A Sequentially Activated Probe for Imaging of Superoxide Anion and Peroxynitrite in PC12 Cells under Oxidative Stress.

Superoxide anion (O2·-) and peroxynitrite (ONOO-), two important oxidants under oxidative stress, coexist in complex cell and organism systems, playing crucial roles in various physiological and pathological processes, particularly in neurodegenerative diseases. Despite the absence of robust molecular tools capable of simultaneously visualizing O2·- and ONOO- in biosystems, the relationship between these two species remains understudied. Herein, we present sequentially activated fluorescent probe, DHX-SP, which exhibits exceptional selectivity and sensitivity toward O2·- and ONOO-. This probe enables precise imaging of these species in living PC12 cells under oxidative stress conditions using distinct fluorescence signal combinations. Furthermore, the probe DHX-SP has the ability to visualize changes in O2·- and ONOO- levels during ferroptosis of PC12 cells and in the Parkinson's disease model. These findings establish a connection between the crosstalk of the phosphorus group of O2·- and ONOO- in PC12 cells under oxidative stress.

Multifunctional fluorescent probe for simultaneous revealing Cys and ONOO- dynamic correlation in the ferroptosis.

Ferroptosis is a type of lipid peroxidation-induced apoptosis brought on by imbalances in iron metabolism and redox. It involves both the thiol-associated anti-ferroptosis pathway and the excessive buildup of reactive oxygen species (ROS), which stimulates the ferroptosis pathway. Determining the precise control mechanism of ferroptosis requires examining the dynamic connection between reactive sulfur species (RSS) and ROS. Cysteine (Cys) and peroxynitrite (ONOO-) are highly active redox species in organisms and play dynamic roles in the ferroptosis process. In this study, a coumarin dye was conjugated with specific response sites for Cys and ONOO-, enabling the simultaneous detection of Cys and ONOO- through the green and red fluorescence channels, respectively (λem = 498 nm for Cys and λem = 565 nm for ONOO-). Using the probe LXB, we monitored the changes in Cys and ONOO- levels in the ferroptosis pathway induced by erastin. The results demonstrate a significant generation of ONOO- and a noticeable decrease in intracellular Cys levels at the beginning upon erastin treatment and finally maintains a relatively low level. This study presents the first probe to investigate the intracellular redox modulation and control between Cys and ONOO- during ferroptosis, providing valuable insights into the potential mutual correlation between Cys and ONOO- in this process.

Shedding light on ONOO- detection: the emergence of a fast-response fluorescent probe for biological systems.

ONOO-, a bioactive molecule, plays a critical role in inflammation-related signaling pathways and pathological mechanisms. Numerous studies have established a direct correlation between elevated ONOO- levels and tumor progression. Therefore, investigating ONOO- levels in inflammation and tumors is of utmost importance. Fluorescence imaging presents a highly sensitive, non-invasive, easily operable, selective, and efficient method for ONOO- detection in situ. In this study, we designed and synthesized a rhodamine-based probe, NRho, which effectively identifies tumors, inflammatory cells, tissues, and organs by detecting ONOO- content. The synthesis process of NRho is simple, yielding a probe with favorable spectral characteristics and rapid response. Our cell imaging analysis has provided novel insights, revealing distinct ONOO- levels among different types of cancer cells, with hepatocellular carcinoma cells exhibiting higher ONOO- content than the others. This observation marks the proposal of such variations in ONOO- levels across cancer cell types. Furthermore, our study has showcased the practicality of our probe in live organ imaging, enabling the identification of tumors from living organs within a brief 5-minute incubation period. Additionally, our findings highlight the rapid detection capability of the probe NRho in various tissue samples, effectively identifying inflammation. This research holds important promise in advancing biomedical research and clinical diagnosis.

Mitochondria-Targeting Multifunctional Fluorescent Probe toward Polarity, Viscosity, and ONOO- and Cell Imaging.

Abnormal changes occurring in the mitochondrial microenvironment are important markers indicating mitochondrial and cell dysfunction. Herein, we designed and synthesized a multifunctional fluorescent probe DPB that responds to polarity, viscosity, and peroxynitrite (ONOO-). DPB is composed of an electron donor (diethylamine group) and electron acceptor (coumarin, pyridine cations, and phenylboronic acid esters), in which the pyridine group with a positive charge is responsible for targeting to mitochondria. D-π-A structure with strong intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT) properties give rise to respond to polarity and viscosity. The introduction of cyanogroup and phenylboronic acid esters increases the electrophilicity of the probe, which is prone to oxidation triggered by ONOO-. The integrated architecture satisfies the multiple response requirements. As the polarity increases, the fluorescence intensity of probe DPB at 470 nm is quenched by 97%. At 658 nm, the fluorescence intensity of DPB increases with viscosity and decreases with the concentration of ONOO-. Furthermore, the probe is not only successfully used to monitor mitochondrial polarity, viscosity, and endogenous/exogenous ONOO- level fluctuations but also to distinguish cancer cells from normal cells by multiple parameters. Therefore, as-prepared probe provides a reliable tool for better understanding of the mitochondrial microenvironment and also a potential approach for the diagnosis of disease.

Novel Boronate Probe Based on 3-Benzothiazol-2-yl-7-hydroxy-chromen-2-one for the Detection of Peroxynitrite and Hypochlorite.

Derivatives of coumarin, containing oxidant-sensitive boronate group, were recently developed for fluorescent detection of inflammatory oxidants. Here, we report the synthesis and the characterization of 3-(2-benzothiazolyl)-7-coumarin boronic acid pinacol ester (BC-BE) as a fluorescent probe for the detection of peroxynitrite (ONOO-), with high stability and a fast response time. The BC-BE probe hydrolyzes in phosphate buffer to 3-(2-benzothiazolyl)-7-coumarin boronic acid (BC-BA) which is stable in the solution even after a prolonged incubation time (24 h). BC-BA is slowly oxidized by H2O2 to form the phenolic product, 3-benzothiazol-2-yl-7-hydroxy-chromen-2-one (BC-OH). On the other hand, the BC-BA probe reacts rapidly with ONOO-. The ability of the BC-BA probe to detect ONOO- was measured using both authentic ONOO- and the system co-generating steady-state fluxes of O2•- and •NO. BC-BA is oxidized by ONOO- to BC-OH. However, in this reaction 3-benzothiazol-2-yl-chromen-2-one (BC-H) is formed in the minor pathway, as a peroxynitrite-specific product. BC-OH is also formed in the reaction of BC-BA with HOCl, and subsequent reaction of BC-OH with HOCl leads to the formation of a chlorinated phenolic product, which could be used as a specific product for HOCl. We conclude that BC-BA shows potential as an improved fluorescent probe for the detection of peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of oxidant-specific products will help to identify the oxidants detected.

Detection of Intracellular Reactive Oxidative Species Using the Fluorescent Probe Hydroxyphenyl Fluorescein.

Various fluorescent probes for the detection of intracellular reactive oxidative species (ROS) have been developed because ROS levels are closely associated with cellular states. Here, we describe a method for detection of intracellular ROS in living cells using the fluorescent probe, hydroxyphenyl fluorescein (HPF), which detects hydroxyl radicals and peroxynitrite. NIH3T3 cells and p53 knockout (p53-/-) mouse embryonic fibroblasts (MEFs) were transformed by expressing oncogenic RAS using a retrovirus system. The cells were treated with HPF at 37 °C for 30 min, and subsequently, images were acquired using a confocal fluorescence microscope at an excitation wavelength of 488 nm after washing with PBS.

Nanoliposomal Ratiometric Fluorescent Probe toward ONOO- Flux.

Peroxynitrite (ONOO-), a powerful biological oxidant, is produced in the mitochondria and reacts with many biomolecular targets under various pathological conditions, leading to a range of disease states. In this work, we developed a nanoliposome-encapsulated ratiometrically fluorescent probe (NRF) based on a hemicyanine structure Cy-O obtained by facile synthesis. Upon reaction with ONOO-, the oxidation and hydrolysis of a π-conjugation system within the nanoliposome triggers a ratiometrically fluorescent response and a large-scale emission shift (238 nm), which provides a specific and sensitive means for the ONOO- detection. Moreover, we have performed DFT calculation at the 6-31+G(d,p) level using a suite of Gaussian 09 programs to obtain insights into the chemical structure optical properties of Cy-O. In addition, the practical applications of the nanoprobe to image exogenous and endogenous ONOO- were achieved further in live cells and animals triumphantly.

Acanthamoeba castellanii as an alternative interaction model for the dermatophyte Trichophyton rubrum.

BACKGROUND: Trichophyton rubrum (Tr) is the main aetiological agent of human dermatophytosis, being isolated from the environment and keratinised tissues. In the environment, Tr can interact with other organisms, such as free-living amoebas (FLA), which can act as an alternative host system to study the interaction between microbes and phagocytic cells. OBJECTIVES: To characterise the Acanthamoeba castellanii (ALX)-Tr interaction. METHODS: Interaction was characterised in three conditions: trophozoites (PYG), late (PYG/NES) and early (NES) encystation stimulus, evaluating encystation kinetics, phagocytosis, exocytosis and fungicidal activity dynamics. RESULTS: Tr was able to induce ALX encystation and be internalised by ALX. The number of internalised conidia was high at 1 hour, and ALX presented fungicidal activity with increased intracellular ROS production and exocytosis. In PYG/NES, phagocytosis and ROS production were reduced, with decreased ALX's fungicidal activity. However, in NES there was an increased fungal engulfment, and a reduced ROS production and higher fungal burden. Furthermore, exogenous mannose decreased phagocytosis of Tr conidia, and divalent cations induced ROS production and increased ALX's fungicidal activity. Interestingly, phagocytosis was reduced in the presence of cytoskeleton inhibitor, but exocytosis was increased, suggesting that Tr conidia may have alternative pathways to escape ALX's cells. CONCLUSION: A castellanii is a proper model for studying Tr-FLA interaction, since ALX can engulf, produce ROS and kill Tr, and all these parameters are influenced by an encystation stimulus and divalent cations. Moreover, this interaction is likely to occur in the environment implicating in the adaptation to environmental stressful conditions in both organisms.

Activatable Two-Photon Near-Infrared Fluorescent Probe Tailored toward Peroxynitrite In Vivo Imaging in Tumors.

A malignant tumor remains one of the leading causes of deaths across the world. Thus, diagnosis of tumor development with noninvasive visualizing methods is significant for tumor therapy. Herein, an activatable two-photon NIR fluorescent probe DHQ-Rd-PN for in vivo imaging of peroxynitrite in a tumor was elaborately designed. The probe demonstrated an increased NIR emission in response to peroxynitrite in vitro, which ensured that the probe detects ONOO- in cell and in vivo. Cellular imaging results disclosed that the probe was competent to detect adscititious ONOO- level change in HeLa cells, as well as endogenous ONOO- concentration in lipopolysaccharides (LPS) and IFN-γ-stimulated RAW 264.7 cells. Additionally, zebrafish in vivo imaging revealed that the probe accumulated in the pancreas and was lightened up by the addition of ONOO-. Remarkably, the probe can be harnessed to image an ONOO- production profile in xenograft 4T1 tumor mice by both one-photon and two-photon in vivo fluorescence imaging. Benefiting with the two-photon excitable properties and NIR emissive properties, the probe can be used for noninvasive in vivo imaging of ONOO- in the onset and development of tumors for the first time. This work provided a noninvasive and efficient detection method for ONOO- in a tumor, which would find more applications in tumor diagnosis and therapies.

High-Selectivity Fluorescent Reporter toward Peroxynitrite in a Coexisting Nonalcoholic Fatty Liver and Drug-Induced Liver Diseases Model.

Peroxynitrite (ONOO-), a highly reactive species, is profoundly involved in many physiological and pathological processes. Change of the ONOO- level usually indicates an abnormal body function. Thus, it is desired to develop a highly reliable ONOO- assay to elucidate its roles in a related disease environment. In this work, we have constructed a ratiometric molecule fluorescent probe RTFP toward ONOO- with high specificity by the combination strategy of probe screening and a rational design method. RTFP displayed excellent detection sensitivity (detection limit: 4.1 nM) and produced a highly ratiometric emission signal (130-fold). Leveraging this probe, we showed the change of ONOO- content in the free-fatty-acid-induced nonalcoholic fatty liver disease (NAFLD) and acetaminophen-induced drug-induced liver injury (DILI) cellular model and for the first time disclosed the involved mechanism of cytochrome P450 2E1 (CYP2E1) enzyme in NAFLD with a DILI pathological environment. Furthermore, RTFP also was utilized to visualize ONOO- fluctuation of living liver tissues in a high-fat-diet-caused NAFLD model. We expected that this probe may help the study of liver injury in the exploration of mechanism and signal path.

Product-boosted fluorescence signal: a new approach for designing small-molecule probes for detection of peroxynitrite.

In situ self-assembled boronate ester comprising commercially available 2-formylphenylboronic acid and 2-(2',3'-bihydroxyphenyl)benzothiazole (BHBT) is explored for the detection of ONOO- with product-boosted fluorescence. The self-assembly can detect ONOO- in the endoplasmic reticulum of living cells.

A novel highly selective fluorescent probe with new chalcone fluorophore for monitoring and imaging endogenous peroxynitrite in living cells and drug-damaged liver tissue.

As a member of the reactive nitrogen species (RNS) family, peroxynitrite (ONOO-) as an oxidant and nitrating mediator plays a significant role in some physiopathologic processes. The excessive production of peroxynitrite anion in a drug-damaged liver is a culprit of hepatotoxicity. The detection of peroxynitrite is of vital importance for the treatment of some diseases including cancer and liver injury. In this study, a novel turn-on fluorescent probe IC-ONOO with new chalcone fluorophore was designed and synthesized for the detection of in vitro and in vivo. The probe responded rapidly towards ONOO- (only within 15 min did the fluorescent intensity maximize), and was endowed with high sensitivity and excellent selectivity. Given the fact that the linear correlation between the fluorescent intensity at 560 nm and the concentrations of the probe ranged from 0 to 9 µM, the limit of detection (LOD) was calculated to be 3.1 × 10-8 M. With all the merits, probe IC-ONOO was qualified as a robust tool to monitor peroxynitrite anion under physiopathologic condition. Moreover, it was successfully applied in the imaging of endogenous peroxynitrite in living MCF-7 cells (Human breast carcinoma cells) and mouse drug-damaged liver tissue with low cytotoxicity. Given all the extraordinary merits, great potential has been seen in its application to other peroxynitrite related diseases.

A Silyl Ether Based Fluorescent Probe for Rapid Monitoring of Endogenous Peroxynitrite Concentration and Imaging in Living Cells through Multicolor Emission.

The peroxynitrite ion (ONOO- ) has important roles in many biological processes. We have developed a multicolor ONOO- -sensing probe (SiONNOH) that undergoes deprotonation and desilylation processes, which result in several changes in the emission wavelengths. In response to different concentrations of ONOO- , the probe exhibits fluorescence changes from pink (595 nm at 2 eq. ONOO- ) to green (540 nm at 6 eq. ONOO- ) via orange (3 eq. ONOO- ) and yellow (4 eq. ONOO- ) under physiological conditions until no fluorescence signal is observed after ONOO- is completely eliminated by lipoic acid. The probe shows the high selectivity for ONOO- and the limit of detection is calculated to be 1.27 µM. Moreover, the probe shows the capacity to monitor the concentration ranges of ONOO- through multicolor fluorescence in living cells, which will greatly facilitate the rapid detection of ONOO- concentration ranges by the naked eye under a UV light without any precision instrumentation.

Lysosome-Targeting Iridium(III) Probe with Near-Infrared Emission for the Visualization of NO/O2•- Crosstalk via In Vivo Peroxynitrite Imaging.

Nitric oxide (NO) and superoxide anions (O2•-) are two noteworthy reactive species implicated in various physiological and pathological processes, such as ROS-induced lysosomal cell death. The interaction ("crosstalk") between them may form a new mediator peroxynitrite (ONOO-) which has implications for cancer, diabetes, Alzheimer's disease, and liver-damage. It is therefore essential to investigate lysosomal NO/O2•- crosstalk in vivo through ONOO--responsive molecular tools in order to fully comprehend the physiological and pathological mechanisms involved. In this study, a lysosome-targeting iridium(III) complex, Ir-NIR, has been investigated as a near-infrared (NIR) phosphorescent probe for visualizing NO/O2•- crosstalk by the phosphorescent detection of endogenous ONOO- levels in vivo. Ir-NIR exhibits a rapid (within 200 s), highly sensitive, and approximately 100-fold enhanced response to ONOO- in phosphorescence intensity. Thus, these characteristics, coupled with good cell permeability and low cytotoxicity, enable the probe to be used to detect intracellular ONOO- living organisms both in vitro and in vivo.

A fast detection of peroxynitrite in living cells.

Peroxynitrite (ONOO-) plays a crucial role in the regulation of diverse pathophysiological processes, and high level of ONOO- is profound association with numerous diseases. Herein, we developed an anthraquinone-based fluorescent probe L for ONOO- determination by a new recognition mechanism: amido oxidized nitroso-group by ONOO-. Probe L with amine-based recognition receptor is more selective to ONOO- than other reactive oxygen species, including H2O2 and ClO-. Furthermore, ONOO- could be rapidly detected by probe L with a Limit of Detection of 13 nM. More importantly, L could be used to monitor intracellular ONOO- in SMMC-7721 cells.

Oxygen-Embedded Pentacene Based Near-Infrared Chemiluminescent Nanoprobe for Highly Selective and Sensitive Visualization of Peroxynitrite In Vivo.

Peroxynitrite (ONOO-) is involved in neurodegenerative, inflammatory, cardiovascular disorders, cancers, and other pathological progress. However, current imaging methods for sensing ONOO- usually suffer from high background/autofluorescence for fluorescent probes and poor selectivity/short emission wavelength for chemiluminescent probes. Herein, we present a novel chemiluminescent molecule (oxygen-embedded quinoidal pentacene) responsive to ONOO- for the first time, on the basis of which we rationally construct a near-infrared nanoprobe for detecting ONOO- via chemiluminescence resonance energy transfer (CRET) mechanism. Notably, our nanoprobe exhibits good selectivity, ultrahigh sensitivity (nanomole level), low background noise, fast response, and high water solubility. Moreover, the near-infrared emission from CRET offers higher tissue penetration of the chemiluminescent signal. Finally, our nanoprobe is further successfully applied to detecting endogenous ONOO- in mice with abdominal inflammation, drug-induced hepatotoxicity, or tumor models in vivo. In summary, the self-luminescing nanoprobes can act as an alternative visualizable tool for illuminating the mechanism of ONOO- involved in the specific pathological process.

AND logic gate based fluorescence probe for simultaneous detection of peroxynitrite and hypochlorous acid.

Hypochlorous acid (HOCl) and peroxynitrite (ONOO-) are two of the most important reactive species and associated with various diseases in various physiological and pathological processes. Nonetheless, many of their roles are still vague due to the shortage of methods for simultaneously detecting HOCl and ONOO-. Herein, three simple yet useful fluorogenic probes, LG-1, LG-2 and LG-3, have been fabricated with facile synthesis route and used to monitor the coexistence of HOCl and ONOO- as AND-based logic gate fluorescent probe firstly. LG-1 and LG-2, which consists of 1,3-oxathiolane group and boronate group respectively, were designed to verify the capacity of monitoring HOCl and ONOO- without interference from each other. The result showed that these two groups are perfect reaction sites of detecting HOCl and ONOO- respectively via specific analyte-induced reactions. Hence, LG-3, which is attached by these two groups to suppress the fluorophore core, can response to HOCl and ONOO- simultaneously without mutual interference and generate the significant time-dependent fluorescence enhancement. By investigating the absorption and fluorescence properties of LG-3 towards HOCl and ONOO- individually and collectively, the result confirmed clearly that LG-3 has the capacity of monitoring the coexistence of HOCl and ONOO-, which could act as a two-input AND-based logic gate fluorescent probe.

A time-resolved near-infrared phosphorescent iridium(iii) complex for fast and highly specific peroxynitrite detection and bioimaging applications.

Peroxynitrite (ONOO-), one of the reactive oxygen/nitrogen species (ROS/RNS) found in vivo, plays crucial roles in many physiological and pathological processes. The ability to selectively and sensitively determine ONOO-in vivo is important for the understanding of its biological roles. Thus, by utilizing the excellent chemical stability and photostability, high luminescence efficiency, and long luminescence lifetime of iridium complexes, we developed a novel near-infrared (NIR) phosphorescent iridium(iii) complex (FNO2) probe to detect ONOO- within seconds. The probe FNO2 showed better selectivity towards ONOO- over other interfering biomolecules, including O2- and ClO-. Moreover, it possessed a long luminescence lifetime, which enabled successful elimination of the interference from background fluorescence in vitro (simulated by Rhodamine B) in time-resolved emission spectra. Finally, in addition to its low cytotoxicity, the probe FNO2 showed emission wavelength in the NIR region and was able to specifically sense ONOO- induced in living cells and inflamed mouse models.

A hepatocyte-targeting near-infrared ratiometric fluorescent probe for monitoring peroxynitrite during drug-induced hepatotoxicity and its remediation.

A novel hepatocyte-targeting near-infrared fluorescent probe named Gal-NIR is developed. Gal-NIR shows ratiometric response to ONOO- with high sensitivity and selectivity. Moreover, the probe can accurately target the hepatocyte, and thus is used for assessing drug-induced hepatotoxicity and its remediation by using hepatoprotective medicines in living cells and mice.

A novel electrochemical sensor based on microporous polymeric nanospheres for measuring peroxynitrite anion released by living cells and studying the synergistic effect of antioxidants.

Peroxynitrite anion (ONOO-) is a crucial reactive nitrogen species (RNS), which has aroused immense research interest in the biological and biomedical fields because aberrant expression levels of ONOO- are related to many diseases. In this work, a novel electrochemical sensor is described for the detection of peroxynitrite anion (ONOO-) released from living cells. It is constructed with a glassy carbon electrode (GCE) decorated with a nanocomposite (CTS-MPNS) synthesized from chitosan (CTS) functionalized microporous polymeric nanospheres (MPNS). The prepared CTS-MPNS/GCE sensor shows a supernormal manifestation in measuring ONOO- in a wide range of concentrations from 3.83 nM to 0.104 mM, and the detection limit is as low as 1.28 nM (S/N = 3), which makes it possible to detect trace amounts of ONOO- released from U87 cells. Significantly, the synergistic effect of different antioxidants on scavenging ONOO- in biological systems is further studied by an electrochemical method for the first time, which provides an efficient strategy for protecting cells against oxidative stress. The developed platform and the efficient strategy may pave the way for their future applications in the field of biomedicine and the treatment of cancer diseases.