Molecular probes for super-resolution imaging of drug dynamics.

Super-resolution molecular probes (SRMPs) are essential tools for visualizing drug dynamics within cells, transcending the resolution limits of conventional microscopy. In this review, we provide an overview of the principles and design strategies of SRMPs, emphasizing their role in accurately tracking drug molecules. By illuminating the intricate processes of drug distribution, diffusion, uptake, and metabolism at a subcellular and molecular level, SRMPs offer crucial insights into therapeutic interventions. Additionally, we explore the practical applications of super-resolution imaging in disease treatment, highlighting the significance of SRMPs in advancing our understanding of drug action. Finally, we discuss future perspectives, envisioning potential advancements and innovations in this field. Overall, this review serves to inform and practitioners about the utility of SRMPs in driving innovation and progress in pharmacology, providing valuable insights for drug development and optimization.

Fluorescence and Lifetime Imaging of Endoplasmic Reticulum Polarity Change During Ferroptosis.

As a new form of regulated cell death, ferroptosis is closely related to various diseases. Tracing ferroptosis related biological behavior is helpful to better understand this process and its related biology. Considering that ferroptosis is featured with remarkable lipid peroxidation which can easily change the membranes' compositions and structures, it is potential to detect intracellular environmental changes for direct assessment of ferroptosis. In view of the close relationship between endoplasmic reticulum (ER) and ferroptosis, we designed an ER-targeted and polarity-sensitive fluorescent probe SBD-CH, which has superior photostability and can respond to polarity with high selectivity without the affection of viscosity. SBD-CH can monitor the trend of ER polarity during ferroptosis by confocal laser scanning microscopy (CLSM), and analyze the distribution of polarity in ferroptosis by fluorescence lifetime imaging microscopy (FLIM). During Erastin induced ferroptosis, the polarity of ER in HT-1080 cells increased and the polarity distribution in ER was more dispersed. Our work provides an effective strategy for evaluating the process of ferroptosis by monitoring the changes of ER polarity.

Biocompatible aggregation-induced emission active polyphosphate-manganese nanosheets with glutamine synthetase-like activity in excitotoxic nerve cells.

Glutamine synthetase (GS) is vital in maintaining ammonia and glutamate (Glu) homeostasis in living organisms. However, the natural enzyme relies on adenosine triphosphate (ATP) to activate Glu, resulting in impaired GS function during ATP-deficient neurotoxic events. To date, no reports demonstrate using artificial nanostructures to mimic GS function. In this study, we synthesize aggregation-induced emission active polyP-Mn nanosheets (STPE-PMNSs) based on end-labeled polyphosphate (polyP), exhibiting remarkable GS-like activity independent of ATP presence. Further investigation reveals polyP in STPE-PMNSs serves as phosphate source to activate Glu at low ATP levels. This self-feeding mechanism offers a significant advantage in regulating Glu homeostasis at reduced ATP levels in nerve cells during excitotoxic conditions. STPE-PMNSs can effectively promote the conversion of Glu to glutamine (Gln) in excitatory neurotoxic human neuroblastoma cells (SH-SY5Y) and alleviate Glu-induced neurotoxicity. Additionally, the fluorescence signal of nanosheets enables precise monitoring of the subcellular distribution of STPE-PMNSs. More importantly, the intracellular fluorescence signal is enhanced in a conversion-responsive manner, allowing real-time tracking of reaction progression. This study presents a self-sustaining strategy to address GS functional impairment caused by ATP deficiency in nerve cells during neurotoxic events. Furthermore, it offers a fresh perspective on the potential biological applications of polyP-based nanostructures.

Iridium(III)-Based PD-L1 Agonist Regulates p62 and ATF3 for Enhanced Cancer Immunotherapy.

Anti-PD-L1 immunotherapy, a new lung cancer treatment, is limited to a few patients due to low PD-L1 expression and tumor immunosuppression. To address these challenges, the upregulation of PD-L1 has the potential to elevate the response rate and efficiency of anti-PD-L1 and alleviate the immunosuppression of the tumor microenvironment. Herein, we developed a novel usnic acid-derived Iridium(III) complex, Ir-UA, that boosts PD-L1 expression and converts "cold tumors" to "hot". Subsequently, we administered Ir-UA combined with anti-PD-L1 in mice, which effectively inhibited tumor growth and promoted CD4+ and CD8+ T cell infiltration. To our knowledge, Ir-UA is the first iridium-based complex to stimulate the expression of PD-L1 by explicitly regulating its transcription factors, which not only provides a promising platform for immune checkpoint blockade but, more importantly, provides an effective treatment strategy for patients with low PD-L1 expression.

Mitochondria-targeted BODIPY dyes for small molecule recognition, bio-imaging and photodynamic therapy.

Mitochondria are essential for a diverse array of biological functions. There is increasing research focus on developing efficient tools for mitochondria-targeted detection and treatment. BODIPY dyes, known for their structural versatility and excellent spectroscopic properties, are being actively explored in this context. Numerous studies have focused on developing innovative BODIPYs that utilize optical signals for imaging mitochondria. This review presents a comprehensive overview of the progress made in this field, aiming to investigate mitochondria-related biological events. It covers key factors such as design strategies, spectroscopic properties, and cytotoxicity, as well as mechanism to facilitate their future application in organelle imaging and targeted therapy. This work is anticipated to provide valuable insights for guiding future development and facilitating further investigation into mitochondria-related biological sensing and phototherapy.

A NIR-II Photoacoustic Probe for High Spatial Quantitative Imaging of Tumor Nitric Oxide in Vivo.

Nitric oxide (NO) exhibits both pro- and anti-tumor effects. Therefore, real-time in vivo imaging and quantification of tumor NO dynamics are essential for understanding the conflicting roles of NO played in pathophysiology. The current molecular probes, however, cannot provide high-resolution imaging in deep tissues, making them unsuitable for these purposes. Herein, we designed a photoacoustic probe with an absorption maximum beyond 1000 nm for high spatial quantitative imaging of in vivo tumor NO dynamics. The probe exhibits remarkable sensitivity, selective ratiometric response behavior, and good tumor-targeting abilities, facilitating ratiometric imaging of tumor NO throughout tumor progression in a micron-resolution level. Using the probe as the imaging agent, we successfully quantified NO dynamics in tumor, liver and kidney. We have pinpointed an essential concentration threshold of around 80 nmol/cm3 for NO, which plays a crucial role in the "double-edged-sword" function of NO in tumors. Furthermore, we revealed a reciprocal relationship between the NO concentration in tumors and that in the liver, providing initial insights into the possible NO-mediated communication between tumor and the liver. We believe that the probe will help resolve conflicting aspects of NO biology and guide the design of imaging agents for tumor diagnosis and anti-cancer drug screening.

A Small-Molecule Ratiometric Photoacoustic Probe for the High-Spatiotemporal-Resolution Imaging of Copper(II) Dynamics in the Mouse Brain.

Copper dysmetabolism is associated with various neurodegenerative disorders, making high-spatiotemporal-resolution imaging of Cu2+ in the brain essential for understanding the underlying pathophysiological processes. Nevertheless, the current probes encounter obstacles in crossing the blood-brain barrier (BBB) and providing high-spatial-resolution in deep tissues. Herein, we present a photoacoustic probe capable of imaging Cu2+ dynamics in the mouse brain with high-spatiotemporal-resolution. The probe demonstrates selective ratiometric and reversible responses to Cu2+ , while also efficiently crossing the BBB. Using the probe as the imaging agent, we successfully visualized Cu2+ in the brain of Parkinson's disease (PD) model mouse with a remarkable micron-level resolution. The imaging results revealed a significant increase in Cu2+ levels in the cerebral cortex as PD progresses, highlighting the close association between Cu2+ alternations in the region and the disease. We also demonstrated that the probe can be used to monitor changes in Cu2+ distribution in the PD model mouse brain during L-dopa intervention. Mechanism studies suggest that the copper dyshomeostasis in the PD mouse brain was dominated by the expression levels of divalent metal transporter 1. The application of our probe in imaging Cu2+ dynamics in the mouse brain offers valuable insights into the copper-related molecular mechanisms underlying neurodegenerative diseases.

An AIE-based monofunctional Pt(ii) complex for photodynamic therapy through synergism of necroptosis-ferroptosis.

Side effects and drug resistance are among the major problems of platinum-based anticancer chemotherapies. Photodynamic therapy could show improved tumor targeting ability and better anticancer effect by region-selective light irradiation. Here, we report an aggregation-induced emission (AIE)-based monofunctional Pt(ii) complex (TTC-Pt), which shows enhanced singlet oxygen production by introduction of a Pt atom to elevate the intersystem crossing (ISC) rate. Moreover, TTC-Pt exhibits decent capacity of inhibition on tumor cell growth upon light irradiation, with negligible dark toxicity compared to the commonly used chemodrug cisplatin. Mechanistic study suggests that TTC-Pt enters HeLa cells via the endocytosis pathway and locates mainly in lysosomes, causing FSP1 down-regulation and intracellular lipid peroxidation accumulation under irradiation, finally leading to ferroptosis and necroptosis. The synergistic dual cell death pathways could help to kill apoptosis-resistant tumor cells. Therefore, TTC-Pt could serve as a potent antitumor photosensitizer, which overcomes the drug resistance with minimum side effects.

ZNT1 and Zn 2+ control TLR4 and PD-L1 endocytosis in macrophages to improve chemotherapy efficacy against liver tumor.

BACKGROUND AND AIMS: HCC is closely associated with inflammation and immune modulation, and combined chemotherapy with other strategies is under extensive investigation to achieve better efficacy. HCC is accompanied by zinc (Zn) deficiency. This study aims to understand how Zn could affect macrophage function and its application for HCC therapy. APPROACH AND RESULTS: Zn 2+ and the Zn transporter 1 (ZNT1, solute carrier family 30 member 1) were markedly reduced in intrahepatic macrophages from patients with HCC and from mouse liver tumors. Lower ZNT1 expression was associated with higher IL-6 production and shorter survival time in patients with HCC. Critically, ZNT1 regulated endosomal Zn 2+ levels for endocytosis of toll-like receptor 4 and programmed cell death ligand 1, thereby decreasing macrophage-induced inflammation and immunosuppression to protect from liver tumors. Myeloid-specific deletion of ZNT1 in mice increased chronic inflammation, liver fibrosis, tumor numbers, and size. Notably, Zn supplementation could reduce inflammation and surface programmed cell death ligand 1 expression in macrophages with the increased CD8 + T cell cytotoxicity, which synergized the antitumor efficacy of Sorafenib/Lenvatinib. CONCLUSIONS: Our study proposes a new concept that ZNT1 and Zn regulate endosome endocytosis to maintain surface receptors, and Zn supplements might be synergized with chemotherapy to treat inflammation-associated tumors, especially those containing programmed cell death ligand 1 + myeloid cells.

Recent advances in Zn2+ imaging: From organelles to in vivo applications.

Zn2+ is involved in various physiological and pathological processes in living systems. Monitoring the dynamic spatiotemporal changes of Zn2+ levels in organelles, cells, and in vivo is of great importance for the investigation of the physiological and pathological functions of Zn2+. However, this task is quite challenging since Zn2+ in living systems is present at low concentrations and undergoes rapid dynamic changes. In this review, we summarize the design and application of fluorescent probes for Zn2+ imaging in organelles, cells, and live organisms reported over the past two years. We aim to provide inspiration for the design of novel Zn2+ probes for multi-level monitoring and deepen the understanding of Zn2+ biology.

Aggregation-Induced Emission (AIE), Life and Health.

Light has profoundly impacted modern medicine and healthcare, with numerous luminescent agents and imaging techniques currently being used to assess health and treat diseases. As an emerging concept in luminescence, aggregation-induced emission (AIE) has shown great potential in biological applications due to its advantages in terms of brightness, biocompatibility, photostability, and positive correlation with concentration. This review provides a comprehensive summary of AIE luminogens applied in imaging of biological structure and dynamic physiological processes, disease diagnosis and treatment, and detection and monitoring of specific analytes, followed by representative works. Discussions on critical issues and perspectives on future directions are also included. This review aims to stimulate the interest of researchers from different fields, including chemistry, biology, materials science, medicine, etc., thus promoting the development of AIE in the fields of life and health.

Proteolytic rewiring of mitochondria by LONP1 directs cell identity switching of adipocytes.

Mitochondrial proteases are emerging as key regulators of mitochondrial plasticity and acting as both protein quality surveillance and regulatory enzymes by performing highly regulated proteolytic reactions. However, it remains unclear whether the regulated mitochondrial proteolysis is mechanistically linked to cell identity switching. Here we report that cold-responsive mitochondrial proteolysis is a prerequisite for white-to-beige adipocyte cell fate programming during adipocyte thermogenic remodelling. Thermogenic stimulation selectively promotes mitochondrial proteostasis in mature white adipocytes via the mitochondrial protease LONP1. Disruption of LONP1-dependent proteolysis substantially impairs cold- or ß3 adrenergic agonist-induced white-to-beige identity switching of mature adipocytes. Mechanistically, LONP1 selectively degrades succinate dehydrogenase complex iron sulfur subunit B and ensures adequate intracellular succinate levels. This alters the histone methylation status on thermogenic genes and thereby enables adipocyte cell fate programming. Finally, augmented LONP1 expression raises succinate levels and corrects ageing-related impairments in white-to-beige adipocyte conversion and adipocyte thermogenic capacity. Together, these findings reveal that LONP1 links proteolytic surveillance to mitochondrial metabolic rewiring and directs cell identity conversion during adipocyte thermogenic remodelling.

A Near-Infrared Fluorescent and Photoacoustic Probe for Visualizing Biothiols Dynamics in Tumor and Liver.

Biothiols, including glutathione (GSH), homocysteine (Hcy) and cysteine (Cys), play crucial roles in various physiological processes. Though an array of fluorescent probes have been designed to visualize biothiols in living organisms, few one-for-all imaging agents for sensing biothiols with fluorescence and photoacoustic imaging capabilities have been reported, since instructions for synchronously enabling and balancing every optical imaging efficacy are deficient. Herein, a new near-infrared thioxanthene-hemicyanine dye (Cy-DNBS) has been constructed for fluorescence and photoacoustic imaging of biothiols in vitro and in vivo. Upon treatment with biothiols, the absorption peak of Cy-DNBS shifted from 592 nm to 726 nm, resulting in a strong NIR absorption as well as a subsequent turn-on PA signal. Meanwhile, the fluorescence intensity increased instantaneously at 762 nm. Then, Cy-DNBS was successfully utilized for imaging endogenous and exogenous biothiols in HepG2 cells and mice. In particular, Cy-DNBS was employed for tracking biothiols upregulation in the liver of mice triggered by S-adenosyl methionine by means of fluorescent and photoacoustic imaging methods. We expect that Cy-DNBS serves as an appealing candidate for deciphering biothiols-related physiological and pathological processes.

Blood-Brain Barrier Permeable Photoacoustic Probe for High-Resolution Imaging of Nitric Oxide in the Living Mouse Brain.

Alternations in the brain nitric oxide (NO) homeostasis are associated with a variety of neurodegeneration diseases; therefore, high-resolution imaging of NO in the brain is essential for understanding pathophysiological processes. However, currently available NO probes are unsuitable for this purpose due to their poor ability to cross the blood-brain barrier (BBB) or to image in deep tissues with spatial resolution. Herein, we developed a photoacoustic (PA) probe with BBB crossing ability to overcome this obstacle. The probe shows a highly selective ratiometric response toward NO, which enables the probe to image NO with micron resolution in the whole brain of living mice. Using three-dimensional PA imaging, we demonstrated that the probe could be used to visualize the detailed NO distribution in varying depth cross-sections (0-8 mm) of the living Parkinson's disease (PD) mouse brain. We also investigated the therapeutic properties of natural polyphenols in the PD mouse brain using the probe as an imaging agent and suggested the potential of the probe for screening therapeutic agents. This study provides a promising imaging agent for imaging of NO in the mouse brain with high resolution. We anticipate that these findings may open up new possibilities for understanding the biological functions of NO in the brain and the development of new imaging agents for the diagnosis and treatment of brain diseases.

BODIPY-Based Multifunctional Nanoparticles for Dual Mode Imaging-Guided Tumor Photothermal and Photodynamic Therapy.

Multifunctional nanoparticles integrating accurate multi-diagnosis and efficient therapy hold great prospects in tumor theranostics. However, it is still a challenging task to develop multifunctional nanoparticles for imaging-guided effective eradication of tumors. Herein, we developed a near-infrared (NIR) organic agent Aza/I-BDP by coupling 2,6-diiodo-dipyrromethene (2,6-diiodo-BODIPY) with aza-boron-dipyrromethene (Aza-BODIPY). Through encapsulating with an amphiphilic biocompatible copolymer DSPE-mPEG5000, well-distributed Aza/I-BDP nanoparticles (NPs) were developed, which exhibited high 1O2 generation, high photothermal conversion efficiency, and excellent photo-stability. Notably, coassembly of Aza/I-BDP and DSPE-mPEG5000 effectively inhibits H-aggregation of Aza/I-BDP in aqueous solution and enhances the brightness simultaneously up to 31-fold. More importantly, in vivo experiments demonstrated that Aza/I-BDP NPs might be used for NIR fluorescent and photoacoustic imaging-guided photodynamic and photothermal therapy.

Manipulating the Subcellular Localization and Anticancer Effects of Benzophenothiaziniums by Minor Alterations of N-Alkylation.

Cationic, water-soluble benzophenothiaziniums have been recognized as effective type I photosensitizers (PSs) against hypoxic tumor cells. However, the study of the structure-property relationship of this type of PS is still worth further exploration to achieve optimized photodynamic effects and minimize the potential side effects. Herein, we synthesized a series of benzophenothiazine derivatives with minor N-alkyl alteration to study the effects on the structure-property relationships. The cellular uptake, subcellular organelle localization, reactive oxygen species (ROS) generation, and photocytotoxicity performances were systematically investigated. NH2NBS and EtNBS specifically localized in lysosomes and exhibited high toxicity under light with a moderate phototoxicity index (PI) due to the undesirable dark toxicity. However, NMe2NBS with two methyl substitutions accumulated more in mitochondria and displayed an excellent PI value with moderate light toxicity and negligible dark toxicity. Without light irradiation, NH2NBS and EtNBS could induce lysosomal membrane permeabilization (LMP), while NMe2NBS showed no obvious damage to lysosomes. After irradiation, NH2NBS and EtNBS were released from lysosomes and relocated into mitochondria. All compounds could induce mitochondria membrane potential (MMP) loss and nicotinamide adenine dinucleotide phosphate (NADPH) consumption under light to cause cell death. NMe2NBS exhibited remarkable in vivo photodynamic therapy (PDT) efficacy in a xenograft mouse tumor (inhibition rate, 89%) with no obvious side effects. This work provides a valuable methodology to investigate the structure-property relationships of benzophenothiazine dyes, which is of great importance in the practical application of PDT against hypoxia tumor cells.

Single-atom engineering of hemicyanine and its amphiphilic derivative for optimized near infrared phototheranostics.

Near-infrared (NIR) dyes are widely used in the field of in vivo phototheranostics. Hemicyanine dyes (HDs) have recently received tremendous attention due to their easy synthesis and excellent NIR features. However, HDs can easily form non-fluorescent aggregates and their potential for phototherapy still needs further exploration due to their poor ability to generate reactive oxygen species (ROS). Herein, a series of hemicyanine dyes with different chalcogen atom (O, S, Se) substitutions were constructed to achieve optimized potential for phototheranostics. By replacing O with the heavy atom Se in the xanthene skeleton, CySe-NEt2 showed much more favourable features such as extended NIR absorption/emission wavelength, boosted 1O2 generation rate and higher photothermal effect. In addition, a poly(ethylene glycol) (PEG) group was introduced into the scaffold and yielded a nanotheranostic agent CySe-mPEG5K, which easily formed nanoparticles with appealing features such as excellent photostability, effective prevention of unpleasant H-aggregation, fast/selective tumor accumulation and minimum dark toxicity. Solid tumor growth was significantly suppressed through combined photodynamic therapy (PDT) and photothermal therapy (PTT) guided by NIR fluorescence (NIRF) and photoacoustic (PA) imaging. This study not only presents the first example of selenium-substituted hemicyanine dyes, but also offers a reliable design strategy for the development of potent NIR phototheranostic agents with multi-mode imaging-guided combination therapeutic ability.

A Photoacoustic Probe with Blood-Brain Barrier Crossing Ability for Imaging Oxidative Stress Dynamics in the Mouse Brain.

Spatiotemporal assessment of the oxidative stress dynamics in the brain is crucial for understanding the molecular mechanism underlying neurodegenerative diseases. However, existing oxidative stress probes have poor blood-brain barrier permeability or poor penetration depth, making them unsuitable for brain imaging. Herein, we developed a photoacoustic probe that enables real-time imaging of oxidative stress dynamics in the mouse brain. The probe not only responds to oxidative stress in a reversible and ratiometric manner, but it can also cross the blood-brain barrier of the mouse brain. Notably, the probe displayed excellent photoacoustic imaging of oxidative stress dynamics in the brains of Parkinson's disease mouse models. In addition, we investigated the antioxidant properties of natural polyphenols in the brain of a Parkinson's disease mouse model using the probe as an imaging agent and suggested the potential of the probe for screening anti-oxidative stress agents.

Fluorescent Probes for Biological Species and Microenvironments: from Rational Design to Bioimaging Applications.

Some important biological species and microenvironments maintain a complex and delicate dynamic balance in life systems, participating in the regulation of various physiological processes and playing indispensable roles in maintaining the healthy development of living bodies. Disruption of their homeostasis in living organisms can cause various diseases and even death. Therefore, real time monitoring of these biological species and microenvironments during different physiological and pathological processes is of great significance. Fluorescent-probe-based techniques have been recognized as one of the most powerful tools for real time imaging in biological samples. In this Account, we introduce the representative works from our group in the field of fluorescent probes for biological imaging capable of detecting metal ions, small bioactive molecules, and the microenvironment. The design strategies of small molecule fluorescent probes and their applications in biological imaging will be discussed. By regulating the design strategy and mechanism (e.g., ICT, PeT, and FRET) of the electronic and spectral characteristics of the fluorescent platforms, these chemical probes show high selectivity and diverse functions, which can be used for imaging of various physiological and pathological processes. Through the exploration of the rational response mechanism and design strategy, combined with a variety of imaging techniques, such as super-resolution imaging, photoacoustic (PA) imaging, etc., we have realized multimode imaging of the important biological analytes from the subcellular level to the in vivo level, which provides powerful means to study the physiological and pathological functions of these species and microenvironments. This Account aims to offer insights and inspiration for the development of novel fluorescent probes for biological imaging, which could provide powerful tools for the study of chemical biology. Overall, we represent a series of turn-on/turn-off/ratiometric fluorescent/PA probes to visually and dynamically trace biological species and microenvironments in cells and even in vivo that seek higher resolution and depth molecular imaging to improve diagnostic methods and clarify new discoveries related to chemical biology. Our future efforts will be devoted to developing multiorganelle targeted fluorescent probes to study the mechanism of subcellular organelle interaction and employing various dual-mode probes of NIR II and PA imaging to investigate the development of related diseases and treat the related diseases at subcellular and in vivo levels.

An ER-targeted "reserve-release" fluorogen for topological quantification of reticulophagy.

The endoplasmic reticulum's (ER) dynamic nature, essential for maintaining cellular homeostasis, can be influenced by stress-induced damage, which can be assessed by examining the morphology of ER dynamics and, more locally, ER properties such as hydrophobicity, viscosity, and polarity. Although numerous ER-specific chemical probes have been developed to monitor the ER's physical and chemical parameters, the quantitative detection and super-resolution imaging of its local hydrophobicity have yet to be explored. Here, we describe a photostable ER-targeted probe with high signal-to-noise ratio for super-resolution imaging that can specifically respond to changes in ER hydrophobicity under stress based on a "reserve-release" mechanism. The probe shows an excellent ability to target ER over commercial ER dyes and can be used to track local changes of hydrophobicity by fluorescence intensity and morphology during the selective autophagy of ER (i.e., reticulophagy). By correlating the level and location of ER damage with the distribution of fluorescence intensity, we were able to assess reticulophagy at the subcellular level. Beyond that, we developed a topological analytical tool adaptable to any ER probe for detecting structural changes in ER and thus quantitatively identifying reticulophagy. The algorithm-assisted tool can also be adapted to a wide range of molecular probes and organelles. Altogether, the new probe and analytical strategy described here show promise for the quantitative detection and analysis of subtle ER damage and stress.