Mitochondrial SO2 Research Articles

Real time precisely tracing the fluctuations of mitochondrial SO2 in cells during ferroptosis and tissues using a mitochondrial-immobilized ratiometric fluorescent probe

Mitochondrial sulfur dioxide (SO2) plays important roles in physiological and pathological activities. Unfortunately, it is lack of a reliable tool to precisely visualize the mitochondrial SO2 and elaborate its complicated functions in various cytoactivities. Here we report a mitochondrial-immobilized fluorescent probe PM-Cl consisting of coumarin and benzyl chloride modified benzothiazole, which enables selective visualization of mitochondrial SO2via chemical immobilization. The spectral results demonstrated that probe PM-Cl could respond to SO2 with high selectivity and sensitivity. Co-localization and the fluorescence of cytolysis extraction verified the excellent mitochondrial targeting and anchoring abilities. Due to the chemical immobilization, probe PM-Cl could firmly retain into mitochondria after stimulation of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and H2O2. Significantly, a series of fluorescence images are indicative of capability for detecting the fluctuations of SO2 in mitochondria during ferroptosis. Furthermore, PM-Cl also could visualize SO2 in myocardium and muscle tissues after the stimulation of CCCP. Taken together, probe PM-Cl is a very potential molecular tool for precisely detecting mitochondrial SO2 to explore its complex functions in physiological and pathological activities.

A mitochondria-specific NIR fluorescence probe for dual-detection of sulfur dioxide and viscosity in living cells and mice.

The level of sulfur dioxide (SO2) and viscosity in mitochondria play vital roles in various physiological and pathological processes. Abnormalities in mitochondrial SO2 and viscosity are closely associated with numerous biological diseases. It is of great significance to develop novel fluorescence probes for simultaneous detection of SO2 and viscosity within mitochondria. Herein, we have developed a water-soluble, mitochondrial-targeted and near-infrared fluorescent probe, CMBT, for the simultaneous detection of SO2 and viscosity. The probe CMBT incorporates benzothiazolium salt as a mitochondrial targeting moiety and 7-diethylaminocoumarin as a rotor for viscosity detection, respectively. Based on the prompt reaction between nucleophilic HSO3-/SO32- and the backbone of the benzothiazolium salt derivative, probe CMBT displayed high sensitivity and selectivity toward SO2 with a limit of detection as low as 0.17 μM. As viscosity increased, the twisted intramolecular charge transfer (TICT) process was restricted, resulting in fluorescence emission enhancement at 690 nm. Moreover, probe CMBT demonstrated exceptional mitochondrial targeting ability and was successfully employed to image variations of SO2 and viscosity in living cells and mice. The work highlights the great potential of the probe as a convenient tool for revealing the relationship between SO2 and viscosity in biological systems.

A remarkable membrane-permeable fluorescent probe for real-time imaging of mitochondrial SO2 with high fidelity during ferroptosis.

Mitochondrial sulfur dioxide (SO2) plays a double-edged role in cells, and the real-time and in situ tracing of its dynamic behaviors to elucidate its complicated functions in detail is of great significance. Here, we developed a simple mitochondria-targeted fluorescent probe ZW for tracing SO2 with good membrane permeability. In probe ZW, the 1-phenylpyrrolidine-decorated benzopyrylium unit is employed as the selective response site for SO2. Besides, it also acts as the main fluorophore for signal conversion. The spectral results displayed that ZW could emit near-infrared (NIR) fluorescence (670 nm) and has a highly sensitive and selective response to SO2 (LOD = 0.19 μM). For biological imaging, compared with the control probe ZE, concentration- and time-dependent results verified that probe ZW has remarkable cell delivery with low concentration (200 nM) and fast response time (3 min). Furthermore, the NIR emission of ZW rendered high-fidelity imaging in living cells. Owing to its positive charge, ZW showed favorable mitochondria-targeting properties by colocalization experiments. Probe ZW could detect SO2 in real-time and in situ with high photostability in cells. Significantly, it has the ability to monitor the changes of endogenous SO2 during ferroptosis.

Bifunctional Single-Molecular Fluorescent Probe: Visual Detection of Mitochondrial SO2 and Membrane Potential.

Mitochondrial membrane potential (MMP) and sulfur dioxide (SO2) significantly affect the mitochondrial state. In this work, TC-2 and TC-8 were constructed through side-chain engineering, in which TC-2 bearing the poorer hydrophobicity could localize on mitochondria better. Interestingly, short-wave emission was captured due to the sensitive response of TC-2 to SO2 (LOD = 13.8 nM). Meanwhile, the probe could bind with DNA, presenting enhanced long-wave emission. Encouragingly, TC-2 could migrate from mitochondria to the nucleus when MMP was decreased, accompanied by the increase of fluorescence lifetime (9-fold). Hence, TC-2 could be used for dual-channel monitoring of mitochondrial SO2 and MMP, which showed a completely different pathway from the commercial MMP detectors JC-1/JC-10. The cellular experiments showed that MMP was gradually decreased due to reactive oxygen species-triggered oxidative stress, and the SO2 level was up-regulated simultaneously. Overall, this work proposed a new method to investigate and diagnose the mitochondrial-related diseases.

A dual-positive charges strategy for sensitive and quantitative detection of mitochondrial SO2 in cancer cells and tumor tissue

Mitochondrial sulfur dioxide (SO2) correlates with various activities of the development and progression of cancer. However, the specific biological function of mitochondrial SO2 in cancerous cells remains amphibolous. Therefore, it is of great significance and urgency to develop a rapid and accurate method to monitor the dynamic fluctuations of mitochondrial SO2 in cancer cells and tumor tissue. Herein, in this work, we introduce a “dual-positive charges” strategy for simultaneously enhancing the sensitivity and mitochondrial targeting ability of SO2 detection in cancer cells for the first time. For proof of concept, the dual positive charged probe DCP was rationally designed and synthesized based on chromenoquinoline fluorophore. Correspondingly, we also synthesized single positive charged SO2 probe MCP as controls. As expected, the detection limit of dual positive charged DCP for SO2 detection was 0.06 μM, which was 7-fold lower than that of the single positive charged probe MCP. Besides, DCP showed a higher mitochondrial co-localization coefficient in cancer cells and it could distinguish cancer cells (HeLa) and normal cells (L929) in co-incubated system. In a word, the evidence suggested that the implementation of dual-positive charges strategy greatly improved the sensitivity to SO2 response and the specificity of mitochondrial targeting in cancer cells. Finally, DCP was successfully applied to monitor SO2 fluctuation in cancer cells, tumor tissue and living zebrafish. Thus, this work provides a powerful tool to investigate the role of mitochondrial SO2 in cancer and other related diseases.

A dual-site and dual-turn-on fluorescence probe for imaging mitochondrial HClO and SO2

Bioactive small molecules, hypochlorous acid (HClO) and sulfur dioxide (SO2), play vital roles in the maintenance of normal physiological function of living organisms. Once their balance is interrupted, it would cause oxidative stress and various diseases. Therefore, monitoring the change of HClO and SO2 in cellular levels and in vivo is of great significant for exploring their physiological and pathological roles. Herein, we present a dual-site and dual-turn-on fluorescence probe PTBI that could simultaneously monitor HClO and SO2 in mitochondria by two well-separated emission channels with a difference of 115 nm. The probe exhibited fast response (HClO: < 5 s, SO2: <1 min) and low detection limits with 45.8 and 53.2 nM for HClO and SO2, respectively. Using the single fluorescence molecule, we not only realized the detection of the basal and exogenous/endogenous levels of HClO and SO2, but also achieved the monitoring mitochondrial oxidative stress induced by HClO in living cells and zebrafish.

Mitochondria-targeted and FRET-based fluorescent probe for the imaging of endogenous SO2 in living cells

Sulfur dioxide (SO2) is an important signal molecule in living systems, and plays a wide range of physiological functions. Real-time and in situ detection of the dynamic balance of SO2 in mitochondria is of great significance to in-depth study its biological roles. Herein, we have developed a mitochondria-targeted fluorescent probe Nap-L based on the FRET mechanism to detect SO2 in living cells. The probe Nap-L employed naphthalimide and positively charged benzopyridine as the donor and acceptor in the FRET system, and emitted green and red fluorescence under excitation. In respond to SO2, the nucleophilic addition of bisulfite to benzopyridine and then interrupted the FRET process from naphthalimide to benzopyridine fluorophore, thereby triggering an obvious change in the fluorescence ratio. The probe Nap-L showed high selectivity to SO2 over the biothiols (Hcy, GSH, Cys) and other biologically related species. Biological experiments suggested that the probe Nap-L mainly distributed in mitochondria, and can be successfully used to detect mitochondrial endogenous SO2 in living cells.

Ratiometric and reversible detection of endogenous SO2 and HCHO in living cells and mice by a near-infrared and dual-emission fluorescent probe

Abstract The small signal transmitters SO2 can elaborately modulate signal transduction pathways. Endogenous formaldehyde (FA) can toxify protein and DNA as a crosslinking agent besides functions in the aspect of spatial memory and cognitive ability. Both SO2 and FA are tightly associated with neurological disorders, cardiovascular diseases. Therefore, it is significant and challenging to monitor the real-time dynamic fluctuation of SO2 and FA in cells or animals. Toward this end, for the first time, a near-infrared (NIR) dual-emission fluorescent probe (BCou-BP) for SO2 and FA was developed based on the rational combination of benzypylium and benzo[g]coumarin cores through tuning the conjugation and rigidity of the fluorophore units. Thus, this rational design affords the new cationic probe the ratiometric detection ability (F604/F694) toward mitochondrial SO2 with a fast response time of 10 s, as well as toward FA in 170 s. Endogenous FA could restore the fluorescence at 694 nm of BCou-BP due to the capture of SO2 from decomposition of the adduct BCou-BP-SO3H. The powerful probe succeeded in real-time visualizing the dynamic change of endogenous mitochondrial SO2 and FA in living cells and mice.

The development of a biotin-guided and mitochondria-targeting fluorescent probe for detecting SO2 precisely in cancer cells

Mitochondrial sulfur dioxide (SO2) is very closely associated with various activities of cancer cell. However, the specific physiological and pathological roles of mitochondrial SO2 in cancer cells are still not well defined. Lacking a powerful molecular tool for detecting mitochondrial SO2 in cancer cells precisely is an essential factor. So it is urgent to develop a specific method for monitoring mitochondrial SO2 in cancer cells. Herein, we described a distinct cancer cell-specific fluorescent probe NS for detecting mitochondrial SO2 accurately in cancer cells. Biotin, possessing of high affinity for cancer cells, was decorated into probe to provide its cancer cell-targeting property. Moreover, the positive charge hemicyanine group was used to anchor mitochondria selectively. A series of spectral results from concentration titration, dynamics and selectivity experiments showed that NS had high sensitivity, fast response and high selectivity to SO2. These properties render NS ability for detecting SO2 in living cells. In biological imaging, the achievements in detecting exogenous and endogenous SO2 displayed the probe had favorable response to SO2 in living cells with well biocompatibility. Significantly, assisted by competitive experiments with excess biotin, NS demonstrated distinct cancer cell-targeting for detecting mitochondrial SO2. Furthermore, NS could locate mitochondria specially and detect mitochondrial SO2 in cancer cells by co-localization. Moreover, NS can trace SO2 in zebrafish with long wavelength emission. Therefore, NS can achieve in tracing mitochondrial SO2 selectively in cancer cells. It would be a powerful tool for well defining the physiological and pathological roles of mitochondrial SO2 in cancer cells.

A multi-signal mitochondria-targetable fluorescent probe for simultaneously discriminating Cys/Hcy/H2S, GSH, and SO2 and visualizing the endogenous generation of SO2 in living cells

Abstract Reactive sulfur species (RSS) include sulfur dioxide (SO2) and various biothiols, such as hydrogen sulfide (H2S), glutathione (GSH), cysteine (Cys), and homocysteine (Hcy). These RSS are closely related to each other through complicated symbiotic networks, and play indispensable roles in multiple pathophysiological processes. Due to their similarity in chemical properties, it is challenging for developing a single fluorescent probe to differentiate them simultaneously. Herein we report the first mitochondria-targetable fluorescent probe NIR-NBD for simultaneous discrimination of Cys/Hcy/H2S, GSH, and SO2 from each other. The probe is characterized of the presence of three different types of electrophilic sites, and displays multiple sets of signal pattern in response to Cys/Hcy/ H2S, GSH, and SO2, including red-green for Cys/Hcy/H2S, red for GSH, and green for SO2. Notably, the probe exhibits excellent selectivity, outstanding sensitivity (detection limit = 3.5 nM) and fast response (within 4 min) toward SO2. Furthermore, the probe has been successfully applied for monitoring mitochondrial SO2 and various biothiols, visualizing the endogenous generation of SO2, and probing into the role of biothiols during apoptosis process.

Developing a ratiometric two-photon probe with baseline resolved emissions by through band energy transfer strategy: Tracking mitochondrial SO2 during neuroinflammation

Sulfur dioxide (SO2) with the largest quantity and widest distribution in the atmosphere is closely related to many nervous system diseases via mitochondria respiration. It is of great significance to monitor this gaseous molecule during various physiological and pathological processes, but currently the task still remains challenging due to the lack of reliable tools. Through-bond energy transfer (TBET) is a relatively new strategy to fabricate ratiometric fluorescent probes, which does not need spectral overlap between the energy donor and acceptor while provides high energy-transfer efficiency. It offers strong dual fluorescence emission peaks as well as large wavelength differences between the two peaks, which increases the bioimaging resolution and reliability. Herein, we developed a TBET-based ratiometric probe (TBET-SO2) with a series of superior properties for in vivo SO2 imaging. Excited by near-infrared pulsed laser (810 nm), the probe undergoes TBET and produces far-red emission (611 nm). It achieved significant energy-transfer efficiency (90.5%) and large spectral gap between two peaks (△λ = 118 nm). Upon reacting with SO2, TBET-SO2 showed ~30-fold enhancement of ratiometric signal contributed by the baseline resolved emissions. A detection limit of as low as 0.09 μM was obtained. Furthermore, TBET-SO2 was successfully applied for visualizing the mitochondrial SO2 in living cells and mice brain tissue during the neuroinflammation process induced by SO2 pollution.

Design of a ratiometric two-photon fluorescent probe for dual-response of mitochondrial SO2 derivatives and viscosity in cells and in vivo

The content of sulfur dioxide (SO2) in mitochondria is related to various physiological and pathological processes. The irregular viscosity in mitochondria would result in mitochondria disorders. In this research, a novel ratiometric two-photon fluorescent probe (MBCB) based on carbazole framework has been strategically designed and synthesized for dual-detection of mitochondrial SO2 derivatives and viscosity. The results show that probe MBCB is capable of responding to SO2 derivatives and viscosity using dual-color imaging. MBCB can not only recognize SO2 derivatives utilizing a ratiometric change of fluorescent intensity (I434 nm/I600 nm), but also shows a high sensitivity and selectivity for monitoring SO2. On the other hand, the fluorescent intensity ratio (I567 nm/I415 nm) of MBCB increased with the increment of viscosity and exhibited a good logarithmic linear relationship between the ratio and viscosity (R2 = 0.99). Satisfactorily, probe MBCB has a good targeting ability to mitochondria and a low cell cytotoxicity, and can be successfully applied to detect HSO3− in living cells and in vivo. Furthermore, the probe can visualize the change of mitochondrial viscosity induced by nystatin.

Dual-detection of mitochondrial viscosity and SO2 derivatives with two cross-talk-free emissions employing a single two-photon fluorescent probe

A versatile two-photon fluorescent probe MCB for dual-detection of viscosity and SO2 derivatives in mitochondria with two cross-talk-free emissions (wavelength difference of 174 nm) has been rationally designed and synthesized. To our delight, probe MCB can not only recognize SO2 derivatives utilizing a blue fluorescence emission at 392 nm with a low detection limit (2 nM) and a fast response time (˜ 60 s), but also monitor viscosity by a red fluorescence emission at 566 nm in the methanol/glycerol mixed viscosity system. The viscosity value showed a good linearity with the fluorescence lifetime of MCB (R = 0.99, x = 0.43). Probe MCB was successfully applied to monitor viscosity and exogenous/endogenous SO2 utilizing the two cross-talk-free channels (red and blue) in mitochondria via two-photon microscopy (TPM) owe to its good two-photon absorption performance and mitochondria-located capability. Probe MCB can detect the change of mitochondrial viscosity during nystatin-induced apoptosis. Mitochondrial viscosity mapping was studied quantificationally via fluorescence lifetime imaging microscopy (FLIM). Furthermore, TP fluorescence dual-color imaging of MCB in live zerbrafishes demonstrate it can realize the dual-response of viscosity and SO2 derivatives in vivo.

A ratiometric fluorescent probe for detection of exogenous mitochondrial SO2 based on a FRET mechanism.

A novel imidazo[1,5-a]pyridine-hemicyanine based ratiometric fluorescent probe for detection of mitochondrial SO2 was designed and synthesized. The probe is based on a fluorescence resonance energy transfer (FRET) mechanism. It exhibits high selectivity and sensitivity towards SO32− with a fast response time (3 min) and detection limit of 0.13 μM. Further, it showed low cytotoxicity and was successfully applied to image exogenous mitochondrial SO2 in cells.

A mitochondria-targeted and deep-red emission ratiometric fluorescent probe for real-time visualization of SO2 in living cells, zebrafish and living mice.

Sulfur dioxide (SO2) plays a vital role in physiological and pathological processes. Excessive inhalation of SO2 is associated with several diseases. However, the potential physiological mechanisms of SO2 in living systems are still unclear due to the lack of an effective technique for imaging SO2 in real time in living mice. Herein, a ratiometric fluorescent probe CSP has been constructed for monitoring SO2. The novel probe CSP displayed fast sensing of SO2, excellent selectivity and photostability. More importantly, CSP was effectively employed for the detection of mitochondrial SO2 not only at the cellular level but also in zebrafish and living mice.

Development of a mitochondrial-targeted ratiometric probe for the detection of SO2 in living cells and zebrafishes.

Sulfur dioxide (SO2), as the fourth gas signal molecule, is closely related to many diseases including respiratory diseases, nervous system diseases, cardiovascular diseases and cancers. Herein, we present a novel mitochondria-targeted ratiometric fluorescent probe (CS) for the detection of SO2 in living cells and zebrafishes. The probe CS was constructed on the base of coumarin and benzopyranium fluorophores, and exhibited intense blue and red fluorescence simultaneously. After the response to SO2, the fluorescence at 433 nm enhanced significantly while the fluorescence at 683 nm decreased obviously. The probe CS has a high sensitivity and selectivity to SO2. Moreover, the probe CS has been successfully applied to the detection of mitochondrial SO2 in living cells and zebrafishes.

Rationally Optimized Fluorescent Probe for Imaging Mitochondrial SO2 in HeLa Cells and Zebrafish.

Organisms have built up immunological systems, where mitochondrial SO2 plays conflicting roles in regulating cell apoptosis. However, no exploration on the influence and regulating principle of mitochondrial SO2 to the specific apoptosis type can be found, which brings about a challenge to fluorescent probes. Herein, we optimize the fluorophore and develop a new fluorescent probe FHMI (( E)-4-(3-formyl-4-hydroxystyryl)-1-methylpyridin-1-iumiodide) by equipping an ICT (intramolecular charge transfer) fluorophore HMII (( E)-4-(4-hydroxystyryl)-1-methylpyridin-1-ium iodide) with an aldehyde group that serves as both fluorescence quencher and reporting group. After the optimization, although the nonconjugated electron donor is formed when sensing SO2, the preset ICT fluorophore HMII is permitted to release the fluorescence at the enlarged wavelength. Compared with the traditional design, the probe FHMI exhibits obvious enhanced fluorescence with large red shift. FHMI is successfully applied to the mechanistic exploration of the dichotomous effects of mitochondrial SO2 to cells apoptosis, showing that mitochondrial SO2 regulates the early apoptosis of HeLa cells via the reduction of mitochondrial membrane potential. FHMI is applied to explore the dichotomous bioinfluence of mitochondrial SO2 to HeLa cells under oxidative stress, visualizing the regulative role of mitochondrial SO2 in the apoptotic process. For the first time, the mitochondrial SO2 is visually found to be closely associated with the early apoptosis of HeLa cells. Moreover, FHMI proves to be readily applicable to monitoring endogenous SO2 in zebrafish. This probe can act as an effective optical tool for exploring SO2 in biospecimen.

A fluorescent and colorimetric probe for imaging the mitochondrial sulfur dioxide in living cells

Sulfur dioxide (SO2), one of active sulfur species (RSS), plays important roles in various physiologies and pathological processes. Hence, it is urgent to monitor the fluctuation of mitochondrial SO2in vivo. In this work, a novel mitochondria-targeting probe CZ was designed and synthesized based on carbazole derivative, which showed that probe was employed to the detection of SO2 with high selectivity, rapid response as well as naked eyes. Besides, an excellent linear relationship (R2 = 0.9977) was observed and the detection limit was calculated as low as 0.504 μM (3σ/k). Surprisingly, with addition of SO2 derivative, fluorescence intensity ratios (F475 nm/F607 nm) of probe CZ increased by 1104-fold ranging from 0.007 to 7.73. Owing to its biocompatibility, probe CZ was also successfully employed for monitoring of mitochondrial SO2 by fluorescence confocal microscopy.

Novel Tumor-Specific and Mitochondria-Targeted near-Infrared-Emission Fluorescent Probe for SO2 Derivatives in Living Cells

Endogenous sulfur dioxide (SO2) is an important gaseous signal molecule, which was also regarded as one of the reactive sulfur spaces (RSS) and closely related to cardiovascular diseases and many neurological disorders. However, the design and synthesis of fluorescent probes with near-infrared-emission which can detect mitochondrial SO2 and its derivatives in living cells still remain unresolved. Herein, a biotin and coumarin-benzoindole conjugate BCS-1 was presented as a ratiometric and colorimetric fluorescent probe for tracing SO2 derivatives with excellent selectivity and rapid responsibility. Notably, it is the first mitochondria-targeted near-infrared-emission probe that could selectively detect SO2 in tumor cells. BCS-1 could selectively enter into mitochondria of tumor cells, and the detection limit for SO2 derivatives was determined as 72 nM.

A mitochondria-targeted ratiometric fluorescent probe to monitor endogenously generated sulfur dioxide derivatives in living cells

Sulfur dioxide (SO2) can be endogenously produced by enzymes in mitochondria during oxidation of H2S or sulphur-containing amino acids, and plays important roles in several physiological processes. However, the design and synthesis of fluorescent probes which can detect mitochondrial SO2 and its derivatives in living cells still remain unresolved. Herein, we report the preparation of a lipophilic cationic dye 1 (Mito-Ratio-SO2), which targets the mitochondria in living cells and is sensitive to the presence of SO2 derivatives. The ratiometric probe Mito-Ratio-SO2 displays a 170 nm blue-shift in emission with two well-resolved emission bands upon addition of sulfite. Mechanistic studies indicate that three probe-SO2 adducts coexist after reaction, as supported by liquid chromatography and density function theory investigations. Importantly, the ratiometric probe is highly selective for sulfite over other bio-species including H2S. Fluorescence co-localization studies indicate that the probe localizes solely in the mitochondria of HeLa cells. Last but not least, fluorescent imaging of HeLa cells successfully demonstrates the detection of intrinsically generated intracellular SO2 derivatives in living cells.