic agent, paclitaxel (PTX), were added to cyanine dye (Cy3)-containing silica NPs utilizing highly efficient stepwise orthogonal click chemistry to afford TGal-PTX @Cy3SiO2NPs. Fluorescence microscopy was used to

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Figure 10. Synthesis of magnetic glyco-NPs via amide coupling and click chemistry. Reprinted with permission from Ref. 65. Copyright (2010) American Chemical Society.

study cellular uptake, and the data indicated that TGal-PTX @Cy3SiO2NPs were taken up by the HepG2 cells through specific binding and ASGP-R-mediated endocytosis. Cytotoxicity assays revealed a dose-dependent cytotoxic effect of TGal-PTX @Cy3SiO2NP after 1 h, with respect to the control, resembling that of the free PTX drug under similar concentrations. In another study, the T-Gal ligand was covalently conjugated to carborane70 and carboraneloaded mesoporous silica NPs (MSNs)71 to generate a target-specific delivery system (T-Gal-Cy3@MSN) for boron neutron capture therapy (BNCT). The increased hydrophilicity and enhanced bioavailability of the glyco-NPs was facilitated by the presence of the polar T-Gal ligand on the NP surface, resulting in an improved BNCT agent for the selective targeting of HepG2 cells. Furthermore, the cell uptake results indicated that T-Gal-Cy3@MSN exhibited an over 50-fold higher delivery efficiency of boron atoms to cells with respect to the clinically used sodium borocaptate. In addition, several unmodified hydrophobic anticancer drugs were noncovalently loaded onto AuNPs by appending suitable macrocyclic molecular receptors including cyclodextrins (CDs).72,73 b-CD and its derivatives are commonly known to form inclusion complexes as the hydrophobic cavities in aqueous solution and have been demonstrated as drug-delivery agents owing to their ability to protect drugs against degradation or inactivation in biological conditions.74 In a recent study, a target-specific drug nano carrier based on b-CD-covered glyco-AuNPs has been reported.75 AuNPs (d 12 nm) were simultaneously decorated with multiple copies of Lac units as the targeting ligands for lectin human galactin-3 (Gal-3) and b-CDs as encapsulating moieties for specific delivery of a synthetic anticancer drug, methotrexate (MTX). The UV–vis spectroscopy was used for measuring the drug carrying capability of this dually functionalized AuNPs, however, the study lacks information about drug-loading amounts on AuNPs surface. Glyco-QDs have also been developed as probes for the in vitro optical imaging of target cells. The first in vitro application of glyco-QDs (d 14–16 nm) as bio-labels was reported by Fang and co-workers.76 CdSe/ZnS core/shell QDs were functionalized with Man and GlcNAc and used to stain pig, mouse, and sea-urchin sperm. While GlcNAc-QDs specifically accumulated only at the sperm heads, as shown by confocal scanning microscope imaging, Man-QDs were spread over the whole sperm body. The specific distribution of the Glyco-QDs may be a result of the difference in the distribution of Man and GlcNAc receptors on a sperm cell. The specific cellular uptake of Gal-QDs by ASGP-R was evaluated by fluorescence microscopy using CdSe/ZnS core/shell QDs (d 10.6 nm)

anchored to Gal residues.77 As evidenced by confocal laser scanning microscopy, the Gal-QDs were preferentially taken up, compared to unfunctionalized QDs, by the lung cancer cells (A549 and H647 cell lines) through receptor-mediated endocytosis after 3 h of incubation. Similar applications using CdSe/ZnS-based glyco-QDs as bioprobes for biosensing and imaging have been reported.78,79 Seeberger and co-workers demonstrated the first in vivo application of glyco-QDs as targeting agents.80 PEGylated glyco-QDs (d 15–20 nm) encapsulated with Man, Gal, and D-galactosamine (GalN) were synthesized and used to study the specific carbohydrate–protein interactions for in vitro imaging and in vivo liver targeting. Gal- and GalN-QDs were preferentially taken up by HepG2 cells via the ASGP-R receptor, as shown by flow cytometry. Fluorescence microscopy indicated that Man- and GalN-encapsulated QDs selectively accumulated in the liver compared to the control (PEGylated-QDs), suggesting that the Man and ASGP-R receptors are responsible for the specific binding to and/or endocytosis into Kupffer cells and hepatocytes, respectively. Recently, highly complex glycan (sialyl N-acetyllactosamine, LacNAc, and LeX)encapsulated glyco-QDs (d 7–10 nm) were prepared using phosphorylcholine (PC)-stabilized QDs as a platform for enzymatic sugar chain elongation.81 The biodistribution of these glyco-QDs was studied by in vivo near-infrared fluorescence imaging. The authors speculated that only the sialylated-QDs were distributed for up to 2 h longitudinally over all tissues, while all other glycans analyzed rapidly accumulated in the liver within 5–10 min of administration. 6. Glyco-NPs as synthetic vaccine candidates Smaller carbohydrates, such as TACAs or glycan fragments, can be conjugated with an immunologically active carrier protein for carbohydrate-based cancer or bacterial vaccine development because these glycans generally do not elicit an immune response.82 Metal NPs (such as colloidal gold) can have an extremely low rate of excretion because of their intrinsic resistance to lysosomal degradation, resulting in long-term bioaccumulation. In addition, the simultaneous antigen-loading, adjuvant co-delivery, and targeting property of NPs have considerably increased their attractiveness as nanocarriers in NP-based vaccines.83,84 Despite the evidence for nanomaterials as antigen carriers, only a few reports related to glyco-NPs as conjugate vaccines have been published.85–87 Barchi and co-workers synthesized AuNPs decorated with the Thomson–Friendenreich (TF) antigen, a cell surface mucin tandem

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Figure 11. Overview of the approach used to develop AuNP-based synthetic anticancer vaccines. Breast cancer cells express aberrant mucins displaying Core 1 glycans such as the Tn-antigen glycan. This ‘multicopy-multivalent’ presentation is mimicked by displaying the Tn antigen glycopolymers on the surface of NPs.87

Figure 12. Direct monitoring of bacterial b-1,4-GalT activity in a cell-free extract with GlcNAc-AuNPs as the acceptor substrate. (a) Experimental configuration for the direct detection of Neisseria meningitidis b-1,4-GalT expressed in E. coli. (b) LDI-TOF mass spectrum of target molecule at m/z 1221.6 ([target molecule+Na]+) generated from glycoAuNPs collected from the crude cell extract. Reproduced with permission from Ref. 89. Copyright (2005) John Wiley and Sons.

repeating unit (MUC-4) peptide (a 28 residue peptide acting as the B cell activating adjuvant), and a non-functional linker with a terminal hydroxyl group.85 Three-week old female Balb/c mice were vaccinated with the glyco-AuNP construct. However, the evaluation of sera produced from vaccinated mice exhibited moderate antibody (IgG and IgM) production. In another example, mice immunized with hybrid AuNPs coated with a synthetic branched tetrasaccharide antigen, an OVA323–339 peptide, and Glc were capable of inducing IgG antibodies against the native polysaccharide of Streptococcus pneumoniae type 14.86 However, low antibody production, with respect to the control antigen, was obtained. Recently, Davis and co-workers described AuNPs covered with a glycopolymer containing the mucin-related carbohydrate antigen a-N-acetyl-D-galactosamine (aGalNAc, Tn antigen) as a cancer vaccine.87 Glycopolymers containing a protected thiol-ending linker of varied length and composition (PEG25Tn25, PEG80Tn20) were prepared by Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization using the Tn-antigen glycan monomer and poly(ethylene glycol) methyl ether methacrylate (PEGMA; Mn = 300 Da) (Fig. 11). The polymer was then conjugated to the AuNP, yielding ‘multicopy multivalent’ nanoscale glycoconjugates. Immunization studies on rabbits indicated that the nanomaterial containing 20–25 Tn antigens per polymer chain produced the highest antibody titer against the Tn-antigen glycan and was cross-reactive against mucin proteins displaying Tn. This work also demonstrates that a synthetic peptide-free carbohydrate-based vaccine is possible.

While B cell activation and antibody production lag far behind the current conjugate vaccine adjuvants obtained from the immunogenic protein carrier, the three previous examples highlight the promising prospects of AuNPs as a nontoxic carrier with a controlled number of carbohydrate antigens on the particle surface. 7. Glyco-AuNPs as a probe for assaying bond formation of glycosyltransferase It has been demonstrated that thiolated molecules chemisorbed onto small AuNPs (d 2–10 nm) can be ionized directly to cleave the AuAS bond using laser irradiation. This effect is due to the higher ionization efficiency of AuNPs compared to organic matrices resulting from the quantum confinement effect.88 This feature provides a unique advantage in which SAMs of thiolated carbohydrates on a AuNP or protein–nanoprobe complex can be directly analyzed by MALDI-TOF MS.57,89 To monitor enzyme activity, the GlcNAc-AuNP (d 3–8 nm) was prepared and incubated with two recombinant human glycosyltransferases, b-1,4-galactosyltransferase (b-1,4-GalT) and a-1,3-fucosyltransferase (a-1,3-FucT), for sequential enzymatic glycosylation.89 LDI-TOF MS analysis of the resulting AuNPs revealed ionization of the trisaccharide, LeX, chemisorbed on the surface of the glyco-AuNPs. The method was then applied to evaluate the activity of a recombinant b-1,4-GalT in an E. coli cell lysate (Fig. 12). Direct measurement of the glycoAuNPs under LDI conditions allowed the identification of the desired product ion at m/z 1221, demonstrating that the crude cell

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Figure 13. An EndoNF-DM-induced glyco-AuNP aggregation-based biosensing strategy for the determination of a-2,8-PST activity. Reproduced from Ref. 93 with permission from The Royal Society of Chemistry.

lysate contains b-1,4-GalT activity that transfers Gal from UDP-Gal to the GlcNAc-AuNPs acceptor. Moreover, the ionization of glycoAuNPs does not require tedious purification, suggesting further potential of this method in the bioanalysis of glycosyltransferase activity. AuNPs possess unique plasmon-enhanced optical properties and are potentially powerful tools in bioanalytical chemistry because of their detection by visual inspection with the naked eye.90 In an investigation of enzyme activity, colorimetric assays based on the aggregation of peptide-coated AuNPs are focused primarily on the elucidation of enzymes with hydrolytic activities.91,92 With respect to specific bond forming activities of glycosyltransferases, a novel glyco-NP-based visual biosensing strategy was illustrated using a-2,8-polysialyltransferase (a-2,8PST) as a model enzyme, which is known for its role in the biosynthesis of polysialic acid (PSA) (Fig. 13).93 AuNPs were functionalized with the GD3-tetrasaccharide substrate, and the glyco-AuNPs (d 13 nm) were used as acceptors in an enzymatic sialylation assay using a-2,8-PST. The aggregation and simultaneous color change of the PSA@AuNP solution was mediated by the presence of a catalytically inactive double mutant of the endosialidase from the K1F bacteriophage (EndoNF-DM). Aggregation did not occur in the untreated (a-2,8-PST) control (GD3-AuNPs), demonstrating the specific cross-linking ability of the EndoNFDM and the need for at least five Sia residues for binding.94 Among other glycan structures tested (Lac and GM3), GD3 is the minimum acceptor sequence required for PST-catalyzed polymerization on the NP surface. Furthermore, the assay has been examined in a complex biological medium, crude cell lysate, and detection sensitivity at submicromolar PST was achieved.

8. Conclusion Glyco-NPs are attractive biomaterials because they mimic carbohydrate clustering on the cell surface and constitute a good bio-mimetic model for presenting glycans in a multivalent fashion.

Glyco-NPs generally enhance binding by several orders of magnitude over their monovalent constituents. Most examples discussed in this review highlight the applications of glycosylated nanomaterials including AuNPs, MNPs, and QDs in glycoscience research (Fig. 1). Particularly promising applications of glyco-NPs include their use as antiadhesion agents and as specific nanoprobes for in vivo imaging, cancer diagnosis, and targeted drug delivery. This review has also attempted to generate interest in glyco-AuNPs, their potential in carbohydrate-based conjugate vaccines used to treat human disease, and the analysis of enzyme activities. Moreover, the multifunctionality of glycosylated nanomaterials is easily generated by incorporating a variety of molecules, fluorescent dyes, and magnetic elements. Glyco-NPs are potentially useful in a variety of biomedical applications. Acknowledgements We gratefully acknowledge the financial support from the National Tsing Hua University and the Ministry of Science and Technology of Taiwan. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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