Fabrication of a Redox-Reversible Near-Infrared Fluorogenic Probe for Ferroptosis Process Monitoring and the Early Diagnosis of Diabetes
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Ferroptosis is a type of cell death triggered by the iron-dependent accumulation of lipid peroxides in cells. Diabetes, a chronic metabolic disorder characterized by hyperglycemia, can lead to various health complications. The process of ferroptosis and the progression of diabetes are closely linked to redox homeostasis, which is regulated by the levels of reactive oxygen and sulfur species. Currently, there are no fluorescent probes available to monitor changes in redox homeostasis during ferroptosis and diabetes. Here, we report the first endeavor to create a reversible near-infrared fluorogenic (NIRF) probe for monitoring the process of ferroptosis reversal and precise diabetes diagnosis. In vitro data demonstrated that NIR-CSTe could cyclically and reversibly detect ONOO– and GSH up to four times with minimal loss in fluorescence intensity. With the help of NIR-CSTe, we observed that HT-1080 cells, induced to undergo ferroptosis by erastin after being washed with PBS for 24 h and then treated with ferrostatin-1, showed a recovery in intracellular GSH levels. In contrast, treatment with deferoxamine did not yield similar results. Lastly, NIR-CSTe was also utilized for the early diagnosis and efficacy assessment of diabetes in relation to ONOO–/GSH redox balance, with results illustrating that the combined administration of metformin and empagliflozin was more effective than using either drug alone. Thus, this smart probe holds significant potential as an essential tool for clinical diagnosis and treatment of diseases associated with redox homeostasis.
Copyright © 2025 American Chemical Society
Analytical Chemistry
Cite this: Anal. Chem. 2025, 97, 4, 2411–2417
https://doi.org/10.1021/acs.analchem.4c05927
Cellular redox homeostasis is critical for maintaining a stable microenvironment that supports the normal function of biomolecules, which is essential for preserving physiological functions within cells. 1, 2 The regulation of redox homeostasis is intricately linked to cellular signal transduction and transcriptional regulation. Disruption of this balance induces oxidative stress, leading to cellular damage and contributing to the development of various diseases, including cancer, cardiovascular diseases, neurodegenerative disorders, and diabetes mellitus. 3-5 Type 2 diabetes is characterized by abnormal glucose metabolism in the body in the form of hyperglycemia, the source of which is related to redox imbalance in the liver. In diabetes, intracellular redox imbalance results in β-cell dysfunction and mediates signaling pathways associated with insulin resistance, which further drives oxidative stress and its related complications. 6-8 These complications include diabetic retinopathy, neuropathy, and cardiovascular disease, all of which stem from oxidative damage to tissues and organs, ultimately culminating in cell death. 9
Ferroptosis is a novel regulated form of programmed cell death that differs from other forms of cell death, such as apoptosis, necrosis, and autophagy. 10, 11 Redox homeostasis plays a pivotal role in the regulation of ferroptosis. It is found that the accumulation of lipid peroxides during ferroptosis with iron-catalyzed Fenton reaction upregulates reactive oxygen species (ROS) levels and causes cellular damage by disrupting redox homeostasis. Activation of antioxidant systems, such as the upregulation of GPx4, can prevent ferroptosis by scavenging lipid peroxides and maintaining redox homeostasis. 12, 13 Therefore, an in-depth study of the relationship between redox homeostasis, ferroptosis, and diabetes will spread new avenues for the prevention, diagnosis, and treatment of these diseases.
Common reactive oxygen and sulfur species jointly regulate redox homeostasis and have a significant role in the physiological and pathological processes within living organisms. For example, intracellular ONOO- is generated from the diffusion reaction between nitric oxide and superoxide anion, the level of which is strictly controlled in the organism and tightly involved with cellular homeostasis. 14 Studies have shown that the excessive production of ONOO- alters the redox state of cells to a pro-oxidant state, which prevents the organism from metabolizing excess ONOO- normally and leads to oxidative stress. ONOO--related oxidative stress is well-linked to various diseases, particularly diabetes and cardiovascular disorders. 15 Glutathione, one of the most important and abundant non-protein thiols in living organisms, is present in the millimolar range in cellular concentrations. A reduction in GSH levels exacerbates intracellular oxidative stress. GSH/GSSG plays a vital role in maintaining cellular redox homeostasis, with abnormal changes in its levels correlated with various diseases, including cancer, aging, and diabetes. 16 Thus, ONOO- and GSH expression levels directly affect the redox homeostasis of organisms, and the development of appropriate tools for real-time monitoring of changes in the ONOO-/GSH redox couple is crucial for the elucidation of redox homeostasis regulatory mechanisms and early diagnosis of diseases.
Compared to fluorescent probes with short absorption and emission wavelengths, near infrared fluorogenic (NIRF) probes offer several advantages, such as high tissue permeability, low photosensitivity, and favorable for multicolor imaging. 17-19 While numerous ONOO- and GSH-targeted NIRF probes have been developed for bioimaging applications, the vast majority of these probes exhibit an irreversible fluorescence response and do not dynamically monitor changes in intracellular redox state. 20-26 A series of fluorescent probes for the detection of ONOO- and GSH during ferroptosis have been designed on the basis of various response strategies; however, almost none have been employed to investigate whether the ferroptosis process is reversible. 27-31 Moreover, reversible fluorescent probes available for monitoring redox homeostasis in diabetes have also not been reported. 32-36
Aiming at these issues mentioned above, we rationally constructed a redox-reversible NIRF probe, NIR-CSTe, based on dihydroxanthene for imaging ONOO-/GSH associated with ferroptosis and diabetes. The response of NIR-CSTe to ONOO- resulted in a pronounced increase in fluorescence intensity at 665 nm, while GSH could reduce the oxidation product of ONOO-. Using NIR-CSTe, Fer-1 was demonstrated to reverse Era-induced ferroptosis and partially recover intracellular GSH levels. Most importantly, NIR-CSTe allowed the assessment of the effects of different drug treatments. These findings suggested that NIR-CSTe was a reliable tool for the detection of ONOO-/GSH in biological processes.
Scheme 1. (a) Our design of redox fluorescent probe NIR-CSTe. (b) Basic synthetic route for NIR-CSTe.
In conclusion, leveraging the reaction selectivity of tellurium, we designed a novel reversible NIRF probe, NIR-CSTe, that could be employed to track the variation of the ONOO-/GSH redox couple. NIR-CSTe reacted specifically with ONOO- and generated its oxidation product NIR-CSTeO, thus resulting in a noticeable fluorescence enhancement at 655 nm. The fluorescence could be subsequently restored by the addition of GSH, confirming the probe's reversible nature. NIR-CSTe demonstrated sufficient sensitivity to image exogenous and endogenous ONOO-/GSH variations in live cells without inducing obvious cytotoxicity. With the assistance of confocal fluorescence imaging, NIR-CSTe demonstrated for the first time that GSH levels in Fer-1 co-incubated cells could be recovered in the presence of Era removal, a process not possible with DFO treatment. Additionally, in vivo imaging showed that NIR-CSTe was a promising tool for early diagnosis of diabetes and evaluation of drug treatment efficacy. We anticipate that our study will not only increase interest in tellurium-containing fluorescent probes but also provide a valuable tool for the prediagnosis and treatment of diseases related to ONOO-/GSH redox imbalances.
