Fibrosis is a hallmark and common outcome of progressive chronic kidney disease (CKD). Noninvasive and accurate diagnosis of kidney fibrosis remains unavailable in clinics, limiting effective patient management. Here, we report the development of fibrogenesis sensing reporters (FSRs) for sensitive in vivo near-infrared fluorescence imaging and ex vivo translational urinalysis of kidney fibrosis. FSR achieves enhanced diagnostic accuracy by engaging two crucial, concurrently up-regulated biomarkers in fibrotic kidneys: lysyl oxidase-derived allysine (LysAld) and transglutaminase 2 (TG2). Engineered to be intrinsically nonfluorescent with excellent renal clearance, injected FSRs preferentially target kidneys through covalent binding to LysAld and are robustly cleaved by TG2, which is elevated in the fibrotic kidney, to unquench a renal-clearable fluorophore, enabling fluorescence "turn-on" for sensitive fibrosis detection by both kidney imaging and urine assays. Leveraging the high specificity of urinary TG2, FSR-based urinalysis distinguished kidney fibrosis from nonfibrotic acute kidney injury and was not confounded by extrarenal fibrosis (such as pulmonary and hepatic), providing diagnostic insights unattainable with routine kidney function tests. In a clinical cohort (n = 35), FSR-based urinalysis discriminated patients with CKD from healthy controls with 84% sensitivity and 94% specificity and provided discrimination between histological mild versus severe fibrosis. In contrast, traditional clinical metrics such as the estimated glomerular filtration rate, serum creatinine, and blood urea nitrogen were not able to classify mild versus severe fibrosis cases. This noninvasive activatable reporter holds translational potential for early identification of renal fibrosis and patient stratification of CKD to improve clinical management and patient outcome.

Real-time monitoring of kidney-infiltrating cytotoxic T lymphocytes (CTLs) is crucial for evaluating immunotherapy in renal cell carcinoma (RCC). However, existing imaging probes often exhibit "always-on" signals and poor renal clearance, limiting their ability to detect renal immune responses. Herein, we report renal-clearable bio-orthogonal near-infrared (NIR) fluorogenic probes (BGRs) that specifically detect granzyme B (GzmB), a biomarker of CTL activation, for dynamic evaluation of RCC immunotherapy. BGRs are built on a nitrile-substituted hemicyanine scaffold with a biothiol-responsive cysteine tail that is dually locked with a GzmB-responsive peptide and conjugated to renal clearable (2-hydroxypropyl)-β-cyclodextrin (HPβCD). Following injection in RCC mouse models under immunotherapy, non-fluorescent BGRM accumulates in the renal tumor, where dual cleavage by elevated glutathione (GSH) and GzmB releases HPβCD and exposes an aminothiol group, triggering nitrile-aminothiol biorthogonal click reaction that activates NIR fluorescence and drives probe self-assembly for enhanced imaging performance. BGRs not only differentiate immunotherapeutic responses in living mice but also enable sensitive optical urinalysis of GzmB in clinical specimens from RCC patients (n = 21), allowing precise stratification of immune activation before and after treatment. This work thus establishes a generalizable strategy for translational optical reporters that addresses the unmet clinical need for dynamic monitoring of immunotherapies in RCC and other urological cancers.

Detecting intracellular genomic G-quadruplexes (G4s) is crucial for understanding their biological functions. Although various G4 recognition probes have been developed, there remains a need for new G4 detection technologies to create detailed and reliable genomic G4 maps. In this study, we developed a small protein (CK13) that specifically recognizes the complementary C-rich single-stranded DNA (ssDNA) released during the formation of G4. Based on CK13 and CUT&Tag technology, we identified tens of thousands of C-rich ssDNA sites within human genomic DNA. These sites contain the vast majority of G4 sites detected by G4 probes, indicating that CK13 can well confirm the results of traditional G4 probes. Since CK13's binding to C-rich ssDNA is minimally influenced by G4-binding proteins, it produces strong signals at the sites where intracellular G4-binding proteins are present. This indicates that, beyond free G4 structures, CK13 can also detect G4s occupied by G4-binding proteins within cells. Our findings demonstrate that C-rich ssDNA complementary to G4 can serve as an indirect marker for G4 formation, offering a promising approach to further explore the regulatory roles of G4s and their interacting proteins.

Second near-infrared (NIR-II) fluorescence imaging holds great promise for studying biopathological processes with high spatial resolution. However, developing activatable NIR-II fluorescent probes (AFPs) remains challenging due to insufficient signal activation in response to biomarkers and labor-intensive probe optimization. Here, we identify the excited-state charge transfer dynamics change ratios (δ) as a critical determinant of the fluorescence "turn-on" ratio of AFPs. We design a series of AFPs and their uncaged counterparts (uAFPs) and systematically analyze their photophysical characteristics and responsiveness. Comprehensive analyses including computational calculations, femtosecond transient absorption spectroscopy, steady-state fluorescence spectra, and fluorescence titration experiments verify a strong correlation between the theoretical and experimental δ values and the fluorescence "turn-on" ratios of activated AFPs. As a proof of concept, the optimal probe AFP2 indicated by δ enables early diagnosis of drug-induced liver injury and ultrasensitive detection of tiny metastatic foci (<2 mm) in mouse models, demonstrating superior sensitivity outperforming conventional methods. This study highlights the potential of δ as a predictor of probe responsiveness, which can streamline and accelerate the development and optimization of NIR-II AFPs for broader preclinical and translational applications.

G-quadruplexes (G4s) are noncanonical DNA structures that play key roles in regulating replication, transcription, and genome stability. Here, we investigate the effects of pyridine-bis(benzimidazole) (PyBI), a selective parallel and hybrid G4 stabilizer, on genome stability in cells. Biophysical and biochemical assays confirm PyBI’s strong affinity for parallel G4s, leading to replication fork stalling and transcriptional repression of G4-associated oncogenes. Cleavage under targets and tagmentation (CUT&Tag) sequencing reveals a PyBI-induced genome-wide increase in G4 peaks, particularly at promoter and transcription start site regions. This G4 induction and stabilization triggers replication stress, G2/M arrest, and apoptosis. DNA repair pathway profiling shows that PyBI-induced G4 stabilization activates both homologous recombination and nonhomologous end joining (NHEJ), with a predominant role for NHEJ, as indicated by higher 53BP1 colocalization at G4 sites. Genome-wide mapping of PyBI-induced DNA breaks further supports a direct link between G4 stabilization and DNA damage. Moreover, PyBI shows synthetic lethality in DNA repair-deficient contexts, highlighting its therapeutic potential. These findings provide mechanistic insights into the genotoxic effects of PyBI and highlight its potential as a novel anticancer agent targeting G4-mediated genome instability.

Bioorthogonal chemistry-mediated self-assembly holds great promise for dynamic molecular imaging in living organisms. However, existing approaches are limited to nanoaggregates with ‘always-on’ signals, suffering from high signal-to-background ratio (SBR) and compromised detection sensitivity. Herein we report a nitrile-aminothiol (NAT) bioorthogonal fluorogenic probe (CyNAP-SS-FK) for ultrasensitive diagnosis of orthotopic hepatocellular carcinoma. This probe comprises a nitrile-substituted hemicyanine scaffold with a cysteine tail dually locked with biomarker-responsive moieties. Upon dual cleavage by tumor-specific cathepsin B and biothiols, the 1,2-aminothiol residue is exposed and spontaneously reacts with nitrile group for in situ intramolecular macrocyclization, enabling near-infrared fluorescence (NIRF) turn-on as well as self-assembly. In living male mice, such ‘cleavage-click-assembly’ regimen allows for real-time and ultrasensitive detection of small cancerous lesions (~2 mm in diameter) with improved SBR (~5) and extended detection window (~36 h), outperforming conventional clinical assays. This study not only presents NAT click reaction-based fluorogenic probes but also highlights a generic dual-locked design of these probes. Existing click chemistry-mediated approaches for dynamic imaging can suffer from low detection sensitivity. Here, the authors report a nitrile-aminothiol bioorthogonal fluorogenic probe with a ‘cleavage-click-assembly’ sensing action for ultrasensitive detection of small cancerous lesions.

G-quadruplexes (G4s) are important in biological processes such as gene transcription, telomere maintenance, and chromosome stability, and they hold promise as therapeutic targets in cancer research. Current G4 probes face challenges, including high background fluorescence, low regulation efficiency, and lack of spatiotemporal control. Photocaged technology offers precise temporal control and minimal background interference, making it a promising solution to these issues. Herein, we developed two photocaged G4 fluorescent probes, Nv-N-CQ and Nv-O-CQ, which use a photoremovable protecting group to block the fluorescence of coumarin-quinazoline (CQ) and its ability to bind to G4s. Upon UV light activation, Nv-O-CQ efficiently converted to CQ with minimal byproduct formation. It selectively bound to G4 structures, such as c-MYC, and enhanced their thermal stability. In cellular experiments, the probe demonstrated light-controlled fluorescence release and spatiotemporal specificity towards G4s in the cytoplasm. These findings highlight the potential of Nv-O-CQ for biological imaging, probe development, and spatiotemporal studies.

Acute kidney injury (AKI) frequently arises as a complication of hepatic ischemia-reperfusion injury (HIRI), yet simultaneous optical imaging of both remains challenging due to the lack of unimolecular dual-responsive probes. Herein, we report a library of hemicyanine-based chemiluminophores (HCLs) with tunable emission to second near-infrared window (725 - 1025 nm) achieved by integrating bicyclic dioxetane onto hemicyanine skeletons to develop multiple-responsive chemiluminescent probes. HCL1 and HCL5 respectively emitting at 725 nm and 1025 nm are selected to construct a cascaded activatable reporter CAR for crosstalk-free duplex chemiluminescence imaging of interlinked biomarkers. Following systemic injection to male mice, CAR preferentially accumulates in the liver and reports HIRI-associated superoxide anion (O2•-), which initiates self-fragmentation and liberates the secondary reporter KIR into kidneys to report AKI-associated N-acetyl-β-D-glucosaminidase (NAG). Such mechanism allows CAR to serve as a reservoir for gradual release of AKI reporters, providing a significantly prolonged imaging window compared to co-administering separate probes. CAR further permits remote detection of HIRI-induced AKI via urinalysis. This study not only offers a powerful tool for simultaneous detection of HIRI and HIRI-induced AKI, but also highlights a unimolecular probe design for ultrasensitive detection of deeply-seated intercorrelated diseases.

Activation of the stimulator of interferon genes (STING) signaling pathway holds great promise for enhancing antitumor immunity. However, its clinical use has been hindered due to the inefficient cytosolic delivery of STING agonists. We herein report an alternative approach that leverages acoustic waves to boost the biosynthesis of endogenous agonist cGAMP (cyclic GMP-AMP) for in situ STING activation in tumor cytoplasm. To transduce ultrasound for bio-intervention, a sono-responsive protein conjugate named SIGMA is constructed by anchoring a sonosensitizer to a tumor-specific nuclear targeting fusion protein. Intravenously injected SIGMA preferentially targets tumor cell nuclei and promotes DNA damage and cGAS (cyclic GMP-AMP synthase) production upon ultrasound stimulation. Acoustic waves concurrently disrupt nuclear integrity, enabling substantial export of both triggers into the cytosol to initiate cGAMP biosynthesis. The resulting cGAMP not only directly activates STING in tumor cells but also diffuses to proximal immune cells, inducing robust anti-tumor immunity that significantly inhibits tumor growth and prolongs survival, as well as establishing long-lasting immune memory that suppresses tumor rechallenge. This study thus highlights an efficient and translatable strategy that harnesses acoustic regulation of natural biosynthetic routes to improve cancer immunotherapy.

Human DNA topoisomerase 1 (Top1) is a crucial enzyme responsible for alleviating torsional stress on DNA during transcription and replication, thereby maintaining genome stability. Previous researches had found that non-working Top1 interacted extensively with chromosomal DNA in human cells. However, the reason for its retention on chromosomal DNA remained unclear. In this study, we discovered a close association between Top1 and chromosomal DNA, specifically linked to the presence of G-quadruplex (G4) structures. G4 structures, formed during transcription, trap Top1 and hinder its ability to relax neighboring DNAs. Disruption of the Top1-G4 interaction using G4 ligand relieved the inhibitory effect of G4 on Top1 activity, resulting in a further reduction of R-loop levels in cells. Additionally, the activation of Top1 through the use of a G4 ligand enhanced the toxicity of Top1 inhibitors towards cancer cells. Our study uncovers a negative regulation mechanism of human Top1 and highlights a novel pathway for activating Top1.

Lethal lipid peroxidation caused by reactive oxygen species occurs in different types of programmed cell death, especially in ferroptosis. Ferroptosis inducers, which serve as small-molecule probes, can provide insight into the mechanism of ferroptosis and facilitate drug discovery. The classical ferroptosis inducers indirectly lead to lipid peroxidation; thus, it is difficult to explore lipid regulation during the ferroptotic process. In this study, we designed two quinazolinone-based lipophilic probes BODIQPy-TPA and QPy-TPA, which proved to directly induce lipid peroxidation by light irradiation in vitro. The probe BODIQPy-TPA, which was mainly distributed in the endoplasmic reticulum (ER), specifically triggered ferroptosis in B16 and HepG2 cells upon light irradiation. As a comparison, the probe QPy-TPA, which was mainly distributed in lipid droplets (LDs), induced cell death by a nonferroptotic pathway. Further lipidomic analysis revealed that these two probes caused different patterns of lipid regulation and lipid peroxidation, suggesting that ferroptosis might activate distinct lipid regulation.

Organelle-targeted photosensitizers (PSs) offer valuable tools for improving photodynamic therapy (PDT), yet systematic studies on how different organelles influence phototherapeutic outcomes are limited. In particular, the connection between organelle targeting and various modes of programmed cell death remains unclear. In this study, we developed a series of PSs using the Coumarin-Quinazolinone (CQ) scaffold, each designed to target different organelles, including the mitochondria, endoplasmic reticulum (ER), lysosome, and nucleolus. Our results show that their PDT performance is highly dependent on their localization, with phototoxic index (PI) ranging from 2 to 245. Notably, the mitochondria-targeted CQ-Mito and ER-targeted CQ-ER exhibited profound phototherapeutic performances, with PI of 167 and 245 respectively. Our further study reveals that CQ-Mito causes cell death by both apoptosis and ferroptosis, while CQ-ER primarily triggers ferroptosis. This study not only provides new agents for PDT but also offers insights into how organelle targeting influences cell death mechanisms, which can shed light on the design of PSs for controlled cell death.

Molecular imaging in the second near-infrared window (NIR-II) provides high-fidelity visualization of biopathological events in deep tissue. However, most NIR-II probes produce "always-on" output and demonstrate poor signal specificity toward biomarkers. Herein, we report a series of hemicyanine reporters (HBCs) with tunable emission to NIR-II window (715-1188 nm) and structurally amenable to constructing activatable probes. Such manipulation of emission wavelengths relies on rational molecular engineering by integrating benz[c,d]indolium, benzo[b]xanthonium, and thiophene moieties to a conventional hemicyanine skeleton. In particular, HBC4 and HBC5 possess bright and record long emission over 1050 nm, enabling improved tissue penetration depth and superior signal to background ratio for intestinal tract mapping than NIR-I fluorophore HC1. An activatable inflammatory reporter (AIR-PE) is further constructed for pH-triggered site-specific release in colon. Due to minimized background interference, oral gavage of AIR-PE allows clear delineation of irritated intestines and assessment of therapeutic responses in a mouse model of inflammatory bowel disease (IBD) through real-time NIRF-II imaging. Benefiting from its high fecal clearance efficiency (>90%), AIR-PE can also detect IBD and evaluate the effectiveness of colitis treatments via in vitro optical fecalysis, which outperforms typical clinical assays including fecal occult blood testing and histological examination. This study thus presents NIR-II molecular scaffolds that are not only applicable to developing versatile activatable probes for early diagnosis and prognostic monitoring of deeply seated diseases but also hold promise for future clinical translations.

Life-threatening hypertension remains inadequately controlled in clinics due to its heterogeneous renin levels. Rapid stratification of hypertension through renin analysis is crucial for effective personalized treatment, yet an ultrasensitive detection approach is currently lacking. Here, we report activatable renin nanoprobes (ARNs) for non-invasive and ultrasensitive profiling of renin activity and guiding antihypertensive treatment decision through near-infrared fluorescence (NIRF) in vivo imaging and in vitro urinalysis. ARNs are intrinsically non-fluorescent due to NIRF reporter connected to a gold nanocluster through a renin-responsive peptide. In hyperreninemia mouse models, ARNs specifically react with renin to liberate the renal clearable NIRF reporter for accurate renin detection that outperforms the gold standard radioimmunoassay. Such specific and sensitive detection also enables imaging-based high-throughput screening of antihypertensive drugs. In hypertensive rat models, ARNs enable ultrasensitive detection of both plasma and urinary renin, facilitating renin-guided precision treatment and significantly improving hypertension control rate (90 % versus 58 %). Our nanoprobe platform holds great potential for assisting clinicians in rapidly and accurately classifying hypertensive patients and improving outcomes through tailored treatment selection.

Tumor vascular disrupting therapy has offered promising opportunities to treat cancer in clinical practice, whereas the overall therapeutic efficacy is notably limited due to the off-target effects and repeated dose toxicity of vascular disrupting agents (VDAs). To tackle this problem, a VDA-free biomimetic semiconducting polymer nanoparticle (SPNP ) is herein reported for precise tumor vascular disruption through two-stage light manipulation. SPNP consists of a semiconducting polymer nanoparticle as the photothermal agent camouflaged with platelet membranes that specifically target disrupted vasculature. Upon the first photoirradiation, SPNP administered in vivo generates mild hyperthermia to trigger tumor vascular hemorrhage, which activates the coagulation cascade and recruits more SPNP to injured blood vessels. Such enhanced tumor vascular targeting of photothermal agents enables intense hyperthermia to destroy the tumor vasculature during the second photoirradiation, leading to complete tumor eradication and efficient metastasis inhibition. Intriguingly, the mechanism study reveals that this vascular disruption strategy alleviates splenomegaly and reverses the immunosuppressive tumor microenvironment by reducing myeloid-derived suppressor cells. Therefore, this study not only illustrates a light-driven self-recruitment strategy to enhance tumor vascular disruption via a single dose of biomimetic therapeutics but also deciphers the immunotherapeutic role of vascular disruption therapy that is conducive to clinical studies.

Chemiluminescence imaging has been recognized as a valuable tool for ultrasensitive detection of physio-pathological events through elimination of background autofluorescence. However, most chemiluminescent nanoprobes suffer from shallow imaging depths and slow clearance from living bodies, which impede their use in clinical settings. We herein report size-transformable nanoreporters (ADN1 and ADN2) that could be activated at disease site by superoxide anion (O 2 ⋅ − ) to trigger nanostructure disassembly into renal excretable fluorescent fragments as well as chemiluminescence turn-on for crosstalk-free duplex chemo-fluorescence imaging and in vitro urinalysis. In peritonitis mouse model, we demonstrate that the representative nanoreporter ADN1 spontaneously accumulates at the disrupted peritoneum and is cleaved by upregulated O 2 ⋅ − to initiate depolymerization and result in red chemiluminescence at 620 nm, enabling sensitive detection of peritonitis at least 19 h earlier than gold standard histological assays. Additionally, the incorporation of a near-infrared (NIR) dye into ADN1 results in ADN2 exhibiting intense and red-shifted chemiluminescence at ≈800 nm, which permits early detection of deeply seated diseases such as drug-induced hepatotoxicity. This study thus showcases a modular design strategy that is not only applicable to developing versatile chemiluminescent nanoprobes with switchable pharmacokinetics for early disease diagnosis, but also promising for future clinical translations.

Targeted prodrug strategy,which utilizes the endogenous biomarkers in cancer cells as activators to release the active drug,has been well established either in the fundamental research or the clinical treatment.However,many prodrugs suffer from safety concern due to"off-target activation".Dual or multiple biomarkers triggered prodrug may provide an effective strategy to overcoming the"off-target effect".Melanoma cells have both high levels of reactive oxygen species(ROS)and tyrosinase(TYR),which makes them significantly different from other tumor cells and normal cells.Here we reported a series of quinazolinone-aryl boronic acid/ester-based prodrugs,which can be activated by the cascade of ROS and TYR and selectively kill melanoma cells.The structure-activity relationship(SAR)analysis revealed that mitochondria-targeting property was vital for their cytotoxicity and the dual activated effector played a significant role in their selectivity towards melanoma cells.Among these candidates,compound 4b showed the highest toxicity to B16,leading to an imbalance of the redox system in melanoma cells,causing mitochondrial DNA damage,and then promoting melanoma cells death.

DNA methylation (mainly at 5-methylcytosine, 5mC) plays an essential role in embryonic development and cellular biology. Alterations in DNA methylation are associated with disease development, especially hematologic malignancies. To investigate the potential of 5mC for diagnosis and treatment, accurate determination of 5mC is essential. Standard bisulfite sequencing-based methodologies or various optical/electrochemical biosensors for identifying 5mC have limitations, such as high cost, severe DNA degradation, over-estimation of the true 5mC level, being able to only display the average 5mC level, etc. Here we propose a single-molecule strategy for the direct identification of whole-genome 5mC by the combination of DNA fiber-based fluorescence in situ hybridization (DNA fiber FISH) and atomic force microscopy (AFM). Using extended DNA fibers and anti-5mC antibody for the detection of 5mC, it is possible to map the physical location of 5mC within the genome DNA. Together with AFM, this method can present the morphology of anti-5mC-DNA complexes and detailed spacing distribution of two neighboring 5mC sites on a single DNA molecule. Furthermore, this approach can be used for reporting other epigenetic modifications, not limited to 5mC or one single epigenetic modification. It can be anticipated to contribute to the development of clinical diagnosis of epigenetic-related diseases.

Optical nanoparticles are promising diagnostic tools; however, their shallow optical imaging depth and slow clearance from the body have impeded their use for in vivo disease detection. To address these limitations, we develop activatable polyfluorophore nanosensors with biomarker-triggered nanoparticle-to-molecule pharmacokinetic conversion and near-infrared fluorogenic turn-on response. Activatable polyfluorophore nanosensors can accumulate at the disease site and react with disease-associated proteases to undergo in situ enzyme-catalysed depolymerization. This disease-specific interaction liberates renal-clearable fluorogenic fragments from activatable polyfluorophore nanosensors for non-invasive longitudinal urinalysis and outperforms the gold standard blood and urine assays, providing a level of sensitivity and specificity comparable to those of invasive biopsy and flow cytometry analysis. In rodent models, activatable polyfluorophore nanosensors enable ultrasensitive detection of tumours (1.6 mm diameter) and early diagnosis of acute liver allograft rejection. We anticipate that our modular nanosensor platform may be applied for early diagnosis of a range of diseases via a simple urine test. Early cancer detection typically involves invasive biopsies. Here the authors designed nanosensors that are depolymerized by disease-associated enzymes in vivo to produce fluorescent urinary signals for non-invasive early diagnosis.

Neural circuitry is typically modulated via invasive brain implants and tethered optical fibres in restrained animals. Here we show that wide-field illumination in the second near-infrared spectral window (NIR-II) enables implant-and-tether-free deep-brain stimulation in freely behaving mice with stereotactically injected macromolecular photothermal transducers activating neurons ectopically expressing the temperature-sensitive transient receptor potential cation channel subfamily V member 1 (TRPV1). The macromolecular transducers, ~40 nm in size and consisting of a semiconducting polymer core and an amphiphilic polymer shell, have a photothermal conversion efficiency of 71% at 1,064 nm, the wavelength at which light attenuation by brain tissue is minimized (within the 400–1,800 nm spectral window). TRPV1-expressing neurons in the hippocampus, motor cortex and ventral tegmental area of mice can be activated with minimal thermal damage on wide-field NIR-II illumination from a light source placed at distances higher than 50 cm above the animal’s head and at an incident power density of 10 mW mm–2. Deep-brain stimulation via wide-field NIR-II illumination may open up opportunities for social behavioural studies in small animals. Deep-brain stimulation in freely behaving mice can be achieved via wide-field near-infrared illumination and stereotactically injected photothermal transducers activating neurons ectopically expressing a temperature-sensitive cation channel.