Polymethine dyes are invaluable tools for studying biological processes in vivo due to their superior fluorescence properties and biological functions. However, despite the availability of excellent polymethine dyes, their uncontrollable interactions with albumin often limits their applications to specialized contexts. In this study, we present a general strategy for modulating the albumin sensitivity of dyes through a hydrophilicity-differentiated bidirectional regulation approach. Experimental data and computational simulations demonstrate that sulfonate positioning is crucial for suppressing hydrophobic interaction-mediated albumin-dye binding. Furthermore, this effect is enhanced when coupled with an optimal length of the polymethine bridge, which governs the postbinding responsiveness of the dye. Together, these factors establish the regulatory basis for differentiating albumin-sensitive and insensitive dyes. Consequently, a hydrophilic and albumin-insensitive Cy5 was identified and constructed a probe, showing potential for detecting hepatic fibrosis in mice without albumin interference (a protein primarily produced in the liver). Conversely, an albumin-sensitive Cy5 was synthesized by reducing the hydrophilicity of a Cy5 framework with a high TICT tendency, which can monitor blood-brain barrier function in vivo.

The sulfur oxidation axis is a core component of the body's sulfur metabolic network, governing the directional interconversion of sulfur species and maintaining metabolic homeostasis. Disruption of this axis can result in the accumulation of toxic intermediates or impaired biosynthetic disorders, both of which are implicated in a range of various diseases. As a central hub in sulfur metabolism, sulfite represents a critically metabolic junction linking upstream reduced sulfur species to downstream oxidized products. However, traditional analytical approaches rely on tissue homogenization, precluding the resolution of sulfite dynamics in vivo spatiotemporal precision. Although fluorescent probes for sulfite detection have been reported, most suffer from limitations in response speed, emission wavelength or biological applicability. To overcome these challenges, we developed three sulfite-responsive fluorescent probes—PNS, QNS and DNS, and systematically evaluated their performance. Among them, DNS emerged as the optimal candidate, exhibiting an ultra-rapid response time (< 2 s) in aqueous media, near-infrared emission at 700 nm for enhanced tissue penetration and excellent selectivity and biocompatibility. Using DNS, we demonstrated in living HepG2 cells that cysteine (Cys) serves as a precursor for sulfite, which is subsequently oxidized to sulfate. Notably, leveraging the superior photophysical properties of DNS, we achieved rapid dynamic tracking of sulfites in a living mouse model. Collectively, this work establishes essential technical support for in vivo sulfite visualization and provides critical technical support for elucidating the physiological functions of sulfite and the pathological mechanisms associated with dysregulated sulfur metabolism.

Azido molecules (azidos) have been extensively investigated for their potential as biomedical tools. However, the precise mechanism underlying their intracellular activation remains elusive. In this study, we adopted experimental and computational approaches to demonstrate that this activation mechanism is a photo-bioactivation process, facilitated by intracellular proteins that interact with azidos. Building on these findings, we developed a passive cell-specific fluorescence imaging tool for non-invasive cell tracking within three-dimensional (3D) scaffolds, an active-targeting subcellular organelle imaging system, and a controllable prodrug delivery platform that enables the spatiotemporal regulation of intracellular release and distribution of therapeutic agents. Overall, our study provides the first comprehensive elucidation of the cellular activation mechanism of azidos, which has significant implications for the development of a diverse array of photo-bioactivated tools and prodrugs.

Amino-functionalized near-infrared (NIR) dyes are crucial precursors for developing fluorescent probes. However, the commonly used amino-functionalized dyes are hindered by visible emission, complicated synthesis processes, small Stokes shifts or poor biocompatibility, which impede the development of high-performance probes. In this study, we present an electron donor engineering design of amino-functionalized NIR xanthene dyes. This design facilitates straightforward synthesis, achieves large Stokes shifts, maintains low molecular weight, exhibits high stability and ensures efficient NIR emission. Among these synthesized candidates, AXN-3 is identified as the optimal fluorophore, demonstrating a maximum emission wavelength of 675 nm and a large Stokes shift of 136 nm. Utilizing this superior scaffold, we developed a novel neutrophil elastase (NE)-activatable probe, AXN-NE, which employs the amino moiety as both the NE recognition site and fluorescence modulator. In vitro studies demonstrated that AXN-NE exhibits high selectivity and sensitivity to NE, showing a significant turn-on response. In a mouse model, the probe enabled high-contrast mapping of neutrophils during drug-induced osteoarthritic inflammation. This work not only provides a versatile NIR xanthene scaffold but also introduces a powerful tool for neutrophil visualization, holding promising applications in biomedical research and clinical diagnosis.

Correction for ‘A colorimetric and near-infrared fluorescent probe with large Stokes shift for biothiol bioimaging’ by Mingli Xie et al., New J. Chem., 2025, 49, 13958–13962, https://doi.org/10.1039/D5NJ01958C.

A novel near-infrared probe, XB-F, has been specifically developed for the detection of biothiols. Utilizing its unique characteristics, we successfully monitored both exogenous and endogenous biothiols in HepG-2 cells and in murine models.

Lipid droplets (LDs) are dynamic and multifunctional organelles that play a crucial role in energy storage, metabolism and lipid signaling. Monitoring the dynamics of LDs is essential for understanding their functions. Twisted intramolecular charge transfer (TICT)-based fluorescent molecules have been widely utilized for LD imaging. However, conventional TICT dyes exhibit sensitivity to both polarity and viscosity, which results in unclear sensing mechanisms for LDs. Additionally, current LD imaging techniques face challenges such as complex washing procedures and limited long-term imaging capabilities. This study presented a far-red coumarin framework designed to modulate the TICT-ICT equilibrium, resulting in the development of two fluorophores that exhibit specialized sensitivity to either polarity or viscosity. The findings suggested that sensitivity to polarity is a crucial factor for LD imaging, as high signal-to-noise ratios (SNR) enable wash-free imaging, while suitable lipophilicity supports long-term imaging. This polarity-specialized TICT probe had the potential to revolutionize LD imaging, facilitating wash-free and extended studies of LD dynamic behaviors and functions during lipolysis.

Biothiols are the main antioxidants in regulating the redox balance and resisting oxidative stress in various biological processes, but the long detection time of current fluorescent probes hinders their rapid imaging in vitro and in vivo. To reveal the influx of biothiols, we rationally develop an ortho-activation approach to accelerate the reaction between the probe and biothiols, by introducing electron-withdrawing fluorine atom into the ortho-site of the phenolic hydroxyl group in the NIR probe to generate an ortho-inductive effect. The ortho-fluorine helps to increase the chemical reactivity of the molecular structure, resulting in a significantly shorter detection time (within 5 min) as compared to previous reports (> 20 min for acrylates-based probes in aqueous solution). Based on this approach, our near-infrared probe 2F-RBX can sensitively and efficiently detect endogenous biothiols in living HepG2 cells and in vivo. These data suggest that ortho-activation is a simple and flexible approach to construct sensitive fluorescent probes for rapid imaging of biothiols, and perhaps other molecules in future, under biological circumstances.

The unique physiological activity and pharmacological properties of nitroxyl (HNO) have led to its use as a drug for treating various diseases, resulting in increased research on its detection and quantification. Although there have been numerous studies on fluorescent probes for detecting HNO, there is a lack of near-infrared (NIR) fluorescent probes that exhibit both a high fluorescence quantum yield and a large Stokes shift for this purpose. Through the optimization and modification of the structure of fluorinated NIR Rhodol, five NIR fluorescent dyes were synthesized, and the most promising candidate with the highest fluorescence quantum yield (Phi f =11.4 % in absolute PBS solution) and a bioapplicable Stokes shift (134 nm) was identified. Subsequently, a novel NIR fluorescent probe NB-HNO was developed. This probe exhibits high sensitivity and selectivity for HNO and has been successfully used to monitor fluctuations in exogenous and endogenous HNO levels in HepG2 cells and mice. Overall, our findings suggest that NB-HNO is a promising probe for imaging HNO and that our NIR fluorophore with high fluorescence quantum yield and large Stokes shift possesses potentially desirable properties for in vivo bioimaging.

Photoactivated fluorophores (PAFs) are highly effective imaging tools that exhibit a removal of caging groups upon light excitation, resulting in the restoration of their bright fluorescence.

The accurate and sensitive in situ imaging of inflammatory bowel disease (IBD) is critical for the precise diagnosis and treatment of this disease, but it remains a challenge for fluorescent probes because of a more dynamical shift of pH in the intestinal tract than under other physical conditions. To address this issue, we report here that the rational introduction of the ortho-halogen bond at the electron acceptor can regulate the protonation and deprotonation of fluorescent dye, resulting in a pH-insensitive dye (pH 5–12). Therefore, constructing a near-infrared (NIR) probe, 2F-RBH, which effectively avoids this pH effect with a stable fluorescent signal and selectively indicates sites of inflammation in situ, by tracking peroxynitrite (ONOO−) in an acute colitis mouse model. Collectively, our results highlight that 2F-RBH is a promising probe for the imaging of IBD and that our pH-insensitive dye has potentially desirable properties for in vivo bioimaging.

BACKGROUND:Metastasis is still a major cause of poor pathological outcome and prognosis in esophageal squamous cell carcinoma (ESCC) patients. NUAK1 has been reported highly expressed in many human cancers and is associated with the poor prognosis of cancer patients. However, the role of NUAK1 and its underlying signaling mechanism in ESCC metastasis remain unclear.METHODS:Expression of NUAK1 in ESCC was detected by real-time quantitative RT-PCR (qRT-PCR), Western blotting and immunohistochemical staining. MTT, colony formation, wound-healing and transwell assays were used to determine the role NUAK1 in vitro. Metastasis was evaluated by use of an experimental pulmonary metastasis model in BALB/c-nu/nu mice. The mechanisms were assessed by using coimmunoprecipitation, immunofluorescence and dual-luciferase reporter gene experiments.RESULTS:NUAK1 was highly expressed in ESCC tissues compared with the adjacent normal esophageal epithelial tissues. Moreover, the elevated expression of NUAK1 positively correlated with tumor invasion depth, lymph node metastasis, pathological TNM stage, and poor survival in ESCC patients. Further experiments showed that NUAK1 overexpression did not change the cell viability and colony formation of ESCC cells, while remarkably promoted the migration and invasion in vitro and experimental pulmonary metastasis in vivo. Mechanistically, NUAK1 enhanced the transcription level of Slug, which enhanced the migratory and invasive capability of ESCC cells. Consistently, silencing Slug almost completely diminished the migration and invasion of NUAK1-overexpressing ESCC cells. Further studies demonstrated that NUAK1 upregulated the transcription activity of Slug through activating the JNK/c-Jun pathway.CONCLUSION:These results demonstrated that NUAK1 promoted the metastasis of ESCC cells through activating JNK/c-Jun/Slug signaling, indicating NUAK1 is a promising therapeutic target for metastatic ESCC.

Although fine particulate matter (FPM) in air pollutants and tobacco smoke is recognized as a strong carcinogen and global threat to public health, its biological mechanism for inducing lung cancer remains unclear. Here, by investigating FPM’s bioactivities in lung carcinoma mice models, we discover that these particles promote lung tumor progression by inducing aberrant thickening of tissue matrix and hampering migration of antitumor immunocytes. Upon inhalation into lung tissue, these FPM particles abundantly adsorb peroxidasin (PXDN) – an enzyme mediating type IV collagen (Col IV) crosslinking – onto their surface. The adsorbed PXDN exerts abnormally high activity to crosslink Col IV via increasing the formation of sulfilimine bonds at the NC1 domain, leading to an overly dense matrix in the lung tissue. This disordered structure decreases the mobility of cytotoxic CD8+ T lymphocytes into the lung and consequently impairs the local immune surveillance, enabling the flourishing of nascent tumor cells. Meanwhile, inhibiting the activity of PXDN abolishes the tumor-promoting effect of FPM, indicating the key impact of aberrant PXDN activity on the tumorigenic process. In summary, our finding elucidates a new mechanism for FPM-induced lung tumorigenesis and identifies PXDN as a potential target for treatment or prevention of the FPM-relevant biological risks.

Hydrogen sulfide (H2S) is an important signaling molecule in various biological processes; however, its real-time monitoring in living cells is hampered by long detection time for current fluorescent probes. To overcome this challenge, we designed a phase-transfer catalyst (PTC) approach to accelerate the reaction between the probe and the analyte by conjugating common fluorescent probes - mostly hydrophobic small molecules - with an amphiphilic PEG-PPG-PEG polymer, enabling the controllable assembly of H2S nanoprobes in an aqueous solution. The PEG block helps to establish a PTC microenvironment that endows the assembled nanoprobes with a significantly reduced detection time (3-10 min; versus 20-60 min for small-molecule probes). Based on this approach, we synthesised two nanoprobes of different wavelengths, DS-Blue-nano and DN-Green-nano, which can sensitively detect H2S in living macrophage cells with bright fluorescence starting at as early as 7 min and reaching stability at 15 min. These data suggest PTC-based nanoprobes as a new and generic approach for constructing sensitive fluorescent probes for the real-time imaging of H2S, and perhaps other molecules in future, under biological conditions.

Scaffolds for tissue repair are designed in an increasingly complicated manner to meet multi-facet biological needs during the healing process. However, overly sophisticated design, especially the use of multiple components and delivery of exogenous cells, hampers the bench-to-bedside translation. Here, a multi-functional - yet mono-compositional - bioactive scaffold is devised to mediate the full-range, endogenous bone repair. Based on immunoactivity screening, a chemically-modified glucomannan polysaccharide is selected and processed into an anisotropic porous scaffold, which accurately stimulates macrophages to produce pro-regenerative cytokines. These cytokines effectively enhance the recruitment ("R") and induced osteogenesis ("IO") of the bone progenitor cells in situ. Meanwhile, the anisotropic porosity and carbohydrate signal of the scaffold facilitate differential adhesion ("A") and distribution ("D") of the macrophages and bone progenitor cells - enabling the former's accumulation at the surface while encouraging the latter's infiltration into the scaffold. Implanted in a rat calvarial defect model, this "RADIO" system effectively promotes healing over 12 weeks, with the obvious formation of hard callus through the scaffold. In summary, RADIO integrates multiple functions into one single scalable system ("all-in-one") to govern the dynamic bone-repair process, by harnessing the power of host macrophages. RADIO represents an open platform to solving the long-lasting complexity-versus-simplicity dilemma in biomaterials design. Statement of Significance Biomaterials as versatile tools for tissue repair are becoming increasingly complicated, yet overly sophisticated design - especially the use of multiple components, exogenous cells, and overdosed growth factors - hampers their clinical application. The pre-requisite for designing a successful integrative scaffold is to identify an inherent biological target responding to biomaterial signals, thereby efficiently and safely promoting tissue repair via the endogenous healing capability instead of extra multifarious biochemical components. For bone regeneration, the pivotal regulator is macrophages. Through activating host macrophages, our single-component scaffold system coordinates the entire bone regenerative cascade in situ and induces successful bone regeneration in a calvarial defect model. This scaffold represents a scalable and multi-functional approach to effectively simplify the sophisticated design in regenerative medicine. (C) 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Near-infrared (NIR) fluorescent probes are among the most attractive chemical tools for biomedical imaging. However, their in vivo applications are hindered by albumin binding, generating unspecific fluorescence that masks the specific signal from the analyte. Here, combining experimental and docking methods, we elucidate that the reason for this problem is an acceptor (A) group-mediated capture of the dyes into hydrophobic pockets of albumin. This pocket-capturing phenomenon commonly applies to dyes designed under the twisted intramolecular charge-transfer (TICT) principle and, therefore, represents a generic but previously unidentified backdoor problem. Accordingly, we create a new A group that avoids being trapped into the albumin pockets (pocket-escaping) and thereby construct a NIR probe, BNLBN, which effectively prevents this backdoor problem with increased imaging accuracy for liver fibrosis in vivo. Overall, our study explains and overcomes a fundamental problem for the in vivo application of a broad class of bioimaging tools.

Switching macrophages from a pro-tumor type to an anti-tumor state is a promising strategy for cancer immunotherapy. Existing agents, many derived from bacterial components, have safety or specificity concerns. Here, we postulate that the structures of the bacterial signals can be mimicked by using non-toxic biomolecules of simple design. Based on bioactivity screening, we devise a glucomannan polysaccharide with acetyl modification at a degree of 1.8 (acGM-1.8), which specifically activates toll-like receptor 2 (TLR2) signaling and consequently induces macrophages into an anti-tumor phenotype. For acGM-1.8, the degree of acetyl modification, glucomannan pattern, and acetylation-induced assembly are three crucial factors for its bioactivity. In mice, intratumoral injection of acGM-1.8 suppresses the growth of two tumor models, and this polysaccharide demonstrates higher safety than four classical TLR agonists. In summary, we report the design of a new, safe, and specific TLR2 agonist that can generate macrophages with strong anti-tumor potential in mice.