Senlian Hong

Cell-Selective Metabolic Glycan Labeling Based on Ligand-Targeted Liposomes

A cell-specific metabolic glycan labeling strategy has been developed using azidosugars encapsulated in ligand-targeted liposomes. The ligands are designed to bind specific cell-surface receptors that are only expressed or up-regulated in target cells, which mediates the intracellular delivery of azidosugars. The delivered azidosugars are metabolically incorporated into cell-surface glycans, which are then imaged via a bioorthogonal reaction.

Live-Cell Stimulated Raman Scattering Imaging of Alkyne-Tagged Biomolecules**

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging.

A bioorthogonal Raman reporter strategy for SERS detection of glycans on live cells.

National Instrumentation Program [2011YQ030124]; National Basic Research Program of China (973 Program) [2012CB917303]; National Natural Science Foundation of China [21172013]

Bifunctional Unnatural Sialic Acids for Dual Metabolic Labeling of Cell-Surface Sialylated Glycans

Sialic acid analogues containing a unique chemical functionality or chemical reporter have been metabolically incorporated into sialylated glycans. This process, termed metabolic glycan labeling, has emerged as a powerful tool for studying sialylation as well as other types of glycosylation. Currently, this technique can install only a single functionality. Here we describe a strategy for dual labeling of sialylated glycans using a new class of bifunctional sialic acid analogues containing two distinct chemical reporters at the N-acyl and C9 positions. These bifunctional unnatural sialic acids were metabolically incorporated into cellular glycans, where the two chemical reporters exerted their distinct functions. This approach expands the capability of metabolic glycan labeling to probe sialylation and glycan-protein interactions.

Targeted Imaging and Proteomic Analysis of Tumor-Associated Glycans in Living Animals†

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor-associated glycans by using ligand-targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer-cell-specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue-specific manner. Subsequently, a copper-free click reaction was performed in vivo to chemoselectively conjugate the azides with a near-infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology.

Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities

Metabolic labeling of biomolecules with bioorthogonal functionalities enables visualization, enrichment, and analysis of the biomolecules of interest in their physiological environments. This versatile strategy has found utility in probing various classes of biomolecules in a broad range of biological processes. On the other hand, metabolic labeling is nonselective with respect to cell type, which imposes limitations for studies performed in complex biological systems. Herein, we review the recent methodological developments aiming to endow metabolic labeling strategies with cell-type selectivity. The cell-selective metabolic labeling strategies have emerged from protein and glycan labeling. We envision that these strategies can be readily extended to labeling of other classes of biomolecules.

SERS Imaging of Cell‐Surface Biomolecules Metabolically Labeled with Bioorthogonal Raman Reporters

Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging.

Live-cell bioorthogonal Raman imaging

Live-cell microscopy demands high specificity, sensitivity, and minimal perturbation to the biomolecules of interest. Meeting all these criteria has been challenging in cellular imaging. Toward this goal, a bioorthogonal Raman imaging method has recently emerged by exploiting small Raman reporters that possess Raman signals that do not overlap with the naturally existing biomolecules in the cells. The Raman reporters are metabolically incorporated into the target biomolecules for direct visualization. Herein, we review recent advances in the methodological development and the proof-of-concept applications of the live-cell bioorthogonal Raman imaging technique.

Mechanistic Investigation and Multiplexing of Liposome-Assisted Metabolic Glycan Labeling

Metabolic labeling of glycans with bioorthogonal reporters has been widely used for glycan imaging and glycoproteomic profiling. One of the intrinsic limitations of metabolic glycan labeling is the lack of cell-type selectivity. The recently developed liposome-assisted bioorthogonal reporter (LABOR) strategy provides a promising means to overcome this limitation, but the mechanism of LABOR has not been investigated in detail. In this work, we performed a mechanistic study on LABOR and explored its multiplexing capability. Our studies support an endocytosis-salvage mechanism. The ligand-targeted liposomes encapsulating azidosugars are internalized into the endosome via the receptor-mediated endocytosis. Unlike the conventional drug delivery, LABOR does not rely on the endosomal escape pathways. Rather, the liposomes are allowed to enter the lysosome, inside which the azidosugars are released from the liposomes. The released azidosugars then intercept the salvage pathways of monosaccharides and get transported into the cytosol by lysosomal sugar transporters. Based on this mechanism, we expanded the scope of LABOR by evaluating a series of ligand-receptor pairs for targeting sialoglycans in various cell types. Different ligand types including small molecules, antibodies, aptamers, and peptides could be easily implemented into LABOR. Finally, we demonstrated that the sialoglycans in two distinct cell populations in a co-cultured system could be selectively labeled with two distinct chemical reporters by performing a multiplexed LABOR labeling.