Xue-Long Sun

Recent Developments in Carbohydrate-Decorated Targeted Drug/Gene Delivery

Targeted delivery of a drug or gene to its site of action has clear therapeutic advantages by maximizing its therapeutic efficiency and minimizing its systemic toxicity. Generally, targeted drug or gene delivery is performed by loading a macromolecular carrier with an appropriate drug or gene, and by targeting the drug/gene carrier to specific cell or tissue with the help of specific targeting ligand. The emergence of glycobiology, glycotechnology, and glycomics and their continual adaptation by pharmaceutical scientists have opened exciting avenue of medicinal applications of carbohydrates. Among them, the biocompatibility and specific receptor recognition ability confer the ability of carbohydrates as potential targeting ligands for targeted drug and gene delivery applications. This review summarizes recent progress of carbohydrate‐decorated targeted drug/gene delivery applications.  © 2009 Wiley Periodicals, Inc. Med Res Rev, 30, No. 2, 270–289, 2010

Site-specific multivalent carbohydrate labeling of quantum dots and magnetic beads.

Cell-surface carbohydrates act as receptors for a variety of protein ligands and thereby play a significant role in a wide range of biological processes, including immune-recognition events and the interaction of viruses and bacteria with host cells as well as tissue growth and repair. As such, binding interactions of carbohydrates and proteins provide a starting point for the development of novel diagnostic agents and a framework for new therapies. It is notable that the low affinity and specificity that are typical of monomeric carbohydrate–protein interactions are dramatically enhanced when the carbohydrate component is presented as a multivalent ligand; a phenomenon referred to as the “cluster-glycoside effect”. In response to this observation, considerable effort has focused on the design of unique, multivalent carbohydrate ligands in the form of linear polymers, liposomes, dendrimers, beads, or nanoparticles. In this regard, we have recently described a useful route for the synthesis of glycopolymers by a cyanoxyl-mediated free-radical polymerization scheme that can be performed under aqueous condition and is tolerant of a wide range of monomer functionalities, including OH, COOH, NH2, and OSO3H groups. Conveniently, this synthetic approach facilitates selective derivatization of the polymer-chain terminus. Herein, we report site-specific multivalent carbohydrate labeling of nanocrystal (quantumdot) and magnetic-bead surfaces using a biotin chain-endfunctionalized glycopolymer and demonstrate the potential value of these multivalent carbohydrate polymers in both imaging and biocapture applications (Figure 1). Semiconductor nanocrystals are a new class of size-tunable optical probe. Recently, nanocrystal surfaces have been functionalized with DNA, peptides, proteins, and other small ligands with intended applications as biological reagents and probes. Nanocrystal–streptavidin conjugates, for example, have been used to stain tissues, cells, and intracellular organelles. Likewise, nanocrystal–avidin–antibody conjugates have improved the sensitivity of conventional fluoroimmunoassays. To the best of our knowledge, carbohydrateconjugated nanocrystals have yet to be explored in bioimaging applications although a few nanocrystal–carbohydrate conjugates have been reported (see also note added in proof). In the present study, nanocrystal–multivalent carbohydrate conjugates were produced by incubating nanocrystal–streptavidin (50 mL, 120 mgmL 1 streptavidin in phosphate buffered saline (PBS), Qdot 565 streptavidin conjugate, Quantum Dot Corp. , Hayward, CA) with biotin end-terminated glycopolymer 1 (50 mL, 1 mgmL 1 in PBS) bearing ten pendant lactose groups for one hour at room temperature. RCA120 is a lectin that binds to terminal b-d-galactose. As a model system, RCA120-immobilized agarose beads (100 mL, 2 mgmL , Sigma) were incubated with nanocrystal–carbohydrate conjugates in PBS (100 mL) for 1 h at room temperature and subsequently washed three times with PBS. Confocal microscopy confirmed fluorescent staining of the lectin-modified bead surfaces (Figure 2A). Of particular interest was that staining intensity was dramatically enhanced by the initial exposure of RCA120 beads to biotin end-terminated glycopolymer 1 followed by incubation of the mixture with streptavidin–nanocrystal conjugates (Figure 2B). The weak-intensity staining observed when using the first approach might have been due to the presence of free glycopolymer along with the nanocrystal–carbohydrate conjugates. As a two-step procedure, the sensitivity of staining was increased through the formation in situ of nanocrystal– carbohydrate complexes on the bead surface without the need to purify the conjugate. The absence of staining on treatment with streptavidin nanocrystals alone or with the use of nonbiotinylated glycopolymer 2 confirms the necessity and specificity of both carbohydrate–lectin and streptavidin–biotin interactions (Figure 2C). Biotin end-terminated glycopolymers were also used to extend the versatility of magnetic-bead-based biocapture assays that have been employed for the rapid isolation of a variety of lectin-bearing cells and biomolecules. Indeed, Rye and Bovin have demonstrated that glycopolymer-derivatized magnetic beads provide a useful tool for the selection of cells expressing a specific carbohydrate-binding phenotype. Bundy and Fenselau have also reported that glycopolymerbased affinity capture surfaces are more sensitive than lectinbased systems for microbial capture. While in both reports glycopolymers were effectively attached to the bead and [a] Dr. X.-L. Sun, Dr. W. Cui, Dr. C. Haller Departments of Surgery and Biomedical Engineering Emory University School of Medicine Atlanta, GA 30322 (USA) E-mail : xsun@emory.edu [b] Dr. E. L. Chaikof Department of Surgery and Biomedical Engineering Emory University School of Medicine Atlanta, GA 30322 (USA) Fax: (+1)404-727-3660 E-mail : echaiko@emory.edu Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Carbohydrate–protein interactions and their biosensing applications

AbstractCarbohydrate recognition is clearly present throughout nature, playing a major role in the initial attachment of one biological entity to another. The important question is whether these prevalent interactions could provide a real suitable alternative to the use of antibodies or nucleic acid for detection and identification. Currently, examples of carbohydrates being employed in biological detection systems are limited. The challenges of using carbohydrate recognition for detection mainly come from the weak affinity of carbohydrate–protein interactions, the lack of versatile carbohydrate scaffolds with well-defined structures, and the less developed high-information-content, real-time, and label-free assay technology. In this review, we focus on discussing the characteristics of carbohydrate–protein interactions in nature and the methods for carbohydrate immobilization based on surface coupling chemistry in terms of their general applicability for developing carbohydrate- and lectin-based label-free sensors. Furthermore, examples of innovative design of multivalent carbohydrate–protein interactions for sensor applications are given. We limit our review to show the feasibility of carbohydrate and lectin as recognition elements for label-free sensor development in several representative cases to formulate a flexible platform for their use as recognition elements for real-world biosensor applications. FigureMultivalent protein–carbohydrate interactions at the cell surface (left) and development of a biosensor using carbohydrates (right)

Chemically-Selective Surface Glyco-Functionalization of Liposomes Through Staudinger Ligation

An efficient and chemoselective liposome surface glyco-functionalization method has been developed based on Staudinger ligation, in which the carbohydrate derivative carrying an azide spacer is conjugated onto the surface of preformed liposomes bearing a terminal triphosphine in PBS buffer (pH 7.4) at room temperature.

Membrane mimetic surface functionalization of nanoparticles: Methods and applications

Nanoparticles (NPs), due to their size-dependent physical and chemical properties, have shown remarkable potential for a wide range of applications over the past decades. Particularly, the biological compatibilities and functions of NPs have been extensively studied for expanding their potential in areas of biomedical application such as bioimaging, biosensing, and drug delivery. In doing so, surface functionalization of NPs by introducing synthetic ligands and/or natural biomolecules has become a critical component in regard to the overall performance of the NP system for its intended use. Among known examples of surface functionalization, the construction of an artificial cell membrane structure, based on phospholipids, has proven effective in enhancing biocompatibility and has become a viable alternative to more traditional modifications, such as direct polymer conjugation. Furthermore, certain bioactive molecules can be immobilized onto the surface of phospholipid platforms to generate displays more reminiscent of cellular surface components. Thus, NPs with membrane-mimetic displays have found use in a range of bioimaging, biosensing, and drug delivery applications. This review herein describes recent advances in the preparations and characterization of integrated functional NPs covered by artificial cell membrane structures and their use in various biomedical applications.

Recent advances in sialic acid-focused glycomics.

Recent emergences of glycobiology, glycotechnology and glycomics have been clarifying enormous roles of carbohydrates in biological recognition systems. For example, cell surface carbohydrates existing as glycoconjugates (glycolipids, glycoproteins and proteoglycans) play crucial roles in cell-cell communication, cell proliferation and differentiation, tumor metastasis, inflammatory response or viral infection. In particular, sialic acids (SAs) existing as terminal residues in carbohydrate chains on cell surface are involved in signal recognition and adhesion to ligands, antibodies, enzymes and microbes. In addition, plasma free SAs and sialoglycans have shown great potential for disease biomarker discovery. Therefore, the development of efficient analytical methods for structural and functional studies of SAs and sialylglycans are very important and highly demanded. The problems of SAs and sialylglycans analysis are vanishingly small sample amount, complicated and unstable structures, and complex mixtures. Nevertheless, in the past decade, mass spectrometry in combination with chemical derivatization and modern separation methodologies has become a powerful and versatile technique for structural analysis of SAs and sialylglycans. This review summarizes these recent advances in glycomic studies on SAs and sialylglycans. Specially, derivatization and capturing of SAs and sialylglycans combined with mass spectrometry analysis are highlighted.

Glycosylation and processing of pro-B-type natriuretic peptide in cardiomyocytes.

B-type natriuretic peptide (BNP) and its related peptides are biomarkers for the diagnosis of heart failure. Recent studies identified several O-glycosylation sites, including Thr-71, on human pro-BNP but the functional significance was unclear. In this study, we analyzed glycosylation and proteolytic processing of pro-BNP in cardiomyocytes. Human pro-BNP wild-type (WT) and mutants were expressed in HEK 293 cells and murine HL-1 cardiomyocytes. Pro-BNP and BNP were analyzed by immunoprecipitation and Western blotting. Glycosidases and glycosylation inhibitors were used to examine carbohydrates on pro-BNP. The effects of furin and corin expression on pro-BNP processing in cells also were examined. We found that in HEK 293 cells, recombinant pro-BNP contained significant amounts of O-glycans with terminal oligosialic acids. Mutation at Thr-71 reduced O-glycans on pro-BNP and increased pro-BNP processing. In HL-1 cardiomyocytes, residue Thr-71 contained little O-glycans, and pro-BNP WT and T71A mutant were processed similarly. In HEK 293 cells, pro-BNP was processed by furin. Mutations at Arg-73 and Arg-76, but not Lys-79, prevented pro-BNP processing. In HL-1 cardiomyocytes, which express furin and corin, single or double mutations at Arg-73, Arg-76 and Lys-79 did not prevent pro-BNP processing. Only when all these three residues were mutated, was pro-BNP processing completely blocked. Our data indicate that pro-BNP glycosylation in cardiomyocytes differed significantly from that in HEK 293 cells. In HEK 293 cells, furin cleaved pro-BNP at Arg-76 whereas in cardiomyocytes corin cleaved pro-BNP at multiple residues including Arg-73, Arg-76 and Lys-79.

Immobilized Sialyloligo-Macroligand and Its Protein Binding Specificity

We report a chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications. Specifically, O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were synthesized by enzymatic α2,3- and α2,6-sialylation of a lactose-containing glycopolymer that was synthesized by cyanoxyl-mediated free radical polymerization. (1)H NMR showed almost quantitative α2,3- and α2,6-sialylation. The O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were printed onto amine-functionalized glass slides via isourea bond formation for glycoarray formation. Specific protein binding activity of the arrays was confirmed with α2,3- and α2,6-sialyl specific binding lectins together with inhibition assays. Further, immobilizing O-cyanate chain-end functionalized sialyllactose-containing glycopolymers onto amine-modified SPR chip via isourea bond formation afforded SPR-based glyco-biosensor, which showed specific binding activity for lectins and influenza viral hemagglutinins (HA). These sialyloligo-macroligand derived glycoarray and SPR-based glyco-biosensor are closely to mimic 3D nature presentation of sialyloligosaccharides and will provide important high-throughput tools for virus diagnosis and potential antiviral drug candidates screening applications.

Liposome surface functionalization based on different anchoring lipids via Staudinger ligation

Liposome surface functionalization facilitates numerous potential applications of liposomes, such as enhanced stability, bioactive liposome conjugates, and targeted drug, gene and image agent delivery. Anchoring lipids are needed for grafting ligands of interest and play important roles in ligand grafting density, liposome stability, and liposome chemical and physical characteristics as well. In this report, glyco-functionalized liposome systems based on two kinds of anchoring lipids, phosphatidylethanolamine (PE) and cholesterol (Chol), were prepared by post chemically selective functionalization via Staudinger ligation. The size and stability of the liposomes were confirmed by dynamic light scattering (DLS). Particularly, the impact of anchor lipids on the stability of glyco-functionalized liposomes was investigated by comparing two different anchor lipids, namely Chol-PEG2000-TP and DSPE-PEG2000-TP. In addition, the encapsulation and releasing capacity of the glycosylated liposome based on the two anchoring lipids were investigated by entrapping 5,6-carboxyfluorescein (CF) dye and monitoring the fluorescence leakage, respectively. Furthermore, the density and accessibility of grafted carbohydrate residues on the liposome surface were evaluated for the two anchoring lipid-derived liposomes with lectin binding, respectively.

End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation

We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4-6 (TM(456)) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM(456) derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM(456) was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM(456) was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM(456) was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and occurs rapidly in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the proteins biological activity.