In situ SERS imaging of protein-specific glycan oxidation on living cells to quantitatively visualize pathogen-cell interactions.

Glycan oxidation on the cell surface occurs in many specific life processes including pathogen-cell interactions. This work develops a surface-enhanced Raman scattering (SERS) imaging strategy for in situ quantitative monitoring of protein-specific glycan oxidation mediated pathogen-cell interactions by utilizing Raman reporter DTNB and aptamer co-assembled platinum shelled gold nanoparticles (Au@Pt-DTNB/Apt). Using Fusarium graminearum (FG) and MCF-7 cells as models, Au@Pt-DTNB/Apt can specifically bind to MUC1 protein on the cell surface containing heavy galactose (Gal) and N-acetylgalactosamine (GalNAc) modification. When FG interacts with cells, the secreted galactose oxidase (GO) can oxidize Gal/GalNAc, and the generated reactive oxygen species (ROS) further oxidizes DTNB to produce TNB for greatly enhancing the SERS signal. This strategy can quantitatively visualize for the first time both the protein-specific glycan oxidation and the mediated pathogen-cell interactions, thus providing key quantitative information to distinguish and explore the pathogen-resistance and pharmacological mechanisms of different drugs.

In Situ Visualization of RNA-Specific Sialylation on Living Cell Membranes to Explore N-Glycosylation Sites.

The small RNAs on living cell membranes were recently found to be N-glycosylated and terminated with sialic acids, although the glycosylation sites and potential functions remain unclear. Herein, we designed a second-generation hierarchical coding strategy (HieCo 2) for in situ visualization of cell surface RNA-specific sialylation. After covalently binding DNA codes to sialic acids and then binding a DNA code to a target RNA via sequence specificity, cascade decoding processes were performed with subsequent signal amplification that enabled sensitive in situ visualization of low-abundance Y5 RNA-specific sialic acids on living cell membranes. The proposed strategy unveils the number of glycosylation sites on a single RNA and reveals the binding preference of glycosylated RNAs to different sialic acid binding-immunoglobulin lectin-type receptors, demonstrating a new route for exploration of the glycosylated RNA-related biological and pathological processes.

Triply Enhanced Immunotherapy via Dual Glycan Reforming Integrated with Perforation.

The enhancement of immunotherapy is an emerging direction to develop highly effective and practical cancer therapeutic methods. Here a triply enhanced immunotherapy drug (TEID) is designed for ingeniously integrating in situ dual glycan reforming with perforation on cell membrane. The TEID is composed of galactose and neuraminidase conjugated streptolysin O (SLO-Gal and SLO-NEU), which are encapsulated in a hyaluronic acid (HA) shell for targeted recognition to tumor tissue via cell surface CD44. After targeted delivery and HAase-mediated degradation in the tumor region, the TEID releases SLO-Gal and SLO-NEU, which can easily anchor Gal and NEU on the tumor cell membrane via the perforation of SLO to perform dual glycan reforming for the introduction of Gal and the cleavage of sialic acid. The former can activate immune cells to secret cytokines for immune-killing, and the latter can weaken the immune inhibition to improve the immunotherapeutic efficacy. Meanwhile, the perforation of SLO can promote the delivery of cytokines into the tumor cells to further enhance the efficacy. The designed triply enhanced immunotherapy strategy opens a significant and promising route to promote clinical immunotherapy of cancer.

Surface-enhanced Raman scattering sensing for detection and mapping of key cellular biomarkers.

Cellular biomarkers mainly contain proteins, nucleic acids, glycans and many small molecules including small biomolecule metabolites, reactive oxygen species and other cellular chemical entities. The detection and mapping of the key cellular biomarkers can effectively help us to understand important cellular mechanisms associated with physiological and pathological processes, which greatly promote the development of clinical diagnosis and disease treatment. Surface-enhanced Raman scattering (SERS) possesses high sensitivity and is free from the influence of strong self-fluorescence in living systems as well as the photobleaching of the dyes. It exhibits rich and narrow chemical fingerprint spectra for multiplexed detection, and has become a powerful tool to detect and map cellular biomarkers. In this review, we present an overview of recent advances in the detection and mapping of different classes of cellular biomarkers based on SERS sensing. These advances fully confirm that the SERS-based sensors and sensing methods have great potential for the exploration of biological mechanisms and clinical applications. Additionally, we also discuss the limitations of present research and the future developments of the SERS technology in this field.

A Versatile G-Quadruplex (G4)-Coated Upconverted Metal-Organic Framework for Hypoxic Tumor Therapy.

Given the complexity of the tumor microenvironment, multiple strategies are being explored to tackle hypoxic tumors. The most efficient strategies combine several therapeutic modalities and typically requires the development of multifunctional nanocomposites through sophisticated synthetic procedures. Herein, the G-quadruplex (G4)-forming sequence AS1411-A (d[(G2 T)4 TG(TG2 )4 A]) is used for both its anti-tumor and biocatalytic properties when combined with hemin, increasing the production of O2 ca. two-fold as compared to the parent AS1411 sequence. The AS1411-A/hemin complex (GH) is grafted on the surface and pores of a core-shell upconverted metal-organic framework (UMOF) to generate a UMGH nanoplatform. Compared with UMOF, UMGH exhibits enhanced colloidal stability, increased tumor cell targeting and improved O2 production (8.5-fold) in situ. When irradiated by near-infrared (NIR) light, the UMGH antitumor properties are bolstered by photodynamic therapy (PDT), thanks to its ability to convert O2 into singlet oxygen (1 O2 ). Combined with the antiproliferative activity of AS1411-A, this novel approach lays the foundation for a new type of G4-based nanomedicine.

In situ evaluation of in vivo sialylation with a dual-color imaging strategy.

This work designs a functional dendrimer probe to conveniently identify newly generated sialic acid groups in vivo with a dual-color imaging strategy, which achieves in situ semiquantitative evaluation of the sialylation difference between tumor and normal tissues to reveal sialylation-related biological events and promote clinical tumor diagnosis.

Tumor identification via in vivo portable Raman detection of sialic acid with a dual gold nanoprobe system.

A dual gold nanoprobe system was designed for in vivo portable Raman detection of sialic acid (SA) for tumor identification. The dual gold nanoprobe system contained two gold nanoprobes, Au10-DTTC/PEG-PBA and Au40-PEG-SA. Au10-DTTC/PEG-PBA was constructed on a 10 nm gold nanoparticle modified with 3,3'-diethylthia tricarbocyanine iodide (DTTCI) as the Raman reporter and 3-aminophenylboronic acid (APBA) through a thiol PEG succinimidyl carboxymethyl ester (HS-PEG-NHS) linker for specific recognition of SA. Au40-PEG-SA was constructed on a 40 nm gold nanoparticle modified with SA through HS-PEG-NHS. For in vivo detection of SA, Au10-DTTC/PEG-PBA and Au40-PEG-SA were subsequently injected into tumor xenografted mice with optimal interval and retention times. Through the specific recognition between PBA and SA, the conjugates of Au10-DTTC/PEG-PBA and Au40-PEG-SA formed in the tumor region emitted strong SERS signals of DTTC, which could be detected by a portable Raman detector. This work provides a convenient and portable method to detect SA in tumor xenografted mice, which is useful for family-stay identification and clinical cleavage of tumors.

O-GlcNAcylation mapping of single living cells by in situ quantitative SERS imaging.

O-GlcNAcylation is involved in many biological processes including cancerization. Nevertheless, its in situ quantification in single living cells is still a bottleneck. Here we develop a quantitative SERS imaging strategy for mapping the O-GlcNAcylation distribution of single living cells. O-GlcNAcylated compounds (OGCs) can be quantified through their in situ azide labeling and then a click reaction competing with azide and Raman reporter labeled 15 nm-gold nanoparticles (AuNPs) for linking to dibenzocyclooctyne labeled 40 nm-AuNPs to produce OGC-negatively correlated SERS signals. The calibration curve obtained in vitro can be conveniently used for detecting OGCs in different areas of single living cells due to the negligible effect of cell medium on the click linkage and Raman signal. This method has been successfully applied in mapping O-GlcNAcylation distribution in different cell lines and monitoring O-GlcNAcylation variation during cell cycling, which demonstrate its great practicability and expansibility in glycosylation related analysis.

Gold Nanostar@Polyaniline Theranostic Agent with High Photothermal Conversion Efficiency for Photoacoustic Imaging-Guided Anticancer Phototherapy at a Low Dosage.

Due to the strong and tunable photothermal effect, metallic nanoparticles are of enormous interest in light-activated biomedical applications, such as photoacoustic imaging (PAI) and photothermal therapy (PTT). However, the photothermal conversion efficiency (PCE) of existing metallic photothermal agents is still unsatisfactory. Herein, we develop an efficient photothermal theranostic agent based on a gold nanostar@polyaniline core-shell nanocomposite with high PCE for PAI-guided PTT at a low dosage. After optimizing the relative composition of polyaniline (PANI) and gold nanostars (AuNSs), this nanocomposite eventually empowers an outstanding PCE of up to 78.6%, which is much better than AuNSs or PANI alone and most of the existing photothermal theranostic agents. Besides, the nanocomposite can act as a targeted probe for tumors by hyaluronic acid (HA) modification without compromising the photothermal performance. The obtained nanoprobes named AuNSPHs exhibit promising biocompatibility and great performance of PAI-guided PTT to treat triple-negative breast cancer both in vitro and in vivo. More importantly, a single injection of AuNSPHs significantly suppresses tumor growth with a low dosage of Au (0.095 mg/kg), which is attributed to the high PCE of AuNSPHs. Taking advantage of the exhilarating photothermal conversion ability, this theranostic agent can safely potentiate the antitumor therapeutic efficacy of laser-induced ablation and holds great potential for future medical applications.

Tumor suppression via diverting intracellular sialylation with multifunctional nanoparticles.

Sialylation plays an important role in tumor-related physiological processes. Therefore, intervention of sialylation has great potential to explore new paths for tumor therapy. In view of the immune modulation of sialic acid (SA) on tumors, this work designs a multifunctional mesoporous silica nanoparticle (MFMSN) to divert intracellular sialylation for tumor suppression. The galactose groups covered on MFMSN act as sialylation substrates to bind intracellular SAs competitively, which inhibits the SA expression on the tumor cell surface. The diverted intracellular sialylation can be visualized on living cells and in vivo by specifically binding the sialylated galactose with a phenylboronic acid labeled ssDNA probe released from the pore of MFMSN to induce DNA strand displacement, which recovers the fluorescence of the dsDNA probe covered on MFMSN surface. The diverting of sialylation efficiently suppresses tumor growth in mice, demonstrating the great potential of the designed strategy for revealing SA-related biological processes and clinical cancer therapy.

Tug-of-war: molecular dynamometers against living cells for analyzing sub-piconewton interaction of a specific protein with the cell membrane.

Protein-membrane interactions play important roles in signal transductions and functional regulation of membrane proteins. Here, we design a molecular dynamometer (MDM) for analyzing protein-membrane interaction on living cells. The MDM is constructed by assembling an artificial lipid bilayer and alkylated Cy3-DNA azide (azide-Cy3-Cx) on a silica bubble. After a functional aptamer is covalently anchored onto the corresponding target protein on a living cell through UV irradiation, azide-Cy3-Cx is conjugated with the aptamer through a click reaction to produce a "tug-of-war" between the MDM and the cell due to the buoyancy of the silica bubble. This induces the detachment of the protein from the cell membrane or the alkane terminal from the MDM enabling sub-piconewton embedding force measurement by changing the alkane chain length and simple fluorescence analysis. The successful analysis of embedding force variation of a protein on the cell membrane upon post-translational modifications demonstrates the practicability and expansibility of this method for mechanics-related research in biological systems.

A pore-forming protein-induced surface-enhanced Raman spectroscopic strategy for dynamic tracing of cell membrane repair.

The plasma membrane repair holds significance for maintaining cell survival and homeostasis. To achieve the sensitive visualization of membrane repair process for revealing its mechanism, this work designs a perforation-induced surface-enhanced Raman spectroscopy (SERS) strategy by conjugating Raman reporter (4-mercaptobenzoic acid) loaded gold nanostars with pore-forming protein streptolysin O (SLO) to induce the SERS signal on living cells. The SERS signal obviously decreases with the initiation of membrane repair and the degradation of SLO pores due to the departure of gold-nanostar-conjugated SLO. Thus, the designed strategy can dynamically visualize the complete cell membrane repair and provide a sensitive method to demonstrate the SLO endocytosis- and exocytosis-mediated repairing mechanism. Using DOX-resistant MCF-7 cells as a model, a timely repair-blocking technology for promoting the highly efficient treatment of drug-resistant cancer cells is also proposed. This work opens an avenue for probing the plasma membrane repairing mechanisms and designing the precision therapeutic schedule.

A Filter Supported Surface-Enhanced Raman Scattering "Nose" for Point-of-Care Monitoring of Gaseous Metabolites of Bacteria.

This work designs a convenient method for fabrication of surface-enhanced Raman scattering (SERS) devices by loading gold nanostars (AuNSs) on a flat filter support with vacuum filtration. The dense accumulation of AuNSs results in a strong sensitization to SERS signal and shows sensitive response to gaseous metabolites of bacteria, which produces a SERS "nose" for rapid point-of-care monitoring of these metabolites. The "nose" shows good reproducibility and stability and can be used for SERS quantitation of a gaseous target with Raman signal. The impressive performance of the proposed SERS "nose" for detecting gaseous metabolites of common foodborne bacteria like Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa from inoculated samples demonstrates its much higher sensitivity than that of human sense and application in distinguishing spoiled food at an early stage and real-time tracing of food spoilage degree. The strong point-of-care testing ability of the designed SERS "nose" and the miniaturization of whole equipment extend greatly the analytical application of SERS technology in food safety and public health.