Immune checkpoint blockade targeting PD-1 has emerged as a frontline therapy for diverse cancers, yet patient responses to anti-PD-1 monoclonal antibodies (mAbs) such as nivolumab and tislelizumab remain highly variable. These differences are closely linked to drug exposure, which affects both therapeutic benefit and adverse outcomes; however, the pharmacokinetic profiles of these antibodies are not well defined. This highlights the need for therapeutic drug monitoring (TDM) to guide precision immunotherapy. Yet, the development of rapid and reliable TDM methods is hindered by the lack of highly specific paratope-targeting biorecognition elements. In this study, we established a positive/negative F(ab')2-based phage display screening strategy to efficiently identify paratope-targeting peptides for anti-PD-1 mAbs. Using this approach, we identified both cyclic and linear peptides, with two candidates showing good affinity and specificity toward anti-PD-1 mAbs while exhibiting minimal IgG binding. Cyclic peptides demonstrated higher specificity than their linear counterparts. Building on these peptides, we further developed a gold nanoparticle (AuNP)-based point-of-care detection platform that produced synchronized colorimetric signals, enabling simple, rapid, and accurate detection of anti-PD-1 mAbs. Application to clinical samples confirmed good accuracy compared to conventional ELISA. This strategy offers an effective tool for peptide screening and TDM of anti-PD-1 mAbs; it provides a broadly applicable framework for monitoring other therapeutic antibodies in clinical practice.

ABSTRACT The aggregate form of interorganelle interaction is essential for the stable operation of cellular energy metabolism, signal transduction, as well as growth and development. Among these, the discovery of lipid droplet–mitochondria interactions (LDMIs) mediated by their aggregation has reshaped the understanding of how intracellular lipid metabolic networks operate within cells. Complex spatiotemporal interactions between lipid droplets and mitochondrial aggregates are commonly observed at the subcellular level, supporting the stable and efficient cycling of lipids and energy within the cell. Advanced techniques enabling organelle aggregation manipulation with high spatiotemporal precision have emerged as critical tools for investigating LDMIs and addressing diseases associated with LDMI dysregulation. This study aimed to summarize and update the distinctive characteristics of LDMIs and highlight the increasing understanding of their operational mechanisms, while also examining diseases caused by their dysfunction. Special emphasis was placed on the current challenges and prospects of strategies for the spatial aggregation manipulation of LDMIs, particularly the remarkable potential of optogenetic tools. With the continuous advancement of subcellular imaging and organelle aggregation manipulation technologies, LDMIs are poised to transform our understanding of cellular lipid metabolism networks and facilitate the effective manipulation of these complex networks at the organelle level.

Metabolic-inflammatory crosstalk orchestrates muscle repair. Although pyroptosis typically aggravates sterile injury, we demonstrated that GSDME-dependent pyroptotic signaling associated with recruited myeloid cells paradoxically supported regeneration. GSDME expression was induced in postsurgical human muscle injury and murine damage models. Gsdme deficiency delayed functional recovery and exacerbated injury-induced myosteatosis, a pathological form of intramuscular ectopic fat deposition. Time-series and scRNA-seq analyses revealed that GSDME loss shifted the transcriptional program from oxidative metabolism to lipid storage and adipogenesis. Lipidomics confirmed aberrant accumulation of triacylglycerols (TAGs) and sphingolipids in Gsdme-deficient muscle. Single-cell profiling further identified divergent fibro-adipogenic progenitor (FAP) states skewed toward adipogenesis, accompanied by impaired expansion of restorative Lyve1+Cd163+Txnip+ tissue-resident macrophages (TRMs), as validated by multiplex flow cytometry. Blocking CCR2-dependent monocyte recruitment produced regenerative defects comparable with those caused by Gsdme deficiency. Myeloid-specific Gsdme reintroduction rescued TRM expansion and function and curbed FAP adipogenic reprogramming, whereas FAP-specific expression proved ineffective. Mechanistically, IL-18 downstream of GSDME-dependent signaling engaged KLF4/JUN signaling in TRMs, sustaining their reparative and lipid-clearing capacity. This GSDME-IL-18-TRM axis was compromised in aged muscle, yet exogenous IL-18 reversed myosteatosis and accelerated regeneration. Together, these findings suggest that GSDME-dependent pyroptotic signaling can act as a metabolic checkpoint that sustains TRM-driven lipid homeostasis to support muscle regeneration.

Monitoring low molecular weight heparin (LMWH) is essential in clinical practice. We present a peptide-based aggregation-induced emission fluorescent probe, Z-BTDPA-Pep, capable of rapid, sensitive, and selective detection of LMWH in serum (0.1-2.0 U ml-1, LOD = 0.00378 U ml-1). The results correlate well with the clinical gold-standard anti-Xa assay, highlighting its potential for real-time LMWH monitoring and safer clinical drug administration.

Surface plasmon resonance (SPR) biosensor has emerged as a transformative tool in high-throughput drug screening and label-free analysis of biomolecular interactions. However, a critical limitation of SPR lies in its stringent requirement for highly purified proteins to ensure reliable quantification of binding affinities and kinetic parameters. In order to address the limitations, the lentiviral particle and styrene-maleic acid polymer have been previously developed to extract and stabilize transmembrane proteins (TMs) indirectly and thus to detect ligands interaction with TMs by SPR biosensor. The present study proposes a high-throughput SPR-based drug screening system that utilizes cell-free protein synthesis (CFPS) to achieve in situ purification and immobilization of TMs on SPR biosensors. First, C−X−C chemokine receptor 4 (CXCR4) protein with His-tag was prepared by CFPS. Then, two types of nickel-nitrilotriacetic acid (Ni-NTA) biosensors, modified with carboxymethylated dextran (CMD) and coated with polycarboxylate hydrogel coating (HC) matrix, were compared with classical carboxymethylated dextran 5 (CM5) biosensor in order to determine the optimal strategy for coupling the CXCR4-CFPS protein. The CMD-NTA/SPR biosensor was next applied to screen for CXCR4 ligands from 96 natural products. Finally, glycyrrhizic acid and ginsenoside Re were proved to be function of CXCR4 inhibitors by affinity test, molecular docking, and cell migration assay. The combination of CFPS with SPR technology facilitates in-situ purification and immobilization of target proteins in a single step, thereby significantly enhancing the efficiency of SPR assay procedures. The system has broad applicability for targeting various challenging TMs and provides potential candidates for subsequent drug development.

Subcellular membrane environments pose major challenges to target-oriented drug discovery due to the structural complexity and dynamic conformational transitions of transmembrane proteins. Conventional affinity chromatography techniques are limited by prolonged preparation period, lack of binding-site resolution, and interference from endogenous membrane proteins. This study introduces in situ synthesized transmembrane-protein affinity chromatography (iSTAC), a biomimetic platform integrating cell-free protein synthesis, engineered silica-phase modification, and tethered lipid bilayers to reconstitute functional multipass transmembrane receptors under native-like conditions. iSTAC enables the rapid column preparation within 5 h (a reduction in time of 33.6-fold), while preserving structural integrity and conformational dynamics. This facilitates high-specificity drug screening and binding-site analysis. We used the 5 hydroxytryptamine receptor 1A (5-HT1A) as a model to identify two natural agonists, crocin I and crocin II, from herbal extracts and validated their anti-insomnia and neuroprotective effects in vivo. Mutational iSTAC analysis precisely mapped their binding interactions at the residues N386, T196, and S199, demonstrating that both compounds activate 5-HT1A via canonical signaling pathways.

Radix Aconiti Lateralis Preparata (Fuzi in Chinese) is an efficient Chinese herbal medicine (CHM) against doxorubicin (DOX)-induced cardiomyopathy (DIC), but the cardioprotective compounds and targets of Fuzi remain unclear. In this work, a novel two-dimensional (2D) polyamidoamine (PAMAM)-modified cardiac mitochondrial membrane chromatography (CMMC) system, with the characteristic of high-density mitochondrial membrane coating and longer column lifespan, was developed for the identification of bioactive components from Fuzi to counteract DIC. Three compounds with strong retention were screened out to have cardioprotective effects against DOX-induced cardiotoxicity in different cardiomyocytes, and one promising compound songorine with the highest affinity was selected for further in vitro and in vivo pharmacodynamic validation and target identification. Hadha, involved in fatty acid oxidation (FAO) and apoptosis, was identified as the functional target of songorine by cardiac mitochondria proteomics, drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) and molecular docking assays. siRNA experiments have also demonstrated that songorine can affect the function of Hadha. It was concluded that songorine could directly bind to the Hadha protein to alleviate the functional impairment of the mitochondrial FAO and oxidative phosphorylation (OXPHOS) and regulate the Bcl-2 family proteins in mice to treat DIC.

Hyaluronidase (HAase), an enzyme responsible for hyaluronic acid (HA) degradation, compromises the structural integrity of HA by cleaving anti-inflammatory high-molecular-weight HA into pro-inflammatory lower molecular weight forms. Consequently, developing simple and efficient strategies for screening HAase inhibitors is of critical importance. Herein, a novel biosensing-based targeted affinity screening method was established for the identification of HAase inhibitors from complex herbal medicines. In the biosensing system, carbon dots served as fluorescent probes, while HA-functionalized gold nanoparticles acted as quenchers, and the addition of HAase induced a marked fluorescence recovery. Under optimized conditions, HAase exhibited a good linear response over the concentration range of 5.3 to 343.8 U mL- 1. This method enables rapid and efficient detection of HAase inhibitory activity in complex herbal extracts. Subsequently, active components were selectively captured, separated, and identified using immobilized HAase affinity chromatography. Following validation with both negative and positive control drugs, the screening model successfully identified Rhodiola rosea L. among 43 herbal medicines as a potent HAase. Gallic acid (GA) was identified as the active constituent, exhibiting an IC50 value of 111.7 & micro;M. Results from molecular docking and molecular dynamics simulations demonstrated a strong binding affinity between GA and HAase. Additionally, cellular experiments confirmed the anti-inflammatory activity of GA, as evidenced by its significant inhibition of LPS-induced inflammatory markers IL-4, IL-5, IL-6, and TNF-a. Collectively, the integrated biosensing and affinity-based screening strategy established in this study provides a precise and efficient platform for the discovery of bioactive compounds from complex natural products.

Monoclonal antibody (mAb) therapeutics are cornerstone treatments for cancers, autoimmune diseases, and infections. However, their efficacy is often compromised by anti-drug antibody (ADA) formation. Accurate and dynamic detection of mAbs and ADAs is essential for early immunogenicity evaluation and individualized dosing. Using the model adalimumab (ADL), we developed a dual biosensing strategy that enables the simultaneous quantification of mAb drugs and ADA. To overcome the lack of paratope-specific recognition elements in mAb assays, a bidirectional positive/negative phage display screening method based on F(ab ')2 fragments was established, removing interference from homologous IgG. Using this approach, a specific ADL-paratope recognition peptide, CP1, was successfully identified, demonstrating high affinity with KD of 7.84 & micro;M. For ADA recognition, an ADL-derived single-chain variable fragment (c-scFv) was used as an ADA paratope-targeting unit. CP1 and c-scFv were conjugated to AuNPs to construct biosensing materials: CP1.1@AuNPs and c-scFv@AuNPs for ADL and ADA detection, respectively, enabling rapid 2-min quantification that highly correlated with results of ELISA detection (R 2 = 0.99). These paratope-specific recognition element-functionalized biosensors were successfully applied for the real-time detection of ADL and ADA in 33 patients with ankylosing spondylitis, supporting personalized therapeutic refinement and offering a practical approach toward the precision management of mAb therapies.

Drug discovery has begun to move beyond traditional targets,turning instead to more complex target landscapes.These complex targets,including membrane proteins,are characterized by their environment and conformational mobility.A limitation of tradi-tional in vitro screening is that the target protein is not in its native environment.As a result,there is a need to go beyond current methods,this will allow you to study a target proteins functional activity in their native environment.To this end,target-based drug screening technologies have evolved through the conver-gence of multiple fields including analytical chemistry[1],biophysics[2],cell biology[3],and computational science[4].This integration can move drug discovery beyond isolated targets and allow for the target protein analysis in their native environment.

The power of drugs lies in their ability to reach their target sites and remain in place for a sufficient duration to exert their therapeutic effects. However, for some drugs, lysosomal phagocytosis presents ongoing challenges. In this study, an engineered organelles visualization drug-delivery system (OVDS) is introduced as a subcellular drug visualization and redistribution framework that facilitates the movement of drug molecules from one organelle, specifically lysosomes, to another, such as the mitochondria. As a proof-of-concept study, an OVDS is developed to facilitate the translocation of 10-hydroxycamptothecin (HCPT) from the lysosomes to mitochondria. This modification of subcellular HCPT distribution allows the evasion of lysosome-mediated HCPT resistance in cancer cells. Unlike traditional chemotherapeutic approaches, when HCPT is incorporated into the OVDS framework (HCPT-OVDS), the positive charge of the OVDS facilitates protonation, thereby enabling HCPT to escape lysosomes and enter mitochondria. Using HCPT-OVDS, substantial drug accumulation is achieved at the target sites in HCPT-resistant cells, with up to 70 ± 6% efficient subcellular translocalization and a 12.8 fold enhancement in cytotoxicity. Overall, the HCPT-OVDS represents an innovative engineering framework for subcellular spatial redistribution and offers a promising solution for addressing cancer drug resistance.

Sini Decoction (SNT) is a traditional formula recognized for its efficacy in warming the spleen and stomach and dispersing cold. However, elucidating the mechanism of action of SNT remains challenging due to its complex multiple components. This study utilized a synergistic approach combining two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE)-based drug affinity responsive target stability (DARTS) with label-free quantitative proteomics techniques to identify the direct and indirect protein targets of SNT in myocardial infarction. The analysis identified 590 proteins, with 30 proteins showing significant upregulation and 51 proteins showing downregulation when comparing the SNT group with the model group. Through the integration of 2D-DIGE DARTS with proteomics data and pharmacological assessments, the findings indicate that protein disulfide-isomerase A3 (PDIA3) may serve as a potential protein target through which SNT provides protective effects on myocardial cells during myocardial infarction.

Anthracycline‐induced cardiotoxicity remains a major limitation in cancer therapy, affecting long‐term cardiovascular health in survivors. Dietary nitrate supplementation has shown cardioprotective effects in preclinical models of doxorubicin (Dox)‐induced and ischemia–reperfusion injury, but it is unclear whether nitrate and/or nitrite (NOx) would have adverse effects on the anticancer efficacy of the drug. To evaluate Dox efficacy against triple‐negative breast cancer (TNBC) in the presence of dietary nitrate and nitrite, tumor‐bearing BALB/c mice ( N = 5 mice per group, 10 mice total) were treated with four weekly intravenous doses of Dox with or without NOx supplementation of their drinking water. Cardiac tissue from the NOx‐treated mice exhibited less fibrosis and lower levels of 4‐hydroxynonenal‐modified proteins, a marker of lipid oxidation and oxidative stress. Tumor sizes varied, but most regressed by the final Dox dose. Importantly, NOx supplementation did not compromise the antitumor efficacy of Dox nor did it promote pulmonary metastasis; instead, a trend toward fewer metastatic lesions was observed. These findings support the potential clinical use of dietary nitrate and nitrite as adjuncts to Dox treatment to mitigate cardiotoxicity without impairing anticancer outcomes.

Saposhnikovia divaricata (SD) is a traditional Chinese medicine (TCM) which has been commonly used for the treatment of rheumatoid arthritis (RA). However, its active components and mechanism of anti-RA are still unclear. Targeting rheumatoid arthritis-fibroblastoid synovial (RA-FLS) and synovial macrophages are promising strategies for RA treatment, and their membrane receptors are important targets for anti-RA active substances. A dual channel 3-mercaptopropyltrimethoxysilane (MPTS) modified 2D cell membrane chromatography (CMC) system was established to characterize dual-cell membrane binding active components in SD. Nine components retained on RAW-CMC column and 8 components retained on FLS-CMC column were screened out. Among them, 8 components retained well on both CMC columns. We further validate the pharmacological activity of 5-O-methylvisammioside, 3’-O-angeloylhamaudol, imperatorin, phellopterin and anomalin. They could efficiently target both inflammatory macrophages and fibroblast synovial cells, reduce the release of inflammatory factors, inhibit abnormal cell proliferation, and promote cell apoptosis. 5-O-methylvisammioside, which exhibited the best pharmacological ability on both target cells, inhibited the NF-κB pathway. Our results showed that the dual channel MPTS modified 2D CMC system is a practical method for rapid screening the active components in TCM binding on multiple target cells’ membrane protein of a disease. The method is very suitable for elucidating the mechanism of TCM with multiple-components and targets, and rapid screening of lead compounds.

Acetylcholinesterase (AChE) plays a crucial role in the activities of the nervous system, and its abnormal function can lead to the occurrence and development of neurodegenerative diseases. Hence, an effective method for real-time monitoring of AChE activity is essential. Very recently, several fluorescence sensors have been developed for the detection of AChE activity, but they are usually imaging in the visible region, relatively small Stokes shifts, or long response times, limiting their application for real-time monitoring in vivo. Inspired by that, a near-infrared (NIR) off-on probe ((E)-4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl dimethylcarbamate, DCM-N) for AChE monitoring with high selectivity and sensitivity is developed. In the probe DCM-N, a bright near-infrared fluorescence emission at 700 nm can be triggered by AChE through the cleavage of amino ester bond in DCM-N, and the resulting fluorescence exhibits a good linear relationship with AChE activity in the range of 0.2-16 U/mL, with a detection limit as low as 0.06 U/mL. For real plasma sample detection, DCM-N demonstrates advantages of accurate detection and fast response compared to the traditional Ellman assay for AChE detection. Moreover, DCM-N can be used for imaging of AChE activity in live cells and tracking of AChE activity in zebrafish models, which is of great significance for medical and physiological research related to AChE. DCM-N possesses several notable features such as light-up NIR emission, fast response, large spectral shifts and strong photostability under physiological conditions. These features enable it to monitor AChE activity both in vivo and in vitro, providing a suitable tool for real-time monitoring and in vivo visualization of AChE activity.

ETHNOPHARMACOLOGICAL RELEVANCE:Bu-Wang San (BWS) is a prominent traditional Chinese medicine known for calming the mind and promoting intelligence. It has been reported to improve learning and memory, enhance memory ability, and promote synaptic plasticity. However, the complexity of the material basis and the diversity of therapeutic targets of BWS on Alzheimer's disease (AD) have not been elucidated. AIM OF THE STUDY:This study aimed to investigate the therapeutic material basis and the mechanism of BWS in AD treatment by comprehensively analyzing multiple GEO datasets of the human hippocampus, network pharmacology, and multi-platform metabolomics validation. MATERIALS AND METHODS:Three GEO datasets of the human hippocampus were utilized to identify AD-associated targets using weighted gene co-expression network analysis (WGCNA) and differential analysis. Network pharmacology analyses were performed to investigate BWS's therapeutic material basis and predict the therapeutic targets of BWS on AD. A rat model was induced through the concurrent administration of AlCl3 and D-galactose to validate BWS's therapeutic potential and underlying mechanisms in AD. To validate the results of GEO data mining and network pharmacology, a comprehensive metabolomics approach integrating gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was conducted on rat serum samples to uncover potential metabolic alterations and their associated pathways. RESULTS:A total of 6367 genes were selected as AD drug targets through WGCNA analysis and enrichment analysis of disease-associated gene expression profiles in the GEO database. Network pharmacology was performed in this study for the identification of potential interactions between the components of BWS and its targets, TP53, STAT3, EGFR, MAOA, NOS3, PPARG, PRKCA, MAPK8, AChE, ARG1, among others, which were among the top 25 highest probable targets of BWS acting on AD. The multi-platform metabolomics indicated that amino sugar and nucleotide sugar metabolism, glycine, serine and threonine metabolism pathways, and other pathways may be associated with the AD model based on AlCl3 and D-galactose. The comparison of differential metabolites between the AD model group and the BWS intervention group revealed that 66 of the 97 differential metabolites exhibited a pullback trend, indicating a potential therapeutic effect of BWS on these metabolites. CONCLUSION:This study builds a systematic strategy combining GEO datasets, network pharmacology, and multi-platform metabolomics and provides valuable insights into the pharmacological mechanism of BWS on AD. The results suggest that BWS may exert its therapeutic effects on AD by modulating the amino sugar and nucleotide sugar metabolism, glycerophospholipid metabolism, glycine, serine and threonine metabolism pathway and acting on the drug targets of ARG1, MAOA, AChE, XDH, GAD2 et al. This strategy provides a deep understanding of the molecular mechanisms of herbal medicine in treating AD at a systematic level.

Membrane receptor affinity chromatography is a practical strategy for screening compounds targeting membrane receptors and determining their binding affinity. The key of these methods is to create a biomimetic cell membrane environment for the immobilization of specific receptors on chromatographic stationary phase. However, its large-scale applications are limited by the labor-intensive and time-consuming procedures including cell culture, recombinant protein expression and column packing. Furthermore, traditional affinity chromatography columns are prone to drawbacks such as heterogeneous composition of the stationary phase and low permeability. In this study, a novel membrane receptor biological affinity chromatographic method based on cell-free protein synthesis (CFPS) and Bis(sulfosuccinimidyl)suberate modified monolithic stationary phase was developed for fast preparation of monolithic micro-affinity column in batches, which realized efficient synthesis and immobilization of immune checkpoint natural killer group 2 family of receptor A (NKG2A) with controlled orientation. Coupling the prepared NKG2A micro-affinity column with an offline-2D-UPLC-QTOF/MS system, two new NKG2A inhibitors, baicalin and wogonoside, were screened out with the KD values of 30.23 and 13.01 mu M respectively, significantly upregulating the gene expression of granzyme B, tumor necrosis factor-alpha and interferon-gamma and the protein expression of CD107a in natural killer (NK) cells. Moreover, the cytotoxic activity of NK cells against tumor cells was enhanced by these two compounds. The proposed CFPS-based monolithic micro- affinity chromatography realizes rapid synthesis and immobilization of transmembrane receptors within one day, achieving homogeneity, good permeability and orientation-controlled high expression. This approach could be extended to any interested transmembrane receptors for rapid drug screening and affinity determination.

The development of programmed death 1 (PD-1) checkpoint/programmed death-ligand 1 (PD-L1) interaction inhibitors has opened a new front in the treatment of carcinoma. An increasing amount of research is devoted to small-molecule compounds that target this PD-1/PD-L1 interaction. In this article, we report the discovery of three new PD-1/PD-L1 inhibitors from Scutellaria baicalensis Georgi and Sophora flavescens Aiton herbal extracts, namely baicalin, maackiain, and oxysophocarpine, using a comprehensive ligand fishing system, which integrates a dual-target surface plasmon resonance biosensor and a magnetic beads method. These three compounds were also confirmed in the serum in vivo, validated by binding affinity evaluation, molecular docking, and competitive enzyme-linked immunosorbent assay (ELISA) assay to act upon the interface of PD-1/PD-L1. Thus, these three ingredients could be potential PD-1/PD-L1 inhibitors and may serve as hit compounds for immunotherapeutic drug discovery. These results also highlight the efficiency of the dual-target surface plasmon resonance (SPR) and magnetic beads ligand fishing system in drug screening for disease treatment.

Biological aggregates play a crucial role in the pathogenesis of thrombotic diseases, especially thrombin-induced biological aggregates. Therefore, the efficient discovery of thrombin inhibitors is of great significance for the prevention and treatment of thrombotic diseases. In this study, the aggregation precursor protein fluorescent probe was successfully prepared for monitoring the production of biological aggregates induced by thrombin. In this program, the aggregation degree of biomolecules can be quickly monitored through a fluorescence sensing technology. To facilitate the modulation of the biological aggregation process, the application of this advanced fluorescence sensing technology was utilized for the screening of thrombin inhibitors, which are pivotal regulatory molecules in biological aggregation. In addition, by combining the target fishing technique, an integrated model for rapid screening of potential inhibitors in complex extracts was further established. This model not only swiftly detects the presence of inhibitory components within complex systems but also precisely captures and identifies active monomers. After positive drug validation of the screening model, three active monomers, namely, ginkgetin, isoginkgetin, and bilobetin, were accurately screened from 30 natural products. These results highlighted the immense potential of the proposed approach for screening active ingredients from a wide range of natural products.

Second near‐infrared (NIR‐II) fluorescence imaging has emerged as a breakthrough technology for accurately revealing complex mechanisms in vivo owing to its high sensitivity, deeper tissue penetration, high spatiotemporal resolution, and high throughput. This review provides a comprehensive overview of NIR‐II fluorescence imaging, specifically focusing on the materials used, including single‐walled carbon nanotubes (SWCNTs), quantum dots (QDs), rare‐earth nanoparticles (RENPs), and organic fluorophores (OFs). It details their development, application, and advantageous performance in NIR‐II fluorescence imaging. Furthermore, this review highlights an approach to dynamic multiplexed NIR‐II fluorescence imaging in vivo that enables multitarget detection, providing a powerful tool for accurately and effectively assessing pathological processes and revealing complex biological mechanisms in vivo. Finally, it explores the aspects of translational medicine for NIR‐II imaging, addressing challenges, and future prospects related to material development, detection equipment, and unmet biomedical applications.