Activatable Fluorescent Probe Research Articles

A Dual-Cascade Activatable Near-Infrared Fluorescent Probe for Precise Intraoperative Imaging of Tumor.

Accurate intraoperative tumor delineation is critical to achieving successful surgical outcomes. However, conventional techniques typically suffer from poor specificity and low sensitivity and are time-consuming, which greatly affects intraoperative decision-making. Here, we report a cascade activatable near-infrared fluorescent (NIRF) probe IR780SS@CaP that can sequentially respond to tumor acidity and elevated glutathione levels for accurate intraoperative tumor localization. Compared with nonactivatable and single-factor activatable probes, IR780SS@CaP with a cascade strategy can minimize nonspecific activation and false positive signals in a complicated biological environment, affording a superior tumor-to-normal tissue ratio to facilitate the delineation of abdominal metastases. Small metastatic lesions that were less than 1 mm in diameter can be precisely identified by IR780SS@CaP and completely excised under NIRF imaging guidance. This study could benefit tumor diagnosis and image-guided tumor surgery by providing real-time information and reliable decision support, thus reducing the risk of both recurrence and complications to improve patient outcomes.

An activatable fluorescence probe for rapid detection and in situ imaging of β-galactosidase activity in cabbage roots under heavy metal stress

β-Galactosidase (β-gal), an enzyme related to cell wall degradation, plays an important role in regulating cell wall metabolism and reconstruction. However, activatable fluorescence probes for the detection and imaging of β-gal fluctuations in plants have been less exploited. Herein, we report an activatable fluorescent probe based on intramolecular charge transfer (ICT), benzothiazole coumarin-bearing β-galactoside (BC-βgal), to achieve distinct in situ imaging of β-gal in plant cells. It exhibits high sensitivity and selectivity to β-gal with a fast response (8 min). BC-βgal can be used to efficiently detect the alternations of intracellular β-gal levels in cabbage root cells with considerable imaging integrity and imaging contrast. Significantly, BC-βgal can assess β-gal activity in cabbage roots under heavy metal stress (Cd2+, Cu2+, and Pb2+), revealing that β-gal activity is negatively correlated with the severity of heavy metal stress. Our work thus facilitates the study of β-gal biological mechanisms.

Alkaline phosphatase activatable near-infrared fluorescent probe for in-situ diagnosis of cholestatic liver injury

Cholestatic liver injury (CLI) is a clinical syndrome caused by impaired bile excretion and is almost asymptomatic in early-stage. If CLI is not recognized timely, it will elicit more severe liver diseases. Therefore, earlier diagnosis of CLI is urgently needed but remains challenging. On the basis of the consensus that elevation of serum alkaline phosphatase (ALP) is the most susceptible indicator of earlier cholestatic diseases. Herein, we conjugated dicyanoisophorone with hydroxyl-substituted coumarin derivative to afford an intramolecular charge transfer (ICT)-based fluorophore TX-OH, and successfully constructed an enzyme activatable fluorescent probe TX-PS, which enables specifically detecting ALP with near-infrared emission (720nm), large stokes shift (198nm) and excellent linear relationship (R2 = 0.9969) between fluorescence intensity and the concentrations of ALP (0-100U/L). Moreover, TX-PS exhibits applicable for imaging of ALP activities in living cells and zebrafish with ignorable toxicity and shows great potential to visually screen ALP inhibitors. Importantly, the liver tissues from CLI mice displayed remarkably higher fluorescence intensity than that from normal mice, suggesting the valuable capability of TX-PS for evaluating CLI degree. Overall, this work affords a powerful tool for earlier diagnosis of CLI and high-throughput screening of ALP inhibitors or new drugs for CLI therapeutics.

Bienzyme-Locked Activatable Fluorescent Probes for Specific Imaging of Tumor-Associated Mast Cells.

Tumor-associated mast cells (TAMCs) have been recently revealed to play a multifaceted role in the tumor microenvironment. Noninvasive optical imaging of TAMCs is thus highly desired to gain insights into their functions in cancer immunotherapy. However, due to the lack of a single enzyme that is specific to mast cells, a common probe design approach based on single-enzyme activation is not applicable. Herein, we reported a bienzyme-locked molecular probe (THCMC) based on a photoinduced electron transfer-intramolecular charge-transfer hybrid strategy for in vivo imaging of TAMCs. The bienzyme-locked activation mechanism ensures that THCMC exclusively turns on near-infrared (NIR) fluorescence only in the presence of both tryptase and chymase specifically coexpressed by mast cells. Thus, THCMC effectively distinguishes mast cells from other leukocytes, including T cells, neutrophils, and macrophages, a capability lacking in single-locked probes. Such a high specificity of THCMC allows noninvasive tracking of the fluctuation of TAMCs in the tumor of living mice during cancer immunotherapy. The results reveal that the decreased intratumoral signal of THCMC after combination immunotherapy correlates well with the reduced population of TAMCs, accurately predicting the inhibition of tumor growth. Thus, this study not only presents the first NIR fluorescent probe specific for TAMCs but also proposes a generic bienzyme-locked probe design approach for in vivo cell imaging.

Activatable near-infrared fluorescence and chemical exchange saturation transfer MRI multimodal imaging probe for tumor detection in vitro and in vivo

Multimodal imaging has emerged as a powerful tool in biomedical research and clinical diagnostics. Ideally, it combines multiple imaging techniques to provide complementary anatomical and molecular information in living subjects. Particularly desirable are multimodal imaging probes capable of providing differential diagnostic signals upon interaction with specific molecular targets. Hydrogen peroxide (H2O2) is a key target in this regard, since it is typically overexpressed in cancer cells. In this study, we present a small-molecule probe that not only selectively detects endogenous H2O2 through multimodal imaging, with a significant H2O2-triggered 15-fold fluorescence enhancement but also turns “on” a chemical exchange saturation transfer (CEST) magnetic resonance (MR) response with 60-fold signal enhancement at pH 7.4. Excellent selectivity against various other biologically relevant species is seen. Using this probe, we observed 3.4–4.5-fold and 2.8–5.8-fold higher H2O2 levels in cancerous cell lines and tumor tissues compared to normal cell lines and tissues, respectively. Time-dependent in vivo fluorescence and CEST imaging in a HeLa (Henrietta Lacks) tumor xenograft mouse model revealed probe-dependent tumor detection by fluorescence and CEST MRI contrast in the tumor area. These observations are attributed to the relatively high endogenous H2O2 levels produced during mitosis. This newly developed probe holds promise for advancing our understanding of H2O2-related biology and for cancer detection both in vitro and in vivo.

ONOO–-activatable and LD-traced NIR fluorescent probe for mechanism study and early diagnosis of NAFLD and diabetes induced liver injury

Lipid metabolism disorder and oxidative stress play an essential role in the occurrence and development of metabolic diseases such as nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). Here, we constructed an ONOO-activatable and lipid drops (LDs) traced near-infrared (NIR) fluorescent probe BDP-NIR-Py+ through structural modification, and evaluated oxidative stress and lipid metabolism disorders in relevant disease by monitoring changes in ONOO and LDs levels in cells and mice. With the dual monitoring of ONOO and LDs, BDP-NIR-Py+ realized real-time visualization of the occurrence and development of NAFLD and T2DM, and early diagnosis of liver injury, which is faster and more sensitive than tissue analysis and blood detection. Collectively, the ability of BDP-NIR-Py+ to simultaneously monitor ONOO and LDs makes it has unlimited potential in the early diagnosis and mechanism research of diseases such as NAFLD and T2DM in which oxidative stress and lipid metabolism disorder interact.

Activity-Based Fluorescence Diagnostics for Cancer.

Fluorescence imaging is one of the most promising approaches to achieve intraoperative assessment of the tumor/normal tissue margins during cancer surgery. This is critical to improve the patients' prognosis, and therefore various molecular fluorescence imaging probes have been developed for the identification of cancer lesions during surgery. Among them, "activatable" fluorescence probes that react with cancer-specific biomarker enzymes to generate fluorescence signals have great potential for high-contrast cancer imaging due to their low background fluorescence and high signal amplification by enzymatic turnover. Over the past two decades, activatable fluorescence probes employing various fluorescence control mechanisms have been developed worldwide for this purpose. Furthermore, new biomarker enzymatic activities for specific types of cancers have been identified, enabling visualization of various types of cancers with high sensitivity and specificity. This Review focuses on recent advances in the design, function and characteristics of activatable fluorescence probes that target cancer-specific enzymatic activities for cancer imaging and also discusses future prospects in the field of activity-based diagnostics for cancer.

Near-infrared II theranostic agents for the diagnosis and treatment of Alzheimer's disease.

Near-infrared II theranostic agents have gained great momentum in the research field of AD owing tothe appealing advantages. Recently, an array of activatable NIR-II fluorescence probes has been developed tospecifically monitor pathological targets of AD. Furthermore, various NIR-II-mediated nanomaterials with desirablephotothermal and photodynamic properties have demonstrated favorable outcomes in the management of AD. We summerized amounts of references and focused on small-molecule probes, nanomaterials,photothermal therapy, and photodynamic therapy based on NIR-II fluorescent imaging for the diagnosis andtreatment in AD. In addition, design strategies for NIR-II-triggered theranostics targeting AD are presented, andsome prospects are also addressed. NIR-II theranostic agents including small molecular probes and nanoparticles have received theincreasing attention for biomedical applications. Meanwhile, most of the theranostic agents exhibited the promisingresults in animal studies. To our surprise, the multifunctional nanoplatforms also show a great potential in thediagnosis and treatment of AD. Although NIR-II theranostic agents showed the great potential in diagnosis and treatment of AD,there are still many challenges: 1) Faborable NIR-II fluorohpores are still lacking; 2) Biocompatibility, bioseurity anddosage of NIR-II theranostic agents should be further revealed; 3) New equipment and software associated withNIR-II imaging system should be explored.

A superoxide anion activatable near-infrared fluorescent probe with a large Stokes shift for imaging of drug-induced liver injury

Drug-induced liver injury (DILI) has become a common liver disease and a major public health problem. Superoxide anion (O2•-) is closely related to DILI, which can be employed as a biomarker of DILI. In this paper, based on a near-infrared fluorescent dye with quinolinium-xanthene structure and a large Stokes shift, a superoxide anion activatable near-infrared fluorescent probe (QL-3F) was designed and synthesized for imaging of drug-induced liver injury. QL-3F displayed a high sensitivity and an excellent selectivity to O2•-, and also the response mechanism of QL-3F to O2•- was verified by calculation of density functional theory (DFT). Moreover, QL-3F has been successfully applied to image endogenous O2•- in RAW264.7 cells and in HepG2 cells with drug-induced hepatotoxicity. More importantly, QL-3F has been used in monitoring O2•- in DILI mice with satisfactory results. The above results indicated that QL-3F is a useful tool to detect O2•- and diagnose drug-induced liver injury.

Fluorescent Probe for Investigating the Mitochondrial Viscosity and Hydrogen Peroxide Changes in Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI), a cause of cerebral dysfunction during cerebral infarction treatment, is closely associated with mitochondrial viscosity and hydrogen peroxide (H2O2). However, the accurate measurement of mitochondrial viscosity and H2O2 levels in CIRI is challenging because of the lack of sufficient selectivity and blood-brain barrier (BBB) penetration of existing monitoring tools related to CIRI, hampering the exploration of the role of mitochondrial viscosity and H2O2 in CIRI. To address this issue, we designed an activatable fluorescent probe, mitochondria-targeting styryl-quinolin-ium (Mito-IQS), with excellent properties including high selectivity, mitochondrial targeting, and BBB penetration, for the visualization of mitochondrial viscosity and H2O2 in the brain. Based on the real-time monitoring capabilities of the probe, bursts of mitochondrial viscosity and H2O2 levels were visualized during CIRI. This probe can be used to monitor the therapeutic effects of butylphthalein treatment. More importantly, in vivo experiments further confirmed that CIRI was closely associated with the mitochondrial viscosity and H2O2 levels. This discovery provides new insights and tools for the study of CIRI and is expected to accelerate the process of CIRI diagnosis, treatment, and drug design.

Development of an activatable far-red fluorescent probe for rapid visualization of hypochlorous acid in live cells and mice with neuroinflammation.

Recent investigations have suggested that abnormally elevated levels of HOCl may be tightly related to the severity of neuroinflammation. Although some successes have been achieved, fluorescent probes with far-red fluorescence emission and capable of detecting HOCl with high specificity in pure aqueous solution are still urgently needed. Herein, a responsive far-red fluorescent probe, DCI-H, has been constructed to monitor HOCl activity in vivo and in vitro. DCI-H could rapidly respond to HOCl within 120s and had a low detection limit for HOCl of 1.5nM. Importantly, physiologically common interfering species, except for HOCl, did not cause a change in the fluorescence intensity of DCI-HOCl at 655nm. The results of confocal imaging demonstrated the ability of DCI-H to visualize endogenous HOCl produced by MPO-catalyzed H2O2/Cl- and LPS stimulation. With the assistance of DCI-H, upregulation of HOCl levels was observed in the mice model of LPS-induced neuroinflammation. Thus, we believed that DCI-H provided a valuable tool for HOCl detection and diagnosis of inflammation-related diseases.

Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing.

Elucidation of biological phenomena requires imaging of microenvironments in vivo. Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.

Monoamine oxidase B activatable red fluorescence probe for bioimaging in cells and zebrafish

A real-time and specific for the detection of Monoamine Oxidase B (MAO-B) to investigate the MAO-B-relevant disease development and treatment process is urgently desirable. Here, we utilized MAO-B to catalyze the conversion of propylamino groups to aldehyde groups, which was then quickly followed by a β-elimination process to produce fluorescent probes (FNJP) that may be used to detect MAO-B in vitro and in vivo. The FNJP probe possesses unique properties, including favorable reactivity (Km = 10.8 μM), high cell permeability, and NIR characteristics (λem = 610 nm). Moreover, the FNJP probe showed high selectivity for MAO-B and was able to detect endogenous MAO-B levels from a mixed population of NIH-3 T3 and HepG2 cells. MAO-B expression was found to be increased in cells under lipopolysaccharide-stimulated cellular oxidative stress in neuronal-like SH-SY5Y cells. In addition, the visualization of FNJP for MAO-B activity in zebrafish can be an effective tool for exploring the biofunctions of MAO-B. Considering these excellent properties, the FNJP probe may be a powerful tool for detecting MAO-B levels in living organisms and can be used for accurate clinical diagnoses of related diseases.

[Development of optical probes with excellent intracellular retention].

Small-molecule based activatable fluorescence probes for detecting specific enzyme activity with high sensitivity can visualize the expression site of marker genes and cancers where the enzyme is highly expressed. However, the enzyme-catalyzed fluorescent hydrolysis product easily leaks out and diffuses from the reaction site, making it difficult to perform long-term tracking and immunohistochemical analysis which needs washing/fixation procedure. Our group have focused on quinone methide chemistry and developed series of activatable fluorescence probes with excellent intracellular retention that are converted to quinone-methide or aza-quinone-methide intermediates upon reaction with enzymes, which are then react with intracellular nucleophiles such as proteins and glutathione to be retained in cells and to exhibit significant increase in fluorescence. Based on this molecular design, we have developed fluorescence probes targeting β-galactosidase and γ-glutamyltranspeptidase with different colors. We also developed photo-functional probes such as activatable photosensitizers and caged fluorophores. These probes can visualize or kill target enzyme-expressing cells with high selectivity by suppressing the leakage of hydrolysis products from target cells, and fluorescence imaging in combination with immunostaining was possible due to the high tolerance of the obtained fluorescence signal even after washing and fixation.

Activatable fluorescent probes for early diagnosis and evaluation of liver injury.

With the increase in people's living standards, the number of patients suffering from liver injury keeps on increasing. Traditional diagnostic methods can no longer meet the needs of early and accurate diagnosis due to their limitations in application. However, fluorescent probes based on different fluorophores and nanomaterials have been gradually lighting up medical research due to their unique properties, such as high specificity and non-invasiveness. In addition, accurate identification of the different types of liver injury biomarkers can significantly improve the level of early diagnosis. Therefore, this review reviews the fluorescent probes used in the detection of biomarkers of liver injury over recent years and briefly summarizes the corresponding biomarkers of different types of liver injury. Impressively, this review also lists the structures and the response mechanisms of the different probes, and concludes with an outlook, suggesting directions in which improvements can be made. Finally, we hope that this review will contribute to the further development of fluorescent probes for the early diagnosis and assessment of liver injury.

Development of an activatable Lysosome-targeted fluorescent probe for the detection of endogenous ONOO− levels

The liver plays a crucial role in several important processes in the human body, including metabolism, detoxification, and immune function. When the liver experiences acute injury, it can cause significant harm and requires prompt detection. Traditional biomarkers lack specificity and cannot detect changes in real-time, making them unsuitable for monitoring pathological processes. Recent studies have shown that acute liver injury (ALI) is closely related to oxidative stress, with peroxynitrite (ONOO−) being a vital byproduct of liver metabolism and become a critical biomarker for detecting liver damage. As a result, this research developed an activatable near-infrared fluorescent probe W-3a that can be used to detect endogenous ONOO− in a mouse model of ALI induced by lipopolysaccharides (LPS). The probe has high selectivity and anti-interference ability, with a reaction time <10 min and a detection limit of 85 nM. It was successfully utilized in detecting endogenous ONOO− in cells and live imaging of ALI mice.

Modular Design Platform for Activatable Fluorescence Probes Targeting Carboxypeptidases Based on ProTide Chemistry.

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.

Bio-orthogonally Activatable Fluorescent Probe for Specific Imaging of Myeloid Cell Leukemia 1 Protein.

The antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) has been increasingly identified as a promising potential therapeutic target attributed to its critical regulation effect in diverse cellar physiopathological events. Current fluorescence imaging strategies tend to be susceptible to the cellular microenvironment, and straightforward mapping of Mcl-1's level variation remains challenging. In this paper, an activatable "off-on" fluorescence strategy for Mcl-1 specific labeling was presented based on bio-orthogonal chemistry by introducing tetrazine-functionalized borondipyrromethene (TB) as a fluorescent reporter and trans-cyclooctyne-derived indole-2-carboxylic acid (TI) as an Mcl-1 targeting moiety. With the click pair of TB and TI, the Mcl-1 expression level in vitro and in vivo was successfully mapped straightforward. Also, the level changes of Mcl-1 upon drug challenge were demonstrated. This work provides a robust fluorescence strategy for Mcl-1 in situ imaging, and the results would further facilitate the comprehensive revelation of the Mcl-1 biological effect.

An Activatable Near-Infrared Fluorescent Probe for Precise Detection of the Pulmonary Metastatic Tumors: A Traditional Molecule Having a Stunning Turn.

An accurate detection of lung metastasis is of great significance for making better treatment choices and improving cancer prognosis, but remains a big challenge in clinical practice. In this study, we propose a reinventing strategy to develop a pH-activatable near-infrared (NIR) fluorescent nanoprobe, pulmonary metastasis tracer (denoted as PMT), based on assembly of NIR dye IR780 and calcium phosphate (CaP). By delicately tuning the intermolecular interactions during the assembly process and dye doping content, as well as the synthetic condition of probe, the fluorescence of PMT could be finely adjusted via the tumor acidity-triggered disassembly. Notably, the selected PMT9 could sharply convert subtle pH variations into a distinct fluorescence signal to generate high fluorescence ON/OFF contrast, dramatically reducing the background signals. Benefiting from such preferable features, PMT9 is able to precisely identify not only the tumor sites in orthotopic lung cancer models but also the pulmonary metastases in mice with remarkable signal-to-background ratio (SBR). This study provides a unique strategy to turn shortcomings of traditional dye IR780 during in vivo imaging into advantages and further expand the application of fluorescent probe to image lung associated tumor lesions.

Engineering an activatable fluorescent probe for studying ONOO− in pyroptotic process

Pyroptosis, a recently discovered form of programmed cell death, plays a pivotal role in oncological treatment. Howbeit, the mechanisms underlying pyroptosis in tumor treatment remain unclear. Previous research has demonstrated that the occurrence of pyroptosis generally accompanies a surge of reactive oxygen species (ROS) generation, with ONOO− being one of these ROS and closely linked to numerous diseases. Therefore, it is imperative to investigate the potential association between ONOO− and pyroptosis. Herein, a highly sensitive and rapidly responsive near-infrared (NIR) probe, Rd700-PN, is fabricated for exploring unrevealed relationships between ONOO− and pyroptosis. We successfully harness Rd700-PN to detect ONOO− fluctuation during cellular pyroptosis for the first time. Furthermore, the results demonstrate that Rd700-PN can scout the chemotherapeutic drug's induction ability of tumor pyroptosis in vivo. Notably, this study provides an excellent means to shed light on the correlation between ONOO− and pyroptosis and to screen antitumor drugs activating pyroptosis.