Shear-Induced Cycloreversion Leading to Shear-Thinning and Autonomous Self-Healing in an Injectable, Shape-Holding Collagen Hydrogel.

In vivo injectable extracellular matrix (ECM) derived hydrogels that are suitable for cell encapsulation have always been the holy grail in tissue engineering. Nevertheless, these hydrogels still fall short today of meeting three crucial criteria: (a) flexibility on the injectability time window, (b) autonomous self-healing of the injected hydrogel, and (c) shape-retention under aqueous conditions. Here we report the development of a collagen-based injectable hydrogel, cross-linked by cycloaddition reaction between furan and maleimide groups, that (a) is injectable up to 48 h after preparation, (b) can undergo complete autonomous self-healing after injection, (c) can retain its shape and size over several years when stored in the buffer, (d) can be degraded within hours when treated with collagenase, (e) is biocompatible as demonstrated by in vitro cell-culture, and (f) is completely resorbable in vivo when implanted subcutaneously in rats without causing any inflammation.

Bioorthogonal Regulated Metabolic Balance for Promotion of Ferroptosis and Mild Photothermal Therapy.

The nanozyme with NADPH oxidase (NOX)-like activity can promote the consumption of NADPH and the generation of free radicals. In consideration of that the upregulation of glucose-6-phosphate dehydrogenase (G6PD) would accelerate the compensation production of NADPH, for inhibition of G6PD activity, our designed bioorthogonal nanozyme can in situ catalyze pro-DHEA to produce G6PD inhibitor and dehydroepiandrosterone (DHEA) drugs to inhibit G6PD activity. Therefore, the well-defined platform can disrupt NADPH homeostasis, leading to the collapse of the antioxidant defense system in the tumor cells. The enzyme-like activity of PdCuFe is further enhanced when irradiated by NIR-II light. The destruction of NADPH homeostasis can promote ferroptosis and, in turn, facilitate mild photothermal therapy. Our design can realize NADPH depletion and greatly improve the therapeutic effect through metabolic regulation, which may provide inspiration for the design of bioorthogonal catalysis.

Stable Cu (I) Complexes for Intracellular Cu-Catalyzed Azide Alkyne Cycloaddition.

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) has heralded a new era of chemical biology and biomedicine. However, caveats of CuAAC include formation of reactive oxygen species (ROS) and other copper-related toxicity. This limits utility in sensitive biological samples and matrices. Towards addressing these caveats, we synthesized and fully characterized two air and water stable trinuclear Cu (I) dimer complexes. The complexes were stable to oxidation in the presence of hydrogen peroxide, acid, base, and other chelators, which was reasoned to be due to the linear benzimidazole-Cu-benzimidazole geometry. Computational investigations of the catalytic cycle implicated two of the three coppers in the trimer complex as the active metal centers. The complexes were shown to catalyze the reaction at far below sub-toxic concentrations for intracellular click reactions to label triple negative breast cancer cells and compared to the current CuSO-4-THPTA standard.

Antibody Modification via Lipoic Acid Ligase A-Mediated Site-Specific Labeling.

Enzymatic modification, particularly utilizing lipoic acid ligase (LplA), has emerged as a transformative approach in biopharmaceuticals, enabling precise and site-specific protein modifications. This review delves into the innovative applications of LplA in antibody modifications, including the creation of antibody-drug conjugates (ADCs) and the advancement of tag-free conjugation techniques. LplA's ability to facilitate the incorporation of bioorthogonal groups and its adaptability to various substrates underscores its versatility. Key developments include the successful generation of dual-labeled antibodies and the application of LplA in modifying antibody fragments. Additionally, the review explores the potential for LplA to enhance the therapeutic efficacy of ADCs through improved drug-to-antibody ratios and site-specific payload attachment. The implications of these advancements are significant, suggesting that LplA-mediated modifications could lead to more effective and targeted antibody-based therapies. This review aims to provide a comprehensive overview of LplA's role in expanding the possibilities of enzymatic conjugation, setting the stage for future research and clinical applications.

'Click' hydrogels from renewable polysaccharide resources: Bioorthogonal chemistry for the preparation of alginate, cellulose and other plant-based networks with biomedical applications.

Click chemistry refers to a class of highly selective reactions that occur in one pot, are not disturbed by water or oxygen, proceed quickly to high yield and generate only inoffensive byproducts. Since its first definition by Barry Sharpless in 2001, click chemistry has increasingly been used for the preparation of hydrogels, which are water-swollen polymer networks with numerous biomedical applications. Polysaccharides, which can be obtained from renewable resources including plants, have drawn growing attention for use in hydrogels due to the recent focus on the development of a sustainable society and the reduction of the environmental impact of the chemical industry. Importantly, plant-based polysaccharides are often bioresorbable and exhibit excellent biocompatibility and biomimicry. This comprehensive review describes the synthesis, characterization and biomedical applications of hydrogels which combine the renewable and biocompatible aspects of polysaccharides with the chemically and biomedically favorable characteristics of click crosslinking. The manuscript focuses on click hydrogels prepared from alginate and cellulose, the most widely used polysaccharides for this type of hydrogel, but also click hydrogels based on other plant-derived polymers (e.g. pectin) are discussed. In addition, the challenges are described that should be overcome to facilitate translation from academia to the clinic.

Engineering of bioorthogonal polyzymes through polymer sidechain design.

Synthetic polymer scaffolds can encapsulate transition metal catalysts (TMCs) to provide bioorthogonal nanocatalysts. These 'polyzymes' catalyze the in situ generation of therapeutic agents without disrupting native biological processes. The design and modification of polymer scaffolds in these polyzymes can enhance the catalytic performance of TMCs in biological environments. In this study, we explore the hydrophobic design space of an oxanorborneneimide-based polymer by varying the length of its carbon side chain to engineer bioorthogonal polyzymes. Activity studies indicate that modulating the hydrophobicity of the polymer scaffold can be used to enhance the catalyst loading efficacy, catalytic activity, and serum stability of polyzymes. These findings provide insight into the structural elements contributing to improving polymeric nanocatalysts for a variety of applications.

In Situ Bioorthogonal Repair of the Vascular Endothelium Glycocalyx to Treat Acute Lung Injury.

In acute lung injury, destruction of the lung endothelial glycocalyx leads to vessel permeabilization and contributes to pulmonary edema and inflammation. Heparan sulfate, which accounts for >70% of glycosaminoglycans in the endothelial glycocalyx, plays a crucial physiological anti-inflammatory role. To treat acute lung injury, it is explored whether a two-step in vivo bioorthogonal chemistry strategy can covalently link intravenously administered heparan sulfate to the lung vascular endothelium and the damaged glycocalyx. First, fusogenic liposomes (EBP-Tz-FLs) carrying the reactive group tetrazine (Tz), and an E-selectin-binding peptide (EBP) to target the lung inflammatory endothelium are administered intravenously. This step aimed to anchor the tetrazine group to the membrane of inflammatory endothelial cells. Second, heparan sulfate (HS-TCO) conjugated to the trans-cyclooctene (TCO) group, which spontaneously reacts with Tz, is injected intravenously, leading to covalent heparan sulfate addition to the vascular endothelium. In a mouse model of acute lung injury, this approach substantially reduced vascular permeability and attenuated lung tissue infiltration. The EBP-Tz-FLs and HS-TCO showed favorable biocompatibility and safety both in vitro and in vivo. The proposed strategy shows good promise in acute lung injury therapy and covalently anchoring functional molecules onto the membrane of target cells.

Chemical recording of pump-specific drug efflux in living cells.

Drug efflux - a process primarily facilitated by efflux pumps such as multidrug resistance proteins (MRPs) - plays a pivotal role in cellular resistance to chemotherapy resistance. Conventional approaches to assess drug efflux are predominantly conducted in vitro and often lack pump specificity. Here we report the bioorthogonal reporter inhibiting efflux (BRIEF) strategy, which enables the recording of pump-specific drug efflux in living cells. In BRIEF, a specific substrate is engineered as a bioorthogonal efflux probe (BEP) for specific pumps. The cellular concentration and protein labeling level of the probe can be augmented when the test drug is transported by the same pumps.  Serendipitously, we discovered that per-O-acetylated unnatural monosaccharides, initially designed for metabolic glycan labeling, are exported by some MRPs. Using Ac4GlcNAl as a BEP, we studied the structure-efflux relationship of flavonoids and identified small molecules, including tannic acid, cholesterol and gallic acid, as novel MRP substrates in high-throughput screening. Tannic acid, known for anti-tumor and anti-SARS-CoV-2 properties, showed increased efficacy upon MRP inhibition. Additionally, BRIEF was adapted to assess p-glycoprotein-mediated efflux using Rhodamine 123 as a BEP, leveraging its light-activatable proximity labeling ability. BRIEF provides a versatile approach to investigate drug efflux and enhance chemotherapy strategies.

In situ chemoproteomic profiling reveals itaconate inhibits de novo purine biosynthesis in pathogens.

Itaconate serves as an immune-specific metabolite that regulates gene transcription and metabolism in both host and pathogens. S-itaconation is a post-translational modification that regulates immune response; however, its antimicrobial mechanism under the physiological condition remains unclear. Here, we apply a bioorthogonal itaconate probe to perform global profiling of S-itaconation in living pathogens, including S. Typhimurium, S. aureus, and P. aeruginosa. Some functional enzymes are covalently modified by itaconate, including those involved in the de novo purine biosynthesis pathway. Further biochemical studies demonstrate that itaconate suppresses this specific pathway to limit Salmonella growth by inhibiting the initiator purF to lower de novo purine biosynthesis and simultaneously targeting the guaABC cluster to block the salvage route. Our chemoproteomic study provides a global portrait of S-itaconation in multiple pathogens and offers a valuable resource for finding susceptible targets to combat drug-resistant pathogens in the future.

Sydnone-based prosthetic groups for radioiodination.

The potential of Strained-Promoted Sydnone-Alkyne Cycloaddition (SPSAC) for radioiodination was evaluated with model cyclooctyne-conjugated peptides. Starting with a series of sydnones with varying N3 and C4 substitution, a preliminary kinetic study with non-radioactive iodinated compounds highlighted the superiority of an arylsydnone substituted by a chlorine atom in C4 position. Interestingly, reaction rate up to 11 times higher than using an azide was achieved with the best system. Access to 125I-labelled sydnones was granted with high efficiency from arylboronic acid precursors by copper catalyzed nucleophilic substitution. Application of SPSAC on the model peptide in radiotracer conditions showed the same trend than in non-radioactive kinetic study and complete reactions could be achieved within less than an hour for the best systems. These results are favorable for use in the production of radiopharmaceuticals with heavy halogens and increase the diversity of available bioorthogonal reaction for nuclear imaging and therapy.

Engineering extracellular vesicles for diagnosis and therapy.

Extracellular vesicle (EV)-based therapeutics have gained substantial interest in the areas of drug delivery, immunotherapy, and regenerative medicine. However, the clinical translation of EVs has been slowed due to limited yields and functional heterogeneity, as well as inadequate targeting. Engineering EVs to modify their inherent function and endow them with additional functions has the potential to advance the clinical translation of EV applications. Bio-orthogonal click chemistry is an engineering approach that modifies EVs in a controlled, specific, and targeted way without compromising their intrinsic structure. Here, we provide an overview of bio-orthogonal labeling approaches involved in EV engineering. We also present the isolation methods of bio-orthogonally labeled vesicles using magnetic beads, microfluidics, and microarray chip technologies. We highlight the in vivo applications of bio-orthogonal labeling EVs for diagnosis and therapy, especially the exciting potential of bio-orthogonal glycometabolic engineered EVs for targeted therapies.

Precise Clearance of Intracellular MRSA via Internally and Externally Mediated Bioorthogonal Activation of Micro/Nano Hydrogel Microspheres.

Traditional high-dose antibiotic treatments of intracellular methicillin-resistant staphylococcus aureus (MRSA) are highly inefficient and associated with a high rate of infection relapse. As an effective antibacterial technology, sonodynamic therapy (SDT) may be able to break the dilemma. However, indiscriminate reactive oxygen species (ROS) release leads to potential side effects. This study incorporates Staphylococcal Protein A antibody-modified Cu2+/tetracarboxyphenylporphyrin nanoparticles (Cu(II)NS-SPA) into hydrogel microspheres (HAMA@Cu(II)NS-SPA) to achieve precise eradication of intracellular bacteria. This eradication is under bioorthogonal activation mediated by bacillithiol (BSH) (internally) and ultrasound (US) (externally). To specify, the US responsiveness of Cu(II)NS-SPA is restored when it is reduced to Cu(I)NS-SPA by the BSH secreted characteristically by intracellular MRSA, thus forming a bioorthogonal activation with the external US, which confines ROS production within the infected MΦ. Under external US activation at 2 W cm-2, over 95% of intracellular MRSA can be cleared. In vivo, a single injection of HAMA@Cu(II)NS-SPA achieves up to two weeks of antibacterial sonodynamic therapy, reducing pro-inflammatory factor expression by 90%, and peri-implant bone trabeculae numbers exceed the control group by five times. In summary, these micro/nano hydrogel microspheres mediated by internal and external bioorthogonal activation can precisely eliminate intracellular MRSA, effectively treating multi-drug resistant intracellular bacterial infections.

High-Throughput Single-Cell Metabolic Labeling, Sorting, and Sequencing of Active Antibiotic-Resistant Bacteria in the Environment.

Active antibiotic-resistant bacteria (ARB) play a major role in spreading antimicrobial resistance (AMR) in the environment; however, they have remained largely unexplored. Herein, we coupled bio-orthogonal noncanonical amino acid tagging with high-throughput fluorescence-activated single-cell sorting (FACS) and sequencing to characterize the phenome and genome of active ARB in complex environmental matrices. Active ARB, conferring resistance to six antibiotics throughout wastewater treatment, were distinguished and quantified. The percentage and concentration of active ARB ranged from 0.28% to 45.3% and from 1.1 × 104 to 2.09 × 107 cells/mL, respectively. Notably, the final effluents retained up to 4.79 × 104 cells/mL of active ARB. Targeted FACS and genomic sequencing revealed a distinct taxonomic composition of active ARB compared with that of the overall population. The coexistence of antibiotic resistome and mobilome in active ARB was also identified, including three high-quality metagenomic assembly genomes assigned to pathogenic bacteria, highlighting the substantial health risks due to their activity, phenotypic resistance, mobility, and pathogenicity. This study advances our understanding of previously overlooked active ARB in the environment by linking their resistance phenotype to their genotype. This high-throughput method will enable efficient quantitative surveillance of active AMR, providing valuable insights into risk control and management.

Engineered Enucleated Mesenchymal Stem Cells Regulating Immune Microenvironment and Promoting Wound Healing.

Persistent excessive inflammation caused by neutrophil and macrophage dysfunction in the wound bed leads to refractory response during wound healing. However, previous studies using cytokines or drugs often suffer from short half-lives and limited targeting, resulting in unsatisfactory therapeutic effects. Herein, the enucleated mesenchymal stem cell is engineered by aptamer bioorthogonal chemistry to modify the cell membrane and mRNA loading in the cell cytoplasm as a novel delivery vector (Cargocyte) with accurate targeting and sustained cytokine secretion. Cargocytes can successfully reduce NETosis by targeting the nuclear chromatin protein DEK protein with aptamers and sustaining interleukin (IL)-4 expression to overcome the challenges associated with the high cost and short half-life of IL-4 protein and significantly prevent the transition of macrophages into the M1 phenotype. Therapeutic effects have been demonstrated in murine and porcine wound models and have powerful potential to improve wound immune microenvironments effectively. Overall, the use of engineered enucleated mesenchymal stem cells as a delivery system may be a promising approach for wound healing.

Repurposing Copper(II)/THPTA as A Bioorthogonal Catalyst for Thiazolidine Bond Cleavage.

Metal-mediated chemical transformations are promising approaches to manipulate and regulate proteins in fundamental biological research and therapeutic development. Nevertheless, unlike bond-forming reactions, the exploration of selective bond cleavage reactions catalyzed by metals that are fully compatible with proteins and living systems remains relatively limited. Here, it is reported that Copper(II)/tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), commonly used in copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, can be repurposed as a new bioorthogonal catalyst for thiazolidine (Thz) bond cleavage. This process liberates an α-oxo-aldehyde group under physiological conditions, without requiring additional additives. To showcase the utility of this method, this simple catalyst system is coupled with genetic code expansion technology to achieve on-demand activation of genetically encoded Thz-caged α-oxo-aldehydes, enabling further functionalization of proteins. For the first time, this cell-compatible Thz uncaging reaction allows for the site-specific installation of α-oxo-aldehydes at the internal positions of proteins in phage and bacterial surface display systems, expanding the chemical space of proteins. Overall, this study expands the toolkit of bioorthogonal catalysts and paves the way for metal-promoted chemical reactions in living systems, potentially benefiting various applications in the future.

Reversing Immune Checkpoint Inhibitor-Associated Cardiotoxicity via Bioorthogonal Metabolic Engineering-Driven Extracellular Vesicle Redirecting.

The cardiotoxicity induced by immune checkpoint inhibitors (ICIs) is associated with high mortality rates. T cells play an important role in ICI-induced cardiac injury. The inhibition of local T-cell activity is considered an effective strategy for alleviating ICI-related cardiotoxicity. Tumor-derived extracellular vesicles (EVs) contribute to immunosuppression via PD-L1 overexpression. In this study, a bioorthogonal metabolic engineering-driven EV redirecting (Biomeder) strategy for in situ engineered EVs with myocardial-targeting peptides is developed. Accumulated tumor-derived EV (TuEVs) reverses the immune environment in the heart by increasing PD-L1 levels in cardiomyocytes and/or by directly inhibiting T-cell activity. More importantly, it is found that the redirection of TuEVs further disrupts immunosuppression in tumors, which facilitates anti-tumor activity. Thus, redirecting TuEVs to the heart simultaneously enhances the antitumor efficacy and safety of ICI-based therapy. Furthermore, the Biomeder strategy is successfully expanded to prevent ICI-induced type 1 diabetes. This Biomeder technique is a universal method for the treatment of various ICI-related adverse events.

A Dual-Purpose Non-Canonical Amino Acid for the Expanded Genetic Code: Combining Metal-Binding and Click Chemistry.

A rationally designed dual-purpose non-canonical amino acid (Trz) has been synthesised and successfully incorporated into a protein scaffold by genetic code expansion. Trz contains a 5-pyridyl-1,2,4-triazine system, which allows for inverse-electron-demand Diels-Alder (IEDDA) reactions to occur on the triazine ring and for metal ions to be chelated both before and after the click reaction. Trz was successfully incorporated into a protein scaffold and the IEDDA utility of Trz demonstrated through the site-specific labelling of the purified protein with a bicyclononyne. Additionally, Trz was shown to successfully coordinate a cyclometallated iridium(III) centre, providing access to a bioorthogonal luminogenic probe. The luminescent properties of the Ir(III)-bound protein blue-shift upon IEDDA click reaction with bicyclononyne, providing a unique method for monitoring the extent and location of the labelling reaction. In summary, Trz is a new dual-purpose non-canonical amino acid with great potential for myriad bioapplications where metal-based functionality is required, for example in imaging, catalysis, and photo-dynamic therapy, in conjunction with a bioorthogonal reactive handle to impart additional functionalities, such as dual-modality imaging or therapeutic payloads.

Transforming Aryl-Tetrazines into Bioorthogonal Scissors for Systematic Cleavage of trans-Cyclooctenes.

Bioorthogonal bond-cleavage reactions have emerged as a powerful tool for precise spatiotemporal control of (bio)molecular function in the biological context. Among these chemistries, the tetrazine-triggered elimination of cleavable trans-cyclooctenes (click-to-release) stands out due to high reaction rates, versatility, and selectivity. Despite an increasing understanding of the underlying mechanisms, application of this reaction remains limited by the cumulative performance trade-offs (i.e., click kinetics, release kinetics, release yield) of existing tools. Efficient release has been restricted to tetrazine scaffolds with comparatively low click reactivity, while highly reactive aryl-tetrazines give only minimal release. By introducing hydroxyl groups onto phenyl- and pyridyl-tetrazine scaffolds, we have developed a new class of 'bioorthogonal scissors' with unique chemical performance. We demonstrate that hydroxyaryl-tetrazines achieve near-quantitative release upon accelerated click reaction with cleavable trans-cyclooctenes, as exemplified by click-triggered activation of a caged prodrug, intramitochondrial cleavage of a fluorogenic probe (turn-on) in live cells, and rapid intracellular bioorthogonal disassembly (turn-off) of a ligand-dye conjugate.

Sulfonated Hydroxyaryl-Tetrazines with Increased pKa for Accelerated Bioorthogonal Click-to-Release Reactions in Cells.

Bioorthogonal reactions that enable switching molecular functions by breaking chemical bonds have gained prominence, with the tetrazine-mediated cleavage of trans-cyclooctene caged compounds (click-to-release) being particularly noteworthy for its high versatility, biocompatibility, and fast reaction rates. Despite several recent advances, the development of highly reactive tetrazines enabling quantitative elimination from trans-cyclooctene linkers remains challenging. In this study, we present the synthesis and application of sulfo-tetrazines, a class of derivatives featuring phenolic hydroxyl groups with increased acidity constants (pKa). This unique property leads to accelerated elimination and complete release of the caged molecules within minutes. Moreover, the inclusion of sulfonate groups provides a valuable synthetic handle, enabling further derivatization into sulfonamides, modified with diverse substituents. Significantly, we demonstrate the utility of sulfo-tetrazines in efficiently activating fluorogenic compounds and prodrugs in living cells, offering exciting prospects for their application in bioorthogonal chemistry.

A Membrane-Anchoring Self-Assembling Peptide Allows Bioorthogonal Coupling of Type-I AIEgens for Pyroptosis-Induced Cancer Therapy.

Enrichment of photosensitizers (PSs) on cancer cell membranes via bioorthogonal reactions is considered to be a very promising therapeutic modality. However, azide-modified sugars-based metabolic labeling processes usually lack targeting and the labeling speed is relatively slow. Moreover, it has been rarely reported that membrane-anchoring pure type-I PSs can induce cancer cell pyroptosis. Here, we report an alkaline phosphatase (ALP) and cholecystokinin-2 receptor (CCK2R) dual-targeting peptide named DBCO-pYCCK6, which can selectively and rapidly self-assemble on cancer cell membrane, and then bioorthogonal enrich type-I aggregation-induced emission luminogens (AIEgen) PSs (SAIE-N3) on the cell membrane. Upon light irradiation, the membrane-anchoring SAIE-N3 could effectively generate type-I reactive oxygen species (ROS) to induce gasdermin E (GSDME)-mediated pyroptosis. In vivo experiments demonstrated that the bioorthogonal combination strategy of peptide and AIEgen PSs could significantly inhibit tumor growth, which is accompanied by CD8+ cytotoxic T cell infiltration. This work provides a novel self-assembly peptide-mediated bioorthogonal reaction strategy to bridge the supramolecular self-assembly and AIE field through strain-promoted azide-alkyne cycloaddition (SPAAC) and elucidates that pure type-I membrane-anchoring PSs can be used for cancer therapy via GSDME-mediated pyroptosis.