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A TCF-Based Carbon Monoxide NIR-Probe without the Interference of BSA and Its Application in Living Cells.

ABSTRACT: As toxic gaseous pollution, carbon monoxide (CO) plays an essential role in many pathological and physiological processes, well-known as the third gasotransmitter. Owning to the reducibility of CO, the Pd⁰-mediated Tsuji-Trost reaction has drawn much attention in CO detection in vitro and in vivo, using allyl ester and allyl ether caged fluorophores as probes and PdCl₂ as co-probes. Because of its higher decaging reactivity than allyl ether in the Pd⁰-mediated Tsuji-Trost reaction, the allyl ester group is more popular in CO probe design. However, during the application of allyl ester caged probes, it was found that bovine serum albumin (BSA) in the fetal bovine serum (FBS), an irreplaceable nutrient in cell culture media, could hydrolyze the allyl ester bond, and thus give erroneous imaging results. In this work, dicyanomethylenedihydrofuran (TCF) and dicyanoisophorone (DCI) were selected as electron acceptors for constructing near-infrared-emission fluorophores with electron donor phenolic OH. An allyl ester and allyl ether group were installed onto TCF-OH and DCI-OH, constructing four potential CO fluorescent probes, TCF-ester, TCF-ether, DCI-ester, and DCI-ether. Our data revealed that ester bonds of TCF-ester and DCI-ester could completely hydrolyze in 20 min, but ether bonds in TCF-ether and DCI-ether tolerate the hydrolysis of BSA and no released fluorescence was observed even up to 2 h. Moreover, passing through the screen, it was concluded that TCF-ether is superior to DCI-ether due to its higher reactivity in a Pd⁰-mediated Tsuji-Trost reaction. Also, the large stokes shift of TCF-OH, absorption and emission at 408 nm and 618 nm respectively, make TCF-ether desirable for fluorescent imaging because of differentiating signals from the excitation light source. Lastly, TCF-ether has been successfully applied to the detection of CO in H9C2 cells.

SUBMITTER: Wu Y

PROVIDER: S-EPMC9268636 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Carbon monoxide (CO) acts as an important gasotransmitter in delivering intramolecular and intermolecular signals to regulate a variety of physiological processes. This lipid-soluble gas can freely pass through the cell membrane and then diffuse to adjacent cells acting as a messenger. Although many fluorescent probes have been reported to detect intracellular CO, it is still a challenge to visualize the release behavior of endogenous CO. The main obstacle is the lack of a probe that can anchor onto the cell membrane while having the ability to image CO in real time. In this work, by grafting a polar head onto a long and linear hydrophobic Nile Red molecule, a cell membrane-anchored fluorophore ANR was developed. This design strategy of a cell membrane-anchored probe is simpler than the traditional one of using a long hydrophobic alkyl chain as a membrane-anchoring group, and endows the probe with better water solubility. ANR could rapidly bind to the cell membrane (within 1 min) and displayed a long retention time. ANR was then converted to a CO-responsive fluorescent probe (ANRP) by complexation with palladium based on a metal palladium-catalyzed reaction. ANRP exhibited a fast response to CO with a 25-fold fluorescence enhancement in vitro. The detection limit was calculated to be 0.23 μM, indicating that ANRP is sensitive enough to image endogenous CO. Notably, ANRP showed excellent cell membrane-anchoring ability. With ANRP, the release of CO from HepG2 cells under LPS- and heme-stimulated conditions was visualized and the cell self-protection effect during a drug-induced hepatotoxicity process was also studied. Moreover, ANRP was successfully applied to the detection of intracellular CO in several cell lines and tissues, and the results demonstrated that the liver is the main organ for CO production, and that cancer cells release more CO from their cells than normal cells. ANRP is the first membrane-anchored CO fluorescent probe that has the ability to reveal the relationship between CO release and diseases. It also has prospects for the studying of intercellular signaling functions of CO.

Project description:Carbon monoxide (CO) is not only known as a toxic gas due to its characteristics as an odorless molecule and its rapid binding to haem-containing molecules, thus inhibiting the respiratory chain in cells resulting in hypoxia. For decades, scientists established evidence about its endogenously production in the breakdown of haem via haem-oxygenase (HO-1) and its physiological effects. Among these, the modulation of various systems inside the body are well described (e.g., anti-inflammatory, anti-oxidative, anti-apoptotic, and anti-proliferative). Carbon monoxide is able to modulate several extra- and intra-cellular signaling molecules leading to differentiated response according to the specific stimulus. With our growing understanding in the way CO exerts its effects, especially in the mitochondria and its intracellular pathways, it is tempting to speculate about a clinical application of this substance. Since HO-1 is not easy to induce, research focused on the application of the gaseous molecule CO by itself or the implementation of carbon monoxide releasing molecules (CO-RM) to deliver the molecule at a time- and dose dependently safe way to any target organ. After years of research in cellular systems and animal models, summing up data about safety issues as well as possible target to treat in various diseases, the first feasibility trials in humans were established. Up-to-date, safety issues have been cleared for low-dose carbon monoxide inhalation (up to 500 ppm), while there is no clinical data regarding the injection or intake of any kind of CO-RM so far. Current models of human research include sepsis, acute lung injury, and acute respiratory distress syndrome as well as acute kidney injury. Carbon monoxide is a most promising candidate in terms of a therapeutic agent to improve outbalanced organ conditions. In this paper, we summarized the current understanding of carbon monoxide's biology and its possible organ targets to treating the critically ill patients in tomorrow's ICU.

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A TCF-Based Carbon Monoxide NIR-Probe without the Interference of BSA and Its Application in Living Cells.

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