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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 4th edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2022. doi: 10.1101/glycobiology.4e.58
Nanomaterials offer tunable chemical and physical properties, such as electronic, photonic, and magnetic properties. Decoration of nanomaterials with glycans increases solubility and biocompatibility and lowers cytotoxicity, while allowing for multivalent glycan presentation. Given the central role of multivalency in glycobiology, glycosylated nanomaterials are interesting probes to study cellular, tissue, and organismal interactions. Nanomaterials purely composed of glycans, such as polysaccharide nanoparticles or nanocrystals, are interesting imaging agents, drug delivery systems, and tissue scaffolds that illustrate the potential of glycans in nanotechnology.
Glycoproteins and glycolipids are natural glycoconjugates that take part in cellular communication, inflammation, and immune response using carbohydrate–protein or carbohydrate–carbohydrate interactions. Certain glycan sequences are characteristic markers of diseases such as cancer, asthma, and diabetes. The elucidation of molecular mechanisms requires tools that mimic the presentation of glycans on the cell surface.
Individual protein–carbohydrate interactions are often of low affinity and broad specificity, complicating the description of glycan function. Nature enhances specificity by utilizing multivalent interactions. The number and the presentation of carbohydrate residues on a biomolecule are major determinants of binding avidity of ligands to cell-surface receptors. The transition from monovalent to multivalent is often associated with a larger variation in affinity/avidity, suggesting a “thresholding” effect, and in some cases cooperativity.
To elucidate those mechanisms, glycans have to be displayed in a scenario closer to that found on the cellular scale. “Nanotechnology” moves from the angstrom to the nanometer range (from ∼10−10 to ∼10−7 m), offering the tools to create, manipulate, and characterize structures on those scales.
Large glycoconjugates bearing multiple copies of a carbohydrate on various scaffolds, such as glycodendrimers or glycopolymers, have been generated to probe carbohydrate–protein interactions. Several glyconanomaterials with inherent high surface/volume ratios have been developed to allow for a greater contact surface area and explore multivalency effects. The integration of nanomaterials in the glycosciences has already enabled biomedical applications such as drug delivery systems, imaging agents, diagnostic platforms, or precise sensing tools that operate through biological mimicry. Further cooperation between the glycosciences and nanotechnology will improve our understanding of glycobiology.
Glyconanomaterials based on metal particles, semiconductors, or carbon-based scaffolds take advantage of the unique physical properties of the nanoscale, such as catalytic, photonic, electronic, or magnetic properties that are not seen in the bulk. The glycan portion ensures water solubility, exceptional stability in water and biological buffers, biocompatibility, structural diversity, and passive and active targeting properties. As a result, biocompatible nanomaterials offering a multivalent glycan presentation are generated` for sensing and drug delivery applications.
Nanoformulations, exclusively based on polysaccharides, have been developed as well. Polysaccharides such as chitosan, dextran, hyaluronic acid, and heparin have given rise to polysaccharide-based nanoparticles (NPs) for pharmaceutical use with superior biocompatibility and biodegradability. Low toxicity, low cost, and easy chemical modifications are additional advantages of polysaccharide-based NPs compared to synthetic polymers. These glyconanomaterials are currently used for drug delivery and tissue engineering, and electronics and device applications are emerging.
Hybrid materials based on inorganic nanostructures and biomolecules are a major focus of nanotechnology. Iron oxide, noble metal, and semiconductor nanoparticles served as synthetic scaffolds to multimerize glycans and enhance the affinity for receptors (Figure 58.1). The physical properties, such as magnetism or fluorescence, of these hybrid materials have given rise to applications in sensing, delivery, and imaging.
Overview of different types of glyconanomaterials created by coupling glycans to the surface of diverse nanomaterials.
The unique optoelectronic properties and facile chemical modification make gold nanoparticles (AuNPs) an important tool to monitor biological binding events. The functionalization of AuNPs with glycans generates materials with high aqueous solubility/dispersibility and biocompatibility. The resonance between collective oscillations of electrons in AuNPs (plasmons) and the incident electromagnetic radiation, gives rise to localized surface plasmon resonance (LSPR). Resonance frequencies of gold surface plasmon bands lie in the visible region (400–750 nm), giving rise to color effects. The high surface area/volume ratio results in high LSPR sensitivity and colorimetric changes, making AuNPs extremely valuable analytical reporters.
AuNPs are produced by reduction of gold salts with sodium citrate and surface modification with “capping” agents. Size, shape, and morphology are tuned by adjusting reaction conditions, allowing access to the near-infrared (NIR) spectrum. Colorimetric carbohydrate–lectin analyses exploiting LSPR of AuNPs have typically used 10-nm particles capped with thiol-poly(ethylene glycol) (thiol-PEG) aldehydes decorated with simple mono- or disaccharides. AuNPs Ricinus communis agglutinin (RCA120) or cholera toxin induced a reversible color change.
Direct visualization is a particularly attractive feature in biology. Stable colloidal gold was first described by Faraday in 1857, but applied in biology only in the 1970s when immunogold-staining procedures were used to observe microorganisms by transmission electron microscopy (TEM). Immunogold staining with mannosylated AuNPs permitted users to probe complement activation and opsonization processes in macrophage-mediated endocytosis. Carbohydrate–protein interactions were visualized by TEM thanks to the mannosylated AuNPs ability to target Escherichia coli type 1 pili mannose-specific receptors.
Very small gold glyconanoparticles can be prepared by reducing a gold salt in the presence of glycosylated thiol ligands (Figure 58.2). Ligand density and composition can be adjusted precisely. These glyconanoparticles conserve the chemical properties of the ligands and can be advantageously characterized by ultraviolet-visible (UV-Vis) spectroscopy, infrared (IR) spectroscopy, elemental analysis, nuclear magnetic resonance (NMR), TEM, and X-ray photoelectron spectroscopy (XPS).
(Left) A calculated representation of a 2-nm-sized gold glyconanoparticle formed by 102 gold atoms and coated with 44 molecules of 5-mercaptopentyl α-D-mannopyranoside and (right) the corresponding transmission electron microscopy (TEM) image. (more...)
The smaller glyco-clusters lack a LSPR band, but can be observed by TEM. Visualization of AuNPs helped to unambiguously demonstrate some weak, previously controversial, effects. For example, Lex-decorated AuNPs provided visual evidence for the existence of Ca-mediated sugar–sugar interactions and were used to explore potential mechanisms of sugar-mediated self-assembly of sponge cells.
Small gold glyconanoparticles were helpful in clarifying mechanistic aspects of multivalent carbohydrate recognition and have been exploited as antiadhesion agents to prevent melanoma metastasis, as vaccine candidates, and for cellular and molecular imaging.
Magnetic nanoparticles (MNPs), including iron oxide and manganese oxide nanoparticles, are interesting contrast agents for magnetic resonance imaging (MRI). MRI can generate internal tomographic tissue images by using a radio frequency (RF)-induced electromagnetic field; modulation of that field's signal by MNPs (so-called “contrast”) helps to detect their location. In clinical practice, gadolinium complexes are commonly used as MRI “contrast” agents. MNP–biomolecule hybrids are typically more sensitive, as they can be loaded with multiple copies of the same ligand for better receptor targeting. Multimodal imaging can be achieved by attaching labels (i.e., a fluorescent dye) that allow for additional detection modes.
The surface functionalization of MNPs is crucial for molecule-specific binding and “molecular MRI.” Antibodies have been widely used as targeting ligands because of their superb specificity, but suffer from high cost, short lifetime due to thermal instability, and potential immunogenicity. Structurally defined ligands, such as glycans, provide an attractive alternative.
Glyco-MNPs allow for detection of early stage disease by successfully mimicking leukocyte recruitment during inflammation (see Figure 58.2). By taking advantage of their high surface/volume ratio, glyco-MNPs can display multiple copies of oligosaccharides, thus increasing multivalency of binding interactions. Tetrasaccharide sialyl-Lewis x (SLex)-functionalized MNPs successfully targeted E-/P-selectins. Notably, SLex-MNPs detected inflammation events, both in vitro and in vivo, without any significant signs of associated cytotoxicity. Specific binding to the activated endothelium of blood vessels allowed for the detection of lesions in clinically relevant brain mouse models of stroke (Figure 58.3). Cross-species efficacy is easier to achieve with sugars than with antibody ligands. Thus, glyco-MNPs may be translated more readily from mammalian models to the clinic.
(A) In vitro binding studies using SLex-MNPs to rat E-selectin; (B) magnetic resonance images (MRIs) and their 3D reconstruction of SLex magnetic nanoparticles. (Adapted from van Kasteren SI, et al. 2009. Proc Natl Acad Sci 106: 18−23.)
Quantum dots (QDs) are luminescent semiconducting nanomaterials, typically made of binary cadmium or zinc selenides or sulfides. QDs can emit light in the entire spectrum, and their optical properties are tunable depending on their size. Compared with organic dyes, QDs also have a broader excitation spectrum and sharper emission bands, allowing for multicomponent analysis with a single excitation source. Glyco-QDs functionalized with carboxymethyldextran and polylysine were used to study carbohydrate–protein interactions. The addition of maltose-modified dendrimers rendered the QDs water-soluble and biocompatible, while enhancing stability.
Using a host–guest strategy, β-cyclodextrin-quantum dots (β-CD-QDs) were prepared. Synthetic β-CD-QDs behaved much like PEGylated QDs and agglutinated lectins such as ConA, Galanthus nivalis agglutinin (GNA), and peanut agglutinin (PNA).
Elemental carbon has several allotropes, including tetravalent diamond and trivalent graphitic structures, which provide potential scaffolds for glycan presentation. Graphene is a two-dimensional carbon allotrope and the basic structure of important carbon-based materials. Buckminsterfullerene (C60) is a discrete spherical construct that, although larger than many small molecules, can be manipulated using techniques common to small molecules. Carbon nanotubes (CNTs) can be considered cylindrical, elongated fullerenes. As a consequence of their curvature, hybridization, and boundary/inner atom ratios, both fullerenes and CNTs possess different reactivity that other carbon allotropes.
Glycosylated-fullerenes such as α-D-mannosyl fullerenes and fullerenols inhibit erythrocyte aggregation. These “sugar balls” are generated by introduction of a reactive group, such as a terminal alkyne, followed by attachment of azido sugars. Thereby, a near-spherical display of glycans is possible. The glycosylated C60s with 0.7-nm diameter can be considered the smallest “nanoparticles” in glyconanotechnology.
CNTs are classified based on the number of graphene-like sheets that make up the sidewalls of the cylinder. Single-walled CNTs (SWCNTs) have a typical diameter of 1–2 nm and multiwalled CNTs (MWCNTs) have diameters of ∼2–25 nm. The CNT length can range from 10s of nanometers to 10s of micrometers or even longer. Both the inner hollow space and the outer surface may be exploited to create functionalized CNTs (f-CNTs) to serve as delivery systems.
Broader use of CNTs has been hampered by their cytotoxicity and poor solubility. CNTs have been coated with glycopolymers. A C18-lipid tail “wrapped” the CNT surface through hydrophobic interaction, to expose α-GalNAc residues. Glycopolymer-coated CNTs were nontoxic in vitro, whereas noncoated CNTs induced death in certain cells. Noncovalent functionalized CNTs have the risk of losing their coating materials once introduced into a biological milieu, and the ultimate fate of such products is unclear.
Covalent surface glycosylation or glycoconjugation of CNTs creates more stable probes for in vivo studies. Oxidation of the CNT surface introduces carboxylic acids that can be used for the covalent attachment of amino-functionalized sugars. Galactosylated SWCNTs can “capture” pathogenic E. coli. Scanning electron microscope (SEM) images show a strongly bound matrix formed by cells binding to the glycosylated nanotubes.
Direct attachment of β-GlcNAc residues via “one-pot” Staudinger reduction and amidation allows for good control of the anomeric configuration. Upon monosaccharide attachment, glycosyltransferases were used for regio- and stereoselective glycan elaboration. The sugar hydroxyl groups were used as “tagging” sites for heavy element-bearing labels to visualize the glycans via TEM.
1,3-Dipolar cycloaddition of reactive azomethine ylides, generated in situ from α-amino acids, created pyrrolidine derivatives of fullerenes and CNTs. This covalent approach avoided oxidative “cutting” and provided filled-and-functionalized glycosylated CNTs for in vivo applications.
CNTs can be considered “1D hollow pores” with an associated capillarity. Through capillary action, molten salts or their solutions can be encapsulated inside CNTs. Glyco-CNTs were used for encapsulation of the radioemitter Na125I and in vivo localization of high levels of this radionuclide. Multiple copies of GlcNAc improved both water dispersibility and biocompatibility. Thanks to the high aspect ratio and surface area to volume ratio of such CNTs, sugars can be efficiently displayed in a multivalent format (Figure 58.4). These glyco-CNTs are alternative radiotracers for in vivo imaging or radiation-delivery systems with high radioisotope-loading capacity and high sensitivity. The rapid uptake of iodide by the mammalian thyroid served as a test of any potential leakage of radioactive iodide “cargo.” Although “free” iodide 125I rapidly entered the thyroid, iodide encapsulated in the glyco-CNT remained at its target site even after a month.
In vivo localization of filled-and-functionalized glyco-single-walled nanotubules (SWNTs). (TH) thyroid, (LU) lungs, (ST) stomach, (LI) liver, (KI) kidney, (BL) bladder. (Adapted from Hong SY, et al. 2010. Nat Mater 9: 485−490.)
The chemical flexibility of graphene-based materials allowed for the formation of dynamic supramolecular constructs. A carbohydrate-functionalized two-dimensional (2D) surface was prepared by decorating thermally reduced graphene sheets with multivalent sugar ligands. Host–guest inclusion provided a versatile strategy to present biofunctional ligands on the carbon surface. The multivalent sugar-functionalized graphene sheet agglutinates bacteria and inhibits their motility. Taking advantage of the supramolecular design, the captured bacteria can be partially released by adding a competitive guest. The unique thermal IR-absorption properties of graphene allow for the killing of the captured bacteria by IR-laser irradiation of the captured graphene–sugar–E. coli complex.
Dendrimers are nanosized branched compounds that can be decorated with ligands, permitting control over their number and orientation. Several scaffolds based on organic molecules, metal complexes, and supramolecular assemblies have been exploited for the formation of multivalent glycodendrimers to study lectin-binding properties. “Click” chemistry and amide bond formation have been used to tether sugar molecules to the dendrimer scaffold. Mannose-conjugated glycodendrimers bind specifically to ConA. Galactose functionalized dendrimeric arms have been conjugated to β-cyclodextrin (βCD). The βCD can incorporate either a drug, such as doxorubicin, or a fluorescent dye, to monitor the uptake of the dendrimeric structure. Specific delivery of doxorubicin to hepatocytes was achieved with the help of targeting galactose units.
Metal complexes like Ru(bpy)3 were also employed as scaffold for the assembly of three-dimensional supramolecular glycodendrimers. βCDs decorated with seven mannose functional groups were bound, via hydrophobic interactions, to the ruthenium core appended with adamantyl groups. The resulting supramolecular assemblies bind E. coli that express mannose receptors in the bacterial pili.
Supramolecular glycodendrimers have been prepared to display carbohydrates in a defined manner, offering controlled multivalency. Supramolecular particles, vesicles, or fibers have been created, offering a dynamic representation of a biological system. Synthetic molecules that mimic glycolipids self-assembled into nanovesicles displaying oligomannosides on their outer surface, resembling the glycocalyx coating of eukaryotic cells, bacteria, and viruses. The supramolecular approach permitted the formation of raft-like nanomorphologies on the synthetic vesicle that could shine light on important binding mechanisms.
Glycodendrimers can also be combined with particles to create multiple levels of multivalency. In one example, glycodendrimers displayed on protein-derived particles (virus-like particles) allowed for picomolar inhibition of Ebola virus–related adhesion events.
Natural polysaccharides are very useful for the preparation of nanosized carriers. The low toxicity, biocompatibility, stability, low cost, hydrophilic nature, and availability of reactive sites for chemical modification render polysaccharides attractive building blocks for pharmaceutical applications. Polysaccharides can be used as NP backbone or coating. Polysaccharide-based nanoparticles are prepared by covalent or ionic cross-linking, polyelectrolyte complexation, or self-assembly of hydrophobically modified polysaccharides.
Chitosan-based NPs are common drug delivery systems. Positively charged chitosan gives rise to ionic cross-linked particles with polyanions to deliver proteins, oligonucleotides, and plasmid DNA. Multifunctional chitosan NPs, incorporating a NIR fluorophore for fluorescence imaging, can encapsulate anticancer drugs or complex small interfering RNA (siRNA) for sequential drug delivery. Hyaluronic acid and heparin-based NPs are promising platforms in cancer therapy; unlike chitosan NPs, they display inherent targeting properties. Hyaluronic acid binds to CD44, a transmembrane glycoprotein that is overexpressed in many types of cancer. This targeting property has promoted the application of hyaluronic acid-based NPs as theranostic agents.
Polysaccharides have also been used to coat polymeric or metallic nanoparticles, improving their water solubility, stability, and long-term circulation. Chitosan-PEG-coated iron oxide NPs improve intracellular delivery of a DNA repair inhibitor (O6-benzylguanine) to glioblastoma multiform cells and enable treatment monitoring by MRI. Dextran was also employed to increase the water solubility and stability of iron oxide MNPs. Sulfated dextran electrostatically interacts with positively charged polycations. Functionalization of dextran-coated iron oxide NPs with sLex tetrasaccharide helped to monitor inflammation events in mouse brains in vivo. Hyaluronic acid–coated superparamagnetic iron oxide NPs have been used for imaging and for drug delivery to cancer cells.
Nanocarriers based on heparin and heparin derivatives have been applied to combat cancer via targeted, magnetic, photodynamic, and gene therapy. Gold and magnetic NPs have been coated or modified with heparin to improve their biocompatibility for applications in heparin-mediated events. Polysaccharide-functionalized gold NPs have given rise to multifunctional NPs with a wide range of applications including imaging, photodynamic therapy, and induction of apoptosis of metastatic cells.
Because of their natural abundance, nanomaterials based on cellulose have become extremely popular, offering biocompatibility, easy surface functionalization, and outstanding mechanical properties. Cellulose nanocrystals (CNCs) are rod-like particles obtained on acid hydrolysis of cellulose fibers. Because of their stiffness, twist, and aspect ratio, CNCs can assemble into chiral nematic phases, resulting in iridescent colors. Applications include functional paper, optoelectronics, engineered tissues, drug delivery, and biosensors.
Glycans are suitable biomarkers for medical diagnostics. Glyconanotechnology aids the development of biosensors and methods for the detection of glycans, lectins, or cancer cells and pathogens. Nanoengineered glycan sensors may help glycoprotein profiling, avoiding labeling or glycan liberation steps. A variety of nanomaterials are being explored as specific probes for label-free lectin or glycan detection. AuNPs and CNTs are the most widely used nanomaterials. Nanoengineered materials can be detected by mass changes with quartz crystal microbalance (QCM) and cantilever sensors, by field-effect transistor (FET) sensors based on carbon nanotubes, or optical sensors based on surface plasmon resonance (SPR) in combination with self-assembled monolayers (SAMs) of glycans or lectins.
Cancer cells and pathogens can be detected using glyconanomaterials. Glycan-functionalized nanodiamonds functioned as cross-linkers for bacteria and permitted water decontamination by filtration of the resulting aggregates through a 10-mm membrane. Mannan-coated gold glyconanoparticles incubated with a human gastric cell line in the presence of the mannose-binding lectin ConA detect and quantify cell-surface mannose glycans and silver signal amplification. The specific detection of cancer cells requires further refinement of the system, such as the substitution of ConA by a panel of lectins that recognize aberrant glycosylation specific to cancer.
The translation of biomolecular binding events into nanomechanics using a nanosized cantilever array of mannosides was applied to detect and discriminate different strains of E. coli. Methods exploiting intrinsic optical properties can be explored for biomedical “nonlabeled” imaging applications. QDs and AuNPs allow for spectral tuning, whereas SWCNTs show characteristic Raman peaks as well as photoluminescence in the NIR range. These intrinsic properties have yet to be systematically explored in vivo, but hold significant potential.
Glyconanomaterials based on different glycan-coated scaffolds have been created. Given the specific roles of endogenous glycoconjugates, nanomaterials that display glycans on similar length scales provide chemical platforms to advance our understanding of carbohydrate-mediated biological events.
Tailored glycomaterials that resemble more closely natural systems are under development to uncover hidden biological mechanisms. The challenge is now to create functional structures based on the appropriate scaffold and combine it with the proper glycan. Systems with increased control on the glycan presentation (i.e., spacing and conformation) is the next goal. At the same time, more complex glycans need to be incorporated into glyconanomaterials. Given the many functions carbohydrates in Nature, the glyconanotechnology field should evolve from simple binding studies involving the prototypical mannose-concanavalin A interaction to the exploration of new and more relevant avenues.
By taking advantage of the physical properties and inducible sizes, translation of fundamental studies to biomedical glyconanotechnology appears to be imminent. Promising examples include the use of glycomaterials for pathogen detection and as diagnostic tools. Biomedical imaging, using multimodal imaging techniques, such as PET/MRI or fluorescence/MRI, is an attractive vision for using glyco-NPs. Still, much effort has to be put to improve glycan stability in biological samples. Glycomimetics offer an interesting approach to avoid enzymatic degradation.
Polysaccharide-based materials are a green alternative to petroleum-based chemicals. A better molecular level understanding of these materials will fuel their application in biotechnology, as tissue scaffolds or nanoformulation for drug delivery. Polysaccharides are major component of the extracellular matrix, playing important roles in cellular communication and evolution. Being able to reproduce these complex natural environments will be game changing for tissue engineering.
The authors acknowledge contributions to previous versions of this chapter by Soledad Penades and appreciate helpful comments and suggestions from Sriram Neelamegham.
The content of this book is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view the terms and conditions of this license, visit https://creativecommons.org/licenses/by-nc-nd/4.0/
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