Ratiometric Fluorescent Probe for Super-Resolution Imaging of Lysosome HClO in Ferroptosis Cells.

Ferroptosis is an iron-dependent programmed cell death that is characterized by the dysregulation of lipid reactive oxygen species (ROS) production, causing abnormal changes in hypochlorous acid (HClO) levels in lysosomes. Super-resolution imaging can observe the fine structure of the lysosome at the nanometer level; therefore, it can be used to detect lysosome HClO levels during ferroptosis at the suborganelle level. Herein, we utilize a ratiometric fluorescent probe, SRF-HClO, for super-resolution imaging of lysosome HClO. Structured-illumination microscopy (SIM) improves the accuracy of lysosome targeting and enables the probe SRF-HClO to be successfully applied to rapidly monitor the up-regulated lysosome HClO at the nanoscale during inflammation and ferroptosis. Importantly, the probe SRF-HClO can also detect HClO changes in inflammatory and ferroptosis mice and evaluate the inhibitory effect of ferroptosis on mice tumors.

Dual-Emissive Detection of ATP and Hypochlorite Ions for Monitoring Inflammation-Driven Liver Injury In Vitro and In Vivo.

Reactive oxygen species play a pivotal role in liver disease, contributing to severe liver damage and chronic inflammation. In liver injury driven by inflammation, adenosine-5'-triphosphate (ATP) and hypochlorite ion (ClO-) emerge as novel biomarkers, reflecting mitochondrial dysfunction and amplified oxidative stress, respectively. However, the dynamic fluctuations of ATP and ClO- in hepatocytes and mouse livers remain unclear, and multidetection techniques for these biomarkers are yet to be developed. This study presents RATP-NClO, a dual-channel fluorescent bioprobe capable of synchronously detecting ATP and ClO- ions. RATP-NClO exhibits excellent selectivity and sensitivity for ATP and ClO- ions, demonstrating a dual-channel fluorescence response in a murine hepatocyte cell line. Upon intravenous administration, RATP-NClO reveals synchronized ATP depletion and ClO- amplification in the livers of mice with experimental metabolic dysfunction-associated steatohepatitis (MASH). Through a comprehensive analysis of the principal mechanism of the developed bioprobe and the verification of its reliable detection ability in both in vitro and in vivo settings, we propose it as a unique tool for monitoring changes in intracellular ATP and ClO- level. These findings underscore its potential for practical image-based monitoring and functional phenotyping of MASH pathogenesis.

A facile near-infrared xanthene fluorescence probe for visualizing of hypochlorous acid in vitro and in vivo.

BACKGROUND: Hypochlorous acid (HClO) is an important biomarker for inflammation, cardiovascular disease, and even cancer. It is of great significance to accurately monitor and quantitatively analyze the fluctuations of HClO to better understand their physiological functions. Traditional HClO detection methods such as high-performance liquid chromatography (HPLC), and mass spectrometry are preferred, but are costly and unsuitable in vivo. Near-infrared (NIR) fluorescence imaging has the advantages of high sensitivity, high temporal and spatial resolutions, minimal autofluorescence, and deep tissue penetration, which facilitates its application in biological systems. Therefore, the development of sensitivity and simple NIR fluorescence monitoring HClO methods in vivo and in vitro is essential and desirable. RESULTS: Herein, we present a NIR probe NOF3 by integrating the rhodamine scaffold and HClO-triggered moiety for the real-time detection of HClO in vitro and in vivo. NOF3 reacts with the HClO and releases the NOF-OH fluorophore of emitted signals at 730 nm, which is in the NIR region. The designed probe detected concentrations of HClO ranging from 0 to 17 µM with a low detection limit of 0.146 µM, presenting excellent sensitivity and selectivity toward HClO over other species. NOF3 manifests significantly turn-on NIR fluorescent signals in response to HClO concentration, which makes it favorable for monitoring dynamic HClO distribution in vivo. We exemplify NOF3 for the tracking of endogenously overexpressed HClO distribution in RAW 264.7 cells, and further realize real-time in vivo bioimaging of HClO activity in inflammation mice. SIGNIFICANCE: The facile NIR NOF3 probe was successfully applied to visualize endogenous and exogenous HClO in living cells and mice. This study provides not only an effective tool for spatial and temporal resolution HClO bioimaging in vivo but also possesses great potential for use in future research on HClO-related biology and pathology.

An efficient PeT based fluorescent probe for mapping mitochondrial oxidative stress produced via the Nox2 pathway.

The human innate immune system eliminates invading pathogens through phagocytosis. The first step of this process is activating the nicotinamide adenine dinucleotide phosphate oxidase (Nox2) that utilizes NADPH to produce superoxide anion radicals and other reactive oxygen species (ROS). These ROS then alter the mitochondrial membrane potential and increase peroxide in the mitochondria. The peroxide reacts with myeloperoxidase (MPO) and chloride ions to produce pro-inflammatory oxidant hypochlorous acid (HOCl), which causes oxidative stress leading to cell death. The adverse effects of HOCl are highly associated with cardiovascular disease, neurodegenerative disorders, acute lung injuries, inflammatory diseases, and cancer. Therefore, mapping HOCl in the Nox2 pathway is crucial for an in-depth understanding of the innate immune system. Herein, we developed a unique pentacyclic pyridinium probe, PM-S, that exhibited efficient photoinduced electron transfer (PeT) with HOCl triggered methyl(phenyl)sulfane. PM-S showed several advantages, including better chemical stability, large Stokes shifts (>6258 cm-1), high sensitivity (∼50 nM) and specificity to mitochondria, compared to its parent pyrylium PY-S derivative. This probe is also efficient in studying the HOCl produced via the Nox2 pathway in HepG2 and HeLa cells. Analysis using a simple microplate reader and FACS analysis with various inhibitors and inducers supported the mechanistic understanding of Nox2, which can offer an advanced platform for monitoring the inflammatory process more efficiently.

Monocomponent Nanodots with Dichromatic Output Regulated by Synergistic Dual-Stimuli for Cervical Cancer Tissue Imaging and Photodynamic Tumor Therapy.

Inflammation exists in the microenvironment of most, if not virtually all, tumors, which greatly exacerbates the difficulty of cancer treatment. Considering the superiority of activatable photosensitizers (PSs), a novel strategy of 'making friends with the enemy' for tumor treatment was proposed. In this strategy, the "enemy" refers to inflammatory cytokines and the tumor site is targeted by detecting the enemy. Upon detection, a dichromatic fluorescence signal is released and the PS is activated specifically by the inflammatory cytokines. In this study, a multifunctional PS (TPE-PTZ-Py) was rationally designed, which can be activated specifically under the synergistic action of hypochlorous acid (HClO) (one kind of inflammatory cytokines) and acid (one typical marker of tumor), and output a ratiometric fluorescence signal simultaneously. The sulfoxide analogue (TPE-PTZO-PyH) as the response product effectively produced 1O2 (1.8-fold higher than that obtained with Rose Bengal) and showed high phototoxicity (IC50 < 7.6 µM). More importantly, imaging analyses confirmed that TPE-PTZ-Py could be activated in human cervical cancer tissue. To date, several phenothiazine (PTZ)-based fluorescent probes have been developed for the selective sensing and imaging of HClO in subcellular organelles; however, this is the first phenothiazine-based nanodrug designed for the treatment of inflammation-associated tumors with a few side effects.

A POSS-assisted fluorescent probe for the rapid detection of HClO in mitochondria with a large emission wavelength in dual channels.

Hypochlorous acid (HClO) is closely related to many diseases and is an inevitable part of the physiological processes. It is significant to detect HClO in mitochondria for getting meaningful physiological and pathological information. However, adequate tools to detect HClO with emissions in two channels are rarely reported. To achieve this target, in this work, a "turn-off" visual and near infrared (NIR) fluorescent dual emission probe D6 based on polyhedral oligomeric silsesquioxanes (POSS) was successfully designed and synthesized. D6 showed high selectivity and sensitivity to HClO. Notably, the emission wavelength of D6 reached 820 nm due to the assistance of the POSS cage. In addition, bioimaging experiments clearly showed that probe D6 promoted the visualization of exogenous and endogenous HClO in living HepG2 cells and zebrafish models.

Synthesis, molecular docking calculation, fluorescence and bioimaging of mitochondria-targeted ratiometric fluorescent probes for sensing hypochlorite in vivo.

Mitochondria are the main sites for the production of hypochlorite (OCl-). The protein adenine nucleotide translocase (ANT) is located in the inner mitochondria membrane, which is mainly participated in the transportation of ions and metabolites. At the cellular organelle level, overexpression of ANT is associated with enhanced production of OCl-, however, abnormal levels of OCl- cause redox imbalance and loss of function of mitochondria. Herein, a novel mitochondria-targeted ratiometric fluorescent probe Mi-OCl-RP has been developed. Molecular docking calculation suggested a potential molecular target for the probe in the ANT, and the high binding energy (-8.58 kcal mol-1) may explain the high mitochondria selectivity of Mi-OCl-RP. The unique probe exhibits excellent spectral properties including ratiometric fluorescence response signals to OCl- (within 7 s), high selectivity and sensitivity, and a large Stokes shift (278 nm). In addition, the colocalization coefficient confirms that Mi-OCl-RP can effectively target mitochondria. Furthermore, Mi-OCl-RP has low toxicity and good permeability, and was successfully employed in ratiometric imaging of OCl-in vivo, affording a robust molecular tool for investigating the biological functions of OCl- in living systems.

Molecular Design toward Heavy-Atom-free Photosensitizers Based on the C═S Bond and their Dual Functions in Hypoxia Photodynamic Cancer Therapy and ClO- Detection.

In this article, we designed and synthesized the thionated NpImidazole derivatives BS and NS, new heavy-atom-free photosensitizers, which efficiently generate a triplet excited state with high singlet oxygen quantum yield. The introduction of the C═S bond to the NpImidazole core is essential for increasing spin-orbit coupling (SOC). The fluorescence emission of BS and NS was quenched at standard ambient temperature, accompanied with the increase in the ISC process from the singlet states to triplet excited states via thionation. BS and NS showed negligible dark cytotoxicity against HeLa cells in working concentration. In contrast, BS and NS rapidly induced cell death under blue light irradiation both under normoxia and hypoxia conditions. Our current study demonstrates that the C═S group can play an important role in type I ROS generation of PSs, which are unprecedented in the previous reports. Finally, the photophysical changes were assigned to the oxidative desulfurization of the C═S group of BS and NS to the C═O group of the corresponding BO and NO via hypochlorite. The combined results demonstrated the dual function of BS and NS as a fluorescent imaging agent for ClO- and an anti-cancer therapeutic by PDT that showed the potential strategy for "one-for-all" and multifunctional agents.

A Fluorescent Probe for the Fast Detection of Hypochlorite and its Applications in Water, Test Strip and Living Cells.

Hypochlorite (ClO-) mediated by oxidative stress play an important role in the body's defense system due to their physiological and pathological significance. In this work, a new and simple probe was designed and synthesized to detect hypochlorite. This probe could rapidly respond to hypochlorite in a short time (20 s) in aqueous media, and showed excellent selectivity and sensitivity, and a wide pH range of 3 ̶ 12, as well as the low detection limit of 1.44 nM. In addition, it was successfully applied to the detection of ClO- in water sample, test paper experiment, and cell imaging.

A novel ratiometric fluorescent probe for differential detection of HSO3- and ClO- and application in cell imaging and tumor recognition.

By connecting 1,8-naphthalimide and indole sulfonate, a ratio fluorescent probe capable of differential detection of hydrogen sulfite and hypochlorite was synthesized for the first time. It was able to achieve the qualitative detection of HSO3- and ClO- with high sensitivity and selectivity, respectively. It provides a multi-purpose probe and is based on different emission channels without mutual interference. The probe has the advantages of larger Stokes shift (ClO-: 115 nm, HSO3-: 88 nm), longer λem (ClO-: 515 nm, HSO3-: 548 nm) and better water solubility (DMF/PBS = 1:99, v/v). In addition, the probe is a ratio fluorescence probe, which can detect fluorescence intensity with two different emission waves. It provides internal self-calibration, reduces interference from the background and increases detection accuracy. In vitro cytotoxicity and imaging experiments show that the probe can effectively perform the detection of exogenous HSO3- and ClO- in cells. It can also achieve the detection of HSO3- and ClO- in the plasma environment. Because the probe can detect endogenous ClO-, it also has a good prospect for biological application in identifying tumor cells. Graphical abstract.

Novel Platinum(II) Complex-based Luminescent Probe for Detection of Hypochlorite in Cancer Cells.

Hypochlorite (ClO⁻) is of great importance either for the metabolism of living organisms or as disinfectant in daily life. However, improper concentration levels of ClO⁻ lead to serious health problems including erythrocyte damage, cardiovascular problems, neuron degeneration, lung/kidney injury and cancer. Therefore, a sensitive and selective detection method is required for the visualization and measurement of ClO⁻. In this work, a novel platinum(II) complex-based luminescent probe Pt-CHO was synthesized and utilized to detect ClO⁻. This "turn-off" probe exhibits high sensitivity, excellent selectivity, good pH stability, low limit of detection and instantaneous response ability. Moreover, the luminescent response is caused by the oxidation of aldehyde into carboxyl groups combined with the coordination of hydroxyl groups at the Pt center, which is rarely reported. The cell imaging of HeLa cells proved the considerable potential of the probe for ClO⁻ imaging in living cells.

Self-Assembly of a Dual-Targeting and Self-Calibrating Ratiometric Polymer Nanoprobe for Accurate Hypochlorous Acid Imaging.

Exploiting an intelligent fluorescent probe, which can precisely target to the lysosome of hepatoma cells and enable accurate molecular imaging, is a key challenge in hepatoma diagnoses. Herein, a single-dye-based polymer nanoprobe (named SPN) with dual-targeting and self-calibrating ratiometric characteristics is rationally fabricated via a simple self-assembly strategy for accurate hypochlorous acid (HClO) imaging in the lysosome of HepG2 cells. Of note, the covalent incorporation of self-calibrating ratiometric fluorophore (pyrene derivatives) into the core of polymer nanoparticles can not only validly avoid the leakage of fluorophores but also greatly enhance their brightness. Besides, this polymer nanoprobe (SPN) displays high water dispersibility, ultrafast response (<1s), favorable selectivity, outstanding long-term stability (>90 days), and good biocompatibility. Furthermore, thanks to the hepatocyte-targeting moiety (galactose) and the interplay of surface charge and size of nanoparticles, the SPN is able to enter into asialoglycoprotein receptor-positive HepG2 cells and further locate at lysosomes, successfully enabling accurate HClO detection in lysosomes of HepG2 cells. This study demonstrates that the versatile SPN can provide more precise dual-targeting and accurate molecular imaging.

Mitochondria-Immobilized Unimolecular Fluorescent Probe for Multiplexing Imaging of Living Cancer Cells.

Cancer incidence and mortality are fast growing worldwide. Recently, multiplexing imaging methods have been reported to be vital for cancer diagnosis and therapy. Fluorescence imaging, which has intrinsic capabilities for multiplexing imaging, is suitable and ripe for cancer imaging. In biomedical research, using a single probe for multiplexing imaging can avoid larger invasive effects and ensure the same spatiotemporal distributions and metabolisms of the probes, which has advantages over using multiple probes. Therefore, developing unimolecular fluorescent probes for multiplexing imaging of living cancer cells is meaningful. We herein report a unimolecular fluorescent probe (ZED) that simultaneously detects cysteine/homocysteine, hypochlorous acid, mitochondrial membrane potential (Δψm), and opening of the mitochondrial permeability transition (MPT) pore in cells. These four analytes are key indicators predominantly associated with multiple aspects of carcinogenesis and cancer therapy in living cells. Besides, ZED also differentiates MCF-7 cells from HBL-100 cells. The sensing process is fast, selective, and sensitive in living cancer cells. As far as we know, ZED is the first probe that simultaneously detects four analytes in cells and the first probe that simultaneously detects Δψm and opening of the MPT pore in mitochondria.

The novel HOCl fluorescent probe CAN induced A549 apoptosis by inhibiting chlorination activity of MPO.

Hypochlorous acid (HOCl) is an important signaling molecule for cell survival. However, it has been reported that excessive HOCl contributes to a variety of diseases such as cancers. And in cancer cells, the level of HOCl is much higher than that in normal cells. Here a coumarin-based fluorescent probe 7-Diethylamino-3-(2,3-dihydro-1H-perimidin-2-yl)-chromen-2-one (CAN) was successfully developed for HOCl detection. The probe could be oxidized by HOCl to induce significant change in its fluorescence profile, which made it feasible for ratiometric detecting HOCl. CAN (below 1 µM) did not affect cell viability and had good capacity in ratiometric detection of HOCl in RAW 264.7 cells. CAN induced A549 apoptosis and inhibited tumor growth in vitro and in vivo. And CAN could decrease the chlorination activity of myeloperoxidase (MPO) in A549. These findings suggested that CAN (below 1 µM) would develop into a HOCl probe. High activity of MPO and level of HOCl might be helpful for A549 survival. A549 could be induced apoptosis by reducing the HOCl level by CAN. It implies a new anticancer strategy by targeting HOCl.

Quantifying the Fast Dynamics of HClO in Living Cells by a Fluorescence Probe Capable of Responding to Oxidation and Reduction Events within the Time Scale of Milliseconds.

The biological roles of reactive oxygen species (ROS) depend highly on their dynamics. However, it has been challenging for measuring the dynamics of ROS in cells. In this study, we address a core challenge in developing fluorescence probes for monitoring ROS dynamics by designing a redox couple that can respond rapidly to both oxidation and reduction events. We show that such molecules can be designed by taking advantage of the steric effects of electron-donating groups at the ortho position relative to the selenium center. We demonstrate this design in a new fluorescence probe named Fl-Se. Results reveal that Fl-Se and its oxidized product Fl-SeO rapidly respond to HClO, an important member of the ROS family, and glutathione (GSH), with t1/2 = 2.7 ms at [HClO] = 1 µM; t1/2 = 61 ms at [GSH] = 1 mM. When applied in cells, Fl-Se satisfactorily tracks the dynamics of intracellular HClO in H2O2-stimulated HL-60 cells, as well as the different dynamic behaviors of HClO fluctuations involved in the phorbol 12-myristate-13-acetate-activated immune response of RAW264.7 cells and the 3-deazaneplanocin A-induced apoptosis of HL 60 cells.

Peptide-capped functionalized Ag/Au bimetal nanoclusters with enhanced red fluorescence for lysosome-targeted imaging of hypochlorite in living cells.

Bioimaging probes for monitoring intracellular reactive oxygen species have important implications for cell biology research. Herein, we developed peptide-capped silver/gold nanoclusters (peptide@Ag/Au NCs) for lysosome-targeted imaging of hypochlorite (ClO-). The peptide@Ag/Au NCs were synthesized via a one-pot method using peptide as both a template and a reducing agent. The fluorescence intensity and absolute quantum yield of peptide@Ag/Au NCs were much higher than those of peptide-capped gold nanoclusters and silver nanoclusters. In the presence of ClO-, the fluorescence of peptide@Ag/Au NCs was quenched, accompanied by a redshift due to ClO--induced oxidation of the peptide ligand and decreased Ag content in Ag/Au NCs. The relative fluorescence intensity F0/F had favourable linearity for ClO- concentrations in the range 0.1-100 µmol/L (R2 = 0.9954), with a detection limit (LOD) of 80 nmol/L. The lysosome-targeted peptide@Ag/Au NCs were applied to detect ClO- in lysosomes in living cells via fluorescence imaging.

Multimodal Imaging Iridium(III) Complex for Hypochlorous Acid in Living Systems.

Biomolecule tracing with different imaging methods is of great significance for more accurately unravelling the fundamental processes in living systems. However, considering the different principles of each imaging method for probe design, it is still a great challenge to apply one molecular probe to achieve two or even more imaging analyses for biomarkers. In general, traditional oxime was reported as a recognition group for fluorescence imaging of HOCl. Herein, for the first time, we designed the oxime decorated iridium(III) complex, which can be directly used for chemiluminescence as well as two-photon luminescence and photoluminescence lifetime imaging of HOCl in living systems. Moreover, the novel chemiluminescence mechanism of Ir-CLFLPLIM for HOCl was also proposed and explored by continuously monitoring chemiluminescence peak shapes and mass spectra, inferring the reaction intermediate and calculating the chemical reaction energy range of the reaction process. This strategy could lead us to expand the chemiluminescence application of transition metal complexes and develop more multimodal imaging probes.

A novel reaction-based fluorescence probe for rapid imaging of HClO in live cells, animals, and injured liver tissues.

Hypochlorous acid (HClO) is an important bioactive molecule, playing vital roles in a large number of physiological and pathological processes. Abnormal concentration of HClO in vivo has close contact with many diseases including inflammatory diseases and cancer. For bioimaging HClO, a new colorimetric and turn-on near-infrared (NIR) fluorescence probe (DDAO-ClO) was designed and synthesized in this work through a specific reaction of HClO with dimethylthiocarbamate. DDAO-ClO proved to show distinct and highly selective colorimetric and NIR fluorescence responses for HClO with fast response time (<3 s) and high sensitivity (LOD = 7.3 nM) in vitro. After confirming the excellent in vitro sensing ability, imaging HClO with DDAO-ClO in living HeLa cells, MCF-7 cells, zebrafish, and mice was all successfully demonstrated. And with this probe, it was further discovered that more endogenous HClO was produced in injured mice liver tissues, which demonstrates that DDAO-ClO not only is effective for in vivo detection of HClO but also has a broad application prospect.

Execution of aggregation-induced emission as nano-sensors for hypochlorite detection and application for bioimaging in living cells and zebrafish.

Hypochlorite (ClO-) could be used as a diagnostic marker for inflammation and related diseases. Although there have been many reports on probes for ClO- imaging, there was still a lack of specificity and anti-interference ability. Herein, carbazole (NEC) and tetraphenylethylene (TPE) equipped with thiobarbituric acid (TBA), NEC-TBA and TPE-TBA, were synthesized and used as a fluorescence biosensor for monitoring ClO- with aggregation-induced emission (AIE) effect. we identified that TPE-TBA, with formed nanoparticles in the mean grain size at 76 nm (5 µM), was a superior probe to target ClO- over other analytes with fluorescence "turn off" strategy. Subsequently, to explore the bioimaging application, TPE-TBA was able to sense exogenous ClO- in living HeLa cells through fluorescence imaging. In zebrafish model, TPE-TBA effectively captured exogenous ClO- in the entire organization of zebrafish. Overall, these AIE-based probes merit further development as organism targeting ClO- sensors.

Single Wavelength Laser Excitation Ratiometric NIR-II Fluorescent Probe for Molecule Imaging in Vivo.

Fluorescence (FL) imaging in the second near-infrared window (NIR-II, 1000-1700 nm) has emerged as a promising bioimaging modality that enables noninvasive visualization of deep tissue with an unprecedented resolution. However, there is a paucity of studies on high-quality responsive NIR-II FL molecules. Herein we report a novel activated NIR-II FL molecule, 4,7-bis(5-(4-(diphenylamine)phenyl)-2-thiophene) [1,2,5]selenadiazolo[3,4-f]benzo[c][1,2,5]thiadiazole (SeTT), which exhibits fast and specific responsive capability to hypochlorous acid (HClO). To obtain the NIR-II ratiometric nanoprobe, SeTT was encapsulated on the surface of Er3+-doped down-conversion nanoparticles (DCNP), achieving the DCNP@SeTT nanoprobe. With a single 980 nm laser excitation, the ratiometric FL signal of SeTT at 1150 nm and DCNP at 1550 nm (I1150 nm/I1550 nm) was linearly correlated with the concentration of HClO with a detection limit of 0.4 µM. The ratiometric nanoprobe was successfully investigated for variations in HClO concentration in the tumor progression, visualization of anatomical structures of the peritoneal cavity in the mice model with inflammation, and quantitative detection of the HClO concentration in a rabbit model of osteoarthritis, achieving a fast response and high selectivity for the detection of HClO. The NIR-II-responsive nanoprobe can serve as a promising and effective tool for highly sensitive monitoring and imaging of HClO in living systems.