Mitochondria-targeted Near-infrared Fluorescent Probe Research Articles

Multifunctional Mitochondria-Targeting Near-Infrared Fluorescent Probe for Viscosity, ONOO-, Mitophagy, and Bioimaging.

Irregularities in mitochondrial viscosity and peroxynitrite (ONOO-) concentration can lead to mitochondrial dysfunction. It is still a great challenge to develop near-infrared (NIR) fluorescent probes to simultaneously detect viscosity, endogenous ONOO-, and mitophagy. Herein, a multifunctional mitochondria-targeting NIR fluorescent probe P-1 was first synthesized for simultaneously detecting viscosity, ONOO-, and mitophagy. P-1 used quinoline cations as a mitochondrial targeting moiety, arylboronate as an ONOO- responsive group, and detected the change of viscosity by the twisted internal charge transfer (TICT) mechanism. The probe has an excellent response to the viscosity during inflammation by lipopolysaccharides (LPSs) and mitophagy induced by starvation at 670 nm. The viscosity changes of the probe induced by nystatin in zebrafish showed that P-1 was able to detect microviscosity in vivo. P-1 also showed good sensitivity with a detection limit of 6.2 nM for ONOO- detection and was successfully applied to the endogenous ONOO- detection in zebrafish. Moreover, P-1 has the ability to distinguish between cancer cells and normal cells. All of these features make P-1 a promising candidate to detect mitophagy and ONOO- -associated physiological and pathological processes.

A mitochondria-targeting near-infrared fluorescent probe for the in vivo detection of peroxynitrite

As a reactive oxygen species (ROS), endogenous peroxynitrite is closely involved in several pathophysiological processes. The development of a peroxynitrite probe would be necessary to clarify the role of peroxynitrite in pathophysiology. In this study, a new fluorescent probe TPPT was synthesized to detect peroxynitrite with high specificity and sensitivity. The results demonstrated that TPPT exhibits a fluorescence turn-on response to peroxynitrite because of the peroxynitrite-triggered oxidative reaction. The reaction product was analyzed by several methods, and the mechanism of the peroxynitrite-triggered oxidative reaction was discussed. The efficacy of TPPT for intracellular peroxynitrite imaging was demonstrated systematically. The results showed that TPPT can successfully visualize intracellular peroxynitrite in RAW264.7 cells, zebrafish, and live mouse tissues.

Selective visualization of cyanide in food, living cells and zebrafish by a mitochondria targeted NIR-emitting fluorescent probe

Cyanide is a highly toxic substance, and the detection of cyanide in the environment and food samples is critical to public health care. Herein, we rationally designed a mitochondria-targeted near-infrared fluorescent probe BTC for ratiometric monitoring of CN– in water, food, living cells, and zebrafish. BTC exhibits a remarkable colorimetric ratiometric fluorescence response to CN– with high selectivity, low detection limit (54.3 nM), and large Stokes shift. The cyanide sensing mechanism was demonstrated by NMR and ESI-MS analysis and density functional theory (DFT). More importantly, BTC was used for efficient naked-eye colorimetric detection of CN– in sprouting potatoes, almonds, and ginkgo fruit samples. Further, the BTC is capable of situ tracking and imaging cyanide in mitochondria of SMMC-7721 cells and in zebrafish via dual emission channels, and was prepared into a kit for convenient and visual on-site sensing of cyanide in food samples.

A mitochondria-targeted near-infrared fluorescent probe for detection and imaging of HSO3- in living cells

Sulfur dioxide, an essential gas signaling molecule mainly produced in mitochondria, plays important roles in many physiological and pathological processes. Herein, a near-infrared fluorescent probe, A1, with good mitochondria targeting ability was developed for colorimetric and fluorescence detection of HSO3-. Probe A1 has a conjugated cyanine structure that can selectively react with HSO3- through the nucleophilic addition. The reaction with HSO3- destroys the conjugated structure of probe A1, resulting in fluorescence quenching, and accompaniedby color change of probe A1 solution from purple-red to colorless. Probe A1 showed high selectivity and good sensitivity to HSO3- in PBS. And the limit of detection was calculated to be 1.28 and 0.037 μM for colorimetry and fluorescence spectrophotometry respectively. In addition, probe A1 mainly entered the mitochondria in living cells, and was successfully used for imaging the exogenous/endogenous HSO3- in cells. These results suggest the potential applications of probe A1 in biological systems.

Research on mitochondrial oxidative stress accompanying the diabetic process under airborne particulate matter pollution by NIR fluorescence imaging of cysteine

Complex airborne particulate matter (APM) pollution seriously destroys the redox balance of organisms. Diabetes is a chronic disorder of the redox system, and its progression is usually accompanied by mitochondrial oxidative stress. The role of cysteine (Cys) in the process of mitochondrial oxidative stress is crucial as an essential scavenger of reactive oxygen species. Consequently, it is of great value to investigate the link between mitochondrial oxidative stress and diabetes by measuring changes in mitochondrial cysteine levels. However, it has not been revealed how the levels of Cys change in the mitochondria during diabetes. Therefore, we designed a novel mitochondria-targeted near-infrared fluorescent probe to assay cysteine. Importantly, by using the new probe, we discovered that mitochondrial Cys levels were significantly decreased in the diabetic cells and mice. We also uncovered that a dramatic reduction of Cys levels in the mice exposed to the presence of excess particulate matter from environmental pollution, for the first time. These findings will provide valuable insights into new treatments for diabetes and offer a practical tool to unravel the connection between mitochondrial oxidative stress and associated diseases. We believe this research will help experts to better understand the role of Cys under the APM threat.

Mitochondria-targeted near-infrared fluorescent probe for monitoring of a Copper(I) in living cells and In vivo

Non-invasive and accurate detection of copper is essential to elucidate its functions. Herein, we report a mitochondria-targeted near-infrared probe (NFL-TPA) for bioimaging of copper. The probe exhibits high sensitivity and selectivity for copper(I) with low detect of limit (14.4 nM). More importantly, the probe can monitor the level of copper(I) in mitochondria, and successfully detect the distribution of copper(I) in living mice by near infrared fluorescence (NIR) imaging in vivo and ex vivo. Furthermore, the probe could assess the therapeutic efficiency of d-penicillamine. Thus, the probe motivates the development of novel NIR platform for copper(I) detection and drug evaluation.

A turn-on mitochondria-targeted near-infrared fluorescent probe with a large Stokes shift for detecting and imaging endogenous superoxide anion in cells

Superoxide anion (O2•-), as a kind of crucial reactive oxygen species (ROS), often causes a series of oxidative damage and various biological dysfunction when it is excessive in cells. Compared with other detection methods, fluorescence detection is often used for cell imaging due to its high temporal and spatial resolution. Hence, a novel “turn-on” fluorescent probe DMPS-O based on N,N-dimethylaniline as a donor and pyridinium group as an acceptor was designed and synthesized to detect superoxide anion. DMPS-O showed a remarkable fluorescence enhancement in the near-infrared region and a large Stokes shift after reacting with superoxide anion and its limit of detection was calculated to be 22.2 nM. Finally, CCK-8 and confocal fluorescence imaging experiments revealed that the fluorescent probe DMPS-O with low cytotoxicity was suitable for detecting endogenous superoxide anions and locating mitochondria in the MCF-7 and HepG2 cells.

A mitochondria-targeting near-infrared fluorescent probe for imaging hypochlorous acid in cells

Hypochlorous acid (HOCl), as an important reactive oxygen species (ROS), plays an important role in various pathological and physiological processes. Therefore, it is of great significance to identify and detect HOCl in organisms. Herein, a mitochondria-targeting near-Infrared fluorescent probe (CVS) has been designed and synthesized for sensing HOCl. Under simulated physiological conditions, CVS can be instantly oxidized by HOCl to CVSO and the fluorescence signal is significantly enhanced. Additionally, CVS shows high selectivity toward HOCl over other ROS and owns low detection limit (94.7 nM) for HOCl. Moreover, CVS can be successfully applied to image HOCl in mitochondria of the HepG2 cells. Thus, CVS is a powerful tool for sensing HOCl and provides a perspective method for further study of the physiological and pathological functions related to HOCl.

Real-Time Evaluation of Hydrogen Peroxide Injuries in Pulmonary Fibrosis Mice Models with a Mitochondria-Targeted Near-Infrared Fluorescent Probe.

Pulmonary fibrosis is a fatal chronic lung disease, leading to poor prognosis and high mortality. Accumulating evidence suggests that oxidative stress characterized by excessive production of hydrogen peroxide (H2O2) is an important molecular mechanism causing pulmonary fibrosis. We conceive a new type of mitochondria-targeted near-infrared fluorescent probe Mito-Bor to investigate changes in the level of endogenous H2O2 in living cells and mice models with pulmonary fibrosis. In the design strategy of the Mito-Bor probe, we selected azo-BODIPY as the fluorophore owing to its near-infrared fluorescence, strong photochemical stability, and low biological toxicity. Under physiological conditions, the response moiety 4-bromomethylphenylboronic acid pinacol ester could easily detect H2O2, and turn the fluorescence switch on. The modification of the lipophilic triphenylphosphine cation on the fluorophore would allow the probe to easily pass through the phospholipid bilayer of cells, and the internal positive charge could contribute to the selectivity of the mitochondria accumulation. The Mito-Bor probe provides high selectivity, low limit of detection, high biocompatibility, and excellent photostability. It can be used to detect changes in the level of H2O2 in living cells and in vivo. Therefore, the probe is applied to investigate the fluctuation of the H2O2 level during the process of inducing pulmonary fibrosis in cells, with changes in its fluorescence intensity correlating with the concentration of H2O2 and indicating the level of oxidative stress in fibroblasts. Conversely, pulmonary fibrosis can be modulated by adjusting the level of H2O2 in cells. A further study in mice models of bleomycin-induced pulmonary fibrosis confirms that NADPH oxidase 4 (NOX4) acts as a "button" to regulate H2O2 levels. The direct inhibition of NOX4 can significantly reduce the level of H2O2, which can delay the progression of lung fibrosis. These results provide an innovative way for the clinical treatment of pulmonary fibrosis.

A mitochondria-targeted near-infrared fluorescent probe for imaging viscosity in living cells and a diabetic mice model.

A mitochondria-targeted near-infrared fluorescent probe NIR-V with 700 nm emission was designed to monitor cell viscosity changes with high selectivity and sensitivity, which was applied to detect the intracellular viscosity and image pancreatic tissue in a diabetic mouse model. Probe NIR-V provides an effective way to diagnose viscosity related diseases.

ICT-based fluorescent ratiometric probe for monitoring mitochondrial peroxynitrite in living cells

Mitochondria-targeted near-infrared fluorescent probe for the detection of peroxynitrite and the bioimaging of peroxynitrite in cells.

A novel “turn-on” mitochondria-targeting near-infrared fluorescent probe for H2S detection and in living cells imaging

Hydrogen sulfide (H2S) has been considered to be involved in cytoprotective processes and redox signaling. It is very meaningful to track and analyze it in mitochondria. Herein, we report a novel “turn-on” mitochondria-targeting near-infrared fluorescent probe (Mito-NIR-SH) for detection of H2S in living cells, which was designed and synthesized by introducing 2,4-dinitrophenyl as fluorescence quenching group and H2S response moiety into Changsha near-infrared fluorophore (CS-OH). The structure of the fluorophore and the probe were characterized by 1H NMR, 13C NMR and mass spectrometry. Meanwhile, Mito-NIR-SH could quantitatively detect H2S at concentrations ranging from 0 to 30 μM with a detection limit as low as 89.3 nM, showing good chemical stability, fast “turn-on” response, selectively mitochondrial location, as well as high sensitivity and selectivity toward H2S. Based on this, it was successfully applied to imaging exogenous and endogenous H2S in living HeLa cells via confocal fluorescence microscopy.

A mitochondria-targeted near-infrared fluorescent probe with a large Stokes shift for real-time detection of hypochlorous acid.

Hypochlorous acid (HOCl) has been known to be intertwined with various pathophysiological processes. In this paper, a novel mitochondria-targeted near-infrared (NIR) fluorescent probe L based on a chromenylium-phenothiazine conjugate has been designed and synthesized for the detection of hypochlorous acid. Due to the HOCl-promoted intramolecular charge transfer processes, the probe L shows near-infrared fluorescence emission at 672 nm, large Stokes shifts of 97 nm, fast response, and high selectivity and sensitivity. Furthermore, the probe L is biocompatible, cell-membrane permeable and mitochondria-targetable, and can be used for endogenous HOCl imaging in living cells.

Imaging of anti-inflammatory effects of HNO via a near-infrared fluorescent probe in cells and in rat gouty arthritis model.

Nitroxyl (HNO) plays a crucial role in anti-inflammatory effects via the inhibition of inflammatory pathways, but the details of the endogenous generation of HNO still remain challenging owing to the complex biosynthetic pathways, in which the interaction between H2S and NO simultaneously generates HNO and polysulfides (H2Sn) in mitochondria. Moreover, nearly all the available fluorescent probes for HNO are utilized for imaging HNO in cells and tissues, instead of the in situ real-time detection of the simultaneous formation of HNO and H2Sn in mitochondria and animals. Here, we have developed a mitochondria-targeting near-infrared fluorescent probe, namely, Mito-JN, to detect the generation of HNO in cells and a rat model. The probe consists of three moieties: Aza-BODIPY as a fluorescent signal transducer, a triphenylphosphonium cation as a mitochondria-targeting agent, and a diphenylphosphinobenzoyl group as an HNO-responsive unit. The response mechanism is based on an aza-ylide intramolecular ester aminolysis reaction with fluorescence emissions on. Mito-JN displays high selectivity and sensitivity for HNO over various other biologically relevant species. Mito-JN was successfully used for the detection of the endogenous generation of HNO, which is derived from the crosstalk between H2S and NO in living cells. The additional generation of H2Sn was also confirmed using our previous probe Cy-Mito. The anti-inflammatory effect of HNO was examined in a cell model of LPS-induced inflammation and a rat model of gouty arthritis. The results imply that our probe is a good candidate for the assessment of the protective effects of HNO in inflammatory processes.

A novel “turn-on” mitochondria-targeting near-infrared fluorescent probe for determination and bioimaging cellular hydrogen sulfide

Hydrogen sulfide (H2S) has been regarded as an important gas transmitter playing vital role in cytoprotective processes and redox signaling. It is very meaningful to monitor and analyze it in biosystem for obtaining important physiological and pathological information. Despite numerous fluorescent probes for cellular H2S have been reported in past decades, only a few have capability to detect mitochondrial H2S with near-infrared (NIR) emission. Therefore, a new mitochondria-targeting NIR fluorescent probe (Mito-NSH) for detection of cellular H2S was developed by introducing 2,4-dinitrophenyl ether into a novel dye (Mito-NOH). A large "turn-on" NIR fluorescence response was obtained due to thiolysis of ether to hydroxyl group when Mito-NSH was treated with NaHS. Moreover, Mito-NSH could quantitatively detect H2S at concentration ranging from 0 to 30 μM with a detection limit of 68.2 nM, and it exerts some superior optical properties, such as large stokes shift (107 nm), highly selectively mitochondria location, fast response and high selectivity to H2S. More impressively, it was successfully applied to imaging exogenous and endogenously generated H2S in living HeLa cells via confocal fluorescence microscopy.

Mitochondria-targeted near-infrared fluorescent probe for the detection of carbon monoxide in vivo

Carbon monoxide is a critical gasotransmitter in the body and related with mitochondrial respiration. To date, various fluorescent probes for CO have been well proposed, but two main problems remain. One is that most of the probes are not mitochondria-targeting, even if the probes claim to be able to detect CO in living cells. The other is that the probes for CO display excitation and emission within the ultraviolet or visible range, which hinders their applications in vivo. Herein, a hemicyanine-based near-infrared (NIR) fluorescent probe named CyAPC is first synthesized and used to detect mitochondrial CO. The characteristics of probe CyAPC are as follows: (1) The fluorescence emission of the sensing system is at 736 nm belonging to NIR region, which is suitable for bioimaging in vivo. (2) CyAPC, a positively charged molecule, would have a high tendency to localize in mitochondria of cells. (3) The fluorescence change of the probe is attributed to the fact that CO with Pd2+ induced cleavage of the allyl formate group from the probe and CyAPC (fluorescence off) is transformed into CyOH (fluorescence on), which is proved by HPLC, MS and DFT calculation. (4) The NIR fluorescent probe is applied for the detection of exogenous and endogenous CO in various biological samples such as cell, tissue and in vivo with satisfactory results.

A highly specific and ultrasensitive near-infrared fluorescent probe for imaging basal hypochlorite in the mitochondria of living cells

The development of highly specific and ultrasensitive fluorescent probes for tracking basal mitochondrial hypochlorite is very important to unravel its diverse cellular functions in the mitochondria of living cells. In this paper, we have developed a water-soluble, mitochondria-targeted near-infrared fluorescent probe NB-OCl for selectively measuring OCl- in the presence of higher concentration (500 μM) other biologically important substances. Surprisingly, the obtained results demonstrated that probe NB-OCl could sensitively determine OCl- in the range of 0–200 pM with the detection limit of 10.8 pM. To the best of our knowledge, NB-OCl is the first fluorescent probe for the specific determination of OCl- at the picomolar level. Moreover, probe NB-OCl exhibits a fast response for OCl- (< 5 s), which would be in favor of tracking the highly reactive and short-lived OCl- in the living systems. The preeminent recognition properties of probe NB-OCl enable its applications in the monitoring of basal OCl- and the fluctuations of endogenous/exogenous OCl- levels in the mitochondria of living cells.

Imaging and evaluation of sulfane sulfur in acute brain ischemia using a mitochondria-targeted near-infrared fluorescent probe.

Ischemia is a pathological condition owing to the deficiency of blood supply to a limited area of tissue. Ischemia can induce burst production of reactive oxygen species and lead to oxidative damage. As a family member of reactive sulfur species, sulfane sulfur plays important physiological roles in many biological events including synthesis of cofactors, modulation of enzyme activities, sulfuration of tRNA, and especially regulation of the intracellular redox state. We hypothesize that the endogenous level of sulfane sulfur will be adjusted to deal with ischemia-induced oxidative damage. Therefore, the bioimaging of sulfane sulfur real-time changes during ischemia is important for better understanding its physiological processes. Herein, we describe the development of a mitochondria-targeted fluorescent probe Mito-SH that allowed for selective and sensitive detection of sulfane sulfur. Mito-SH is designed on the basis of the tautomerization of sulfane sulfur to thiosulfoxide, which ensures its high selectivity and sensitivity. A lipophilic triphenylphosphonium cation is selected as the mitochondria-targeted moiety, which can precisely navigate Mito-SH into mitochondria. The emission profile of azo-BODIPY fluorophore locates at the near-infrared region, which deeply penetrates tissue and effectively avoids the interference of biological background. Mito-SH exhibits the desirable combination of selectivity, sensitivity and excellent fluorescence response upon reaction with sulfane sulfur in cells. By employing Mito-SH, we evaluate the real-time sulfane sulfur dynamic changes under oxygen-glucose deprivation. Finally, Mito-SH has been successfully used for imaging sulfane sulfur changes caused by acute ischemia in mice.

Construction of a fluorine substituted chromenylium-cyanine near-infrared fluorophore for ratiometric sensing

Abstract Near-infrared (NIR) fluorescent probes have been considered as powerful tools for exploring bioactive species and biological processes in living systems by virtue of the substantial merits of the NIR window (such as minimal photo-toxicity, deep tissue penetration, and low background interference). However, the development of effective NIR fluorescent probes for biological application is still facing great challenges due to some weakness (for example, pH sensitivity, poor chemical and photo-stability) of mainly current NIR fluorophores. Herein, we synthesized a novel fluorine atom substituted NIR chromenylium-cyanine fluorophore, termed CCF-OH, with an optically tunable hydroxyl group. Compound CCF-OH possess several optical advantages, including high chemical stability, high photo-stability, and high fluorescence quantum yield (Φ = 0.47). Significantly, a low pKa value (6.3) of CCF-OH was obtained, making its fluorescence signal steady around the pH ranging from 6.8 to 8.1. To demonstrate the practicability of CCF-OH, a mitochondria-targeted near-infrared fluorescent probe, N-Cys, was developed by masking the hydroxyl group of CCF-OH with an acrylate group as the recognition unit for cysteine (Cys). N-Cys responded to Cys with an obvious ratiometric signal. Furthermore, N-Cys has also been successfully applied for ratiometric NIR imaging of Cys in living cells, suggesting the great potential of the new NIR fluorophore, CCF-OH, for practical applications in living systems.

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.