The Dual-Response-Single-Amplification Fluorescent Nanomachine for Tumor Imaging and Gastric Cancer Diagnosis.

Gastric cancer (GC) is one of the most common tumors worldwide and is the leading cause of tumor-related mortality. Traditional biomarkers and screening methods cannot meet the clinical demands. There is an urgent need for highly sensitive diagnostic markers as well as accurate quantification methods for early gastric cancer (EGC) screening. Here a dual-target cooperatively responsive fluorescent nanomachine by the innovative application of two targets─responsive strand migration system with a single-amplification-cycle element was developed for the simultaneous detection of GC biomarkers miR-5585-5p and PLS3 mRNA, which were selected by next-generation sequencing and RT-qPCR. It was also an RNA extraction-free, PCR-free, and nonenzymatic biosensor to achieve tumor cell imaging and serum diagnosis. Requiring only a 20 µL serum sample and 20 min incubation time, the nanomachine achieved an ultrasensitive detection limit of fM level with a broad linear range from fM to nM. More importantly, a higher AUC value (0.884) compared to the clinically used biomarker CA 72-4 was obtained by the nanomachine to distinguish GC patients successfully. Notably, for the key concerns of diagnosis of EGC patients, the nanomachine also achieved a satisfactory AUC value of 0.859. Taken together, this work has screened and obtained multiple biomarkers and developed a fluorescent nanomachine for combination diagnosis of GC, providing an ingenious design of a functionalized DNA nanomachine and a feasible strategy for the transformation of serum biomarkers into clinical diagnosis.

Hydrogen selenide, a vital metabolite of sodium selenite, uncouples the sulfilimine bond and promotes the reversal of liver fibrosis.

Sodium selenite has alleviating effects on liver fibrosis; however, its therapeutic molecular mechanism remains unclear. Herein, hydrogen selenide, a major metabolite of Na2SeO3, was tested to uncouple the sulfilimine bond in collagen IV, the biomarker of liver fibrosis. A mouse model of liver fibrosis was constructed via a CCl4-induced method, followed by the administration of 0.2 mg kg-1 Na2SeO3 via gavage three times per week for 4 weeks. Changes in H2Se, NADPH, and H2O2 levels were monitored in real time by using NIR-H2Se, DCI-MQ-NADPH, and H2O2 probes in vivo, respectively. H2Se continuously accumulated in the liver throughout the Na2SeO3 treatment period, but the levels of NADPH and H2O2 decreased. The expression of collagen IV was analyzed through Western blot and liquid chromatography-mass spectrometry. Results confirmed that the sulfilimine bond of collagen IV in the fibrotic mouse livers could be broken by H2Se with the Na2SeO3 treatment. Therefore, the therapeutic effect of Na2SeO3 on liver fibrosis could be mainly attributed to H2Se that uncoupled the sulfilimine bond to induce collagen IV degradation. This study provided a reasonable explanation for the molecular mechanism of the in vivo function of Na2SeO3 and the prevention of liver fibrosis by administering inorganic selenium.

Fishing out Methionine-Containing Proteins from Complex Biological Systems Based on a Non-Enzymatic Biochemical Reaction.

Nowadays, it still remains a great challenge to develop a simple, fast, and low-toxic method for identification and separation of proteins from complex biological systems. Herein, a nanocomposite (Fe3O4@Au-Se-peptide) was designed and synthesized to fish out methionine-containing proteins based on a non-enzymatic biochemical reaction, which couples amino groups of lysine with the S-methyl group of methionine in the presence of HOBr. Peptides which contain four lysine residues (Lys-Lys-Lys-Lys-{Se-Cys}) linked to the Fe3O4@Au nanocomposites were used to capture methionine residues efficiently via a S═N cross-linking. The methionine-containing protein was obtained by magnetic separation and released from the Fe3O4@Au-Se-peptide nanocomposites with the influence of H2Se. The HRMS and SDS-PAGE results confirmed the methionine-containing protein could be successfully fished out from a mixture solution. This work provides a useful strategy for recognition and separation of a category of proteins from complex biological systems.

H2Se Induces Reductive Stress in HepG2 Cells and Activates Cell Autophagy by Regulating the Redox of HMGB1 Protein under Hypoxia.

Rationale: Selenium has been shown to have chemotherapeutic effects against cancer. However, the anti-cancer mechanism of selenium is not fully understood, and the role of hydrogen selenide (H2Se), which is a common metabolite of dietary selenium compounds, has not been elucidated due to the lack of detection methods. In this study, we revealed a new anti-cancer mechanism of selenite with the help of a H2Se fluorescent probe. Methods: HepG2 cells were cultured under a simulated tumor hypoxic microenvironment. The H2Se and H2O2 levels were detected by fluorescent probes in living cells and in mice. Autophagic and apoptotic proteins were detected by Western blotting. The redox of HMGB1 protein were analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results: After pharmacological doses of Na2SeO3 treatment of HepG2 cells under hypoxic conditions, high levels of H2Se were produced before cell death. The H2Se accumulation resulted in reductive stress instead of oxidative stress, which was induced by Na2SeO3 treatment under normoxic conditions. Furthermore, H2Se targeted the HMGB1 protein and induced cell autophagy. H2Se could interrupt the disulfide bond in HMGB1 and promote its secretion. The reduced HMGB1 outside the cells stimulated cell autophagy by inhibiting the Akt/mTOR axis. Here, autophagy played a dual role, i.e., mild autophagy inhibited apoptosis, while excessive autophagy led to autophagy-associated cell death. Conclusions: These results show that H2Se plays a key role during HepG2 cell death induced by selenite. Our findings reveal a new anti-cancer mechanism of selenite and provide a new research area for selenium studies.

Real-Time in Situ Visualizing of the Sequential Activation of Caspase Cascade Using a Multicolor Gold-Selenium Bonding Fluorescent Nanoprobe.

The caspase cascade is an ensemble of very important signaling molecules that plays a critical role in cell apoptosis. Real-time monitoring of the upstream and downstream activation relationships of the caspases in the signal pathway is of great significance for understanding the regulatory mechanisms of these signaling molecules in the development of various diseases. Herein, a multicolor fluorescent nanoprobe, GNP-Se-Casp, has been developed based on Au-Se bonding for real-time in situ monitoring caspase- (casp-) 3, 8, and 9 during cell apoptosis. In the real-time fluorescence imaging of apoptotic HeLa cells induced by staurosporine using GNP-Se-Casp, the fluorescence signals corresponding to casp-8 and casp-9 sequentially turn on, followed by the appearance of the fluorescence of casp-3, which visualizes the upstream and downstream relationships of casp-3, -8, and -9. Thus, GNP-Se-Casp is an effective tool for real-time in situ monitoring of caspase cascade activation in the apoptosis process of tumor cells. This design strategy is easily adaptable to in situ detection of other signal molecules, especially those with upstream and downstream activation relationships.

Cyclic Regulation of the Sulfilimine Bond in Peptides and NC1 Hexamers via the HOBr/H2Se Conjugated System.

The sulfilimine bond (-S═N-), found in the collagen IV scaffold, significantly stabilizes the architecture via the formation of sulfilimine cross-links. However, precisely governing the formation and breakup process of the sulfilimine bond in living organisms for better life functions still remains a challenge. Hence, we established a new way to regulate the breaking and formation of the sulfilimine bond through hydrogen selenide (H2Se) and hypobromous acid (HOBr), which can be easily controlled at simulated physiological conditions. This novel strategy provides a circulation regulation system to modulate the sulfilimine bond in peptides and NC1 hexamers, which can offer a substantial system for further study of the physiological function of collagen IV.

High-Quantum-Yield Mitochondria-Targeting Near-Infrared Fluorescent Probe for Imaging Native Hypobromous Acid in Living Cells and in Vivo.

The discovery that hypobromous acid (HOBr) can regulate the activity of collagen IV has attracted great attention. However, HOBr as an important reactive small molecule has hardly ever been studied using a detection method suitable for organisms. Herein, a high-quantum-yield mitochondria-targeting near-infrared (NIR) fluorescent probe for HOBr, RhSN-mito, was designed. RhSN-mito was easily obtained by the Suzuki cross-coupling reaction. The test results show that RhSN-mito can rapidly respond to HOBr with ultrasensitivity and high selectivity. The achievement of ultrasensitivity lies in the high signal-to-noise ratio and the highest fluorescence quantum yield of the reaction product (ΦF = 0.68) in the near-infrared region, as far as we know. RhSN-mito is successfully applied to image native HOBr in mitochondria of HepG2 cells and zebrafish. Thus, RhSN-mito is a powerful tool for detecting native HOBr in vivo and is expected to provide a method to further study the physiological and pathological functions related to HOBr.

Highly Selective Fluorescent Probe for Imaging H2Se in Living Cells and in Vivo Based on the Disulfide Bond.

Hydrogen selenide (H2Se) is an important metabolite of dietary Se compounds and has been implicated in various pathological and physiological processes. The development of highly sensitive and selective methods for the sensing of H2Se is therefore very important. Herein, we developed a fluorescent probe (hemicyanine (Hcy)-H2Se) for detecting H2Se based on a new H2Se-specific receptor unit, 1,2-dithiane-4,5-diol. Hcy-H2Se showed high selectivity toward H2Se over thiols (RSH), hydrogen sulfide (H2S), and selenocysteine (Sec) and was further exploited for the fluorescence imaging of H2Se both in living cells and in vivo. Furthermore, with the aid of Hcy-H2Se, we demonstrated that H2Se can be generated and gradually accumulated in HepG2 cells under hypoxic conditions and in the solid tumor after treatment with Na2SeO3.

An Ultrasensitive Cyclization-Based Fluorescent Probe for Imaging Native HOBr in Live Cells and Zebrafish.

Bromine has been reported recently as being the 28(th) essential element for human health. HOBr, which is generated in vivo from bromide, is a required factor in the formation of sulfilimine crosslinks in collagen IV. However, to date, no method for the specific detection of native HOBr in vivo has been reported. Herein, we develop a simple small molecular probe for imaging HOBr based on a specific cyclization catalyzed by HOBr. The probe can be easily synthesized in high yield through a Suzuki cross-coupling reaction. The probe exhibits ultrahigh sensitivity at the picomole level, in addition to specificity for HOBr and real-time response. Importantly, without Br(-) stimulation, this probe reports native HOBr levels in HepG2 cells. Thus, the probe is a promising new tool for imaging endogenous HOBr and may provide a means for finding new physiological functions of HOBr in living organisms.

A Glutathione (GSH)-Responsive Near-Infrared (NIR) Theranostic Prodrug for Cancer Therapy and Imaging.

To reduce the side effects of chemotherapy, nontoxic prodrugs activated by the tumor microenvironment are urgently required for use in cancer treatment. In this work, we developed prodrug 4 for tumor-targeting treatment and imaging of the anticancer drug release in vivo. Taking advantage of the high glutathione (GSH) concentration in cancer cells, the disulfide bond in prodrug 4 was cleaved, resulting in the release of an active anticancer drug and a near-infrared (NIR) fluorescence dye turn-on. Furthermore, contrast to the free anticancer drug, the prodrug exhibited higher cytotoxicity to hepatoma cells than that to normal HL-7702 cells. Thus, prodrug 4 is a promising platform for specific tumor-activatable drug delivery system, because of its favorable features of in situ and in vivo monitoring of the drug release and therapeutic efficacy.