Mitochondria-targeted Fluorescent Probe Research Articles

Accurate visualization colorectal cancer by monitoring viscosity variations with a novel mitochondria-targeted fluorescent probe

Colorectal cancer (CRC) is one of the most prevalent malignant tumors worldwide, exhibiting high morbidity and mortality. Lack of efficient tools for early diagnosis and surgical resection guidance of CRC have been a serious threat to the long-term survival rate of the CRC patients. Recent studies have shown that relative higher viscosity was presented in tumor cells compared to that in normal cells, leading to viscosity as a potential biomarker for CRC. Herein, we reported the development of a series of novel viscosity-sensitive and mitochondria-specific fluorescent probes (HTB, HTI, and HTP) for CRC detection. Among them, HTB showed high sensitivity, minimal background interference, low cytotoxicity, and significant viscous response capability, making it an ideal tool for distinguishing colorectal tumor cells from normal cells. Importantly, we have successfully utilized HTB to visualize in a CRC-cells-derived xenograft (CDX) model, enriching its medical imaging capacity, which laid a foundation for further clinical translational application.

Multifunctional fluorescence probe for simultaneous detection of viscosity, polarity, and ONOO− and its bioimaging in vitro and vivo

Intracellular microenvironment (viscosity and polarity) and peroxynitrite ions (ONOO−) are involved in maintaining cell morphology, cell function, and signaling so that it is crucial to explore their level changes in vitro and vivo. In this work, we designed and synthesized a mitochondria-targeted fluorescence probe XBL for monitoring the dynamic changes of viscosity, polarity, and ONOO− based on TICT and ICT mechanism. The fluorescence spectra showed obvious changes for polarity at 500 nm as well as ONOO− and viscosity at 660 nm, respectively. The XBL can image simultaneously viscosity, polarity, and ONOO− in cells, and the results showed excess ONOO− leaded to the increase of viscosity in mitochondrial. The ferroptosis process was accompanied by increase of intracellular viscosity and ONOO− levels (or decrease of polarity), which allowed us to better understand the relevant physiological and pathological processes. The XBL can distinguish normal cells and cancerous cells by the fluorescence intensity changes in green and red channels, and image viscosity in inflamed mice. Thus, XBL can provided the chemical tool to understand the physiological and pathological mechanisms of disease by simultaneous detection of viscosity, polarity and ONOO−.

A mitochondria-targeting fluorescent probe for the dual-emission fluorescence-enhanced detection of hydrogen sulfide and turn-on detection of hydrazine

A fluorescent probe (probe 1) with selectivity and sensitivity for H2S and N2H4 has been developed. Probe 1 exhibits dual-excitation and dual-emission fluorescence response to H2S and fluorescence enhancement toward N2H4 with distinct naked-eye color changes. Moreover, probe 1 displayed low detection limits for H2S and N2H4 of 0.90 nM and 23.6 nM, respectively. The reaction mechanism of probe 1 toward H2S and N2H4 was established using 1H NMR and HR-MS spectroscopy. Additionally, probe 1 was used for monitoring and differentiating H2S and N2H4 in HeLa cells and displays good biocompatibility and low cytotoxicity.

Real-time monitoring of abnormal mitochondrial viscosity in glioblastoma with a novel mitochondria-targeting fluorescent probe.

Glioblastoma is the most fatal and insidious malignancy, due to the existence of the blood-brain barrier (BBB) and the high invasiveness of tumor cells. Abnormal mitochondrial viscosity has been identified as a key feature of malignancies. Therefore, this study reports on a novel fluorescent probe for mitochondrial viscosity, called ZVGQ, which is based on the twisted intramolecular charge transfer (TICT) effect. The probe uses 3-dicyanomethyl-1,5,5-trimethylcyclohexene as an electron donor moiety and molecular rotor, and triphenylphosphine (TPP) cation as an electron acceptor and mitochondrial targeting group. ZVGQ is highly selective, pH and time stable, and exhibits rapid viscosity responsiveness. In vitro experiments showed that ZVGQ could rapidly recognize to detect the changes in mitochondrial viscosity induced by nystatin and rotenone in U87MG cells and enable long-term imaging for up to 12 h in live U87MG cells. Additionally, in vitro 3D tumor spheres and in vivo orthotopic tumor-bearing models demonstrated that the probe ZVGQ exhibited exceptional tissue penetration depth and the ability to penetrate the BBB. The probe ZVGQ not only successfully visualizes abnormal mitochondrial viscosity changes, but also provides a practical and feasible tool for real-time imaging and clinical diagnosis of glioblastoma.

A mitochondria-targeted fluorescent probe with viscosity sensitivity to distinguish between normal and cancer cells

Viscosity is one of the key factors of the mitochondrial microenvironment, and any abnormal change in the viscosity is not conducive to the normal functioning of the mitochondria.

A red-emitting mitochondria targetable fluorescent probe for detecting viscosity in HeLa, zebrafish, and mice.

Viscosity, an essential parameter of the cellular microenvironment, has the ability to indicate the condition of living cells. It is closely linked to numerous diseases like Alzheimer's disease, diabetes, and cardiovascular disorders. Therefore, it is necessary to design tools to effectively monitor viscosity changes, which could provide promising avenues for therapeutic interventions in these diseases. Herein, we report a novel mitochondria-targeting fluorescent probe GX-VS which was suitable for the detection of viscosity changes in vivo and in vitro. The probe GX-VS had many advantages such as long emission wavelength (650 nm), large Stokes shift (105 nm), significant fluorescence enhancement (59-fold), high sensitivity, good biocompatibility and so on. Biological experiments showed that the probe could target mitochondria and detect viscosity alterations in HeLa cells. Moreover, it has been successfully utilized to monitor viscosity changes induced by lipopolysaccharides (LPS) in inflammatory zebrafishes and living mice, which further underscored the capacity of GX-VS to explore fluctuations in viscosity within living organisms.

Discovery of a mitochondria-targeted fluorescent probe as TrxR inhibitors for cancer therapy

Developing fluorescent inhibitors to target subcellular organelles is a promising approach for precision therapy.

Mitochondria-targeted fluorescent probe for simultaneously imaging viscosity and sulfite in inflammation models.

Many diseases in the human body are related to the overexpression of viscosity and sulfur dioxide. Therefore, it is essential to develop rapid and sensitive fluorescent probes to detect viscosity and sulfur dioxide. In the present work, we developed a dual-response fluorescent probe (ES) for efficient detection of viscosity and sulfur dioxide while targeting mitochondria well. The probe generates intramolecular charge transfer by pushing and pulling the electron-electron system, and the ICT effect is destroyed and the fluorescence quenched upon reaction with sulfite. The rotation of the molecule is inhibited in the high-viscosity system, producing a bright red light. In addition, the probe has good biocompatibility and can be used to detect sulfite in cells, zebrafish and mice, as well as upregulation of viscosity in LPS-induced inflammation models. We expect that the dual response fluorescent probe ES will be able to detect viscosity and sulfite efficiently, providing an effective means of detecting viscosity and sulfite-related diseases.

A single mitochondria-targetable fluorescent probe for visualizing cysteine and glutathione in ferroptosis of myocardial ischemia/reperfusion injury

Ferroptosis plays an important role in the early stage of myocardial ischemia/reperfusion (MI/R) injury, which is closely associated with the antioxidant damage of mitochondrial cysteine (Cys)/glutathione (GSH)/glutathione peroxidase 4 (GPX4) axis. Visualization of Cys and GSH in mitochondria is meaningful to value ferroptosis and further contributes to understanding and preventing MI/R injury. Herein a mitochondria-targetable thiols fluorescent probe (MTTP) was designed and synthesized based on sulfonyl benzoxadiazole (SBD) chromophore with a triphenylphosphine unit as the mitochondria-targeted functional group. Cys and GSH can be differentiated by MTTP with two distinguishable emission bands (583 nm and 520 nm) through the controllable aromatic substitution-rearrangement reaction. Importantly, MTTP is capable of monitoring ferroptosis and its inhibition by measuring mitochondrial Cys and GSH. MTTP was also employed to non-invasively detect ferroptosis during oxygen and glucose deprivation/reoxygenation (OGD/R)-induced MI/R injury in H9C2 cells. In a word, MTTP provides a visual tool that can simultaneously detect Cys and GSH to monitor ferroptosis processes during MI/R injury, which helps for more deeper understanding of the role of ferroptosis in MI/R injury-related diseases.

Carbazole-based mitochondria-targeted fluorescent probes for in vivo viscosity and cyanide detection in cells and zebrafish

Cells of most eukaryotic species contain mitochondria, which play a role in physiological processes such as cellular senescence, metabolism, and autophagy. Viscosity is considered a key marker for many illnesses and is involved in several crucial physiological processes. Cyanide (CN−) can target cytochrome-c oxidase, disrupting the mitochondrial electron transport chain and causing cell death through asphyxiation. In this study, a fluorescent probe named HL-1, which targets mitochondria and measures viscosity and CN− levels, was designed and synthesized. HL-1 is viscosity-sensitive, with a linear correlation coefficient of up to 0.992. In addition, HL-1 was found to change color substantially during a nucleophilic addition reaction with CN−, which has a low detection limit of 47 nM. HL-1 not only detects viscosity and exogenous CN− in SKOV-3 cells and zebrafish but also monitors viscosity changes during mitochondrial autophagy in real time. Furthermore, HL-1 has been used successfully to monitor changes in mitochondrial membrane potential during apoptosis. Endogenous CN− in plant samples was quantified. HL-1 provides new ideas for studying viscosity and CN−.

A large Stokes shift and emission shift ratiometric mitochondria-targeted fluorescent probe for sensing HClO with an ESIPT-ICT integrated platform

Hypochlorous acid (HClO) is regarded as a significant oxidative stress marker and plays key factor in various pathological and physiological processes. However, the overexpression of HClO is relation to variety of diseases due to cellular damage and oxidative stress. Studies have described that the HClO level in mitochondria is crucial to maintain the normal function of mitochondrial. Herein, we presented a simple mitochondria-targeted fluorescent probe (BA-MR), which consists of the benzothiazole dye as the fluorescent signaling unit, the methyl thioether as the HClO recognition group, and the imine pyridinium salt moiety as the mitochondria targeting group. The free probe BA-MR possessed a super large Stokes shift (260 nm) and displayed a red fluorescent at 600 nm. Upon addition of HClO, the methyl thioether of BA-MR was oxidezed exclusively into a sulfoxide group, thereby leading to the generation of compound BAO-MR with a prominent blueshift of emission at 450 nm. As expected,the probe displayed highly sensitive and selective fluorescence response to HClO with fast response (120 s) and a low detection limit (86 nM). Importantly, the newly-synthesized probe was displayed to have low cytotoxicity, and could be successfully used to imaging HClO in mitochondria, which demonstrated the probe BA-MR can act as an effective tool for researching HClO-related physiological and pathological processes.

A multi-signal mitochondria-targeted fluorescent probe for simultaneously distinguishing biothiols and realtime visualizing its metabolism in cancer cells and tumor models

Biothiols and its metabolite SO2 derivatives play vital roles in various physiological processes. Although a few probes have been designed for monitoring the metabolism of biothiols, developing multi-signal fluorescent probes with practicability for simultaneously distinguishing biothiols (GSH, Cys and Hcy) and real-time visualizing SO2 derivatives is an enormous challenge. To better visualize biothiols metabolism in vitro and vivo, we developed a novel multi-signal NIR fluorescent probe (probe 2) with mitochondria-targeted for distinguishing biothiols and its metabolism, based on an ICT-PET synergetic mechanism. Probe 2 with dual recognition sites distinguishing detected Cys/Hcy (Red-Green), GSH (Green) and SO32− (Blue) via three channels. First probe 2 distinguished Cys and GSH to estimate main biothiols in living cells through the ratio changes of two well-defined emission bands (Red-Green), and then imaged its metabolite SO2 with ratiometric fluorescence (Red-Blue), eliminating the interference by different biothiols. Notably, probe 2 exhibits satisfactory sensitivity (detection limit: 0.21, 0.13, 0.14 and 3.06 μM for Cys, Hcy, GSH and SO32−, respectively), high selectivity, reliability at physiological pH, and rapid fluorescence response (within 10 min). Given these advantages, probe 2 has been successfully applied to the real-time monitor GSH metabolic process in MCF-7 cells and biothiols metabolism in breast cancer, suggesting biothiols metabolic changes might be a diagnostic indicator during cancer treatment. So probe 2 is a convenient and efficient tool for understanding the physiological functions of biothiols and its metabolism.

Mitochondrial-targeting fluorescent probe awakened by nitroreductase for hypoxic imaging and surgical navigation of solid tumors

As a typical signal of hypoxia, the up-regulation of nitroreductase (NTR) expression has been widely used to evaluate the anoxic microenvironment of solid tumors. Herein, we propose a near infrared (NIR) fluorescent probe Q-NTR wakened up by NTR in mitochondria. The presence of strong electron-absorbing group nitro can quench fluorescence effectively and provide a sufficiently low background signal. Probe Q-NTR can detect the activity of NTR to evaluate the degree of hypoxia in tumor tissues. Due to its NIR emission, subcellular structure level imaging, and rapid response characteristics, it provides a practical tool for studying NTR activity and hypoxia related biological processes. This sensing scheme has been successfully applied to monitor endogenous NTR activity in subcellular structures (in mitochondria) in anoxic microenvironments and selectively stain cells in anoxic conditions, demonstrated its great potential in early cancer diagnosis. In addition, probe Q-NTR could accurately locate the hypoxic environment, achieving precise navigation during solid tumor resection. In summary, probe Q-NTR will lay the foundation for further research on cell metabolism, tumor metabolism, and solid tumor resection under hypoxic conditions in the future.

Oxidative cleavage of alkene: A new strategy to construct a mitochondria-targeted fluorescent probe for hydrogen peroxide imaging in vitro and in vivo

Hydrogen peroxide (H2O2) is a significant reactive oxygen species (ROS) mainly produced in mitochondria, and it plays crucial roles in many physiological processes. Till now, a plenty of fluorescent probes relying on various organic reactions have been explored for H2O2. However, these probes still suffered from some problems, including the interference from ONOO– and ClO–, unsatisfying limit of detection (LOD > 100 nM), long response time (over 30 min) and lacking mitochondria-targeting ability. Herein, we employ the oxidative cleavage reaction of alkene as a novel sensing strategy, and successfully construct a turn-on fluorescent probe TBBP–Pro for detecting H2O2 in biological system. In this probe, the cyanovinyl-pyridinium moiety not only acts as a specific response site for H2O2, but also targets mitochondria efficiently. Comparing with previous H2O2 probes, TBBP–Pro possesses the advantages of high selectivity, excellent sensitivity (LOD = 47 nM) and fast response (within 5 min) toward H2O2. Additionally, TBBP–Pro successfully visualizes the endogenous H2O2 in mitochondria of HepG2 cells and zebrafish larvae. More importantly, TBBP–Pro is promising to monitor the dynamic change of H2O2 level for the diagnosis and treatment of inflammatory mice.

A dual-lock-controlled mitochondria-targeted ratiometric fluorescence probe for simultaneous detection of atherosclerosis-related HClO and viscosity

Precise detection of inflammatory microenvironment-related viscosity and hypochlorous acid (HClO) contributes to illuminating the pathogenesis and further diagnosing of atherosclerosis (AS). Herein, a dual-lock-controlled mitochondria-targeted fluorescence probe (NS) for simultaneous imaging of HClO and viscosity in AS-related foam cells is presented. NS performs linear increase in green-fluorescence along with increased viscosity (excited at 425 nm), permitting “off–on” fluorescence imaging of viscosity. Meanwhile, upon HClO activation, NS exhibits red-shifted and enhanced fluorescence in orange, thus leading to ratiometric fluorescence quantification of HClO (excited at 465 nm). Such dual-lock-controlled effect makes NS realize simultaneous imaging of viscosity and HClO with high sensitivity and selectivity via “off–on” and ratiometric fluorescence readouts, respectively. Besides, endowed with mitochondria-targeting capacity, NS achieves in situ imaging of mitochondria viscosity and HClO in living RAW264.7 cells. Importantly, for the first time, NS realizes simultaneous imaging of mitochondria viscosity and HClO in macrophage-derived foam cells, revealing the close association between HClO level and viscosity change in mitochondria during foaming translation of macrophages in atherogenesis. This work not only provides a novel strategy and tool to image organelle-located viscosity and HClO in living systems, but also holds great potential in early diagnosis of AS.

Intracellular biothiols assisted triple-channel imaging of mitochondrial HClO in living cells

A robust mitochondria-targetable fluorescent probe, MBP, for HClO was judiciously constructed by directly connecting a leucomethylene moiety and a pyronin dye through an o-phenylenediamino linker. Under the assistance of intracellular Cys and GSH, MBP can selectively sense HClO through an unrivalled triple-channel fluorescence (peaked at 541, 620, and 690 nm, respectively) turn-on manner for the first time, and the related LOD based on distinct channels data were calculated to be 17, 9, and 31 nM, respectively. This distinctive fluorescence signal response pattern can provide mutual signals for corroboration, which may have the potential to significantly diminish the interferences aroused from external factors, thereby guaranteeing more reliable experimental data outputting. Moreover, MBP also displayed some other powerful properties, including good selectivity, fast response (within 180 s) toward HClO, and excellent mitochondrial-targeting potency (the corresponding Pearson's coefficient was determined to be 0.96). Noteworthily, the application of MBP for triple channel fluorescence image of exogenous/endogenous HClO in living cells has been successfully achieved, thereby holding the potential for biological investigations.

Screening of H2S donors with a red emission mitochondria-targetable fluorescent probe: Toward discovering a new therapeutic strategy for Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder caused by various factors such as neuroinflammation, oxidative stress, mitochondrial dysfunction, and neuronal apoptosis. Recent studies have shown that H2S supplementation reverses neuronal loss and mitigates motor deficits in PD patients through anti-inflammatory, antioxidant, improved mitochondrial function and proautophagic. Therefore, the discovery and use of H2S donors may be an exciting and intriguing strategy for the treatment of PD. Herein, we report a red emission mitochondria-targetable fluorescent probe, Rho-H2S, which can specifically and sensitively detect H2S with a limit of detection of 62.5 nM. Bioimaging experiments have shown that the probe has excellent mitochondrial targeting and good imaging capabilities for the detection of exogenous and endogenous H2S in cells. More importantly, based on the Rho-H2S probe, we first confirmed the sulforaphane (SFN) among 15 glucosinolate and isothiocyanate compounds from cruciferous vegetables with an outstanding ability to release H2S and we further proved that SFN could alleviate the symptoms of PD in vivo. All results demonstrate that Rho-H2S could be an effective tool for screening H2S donors and can contribute to the development of new therapeutic strategies for PD.

Engineering a mitochondria-targeted fluorescent probe with long wavelength for rapid detection of H2S in beer, water and living cells

Hydrogen sulfide (H2S) is involved in the regulatory mechanisms of the human physiological system, it can also have adverse effects on the environment and food, resulting in health hazards. In this study, a novel fluorescent probe HMTI ((E)-2-(4-(diethylamino)-2-hydroxystyryl)-3-methylbenzo[d]thiazol-3-ium iodide)) for the discrimination of H2S was designed and synthesized. HMTI showed fluorescence signal quenching at 622 nm of long wavelength, fast response to H2S (30 s), low limit of detection (LOD = 99.68 nM), excellent ability of specific recognition and anti-interference. In addition, probe HMTI not only could be capable of detecting H2S in environmental water samples and beer, but also untilized for exogenous and endogenous H2S imaging in living cells and effectively targeting mitochondria. Collectively, these results laid the foundation for the successful development of a sensor that could be applicable for monitoring H2S in the environment, food and living cells.

A two-photon mitochondria-targeting azo reductase probe for imaging in tumor cells and mice

Herein, a two-photon mitochondria-targeting fluorescent probe (TPNP) which can be activated by the azo reductase (AzoR) has been developed for imaging the hypoxic microenvironment of tumors in vivo. The probe TPNP showed a low detection limit (LOD = 1.85 eq), high selectivity, and considerable steadiness in different pH conditions (3.0–11.0). In living cells, it could co-locate with the commercial dye in the mitochondria (co-localization coefficient: 0.92) and indicate hypoxia in both one-photon and two-photon fluorescence imaging. Its AzoR-activation mechanism was further verified by the docking simulation as well as the experiments of adding AzoR or electron transporter inhibitors. Moreover, the in vivo real-time fluorescence imaging proved that TPNP could monitor hypoxia and the tumor progression in mouse tumor models. Overall, this designed probe exhibited high sensitivity, synthetic ease, and excellent fluorescent properties, which might therefore enlighten new ideas for the diagnosis of tumors in future studies.

Mitochondrial targeting fluorescent probe for specific detection of nickel ions and biological imaging

Nickel ion, a kind of essential trace element, is indispensable for maintaining life health. Nickel ions in excess will induce a series of reactions in the body and be correlative with the diseases and even cancer. As one of the most important organelles, mitochondrion is responsible for maintaining the homeostasis of metal ions in cells. Therefore, sensitive detection of nickel ions in mitochondria contributes to further study the physiological function of mitochondria, and is of great significance for investigating body health. In this study, a simple and effective fluorescence probe method for the detection of nickel ions in organisms and environment was proposed. The probe has shown excellent spectral performance in vitro experiments, and the detection limits was as low as 17 nM. And it can be applied to the detection of nickel ions in environmental water samples with satisfactory results, the recovery rate is higher than 85%. More importantly, the probe can specifically target to mitochondria to realize the imaging of nickel ions in cells and provide an effective auxiliary tool for in-depth study of the relationship between mitochondrial function and metal ions. In addition, the probe has been successfully applied to the detection of nickel ions in zebrafish model, which further demonstrates the great potential and value of the probe in biological applications.