Construction of a Label-Detection Integrated Visual Probe to Reveal the Double-Edged Sword Principle of Ferroptosis in Liver Injury.

Ferroptosis has been confirmed as a potential mediator and an indicator of the severity of liver injury. Despite the fruitful results, there are still two deficiencies in the research on the association between ferroptosis and liver injury. First, iron ions are usually selected as the target bioanalyte, but its detection based on a fluorescent probe is interfered with by specific chemical reaction mechanisms, leading to low sensitivity and poor physiological stability. Second, more efforts were focused on the harmful effects of ferroptosis on liver injury and less involved in the therapeutic value of ferroptosis for liver injury. Hence, in this work, we proposed a new nonreactive analyte (mitochondrial viscosity) as an analysis marker, which can circumvent the challenges caused by specific reaction mechanisms of iron ions. Meanwhile, we constructed a novel label-detection integrated visual probe (VPF) to explore the feasibility of ferroptosis in the treatment of liver injury. As expected, we not only successfully traced the dynamic changes in mitochondrial viscosity but also visualized the changes in cell morphology during induced and inhibited ferroptosis. Conspicuously, this work revealed that liver injury can be alleviated by regulating ferroptosis, confirming the therapeutic value of ferroptosis in liver injury. In addition, a complex biological communication network between ferroptosis and liver injury was constructed by western blotting, providing an important theoretical mechanism for revealing their double-edged sword relationship. This study not only provides a new strategy for studying the complex relationship between ferroptosis and liver injury but also facilitates the future treatment of liver injury.

Fluorescent probes for ferroptosis bioimaging: advances, challenges, and prospects.

Ferroptosis is a form of regulatory cell death distinct from caspase-dependent apoptosis and plays an important role in life entities. Since ferroptosis involves a variety of complex regulatory factors, the levels of certain biological species and microenvironments would change during this process. Thus, the investigation of the level fluctuation of key target analytes during ferroptosis is of great significance for disease treatment and drug design. Toward this aim, multiple organic fluorescent probes with simple preparation and non-destructive detection have been developed, and research over the past decade has uncovered a vast array of homeostasis and other physiological characteristics of ferroptosis. However, this significant and cutting-edge topic has not yet been reviewed. In this work, we aim to highlight the latest breakthrough results of fluorescent probes for monitoring various bio-related molecules and microenvironments during ferroptosis at the cellular, tissue and in vivo levels. Accordingly, this tutorial review has been organized according to the target molecules identified by the probes including ionic species, reactive sulfur species, reactive oxygen species, biomacromolecules, microenvironment, and others. In addition to providing new insights into the findings of each fluorescent probe in ferroptosis studies, we also discuss the defects and limitations of the probes developed, and highlight the potential challenges and further prospects in this domain. We anticipate that this review will convey profound implications for designing powerful fluorescent probes to decrypt changes in key molecules and microenvironments during ferroptosis.

Investigating the Therapeutic Effects of Ferroptosis on Myocardial Ischemia-Reperfusion Injury Using a Dual-Locking Mitochondrial Targeting Strategy.

Research on ferroptosis in myocardial ischemia/reperfusion injury (MIRI) using mitochondrial viscosity as a nexus holds great promise for MIRI therapy. However, high-precision visualisation of mitochondrial viscosity remains a formidable task owing to the debilitating electrostatic interactions caused by damaged mitochondrial membrane potential. Herein, we propose a dual-locking mitochondria-targeting strategy that incorporates electrostatic forces and probe-protein molecular docking. Even in damaged mitochondria, stable and precise visualisation of mitochondrial viscosity in triggered and medicated MIRI was achieved owing to the sustained driving forces (e.g., pi-cation, pi-alkyl interactions, etc.) between the developed probe, CBS, and the mitochondrial membrane protein. Moreover, complemented by a western blot, we confirmed that ferrostatin-1 exerts its therapeutic effect on MIRI by improving the system xc-/GSH/GPX4 antioxidant system, confirming the therapeutic value of ferroptosis in MIRI. This study presents a novel strategy for developing robust mitochondrial probes, thereby advancing MIRI treatment.

Permeability-Controlled Probe for Directly Visualizing the Opening of Mitochondrial Permeability Transition Pore in Native Status.

The opening of mitochondrial permeability transition pore (mPTP) plays a fundamental role in cell apoptosis regulation, ischemia-reperfusion injury, and neurodegenerative disorders. However, the molecular tools for detecting mPTP open in cellular native status have not been reported yet. Herein, we de novo designed a robust fluorescent probe mPTP-F to monitor mPTP opening in cellular native status for the first time. The membrane-permeable probe could accumulate into mitochondria and convert to a product poorly permeable to biomembranes, which was trapped in mitochondria to form near-infrared (NIR)-emissive aggregates. After mPTP opening, the product was released from mitochondria through the pore to form green-emissive monomers. Significantly, with mPTP-F, we discovered that formaldehyde, a signaling molecule, could induce mPTP opening. Therefore, the new probe could serve as a desirable molecular tool for the study of ischemia-reperfusion injury, cell apoptosis, and relative areas.

Organic fluorescent probes for monitoring autophagy in living cells.

As a ubiquitous degradation process in cells, autophagy plays important roles in various biological activities. However, the abnormality of autophagy is closely related to many diseases, such as aging, neurological disorder, and cancer. Thus, monitoring the process of autophagy in living cells has high significance in biological studies and diagnosis of related diseases. In order to real-time and in situ monitor the process of autophagy, various organic fluorescent probes have been explored in recent years owing to the advantages such as handy staining processes, flexible molecular design strategies, and near-nondestructive detection. However, this interesting and frontier topic has not been reviewed so far. In this tutorial review, we will focus on the latest breakthrough results of organic fluorescent probes in monitoring autophagy of living cells, especially the probe design strategies based on the several microenvironment changes of the autophagy process, and the responding mechanisms and bio-imaging applications in the autophagy process. In addition, we will discuss the shortcomings and limitations of the probes developed, such as susceptible to interference, unable to monitor the whole process, and lack of clinical applications. Finally, we will highlight some challenges and further opportunities in this field. This tutorial review may promote the development of more robust fluorescent probes to further reveal the mechanisms of autophagy, which is the basis of degradation and recycling of cell components.

Small molecule based fluorescent chemosensors for imaging the microenvironment within specific cellular regions.

The microenvironment (local environment), including viscosity, temperature, polarity, hypoxia, and acidic-basic status (pH), plays indispensable roles in cellular processes. Significantly, organelles require an appropriate microenvironment to perform their specific physiological functions, and disruption of the microenvironmental homeostasis could lead to malfunctions of organelles, resulting in disorder and disease development. Consequently, monitoring the microenvironment within specific organelles is vital to understand organelle-related physiopathology. Over the past few years, many fluorescent probes have been developed to help reveal variations in the microenvironment within specific cellular regions. Given that a comprehensive understanding of the microenvironment in a particular cellular region is of great significance for further exploration of life events, a thorough summary of this topic is urgently required. However, there has not been a comprehensive and critical review published recently on small-molecule fluorescent chemosensors for the cellular microenvironment. With this review, we summarize the recent progress since 2015 towards small-molecule based fluorescent probes for imaging the microenvironment within specific cellular regions, including the mitochondria, lysosomes, lipid drops, endoplasmic reticulum, golgi, nucleus, cytoplasmic matrix and cell membrane. Further classifications at the suborganelle level, according to detection of microenvironmental factors by probes, including polarity, viscosity, temperature, pH and hypoxia, are presented. Notably, in each category, design principles, chemical synthesis, recognition mechanism, fluorescent signals, and bio-imaging applications are summarized and compared. In addition, the limitations of the current microenvironment-sensitive probes are analyzed and the prospects for future developments are outlined. In a nutshell, this review comprehensively summarizes and highlights recent progress towards small molecule based fluorescent probes for sensing and imaging the microenvironment within specific cellular regions since 2015. We anticipate that this summary will facilitate a deeper understanding of the topic and encourage research directed towards the development of probes for the detection of cellular microenvironments.

Noninvasive Cancer Diagnosis In Vivo Based on a Viscosity-Activated Near-Infrared Fluorescent Probe.

Effective and noninvasive cancer diagnosis is expected to ease the burden of continued increased deaths worldwide. Herein, we proposed viscosity of the tumor microenvironment as a biomarker and further develop a versatile optical agent, TBM-V, for monitoring tumor microenvironmental viscosity alterations to achieve cancer diagnosis, therapeutic effect tracking, and anticancer drug screening. When in highly viscous media, near-infrared signals of TBM-V are specifically activated, endowing the probe with the capacity of avoiding biological autofluorescence and achieving high signal-to-noise ratio imaging. The results of vascular imaging disclosed higher fluorescence of the blood vessels in the tumor than the normal ones, implying tumors being pointed out with brighter fluorescence. With the assistance of fluorescence imaging technology, TBM-V achieved noninvasively identifying cancer in vivo with high signal-to-noise ratio imaging. In addition, the capability of TBM-V to evaluate anticancer drug efficacy with viscosity as a robust biomarker was explored. Furthermore, as a proof of concept, screening of the anticancer drugs is also realized through in situ monitoring of the microenvironmental viscosity fluctuations of the tumor with TBM-V. Note that this proposed fluorescence imaging method outperforms the clinical hematoxylin and eosin (H&E) staining assay with the advantageous features of noninvasive and in vivo characteristics. We expected that this unique strategy will reinvigorate the continued perfection of the cancer diagnosis systems.

Strategies for designing organic fluorescent probes for biological imaging of reactive carbonyl species.

Reactive carbonyl species (RCS) are involved not only in diverse physiological pathways but also in various pathological processes, including Alzheimer's disease, diabetes, cardiovascular disease, and other malignant diseases. Therefore, it is essential to develop a simple and sensitive technology which can be employed to selectively monitor RCS in living biological samples, such as living cells, tissues, and animals. The subtle changes in the concentration of RCS in organisms can be detected by this technique. In this review, the design strategies of the typical examples of RCS fluorescent probes are highlighted and discussed. These advanced RCS probes may set the foundation for biomedical research on dynamic real-time monitoring of RCS in living systems.

Visualization of Mitochondrial Viscosity in Inflammation, Fatty Liver, and Cancer Living Mice by a Robust Fluorescent Probe.

Elucidating the internal relationship between diseases and mitochondrial viscosity remains a great challenge due to the lack of the studies on multidisease living animals. So far, demonstrating abnormal mitochondria viscosity in the fatty liver and tumor living mice has not been achieved. To address this critical challenge, the powerful two-photon and mitochondria-targeted fluorescence probe CBI-V was designed herein for viscosity detection with deep red emission. Utilizing the new probe CBI-V, the mitochondrial viscosity alteration in living systems caused by monensin or nystatin has been monitored sensitively and selectively in real time. Moreover, the probe is capable of imaging mitochondrial viscosity in the inflammatory models at the cell, zebrafish, and mice level. Importantly, the visualization of mitochondrial viscosity has been achieved in both fatty liver and tumor living mice for the first time. This work not only advances the study of the relationship between disease and mitochondrial viscosity but also opens up an efficient way for diagnosing mitochondrial viscosity related diseases.

ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m6A demethylation.

Zinc-finger homeobox 3 (ZFHX3, also known as ATBF1) suppresses prostatic tumorigenesis. ZFHX3 is frequently found to have numerous deletions in human prostate cancer (PCa). However, the underlying molecular function of ZFHX3 during prostatic tumorigenesis is not well understood. N6-methyladenosine (m6A) modification in RNA plays a critical role in the development of cancers; however, the relationship between ZFHX3 and m6A modification is largely unknown in PCa. In this study, we found that ZFHX3 knockdown decreased total m6A levels through enhancing the transcriptional activity of FTO in PCa cells. Importantly, FTO inhibition suppressed cell proliferation and rescued the promoting function of ZFHX3 knockdown on cell proliferation. In vivo, we verified that FTO was upregulated and ZFHX3 was decreased in PCa patients and that a high level of ZFHX3 is indispensable for low FTO expression and is correlated with better patient survival. Through transcriptome sequencing and MeRIP sequencing, we revealed that E2F2 and CDKN2C were the direct targets of FTO-mediated m6A modification and ZFXH3 was required for the regulation of FTO on E2F2 and CDKN2C expression. Unexpectedly, we uncovered that ZFHX3 expression was in return regulated by FTO in an m6A-dependent way. These findings establish a novel crosstalk mechanism between ZFHX3 and FTO in prostatic tumorigenesis.

Lipid droplet polarity decreases during the pathology of muscle injury as revealed by a polarity sensitive sensor.

Revealing the relationship between lipid droplets (LDs)polarity and disease is indispensable in clinicopathological diagnosis. So far, muscle injury is often ignored as it is not life-threatening as cardiovascular and cerebrovascular diseases, making the exploration of the internal relationship between muscle injury and LDs polarity a gray area. Herein, a fluorescent probe (CCB) with powerful polar-sensitive as well as precise LDs targeting was designed for visualizing the LDs polarity in the pathology of muscle injury. By means of the probe CCB, the identification of cancer cells and the monitoring of LDs polarity changes in dysfunctional cells were successfully realized. Furthermore, the penetration ability of CCB in tissues of mice was tested to verify the applicability of the probe in organisms. Importantly, by CCB, the relationship between muscle damage and LDs polarity was explored, revealing that muscle damage caused a significant decrease in LDs polarity accompanied by a significant increase in fluorescence. Most importantly, it is the first time to reveal the relationship between muscle damage and LDs polarity. Therefore, the probe CCB will be a powerful monitoring platform for diagnosing related diseases caused by abnormal LDs polarity.

Utilizing a Solvatochromic Optical Agent to Monitor the Polarity Changes in Dynamic Liver Injury Progression.

Unraveling the changing rule of endoplasmic reticulum (ER) polarity is of significance for liver injury. However, the rule of the ER polarity changes during the occurrence and progression of liver injury remains a mystery. Toward that, a unique fluorescent probe, ERNT, capable of imaging ER polarity in multiple liver injury models with high accuracy and fidelity was designed herein. In light of its excellent solvatochromism, the ER polarity was determined to be higher in the case of endoplasmic reticulum stress (ERS) induced by tunicamycin and dithiothreitol than that of the normal state at the cell level. Importantly, with the assistance of the PerkinElmer IVIS Spectrum imaging system and the powerful tool of ERNT, our work first revealed that the ER polarity increases with the evolution of liver injuries. Moreover, as a demonstration, ERNT achieved evaluating hepatoprotective drug efficacy by detecting ER polarity, confirming its high clinical application prospect. Thus, our work not only first unravels the rule of ER polarity in dynamic liver injury progression but may also inspire more diagnostic and therapeutic programs for liver diseases shortly.

Development of a two-photon fluorescent probe to monitor the changes of viscosity in living cells, zebra fish and mice.

The detection of viscosity is of great significance for medical research. Herein, we have developed a two-photon fluorescent probe CB-V for monitoring micro-viscosity changes. The fluorescence emission intensity of CB-V increased 9.6-fold from methanol to glycerol exhibiting an excellent fluorescence response. With excellent properties of CB-V, monitoring the viscosity variations has been achieved not only in living cells but also in zebra fish and mice.

Tracking lysosomal polarity variation in inflamed, obese, and cancer mice guided by a fluorescence sensing strategy.

Elucidating lysosome polarity effect in complicated biosystems was impeded with the deficiency of lacking multi-disease models for researching the relation between lysosomal polarity and diseases. So far, dissecting the abnormal lysosome polarity in the inflamed and obese living mice has not been realized. To overcome this challenge, a robust probe MND-Lys was proposed for monitoring lysosomal polarity with two-photon emission. Using the probe, monitoring the intrinsic polarity variance in embryos and adult zebrafish has been achieved for the first time. Moreover, besides obviously discriminating tumors from normal ones, the probe also enabled tracing polarity changes in inflammatory and obese mice for the first time. The unique tracking and distinguishing polarity in lysosome make the probe a promising agent for fluorescence visualization studies of LD-lysosome related bioprocess and metabolism diseases.

Rational design of a lipid-droplet-polarity based fluorescent probe for potential cancer diagnosis.

A robust fluorescent probe CTPA has been rationally designed for cancer diagnosis by monitoring lipid drop (LD) polarity and number variation. With the outstanding properties of CTPA, we have shown that the diagnosis of cancer can be achieved not only at the cellular levels but also in organs and living mice for the first time.

A novel mitochondria-targeted rhodamine analogue for the detection of viscosity changes in living cells, zebra fish and living mice.

Monitoring the intracellular viscosity changes is crucial for better understanding diffusion-controlled cellular processes. Herein, we synthesized a novel phenyl-substituted imidazole-fused rhodamine analogue RV-1 with a long absorption wavelength at 573 nm and a large Stokes shift of about 82 nm for monitoring the changes in cellular viscosity. The new probe RV-1 showed a very strong fluorescence emission at around 655 nm, with a 48.5-fold enhancement of fluorescence intensity from methanol to 99% glycerol. Significantly, the innovative probe RV-1 was successfully applied for the detection of viscosity changes not only in living cells, but also in zebra fish and living mice.