Bioorthogonal Reactions Research Articles

In Situ Sprayed Exosome-Cross-Linked Gel as Artificial Lymph Nodes for Postoperative Glioblastoma Immunotherapy.

One key challenge in postoperative glioblastoma immunotherapy is to guarantee a potent and durable T-cell response, which is restricted by the immunosuppressive microenvironment within the lymph nodes (LNs). Here, we develop an in situ sprayed exosome-cross-linked gel that acts as an artificial LN structure to directly activate the tumor-infiltrating T cells for prevention of glioma recurrence. Briefly, this gel is generated by a bio-orthogonal reaction between azide-modified chimeric exosomes and alkyne-modified alginate polymers. Particularly, these chimeric exosomes are generated from dendritic cell (DC)-tumor hybrid cells, allowing for direct and robust T-cell activation. The gel structure with chimeric exosomes as cross-linking points avoids the quick clearance by the immune system and thus prolongs the durability of antitumor T-cell immunity. Importantly, this exosome-containing immunotherapeutic gel provides chances for ameliorating functions of antigen-presenting cells (APCs) through accommodating different intracellular-acting adjuvants, such as stimulator of interferon genes (STING) agonists. This further enhances the antitumor T-cell response, resulting in the almost complete elimination of residual lesions after surgery. Our findings provide a promising strategy for postsurgical glioma immunotherapy that warrants further exploration in the clinical arena.

In Situ Engineering Cancer Mask to Immobilize Tumor Cells and Block Metastasis.

This work reports a new concept of cancer mask in situ to alter the specific biological functions of cancer cells. Metastatic cancer cells are highly invasive in part due to the presence of the glycan matrix in the cell membrane. Using a rational designed bio-orthogonal reaction, the cancer cell surface is reconstructed in situ by incorporating endogenous polysialic acids in the glycan matrix on the cell membrane to form a mesh-like network, called cancer mask. The network of the glycan matrix can not only immobilize cancer cells but also effectively block the stimulation of metastasis promoters to tumor cells and inhibit the formation of epithelial to mesenchymal transition (EMT), causing metastatic cancer cells incarceration. The results demonstrate a new strategy to control and even eliminate the cancer metastasis that is a major cause of treatment failure and poor patient outcome.

Abstract PO4-05-05: Immune Activation Signatures for predicting CDKi primary response in advanced breast cancer patients

Abstract Background Cyclin Dependent Kinase inhibitors (CDKi’s) in combination with Endocrine Therapy (ET) have changed the first line management of patients with the most common subtype (HR+, HER2-) of metastatic breast cancer (MBC), improving progression-free survival (PFS) and overall survival. However, in approved indications for advanced breast cancer, approximately 20% of patients have been observed to have no response to CDKi’s and ET, and to progress after starting these therapies. Unlike other targeted therapeutics, there is no companion diagnostic or other clinically reliable biomarker for clinical decision support to indicate which patients will and will not benefit from CDKi therapy. Methods We have developed a novel platform that measures the Immune Activation Signature of patient plasma samples. Via simple measurements of the total concentration of protein-incorporated amino acid residues within patient plasma samples, the platform is designed to reveal changes in the proportion of immunoglobulins and albumin, and class-switching among immunoglobulins. For that purpose, we use Bioorthogonal chemical labelling reactions to label the protein-incorporated amino acids residues within the plasma. We measured the plasma samples of N=30 HR+, HER2- MBC patients, median age 54 years old, who were baseline naïve for CDKi’s treatment (16 on palbociclib, 11 on ribociclib, and 3 on abemaciclib) and ET. Eighteen patients had metastatic visceral disease and 12 patients had bone-only MBC. All patients were prospectively followed, and response assessed according to RECIST criteria on a CT scan every 3-months. Results Non-responding patients developed objective progressive disease during the first 6 months after starting CDKis and ET. In this cohort, 4 patients (13%) were classified as non-responders. The median PFS in non-responding patients was 4.5 months (4.03-5.02) and the median PFS in the cohort of responding patients was 16.6 months (6.8 – 40.9). The Immune Activation Signatures of the Responding and Non-Responding patients are shown in table 1. We analyzed the results with classical statistics and machine learning. A multivariate analysis of variance (MANOVA) test of the null hypothesis that the Immune Activation Signatures of Non-Responders and Responders are the same gave p = 0.00388. We analyzed the measured Immune Activation Signatures using a supervised machine learning classifier and observed in a held-back validation set correct prediction of 100% of non-responding patients and 95% accurate predictions overall. Conclusions According to these results, this platform may provide a clinically helpful biomarker for clinical decision support in the advanced or aggressive breast cancer context (patients with visceral metastasis bordering on visceral crisis), or for patient stratification in new indications. Table. Immune Activation Signature measurements (mean values in Clinical Outcome) Citation Format: Luis Costa, Cong Tang, Emma Yates, Qi Shi, Wesley Sukdao, Patricia Corredeira, Gonçalo Costa, Helena Pais, Catarina Abreu, Leonor Ribeiro, Rita Teixeira de Sousa, Sofia Torres, André Mansinho, Sandra Casimiro, Ana Cavaco, Patricia Alves, Angela Rodrigues, Lisiana Szeneszi, Gonçalo Bernardes. Immune Activation Signatures for predicting CDKi primary response in advanced breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-05-05.

Intrahippocampal Supramolecular Assemblies Directed Bioorthogonal Liberation of Neurotransmitters to Suppress Seizures in Freely Moving Mice.

The precise delivery of anti-seizure medications (ASM) to epileptic loci remains the major challenge to treat epilepsy without causing adverse drug reactions. The unprovoked nature of epileptic seizures raises the additional need to release ASMs in a spatiotemporal controlled manner. Targeting the oxidative stress in epileptic lesions, here the reactive oxygen species (ROS) induced in situ supramolecular assemblies that synergized bioorthogonal reactions to deliver inhibitory neurotransmitter (GABA) on-demand, are developed. Tetrazine-bearing assembly precursors undergo oxidation and selectively self-assemble under pathological conditions inside primary neurons and mice brains. Assemblies induce local accumulation of tetrazine in the hippocampus CA3 region, which allows the subsequent bioorthogonal release of inhibitory neurotransmitters. For induced acute seizures, the sustained release of GABA extends the suppression than the direct supply of GABA. In the model of permanent damage of CA3, bioorthogonal ligation on assemblies provides a reservoir of GABA that behaves prompt release upon 365nm irradiation. Incorporated with the state-of-the-art microelectrode arrays, it is elucidated that the bioorthogonal release of GABA shifts the neuron spike waveforms to suppress seizures at the single-neuron precision. The strategy of in situ supramolecular assemblies-directed bioorthogonal prodrug activation shall be promising for the effective delivery of ASMs to treat epilepsy.

Injectable bioactive poly(propylene fumarate) and polycaprolactone based click chemistry bone cement for spinal fusion in rabbits.

Degenerative spinal pathology is a widespread medical issue, and spine fusion surgeries are frequently performed. In this study, we fabricated an injectable bioactive click chemistry polymer cement for use in spinal fusion and bone regrowth. Taking advantages of the bioorthogonal click reaction, this cement can be crosslinked by itself eliminating the addition of a toxic initiator or catalyst, nor any external energy sources like UV light or heat. Furthermore, nano-hydroxyapatite (nHA) and microspheres carrying recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) were used to make the cement bioactive for vascular induction and osteointegration. After implantation into a rabbit posterolateral spinal fusion (PLF) model, the cement showed excellent induction of new bone formation and bridging bone, achieving results comparable to autograft control. This is largely due to the osteogenic properties of nano-hydroxyapatite (nHA) and the released rhBMP-2 and rhVEGF growth factors. Since the availability of autograft sources is limited in clinical settings, this injectable bioactive click chemistry cement may be a promising alternative for spine fusion applications in addressing various spinal conditions.

Cyclopropenes as Chemical Reporters for Dual Bioorthogonal and Orthogonal Metabolic Labeling of DNA.

Dual bioorthogonal labeling enables the investigation and understanding of interactions in the biological environment that are not accessible by a single label. However, applying two bioorthogonal reactions in the same environment remains challenging due to cross-reactivity. We developed a pair of differently modified 2'-deoxynucleosides that solved this issue for dual and orthogonal labeling of DNA. Inverse-electron demand Diels-Alder and photoclick reactions were combined to attach two different fluorogenic labels to genomic DNA in cells. Using a small synthetic library of 1- and 3-methylcyclopropenyl-modified 2'-deoxynucleosides, two 2'-deoxyuridines were identified to be the fastest-reacting ones for each of the two bioorthogonal reactions. Their orthogonal reactivity could be evidenced in vitro. Primer extension experiments were performed with both 2'-deoxyuridines investigating their replication properties as substitutes for thymidine and evaluating subsequent labeling reactions on the DNA level. Finally, dual, orthogonal and metabolic fluorescent labeling of genomic DNA was demonstrated in HeLa cells. An experimental procedure was developed combining intracellular transport and metabolic DNA incorporation of the two 2'-deoxyuridines with the subsequent dual bioorthogonal labeling using a fluorogenic cyanine-styryl tetrazine and a fluorogenic pyrene-tetrazole. These results are fundamental for advanced metabolic labeling strategies for nucleic acids in the future, especially for live cell experiments.

Cyclopropene als Chemische Reporter für die Duale Bioorthogonale und Orthogonale Metabolische Markierung von DNA

AbstractDie duale bioorthogonale Markierung ermöglicht die Untersuchung und das Verständnis von Wechselwirkungen in der biologischen Umgebung, die mit einer einzigen Markierung nicht zugänglich sind. Eine Herausforderung bleibt jedoch die Anwendung von zwei bioorthogonalen Reaktionen in der gleichen Umgebung aufgrund von Kreuzreaktivität. Um dieses Problem für die duale und orthogonale Markierung von DNA zu lösen, haben wir zwei unterschiedlich modifizierte 2′‐Desoxynukleoside entwickelt. Mit Hilfe der Diels‐Alder‐Reaktion mit inversem Elektronenbedarf und Photoklick‐Reaktion wurde genomische DNA in Zellen mit zwei verschiedenen fluorogenen Markern verknüpft. Aus einer kleinen synthetischen Bibliothek aus 1‐ und 3‐methylcyclopropenyl‐modifizierten 2′‐Desoxynukleosiden wurden zwei 2′‐Desoxyuridine identifiziert, die am schnellsten in jeweils einer der beiden bioorthogonalen Reaktionen reagieren. Ihre orthogonale Reaktivität konnte in vitro nachgewiesen werden. Ihre Replikationseigenschaften als Ersatz für Thymidin wurden in Primerverlängerungsexperimenten untersucht, um die anschließenden Markierungsreaktionen an DNA abzuschätzen. Schließlich wurde eine duale, orthogonale und metabolische Fluoreszenzmarkierung von genomischer DNA in HeLa‐Zellen nachgewiesen. Es wurde ein experimentelles Protokoll entwickelt, das den intrazellulären Transport und den metabolischen DNA‐Einbau der beiden 2′‐Desoxyuridine mit der anschließenden dualen bioorthogonalen Markierung unter Verwendung eines fluorogenen Cyanin‐Styryl‐Tetrazins und eines fluorogenen Pyren‐Tetrazols kombiniert. Diese Ergebnisse sind von grundlegender Bedeutung für fortgeschrittene metabolische Markierungsstrategien für Nukleinsäuren in der Zukunft, insbesondere in lebenden Zellen.

Living Artificial Skin: Photosensitizer and Cell Sandwiched Bacterial Cellulose for Chronic Wound Healing.

Chronic wounds pose a significant global public health challenge due to their suboptimal treatment efficacy caused by bacterial infections and microcirculatory disturbances. Inspired by the biofunctionality of natural skin, an artificial skin (HV@BC@TBG) is bioengineered with bacterial cellulose (BC) sandwiched between photosensitizers (PS) and functionalized living cells. Glucose-modified PS (TBG) and vascular endothelial growth factor (VEGF)-functionalized living cells (HV) are successively modified on each side of BC through biological metabolism and bio-orthogonal reaction. As the outermost layer, the TBG layer can generate reactive oxygen species (ROS) upon light illumination to efficiently combat bacterial infections. The HV layer is the inner layer near the diabetic wound, which servs as a living factory to continuously secrete VEGF to accelerate wound repair by promoting fibroblast proliferation and angiogenesis. The sandwiched structural artificial skin HV@BC@TBG is nontoxic, biocompatible, and demonstrated its ability to significantly accelerate the healing process of infected diabetic wounds, rendering it a promising next-generation medical therapy for chronic wound management.

Development of Biocompatible Cu(I)-Microdevices for Bioorthogonal Uncaging and Click Reactions.

Transition-metal-catalyzed bioorthogonal reactions emerged a decade ago as a novel strategy to implement spatiotemporal control over enzymatic functions and pharmacological interventions. The use of this methodology in experimental therapy is driven by the ambition of improving the tolerability and PK properties of clinically-used therapeutic agents. The preclinical potential of bioorthogonal catalysis has been validated in vitro and in vivo with the in situ generation of a broad range of drugs, including cytotoxic agents, anti-inflammatory drugs and anxiolytics. In this article, we report our investigations towards the preparation of solid-supported Cu(I)-microdevices and their application in bioorthogonal uncaging and click reactions. A range of ligand-functionalized polymeric devices and off-on Cu(I)-sensitive sensors were developed and tested under conditions compatible with life. Last, we present a preliminary exploration of their use for the synthesis of PROTACs through CuAAC assembly of two heterofunctional mating units.

Site-Specific Conjugation of Bottlebrush Polymers to Therapeutic Protein via Bioorthogonal Chemistry.

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.

An all-in-one tetrazine reagent for cysteine-selective labeling and bioorthogonal activable prodrug construction

The prodrug design strategy offers a potent solution for improving therapeutic index and expanding drug targets. However, current prodrug activation designs are mainly responsive to endogenous stimuli, resulting in unintended drug release and systemic toxicity. In this study, we introduce 3-vinyl-6-oxymethyl-tetrazine (voTz) as an all-in-one reagent for modular preparation of tetrazine-caged prodrugs and chemoselective labeling peptides to produce bioorthogonal activable peptide-prodrug conjugates. These stable prodrugs can selectively bind to target cells, facilitating cellular uptake. Subsequent bioorthogonal cleavage reactions trigger prodrug activation, significantly boosting potency against tumor cells while maintaining exceptional off-target safety for normal cells. In vivo studies demonstrate the therapeutic efficacy and safety of this prodrug design approach. Given the broad applicability of functional groups and labeling versatility with voTz, we foresee that this strategy will offer a versatile solution to enhance the therapeutic range of cytotoxic agents and facilitate the development of bioorthogonal activatable biopharmaceuticals and biomaterials.

Selective activation of prodrugs in breast cancer using metabolic glycoengineering and the tetrazine ligation bioorthogonal reaction

Increasing the selectivity of chemotherapies by converting them into prodrugs that can be activated at the tumour site decreases their side effects and allows discrimination between cancerous and non-cancerous cells. Herein, the use of metabolic glycoengineering (MGE) to selectively label MCF-7 breast cancer cells with tetrazine (Tz) activators for subsequent activation of prodrugs containing the trans-cyclooctene (TCO) moiety by a bioorthogonal reaction is demonstrated. Three novel Tz-modified monosaccharides, Ac4ManNTz 7, Ac4GalNTz 8, and Ac4SiaTz 16, were used for expression of the Tz activator within sialic-acid rich breast cancer cells’ surface glycans through MGE. Tz expression on breast cancer cells (MCF-7) was evaluated versus the non-cancerous L929 fibroblasts showing a concentration-dependant effect and excellent selectivity with ≥35-fold Tz expression on the MCF-7 cells versus the non-cancerous L929 fibroblasts. Next, a novel TCO-N-mustard prodrug and a TCO-doxorubicin prodrug were analyzed in vitro on the Tz-bioengineered cells to probe our hypothesis that these could be activated via a bioorthogonal reaction. Selective prodrug activation and restoration of cytotoxicity were demonstrated for the MCF-7 breast cancer cells versus the non-cancerous L929 cells. Restoration of the parent drug’s cytotoxicity was shown to be dependent on the level of Tz expression where the Ac4ManNTz 7 and Ac4GalNTz 8 derivatives (20 µM) lead to the highest Tz expression and full restoration of the parent drug’s cytotoxicity. This work suggests the feasibility of combining MGE and tetrazine ligation for selective prodrug activation in breast cancer.

Bioactive Moldable Click Chemistry Polymer Cement with Nano-Hydroxyapatite and Growth Factor-Enhanced Posterolateral Spinal Fusion in a Rabbit Model.

Spinal injuries or diseases necessitate effective fusion solutions, and common clinical approaches involve autografts, allografts, and various bone matrix products, each with limitations. To address these challenges, we developed an innovative moldable click chemistry polymer cement that can be shaped by hand and self-cross-linked in situ for spinal fusion. This self-cross-linking cement, enabled by the bioorthogonal click reaction, excludes the need for toxic initiators or external energy sources. The bioactivity of the cement was promoted by incorporating nanohydroxyapatite and microspheres loaded with recombinant human bone morphogenetic protein-2 and vascular endothelial growth factor, fostering vascular induction and osteointegration. The release kinetics of growth factors, mechanical properties of the cement, and the ability of the scaffold to support in vitro cell proliferation and differentiation were evaluated. In a rabbit posterolateral spinal fusion model, the moldable cement exhibited remarkable induction of bone regeneration and effective bridging of spine vertebral bodies. This bioactive moldable click polymer cement therefore presents a promising biomaterial for spinal fusion augmentation, offering advantages in safety, ease of application, and enhanced bone regrowth.

A Synthetic Strategy toward S-, N-, and O-Heterocyclooctynes Facilitates Bioconjugation Using Multifunctional Handles.

Over the past two decades, the introduction of bioorthogonal reactions has transformed the ways in which chemoselective labeling, isolation, imaging, and drug delivery are carried out in a complex biological milieu. A key feature of a good bioorthogonal probe is the ease with which it can be attached to a target compound through bioconjugation. This paper describes the expansion of the utility of a class of unique S-, N-, and O-containing heterocyclooctynes (SNO-OCTs), which show chemoselective reactivity with type I and type II dipoles and divergent reactivities in response to electronic tuning of the alkyne. Currently, bioconjugation of SNO-OCTs to a desired target is achieved through an inconvenient aryl or amide linker at the sulfamate nitrogen. Herein, a new synthetic approach toward general SNO-OCT scaffolds is demonstrated that enables the installation of functional handles at both propargylic carbons of the heterocycloalkyne. This capability increases the utility of SNO-OCTs as labeling reagents through the design of bifunctional bioorthogonal probes with expanded capabilities. NMR kinetics also revealed up to sixfold improvement in cycloaddition rates of new analogues compared to first-generation SNO-OCTs.

Development and Application of Bioorthogonal Reactions for Drug Imaging and Activation

Chemical transformations compatible with biological systems serve as invaluable tools for probing biomolecules, drug compounds, and biological processes in their native environments. The complex environment of living organisms requires these reactions to be highly selective and efficient, posing a formidable challenge to the field of organic chemistry. A number of chemical reactions have recently emerged to meet this challenge, providing a diverse toolkit for researchers. This manuscript presents a comprehensive survey of current bioorthogonal reactions, emphasizing their applications in bioimaging, diagnostics, and medicine.

Single-Atom Catalysts Mediated Bioorthogonal Modulation of N6-Methyladenosine Methylation for Boosting Cancer Immunotherapy.

Bioorthogonal reactions provide a powerful tool to manipulate biological processes in their native environment. However, the transition-metal catalysts (TMCs) for bioorthogonal catalysis are limited to low atomic utilization and moderate catalytic efficiency, resulting in unsatisfactory performance in a complex physiological environment. Herein, sulfur-doped Fe single-atom catalysts with atomically dispersed and uniform active sites are fabricated to serve as potent bioorthogonal catalysts (denoted as Fe-SA), which provide a powerful tool for in situ manipulation of cellular biological processes. As a proof of concept, the N6-methyladensoine (m6A) methylation in macrophages is selectively regulated by the mannose-modified Fe-SA nanocatalysts (denoted as Fe-SA@Man NCs) for potent cancer immunotherapy. Particularly, the agonist prodrug of m6A writer METTL3/14 complex protein (pro-MPCH) can be activated in situ by tumor-associated macrophage (TAM)-targeting Fe-SA@Man, which can upregulate METTL3/14 complex protein expression and then reprogram TAMs for tumor killing by hypermethylation of m6A modification. Additionally, we find the NCs exhibit an oxidase (OXD)-like activity that further boosts the upregulation of m6A methylation and the polarization of macrophages via producing reactive oxygen species (ROS). Ultimately, the reprogrammed M1 macrophages can elicit immune responses and inhibit tumor proliferation. Our study not only sheds light on the design of single-atom catalysts for potent bioorthogonal catalysis but also provides new insights into the spatiotemporal modulation of m6A RNA methylation for the treatment of various diseases.

Ingestible Artificial Urinary Biomarker Probes for Urine Test of Gastrointestinal Cancer.

Although colorectal cancer diagnosed at an early stage shows high curability, methods simultaneously possessing point-of-care testing ability and high sensitivity are limited. Here, an orally deliverable biomarker-activatable probe (termed as HATS) for early detection of orthotopic tumors via remote urinalysis is presented. To enable its oral delivery to the colon, HATS is designed to have remarkable resistance to acidity and digestive enzymes in the stomach and small intestine and negligible intestinal absorption. Upon reaction with a cancer biomarker in the colon segment, HATS releases a small fragment of tetrazine that can transverse the intestinal barrier, enter blood circulation, and ultimately undergo renal clearance to urine. Subsequently, the urinary tetrazine fragment is detected by bioorthogonal reaction with trans-cyclooctene-caged resorufin (TCO-Reso) to afford a rapid and specific fluorescence enhancement of TCO-Reso. Such signal readout is correlated with the urinary tetrazine concentration and thus measures the level of cancer biomarkers in the colon. HATS-based optical urinalysis detects orthotopic colon tumors two weeks earlier than clinical serological tests and can be developed to a point-of-care paper test. Thereby, HATS-based urinalysis provides a non-invasive and sensitive approach to cancer screening at low-resource settings.

Gas Evolution as a Tool to Study Reaction Kinetics Under Biomimetic Conditions.

The field of bioorthogonal chemistry is rapidly growing, presenting successful applications of organic and transition metal-catalysed reactions in cells and living systems (in vivo). The development of such reactions typically proceeds through many iterative steps focused on biocompatibility and fast reaction kinetics to ensure product formation. However, obtaining kinetic data, even under simulated biological (biomimetic) conditions, remains a challenge due to substantial concentrations of salts and biomolecules hampering the use of typically employed solution-phase analytical techniques. In this study, we explored the suitability of gas evolution as a probe to study kinetics under biomimetic conditions. As proof of concept, we show that the progress of two transition metal-catalysed bioorthogonal chemical reactions can be accurately monitored, regardless of the complexity of the medium. As such, we introduce a protocol to gain more insight into the performance of a catalytic system under biomimetic conditions to further progress iterative catalyst development for in vivo applications.

Injectable and Multifunctional Hydrogels Based on Poly(N-acryloyl glycinamide) and Alginate Derivatives for Antitumor Drug Delivery.

Chemotherapy is a conventional treatment that uses drugs to kill cancer cells; however, it may induce side effects and may be incompletely effective, leading to the risk of tumor recurrence. To address this issue, we developed novel injectable thermal/near-infrared (NIR)-responsive hydrogels to control drug release. The injectable hydrogel formulation was composed of biocompatible alginates, poly(N-acryloyl glycinamide) (PNAGA) copolymers with an upper critical solution temperature, and NIR-responsive cross-linkers containing coumarin groups, which were gelated through bioorthogonal inverse electron demand Diels-Alder reactions. The hydrogels exhibited quick gelation times (120-800 s) and high drug loading efficiencies (>90%). The hydrogels demonstrated a higher percentage of drug release at 37 °C than that at 25 °C due to the enhanced swelling behavior of temperature-responsive PNAGA moieties. Upon NIR irradiation, the hydrogels released most of the entrapped doxorubicin (DOX) (97%) owing to the cleavage of NIR-sensitive coumarin ester groups. The hydrogels displayed biocompatibility with normal cells, while induced antitumor activity toward cancer cells. DOX/hydrogels treated with NIR light inhibited tumor growth in nude mice bearing tumors. In addition, the injected hydrogels emitted red fluorescence upon excitation at a green wavelength, so that the drug delivery and hydrogel degradation in vivo could be tracked in the xenograft model.

Enzyme-Mediated Bioorthogonal Cascade Catalytic Reaction for Metabolism Intervention and Enhanced Ferroptosis on Neuroblastoma.

It remains a tremendous challenge to explore effective therapeutic modalities against neuroblastoma, a lethal cancer of the sympathetic nervous system with poor prognosis and disappointing treatment outcomes. Considering the limitations of conventional treatment modalities and the intrinsic vulnerability of neuroblastoma, we herein develop a pioneering sequential catalytic therapeutic system that utilizes lactate oxidase (LOx)/horseradish peroxidase (HRP)-loaded amorphous zinc metal-organic framework, named LOx/HRP-aZIF, in combination with a 3-indole-acetic acid (IAA) prodrug. On the basis of abnormal lactate accumulation that occurs in the tumor microenvironment, the cascade reaction of LOx and HRP consumes endogenous glutathione and a reduced form of nicotinamide adenine dinucleotide to achieve the first stage of killing cancer cells via antioxidative incapacitation and electron transport chain interference. Furthermore, the generation of reactive oxygen species induced by HRP and IAA through bioorthogonal catalysis promotes ferritin degradation and lipid peroxidation, ultimately provoking self-enhanced ferroptosis with positive feedback by initiating an endogenous Fenton reaction. This work highlights the superiority of the natural enzyme-dependent cascade and bioorthogonal catalytic reaction, offering a paradigm for synergistically enzyme-based metabolism-ferroptosis anticancer therapy.