Bioconjugate Chemistry最新文献

High glutathione levels in the tumor microenvironment drive tumor resistance and proliferation, making glutathione depletion a key cancer therapeutic strategy. Existing inhibitors face issues such as poor specificity and biocompatibility, creating a need for new formulations with high targeting, low toxicity, and efficient depletion. This study synthesized carbon dots (PCDs) via the hydrothermal method using 3,4,9,10-perylenetetracarboxylic dianhydride as a precursor. PCDs oxidize glutathione for depletion via intrinsic redox properties and act as efficient glutathione probes with fluorescence and colorimetric detection limits of 0.527 and 13.11 μM, respectively, showing excellent stability in complex biological environments. Under an 808 nm near-infrared laser, PCDs exhibit 41.88% photothermal conversion efficiency. PCDs induce tumor cell apoptosis by depleting glutathione with enhanced antitumor effects under photothermal therapy synergy. They disrupt tumor redox homeostasis to trigger immunogenic cell death, promote dendritic cell maturation, polarize M2 macrophages to M1, and activate T cell-mediated immunity. In vivo dual-tumor models confirmed that PCDs combined with αPD-L1 efficiently ablate primary tumors, inhibit distal growth, and exert systemic antitumor immune effects. This simple-synthesized, biocompatible PCDs integrate detection and antitumor functions, offering new ideas for next-generation nanodiagnostic/therapeutic materials and combination therapy.

Fluorescence-guided surgery (FGS) using fluorescent imaging probes is an emerging technology that enables tumor margin detection at potentially higher accuracy than is possible with traditional means. Most FGS imaging probes are administered systemically, many hours before a planned surgery, and must have cleared the bloodstream once surgery is initiated, so as not to contaminate the operative field. Topical administration of specially designed conjugates in the form of spray-on probes (SOP) offers the advantage of much lower doses, on-demand use, and result in far higher achievable image contrast. Here we report the discovery and characterization of a far-red SOP imaging agent targeting fibroblast activation protein alpha. We show that only 1 out of 10 synthesized probes has appropriate imaging and biological characteristics, increasing tumor-to-background ratio by 5–15 fold. The optimal SOP (FTF-Cy5) was able to delineate tumor margins rapidly (<10 min) when sprayed on at a low dose (5 μM) followed by a short washing step. These results suggest that the lead far-red SOP has the potential to be utilized as a spray-on intraoperative tumor margin delineation tool using far-red fluorescence imaging systems in the operating room, thereby simplifying surgical workflows and enabling more complete cancer resections in the future.

STING activates the innate immune system by inducing type-1 interferon (IFN) production and has been pursued as a therapeutic option in immuno-oncology. The targeted delivery of STING agonists to CCR2+ immune cells could enhance the therapeutic window of the agonists by selectively activating the STING pathway within targeted immune cells. The chemistry strategy was established to enable the targeted delivery of the cyclic dinucleotide STING agonist dazostinag to CCR2+ cells through an antibody-drug conjugate (ADC) approach. A self-immolative spacer between the adenine of dazostinag and the Cathepsin-B cleavable Val-Ala dipeptide linker rendered a linker payload that exhibits strong plasma stability while allowing the rapid payload release upon internalization into lysosomes. The stochastic cysteine conjugation of the dazostinag containing these linkers provided ADC TAK-500 and its mouse surrogate mTAK-500 with DAR = 4. In syngeneic tumor-bearing mouse models, mTAK-500 showed target specific antitumor activity as well as the induction of immune-stimulating cytokines.

Although targeted therapy and immunotherapy have shown encouraging clinical results in treating lung cancer, they can only benefit a handful of patients. Therefore, there is an urgent need to develop novel anticancer strategies to treat lung cancer. Here, we report a cancer-targeting strategy for the treatment of lung cancer via coating pretreated neutrophil membranes (pM) stimulated with lung cancer cells onto hollow manganese dioxide (HMnO₂) cores, and further development of sono-immunotherapeutic nanoparticles (MTpMAb@R) by decoration of sonosensitizers and anti-CTLA-4 antibodies on the HMnO₂ nanoshells for activatable tumor sono-immunotherapy. The pM-coated nanoparticles with high expression of chemokine receptors target tumors more efficiently. With ultrasound irradiation, MTpMAb@R nanoparticles can both generate immunogenic cell death through the sonodynamic effect and block the immune checkpoint by releasing anti-CTLA-4 antibodies, which concurrently induces a sequence of antitumor immune responses. As a result, such a synergistic therapeutic action is achieved by combined sono-immunotherapy. Therefore, this study represents a promising strategy for treating lung cancer with high precision.

Mesothelin (MSLN) is a tumor biomarker expressed at high levels on the surface of numerous cancers with extremely limited expression in healthy tissues. MSLN-targeting agents developed for diagnosis and therapy could have a significant impact on the management of MSLN-expressing cancers. Pleural mesothelioma (PM) is a deadly cancer that arises from mesothelial cells lining the pleura and is predominantly linked to asbestos exposure. There are currently no effective treatments, and diagnosis occurs in late stages of disease due to the lack of clinical symptoms in the early stages. Recent efforts to diagnose and treat PM have focused on identifying and targeting relevant biomarkers, including MSLN. We engineered proteins based on the nonantibody fibronectin type III (Fn3) protein scaffold that bind MSLN with high affinity and specificity, using yeast-surface display and directed evolution. Previous work with Fn3 scaffold proteins has demonstrated tissue distribution desirable for applications in molecular imaging and targeted radiotherapy, which may overcome limitations encountered thus far with antibody-based approaches to treat PM. The MSLN-targeting Fn3 was further developed for bioconjugation with the 1,4,7,10-tetraazacyclododecane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) radiometal chelator. MSLN-binding Fn3 specifically binds to the MSLN-expressing PM lines, colocalizes with MSLN, and internalizes upon binding. Fn3-DOTAGA was further coupled with cold metal gallium-69, and the resulting conjugate maintained binding with high affinity to MSLN-expressing PM cells. MSLN-binding Fn3-DOTAGA-⁶⁹Ga is a promising molecule with diagnostic and therapeutic relevance, toward applications in molecular imaging and targeted radiotherapy.

Cysteine sulfenylation (Cys–SOH) is a transient redox-sensitive post-translational modification that regulates protein activity and cellular stress responses, yet its in vivo dynamics remain difficult to capture due to short half-life and low abundance. Here, we report the design and application of BTD-Az, a cell-permeable probe that incorporates an azide handle into the benzothiazine scaffold, enabling rapid bioorthogonal labeling of Cys–SOH both in vivo and in vitro, followed by efficient enrichment through strain-promoted azide-alkyne cycloaddition. This approach streamlines the detection and analysis of Cys–SOH with exceptional specificity and convenience. BTD-Az exhibited negligible cytotoxicity, efficiently enriched sulfenylated proteins in vitro, and achieved direct in vivo labeling in mouse tissues through SPAAC-mediated pull-down. Coupling BTD-Az with 4D-DIA proteomics allowed global sulfenylome profiling, revealing >5000 labeled proteins across tissues. As a proof-of-concept biological application, we applied BTD-Az to aging cartilage and identified 95 proteins with differential sulfenylation, including the mitochondrial enzyme IDH2, whose Cys–SOH modification promoted proteasomal degradation and exacerbated redox imbalance. Collectively, this study establishes BTD-Az as a robust chemical tool for in vivo sulfenylation profiling, providing a broadly applicable platform for redox proteomics and the discovery of redox-sensitive regulatory mechanisms in health and disease.

Following global efforts to decipher the glycome of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), we further explored the immunologically potent carbohydrates in the viral glycan shield. Specifically, we constructed a large panel of glycoconjugates, utilizing soluble protein carriers and bacteriophage Qβ viral-like particles (Qβ-VLPs) to display oligomannoses in multiple cluster configurations for immune recognition. Using a broad-spectrum virus-neutralizing agent, Galanthus nivalis agglutinin (GNA), and SARS-CoV-2-neutralizing antisera elicited in nonhuman primates (NHPs), we performed a carbohydrate-microarray-based glyco-epitope-mapping analysis of these synthetic glyco-conjugates. We found that several oligomannose-conjugates, including members of the Bovine Serum Albumin (BSA)- and Qβ-series of high-mannose-conjugates, reestablished the GNA epitopes with potency comparable to that of those expressed by the native viral glycoproteins. However, the NHP antisera differentially react with these glyco-conjugates and exhibit high selectivity for the GNA⁺-Qβ-VLPs. These findings suggest that the high-density oligomannose clusters presented by the Qβ-VLPs may resemble the virion-surface-exposed oligomannoses, thereby supporting glycan-specific immune recognition by antibodies elicited through natural infection.

Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that regulates various biological processes and has been implicated in neurological diseases, cancer, and autoimmune diseases. However, current methods for detecting A-to-I sites, including inosine chemical erasing and acrylonitrile-derivative labeling, suffer from compromised sensitivity and specificity due to two critical limitations: cross-reactivity with pseudouridine and suboptimal enrichment efficiency. Here, we introduce a novel chemical labeling strategy using propiolates as selective inosine-binding agents, coupled with biotin–streptavidin enrichment, enabling precise transcriptome-wide profiling of A-to-I editing sites. Through screening a range of propiolates and optimizing the reaction conditions, we demonstrated that tert-butyl propiolate functions as a highly selective probe, achieving 6-fold higher specificity for I compared to pseudouridine (Ψ) in RNA editing detection. This scaffold represents the first application of propiolates in RNA editing detection. Subsequent RT-qPCR analysis revealed that the optimized protocol achieved a 55-fold enrichment efficiency of inosine-containing RNAs through copper-free click chemistry conjugation and streptavidin magnetic bead pulldown. Compared to acrylonitrile-derivative labeling methods, this protocol represents a 3.7-fold improvement in enrichment efficiency. Applied to human cellular RNA, this method robustly identified A-to-I editing sites with enhanced accuracy and coverage. By reducing pseudouridine cross-reactivity and enabling efficient RNA enrichment, our strategy provides a universal platform for studying RNA editing dynamics in development, disease, and therapeutic contexts, thereby opening new avenues for epitranscriptomic biomarker discovery. This work advances the molecular toolbox for epitranscriptomics, offering broad utility in dissecting the functional roles of A-to-I editing in health and pathology.

Pneumococcal conjugate vaccines (PCVs) have effectively enhanced immunogenicity by conjugating a carrier protein to a purified capsular polysaccharide. The degree of conjugation influences the effective size of the final conjugate, and control of this reaction is critical in developing a robust process. Sodium triacetoxyborohydride (STAB) is a common reducing agent used to perform reductive aminations to provide a means for conjugation and can be utilized as an in situ preparation in the PCV conjugation process. Robust analytical methods for characterizing STAB were not previously available. Herein, we develop methods to rapidly assess STAB for both activity and composition using quantitative NMR methodologies and apply these learnings to improve our understanding of the bioconjugation process. It was determined that decreasing the reaction temperature to synthesize STAB resulted in a more active reducing reagent enriched with sodium diacetoxyborohydride (SDAB). Conjugation reactions performed with a model polysaccharide and carrier protein found that an increased SDAB content led to larger conjugation sizes. Moreover, we established a correlation between the conjugate size and SDAB concentration by charging the reaction with varying molar equivalents of SDAB. Through this work, a deeper understanding of the critical attributes of STAB was developed using diverse analytical methods, and these learnings can be applied to develop a more appropriate control strategy for producing glycoconjugate therapeutics.

Targeted perturbation of redox balance through concurrent elevation of reactive oxygen species (ROS) production and glutathione (GSH) depletion has emerged as a therapeutic paradigm for triggering tumor cell apoptosis. Nevertheless, the conventional single-agent system demonstrates limited therapeutic efficacy due to insufficient oxidative stress amplification within tumor cells. Herein, we designed pH- and GSH double-responsive metallic magnetic Ag-NH₂–CoFe₂O₄@C@DOX nanoparticles (ANFCD NPs), which disrupted the redox balance within the tumor microenvironment (TME) to achieve synergistic chemodynamic therapy (CDT), photothermal therapy (PTT), and chemotherapy (CT) effects. In the acidic TME, ANFCD NPs functioned as both a Fenton catalyst and GSH depletor through the reversible redox property of Fe (II/III) and Co (I/II), inducing oxidative stress and exerting a “leverage” rebalancing to potentiate CDT. Additionally, ANFCD NPs showed high photothermal conversion efficiency, enhancing PTT efficacy via magnetic targeting-driven tumor accumulation. Meanwhile, they could also responsively release DOX to achieve CT. More importantly, the hyperthermia generated by ANFCD NPs not only effectively eradicated tumor cells but also boosted the CDT effect and promoted DOX release, ultimately achieving the aim of combined therapy. Therefore, such a nanomaterial is a promising therapeutic agent for disrupting redox homeostasis to augment multimodal collaborative therapy, which might show further applications in nanomedical science.