Understanding the molecular machinery regulating ferroptosis and leveraging this accumulating knowledge could facilitate disease prevention, diagnosis, treatment, and prognosis. The emerging approaches endowing in situ detection of the major regulators of ferroptosis at the cellular and tissue levels, and in living subjects, provide a multi-scale perspective to study ferroptosis. More importantly, ferroptosis-based applications integrating the rationality of ferroptosis detection with technological platforms have shown great promise in preclinical research. In this review, we first briefly summarized the mechanisms and key regulators involved in ferroptosis. Ferroptosis detection approaches were then presented to delineate their design, mechanisms of action, and applications. Special interest was placed on ferroptosis-based applications integrating multifunctional platforms. We hope that this review is timely to spur research interest in ferroptosis-based disease management.

Ferroptosis Detection: From Approaches to Applications.

Polarity is an integral microenvironment parameter which is crucial for establishing and reflecting a large number of sophisticated physiological functions and pathological effects. Monitoring the changes of intracellular polarity is...

Red Emissive Carbon Dots as Fluorescent Sensor for Fast Specific Monitoring and Imaging of Polarity in Living Cells

Mitophagy is a vital cellular process playing vital roles in regulating cellular metabolism and mitochondrial quality control. Mitochondrial viscosity is a key microenvironmental index, closely associated with mitochondrial status. To monitor mitophagy and mitochondrial viscosity, three molecular rotors (Mito-1, Mito-2, and Mito-3) were developed. All probes contain a cationic quinolinium unit and a C12 chain so that they can tightly bind mitochondria and are not affected by the mitochondrial membrane potential. Optical studies showed that all probes are sensitive to viscosity changes with an off-on fluorescence response, and Mito-3 shows the best fluorescence enhancement. Bioimaging studies showed that all these probes can not only tightly locate and visualize mitochondria with near-infrared fluorescence but also effectively monitor the mitochondrial viscosity changes in cells. Moreover, Mito-3 was successfully applied to visualize the mitophagy process induced by starvation, and mitochondrial viscosity was found to show an increase during mitophagy. We expect Mito-3 to become a useful imaging tool for studying mitochondrial viscosity and mitophagy.

Mitochondrial Membrane Potential Independent Near-Infrared Mitochondrial Viscosity Probes for Real-Time Tracking Mitophagy.

Currently, microbial infections have posed an arduous challenge to global public health, whereas the rise of antibiotic resistance is rendering traditional antibiotic therapies futile, prompting the development of new antimicrobial technologies. Photoactive nanomaterials have thus garnered a thriving interest for disinfection owing to their superior antibacterial efficaciousness, favorable biosafety, and rapidness and spatiotemporal precision in excreting bactericidal actions. The review summarizes recent advances and emerging trends in the design, nanoengineering, and bioapplications of photoactive antimicrobials. It commences by elaborating fundamental theories on bacterial resistance, and antibacterial mechanisms of nanomaterials and phototherapy. Subsequently, the regulation of the antibacterial effectiveness of photoactive nanomaterials is comprehensively discussed, centering on criteria and strategies for tuning photoabsorption spectra, photothermal conversion, and photocatalytic efficiency, alongside tactics for enabling synergistic therapies. This is followed by comparative analyses of techniques and modalities for synthesizing and engineering photoactive nanomaterials with diverse structures, forms, and functionalities. Thereafter, the state‐of‐the‐art applications of phototherapies across various medical sectors are portrayed, and key challenges and opportunities are finally discussed to spur future innovations and translation. This review is envisaged to provide useful guidance for devising and developing nanomaterials‐based photoresponsive antimicrobials with application‐specific materials properties and biological functions.

Design and Nanoengineering of Photoactive Antimicrobials for Bioapplications: from Fundamentals to Advanced Strategies

Lipid droplets (LDs) are closely associated various diseases, such as liver diseases and cancers. In the last decade, a mountain of fluorescent probes has been developed for LDs, while the ones bearing near-infrared (NIR) emission, superb specificity, and high stability are still lacking. In this work, we prepare a phenothiazine-derived probe that shows a suitable hydrophobicity with ClogP as 6.25 and a strong emission at 681 nm in the low polarity solvents such as 1,4-dioxane, enabling the NIR imaging of LDs. The probe is verified to showcase high chemostability, superb pH tolerance, and excellent anti-photobleaching capacity. Using the probe, the changes in the polarity, size, and number of LDs can be monitored in living cells under different stimuli. Moreover, a variety of morphological dynamics of LDs is visualized over a period of time, including fusion, fission, transfer of lipid, LD growth, and lipolysis. Utilizing its NIR feature, in vivo tumor imaging is achieved and the therapeutic efficacy of cisplatin is primarily assessed in a triple-negative breast cancer mice model using LDs as the biomarker for the first time. These results suggest that NIR-LD is potential to act as a potent imaging agent for LDs-related diseases. 

A stable NIR fluorescent probe for imaging lipid droplets in triple-negative breast cancer

As a new form of regulated cell death, ferroptosis is closely related to various diseases. To interpret this biological behavior and monitor related pathological processes, it is necessary to develop appropriate detection strategies and tools. Considering that ferroptosis is featured with remarkable lipid peroxidation of various cell membranes, it is logical to detect membranes' structural and environmental changes for the direct assessment of ferroptosis. For this sake, we designed novel polarity-sensitive fluorescent probes Mem-C1C18 and Mem-C18C18, which have superior plasma membrane anchorage, high brightness, and sensitive responses to environmental polarity by changing their fluorescence lifetimes. Mem-C1C18 with much less tendency to aggregate than Mem-C18C18 outperformed the latter in high resolution fluorescence labeling of artificial vesicle membranes and plasma membranes of live cells. Thus, Mem-C1C18 was selected to monitor plasma membranes damaged along ferroptosis process for the first time, in combination with the technique of fluorescence lifetime imaging (FLIM). After treating HeLa cells with Erastin, a typical ferroptosis inducer, the mean fluorescence lifetime of Mem-C1C18 displayed a considerable increase from 3.00 to 4.93 ns, with a 64% increase (corresponding to the polarity parameter Δf increased from 0.213 to 0.232). Therefore, our idea to utilize a probe to quantitate the changes in polarity of plasma membranes proves to be an effective method in the evaluation of the ferroptosis process.

Polarity-Sensitive and Membrane-Specific Probe Quantitatively Monitoring Ferroptosis through Fluorescence Lifetime Imaging.

Multidrug-resistant (MDR) bacteria cause severe clinical infections and a high mortality rate of over 40% in patients with immunodeficiencies. Therefore, more effective, broad-spectrum, and accurate treatment for severe cases of infection is urgently needed. Here, we present an adoptive transfer of macrophages loaded with a near-infrared photosensitizer (Lyso700D) in lysosomes to boost innate immunity and capture and eliminate bacteria through a photodynamic effect. In this design, the macrophages can track and capture bacteria into the lysosomes through innate immunity, thereby delivering the photosensitizer to the bacteria within a single lysosome, maximizing the photodynamic effect and minimizing the side effects. Our results demonstrate that this therapeutic strategy eliminated MDR Staphylococcus aureus (MRSA) and Acinetobacter baumannii (AB) efficiently and cured infected mice in both two models with 100% survival compared to 10% in the control groups. Promisingly, in a rat model of central nervous system bacterial infection, we performed the therapy using bone marrow-divided macrophages and implanted glass fiber to conduct light irradiation through the lumbar cistern. 100% of infected rats survived while none of the control group survived. Our work proposes an efaficient and safe strategy to cure MDR bacterial infections, which may benefit the future clinical treatment of infection. 

Adoptive macrophage directed photodynamic therapy of multidrug-resistant bacterial infection

Rapidly capturing slight changes in cell surface pH is extremely important to evaluate the rapid diffusion of acidic metabolites into the extracellular environment caused by disease and physiological pH fluctuations of cells. In this work, we designed a membrane-targeted pH probe, Mem-C0C18, based on a novel heterocyclic xanthene-analogous backbone. Mem-C0C18 shows specific and stable staining ability towards membrane. Importantly, the fluorescence lifetime of Mem-C0C18 is highly sensitive against acidity within membrane, which is in favor of quantifying pH through fluorescence lifetime imaging. Using Mem-C0C18, we recorded pH changes of 0.61 units on the surface of human cervical cancer cells (Hela) during glycolysis. Further on, we observed a robust pH-regulating mechanism of the plasma membrane that the pH fluctuation range within membrane (5.32-6.85) is much smaller than the change in extracellular environment (4.00-8.00). Consequently, we demonstrate a pH probe for quantifying small pH fluctuations within cell membrane that merits further evaluation for biology applications.

A Novel Heterocyclic Xanthene-Analogous pH Probe for Quantitative Monitoring of Cell Surface pH by Fluorescence Lifetime Imaging

Although super-resolution imaging provides a great opportunity to disclose the structures of living cells at the nanoscale level, resolving the structural details of organelles is highly dependent on the targeting accuracy and photophysical properties of fluorescence trackers. Herein, we report a series of ultrabright and photostable trackers of lysosomal membranes for super-resolution imaging using stimulated emission depletion microscopy (STED). These trackers are composed of lipophilic NIR BODIPY derivatives and ionizable tertiary amines. This structural feature enables accurate targeting of the lysosomal membrane through the formation of transient amphiphilicity driven by the acidity in the lysosome. As a representative, Lyso-700 is applied for STED-based super-resolution imaging of the lysosomal membrane of living macrophages. By use of Lyso-700, the interaction details between lysosomes of macrophages and fungi are visualized. Overall, these trackers display great potential as advanced lysosome trackers and merit further evaluation for lysosome-related studies.

BODIPY-Based NIR Trackers with Acidity-Driven Amphiphilicity for STED Super-Resolution Imaging of Lysosomal Membranes.

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Polarity-Sensitive and Membrane-Specific Probe Quantitatively Monitoring Ferroptosis through Fluorescence Lifetime Imaging.

Adoptive macrophage directed photodynamic therapy of multidrug-resistant bacterial infection

A Novel Heterocyclic Xanthene-Analogous pH Probe for Quantitative Monitoring of Cell Surface pH by Fluorescence Lifetime Imaging

BODIPY-Based NIR Trackers with Acidity-Driven Amphiphilicity for STED Super-Resolution Imaging of Lysosomal Membranes.