Manipulating complex chromatin folding via CRISPR-guided bioorthogonal chemistry.

Precise manipulation of chromatin folding is important for understanding the relationship between the three-dimensional genome and nuclear function. Existing tools can reversibly establish individual chromatin loops but fail to manipulate two or more chromatin loops. Here, we engineer a powerful CRISPR system which can manipulate multiple chromatin contacts using bioorthogonal reactions, termed the bioorthogonal reaction-mediated programmable chromatin loop (BPCL) system. The multiinput BPCL system employs engineered single-guide RNAs recognized by discrete bioorthogonal adaptors to independently and dynamically control different chromatin loops formation without cross-talk in the same cell or to establish hubs of multiway chromatin contacts. We use the BPCL system to successfully juxtapose the pluripotency gene promoters to enhancers and activate their endogenous expression. BPCL enables us to independently engineer multiway chromatin contacts without cross-talk, which provides a way to precisely dissect the high complexity and dynamic nature of chromatin folding.

MOFs Modulate Copper Trafficking in Tumor Cells for Bioorthogonal Therapy.

In situ drug synthesis using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has attracted considerable attention in tumor therapy because of its satisfactory effectiveness and reduced side-effects. However, the exogenous addition of copper catalysts can cause cytotoxicity and has hampered biomedical applications in vivo. Here, we design and synthesize a metal-organic framework (MOF) to mimic copper chaperone, which can selectively modulate copper trafficking for bioorthogonal synthesis with no need of exogenous addition of copper catalysts. Like copper chaperones, the prepared ZIF-8 copper chaperone mimics specifically bind copper ions through the formation of coordination bonds. Moreover, the copper is unloaded under the acidic environment due to the dissipation of the coordination interactions between metal ions and ligands. In this way, the cancer cell-targeted copper chaperone mimics can selectively transport copper ions into cells. Regulation of intracellular copper trafficking may inspire constructing bioorthogonal catalysis system with reduced metal cytotoxicity in live cells.

A Hydrogen-Bonded Organic Framework-Based Mitochondrion-Targeting Bioorthogonal Platform for the Modulation of Mitochondrial Epigenetics.

Bioorthogonal chemistry represents a powerful tool in chemical biology, which shows great potential in epigenetic modulation. As a proof of concept, the epigenetic modulation model of mitochondrial DNA (mtDNA) is selected because mtDNA establishes a relative hypermethylation stage under oxidative stress, which impairs the mitochondrion-based therapeutic effect during cancer therapy. Herein, we design a new biocompatible hydrogen-bonded organic framework (HOF) for a HOF-based mitochondrion-targeting bioorthogonal platform TPP@P@PHOF-2. PHOF-2 can activate a prodrug (pro-procainamide) in situ, which can specifically inhibit DNA methyltransferase 1 (DNMT1) activity and remodel the epigenetic modification of mtDNA, making it more susceptible to ROS damage. In addition, PHOF-2 can also catalyze artemisinin to produce large amounts of ROS, effectively damaging mtDNA and achieving better chemodynamic therapy demonstrated by both in vitro and in vivo studies. This work provides new insights into developing advanced bioorthogonal therapy and expands the applications of HOF and bioorthogonal catalysis.

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.

Bioorthogonal Aptamer-ATTEC Conjugates for Degradation of Alpha-Synuclein via Autophagy-Lysosomal Pathway.

Autophagosome-tethering compound (ATTEC) technology has recently been emerging as a novel approach for degrading proteins of interest (POIs). However, it still faces great challenges in how to design target-specific ATTEC molecules. Aptamers are single-stranded DNA or RNA oligonucleotides that can recognize their target proteins with high specificity and affinity. Here, ATTEC is combined with aptamers for POIs degradation. As a proof of concept, pathological protein α-synuclein (α-syn) is chosen as the target and an efficient α-syn degrader is generated. Aptamer as a targeting warhead of α-syn is conjugated with LC3B-binding compound 5,7-dihydroxy-4-phenylcoumarin (DP) via bioorthogonal click reaction. It is demonstrated that the aptamer conjugated with DP is capable of clearing α-syn through LC3 and autophagic degradation. These results indicate that aptamer-based ATTECs are a versatile approach to degrade POIs by taking advantage of the well-defined different aptamers for targeting diverse proteins, which provides a new way for the design of ATTECs to degradation of targeted proteins.

Cell membrane-camouflaged liposomes for tumor cell-selective glycans engineering and imaging in vivo.

The dynamic change of cell-surface glycans is involved in diverse biological and pathological events such as oncogenesis and metastasis. Despite tremendous efforts, it remains a great challenge to selectively distinguish and label glycans of different cancer cells or cancer subtypes. Inspired by biomimetic cell membrane-coating technology, herein, we construct pH-responsive azidosugar liposomes camouflaged with natural cancer-cell membrane for tumor cell-selective glycan engineering. With cancer cell-membrane camouflage, the biomimetic liposomes can prevent protein corona formation and evade phagocytosis of macrophages, facilitating metabolic glycans labeling in vivo. More importantly, due to multiple membrane receptors, the biomimetic liposomes have prominent cell selectivity to homotypic cancer cells, showing higher glycan-labeling efficacy than a single-ligand targeting strategy. Further in vitro and in vivo experiments indicate that cancer cell membrane-camouflaged azidosugar liposomes not only realize cell-selective glycan imaging of different cancer cells and triple-negative breast cancer subtypes but also do well in labeling metastatic tumors. Meanwhile, the strategy is also applicable to the use of tumor tissue-derived cell membranes, which shows the prospect for individual diagnosis and treatment. This work may pave a way for efficient cancer cell-selective engineering and visualization of glycans in vivo.

Hydrogen-bonded organic frameworks: new horizons in biomedical applications.

Hydrogen-bonded organic frameworks (HOFs) are an emerging attractive class of highly crystalline porous materials characterized by significant biocompatibility, rich chemical functionalities and well-defined porosity. The unique advantages including metal-free nature and reversible binding manner significantly distinguish HOFs from other porous materials in the biotechnology and biomedical field. However, the relevant HOF studies still remain in their infancy despite the promising and remarkable results that have been presented in recent years. Due to the intricate and dynamic nature of physiological conditions, the major challenge lies in the stability and structural diversity of HOFs in vivo. In this Tutorial Review, we summarize the common building blocks for the construction of HOF-based functional biomaterials and the latest developments in the biological field. Moreover, we highlight current challenges regarding the stability and functionalization of HOFs along with the corresponding potential solutions. This Tutorial Review will have a profound effect in future years on the design and applications of HOF-based biomaterials.

An Antibody Engineered Metalloenzyme for Mediating Cell-Cell Communication and Activation of Immuno- and Chemotherapy.

Artificial metalloenzymes (ArMs) are gaining much attention in life sciences. However, the function of the present ArMs for disease treatment is still in its infancy, which may impede the possible therapeutic potential. Herein, we construct an antibody engineered ArM by using the Fc region of IgG and bioorthogonal chemistry, which endows the ArM with the capability of manipulating cell-cell communication and bioorthogonal catalysis for tumor immuno- and chemotherapy. Specially, Fc-Pd ArM is modified on the cancer cell surface by metabolic glycoengineering to catalyze the bioorthogonal activation of prodrug for tumor chemotherapy. More importantly, the antibody-based ArM can mediate cell-cell communication between cancer cells and NK cells, activating the ADCC effect for immunotherapy. In vivo antitumor applications suggest that the ArM can not only eliminate primary tumor but also inhibit tumor lung metastasis. Our work provides a new attempt to develop artificial metalloenzymes with cell-cell communication the ability for bioorthogonal catalysis and combination therapy.

Bioorthogonally Activatable Autophagy-Tethering Compounds for Aptamer-Guided Mitochondrial Degradation.

Although macroautophagy degradation targeting chimeras (MADTACs) have been demonstrated to be efficient in a broad spectrum from intracellular proteins to macromolecular complexes such as lipid droplets and the mitochondrion, MADTACs still face degradation of uncontrolled protein in normal cells and cause systemic toxicity, thus limiting their therapeutic applications. Herein, we employ bioorthogonal chemistry to develop a spatially controlled MADTACs strategy. Separated warheads display no activity in normal cells but can be activated by aptamer-based Cu nanocatalyst (Apt-Cu30) in tumors specifically. These in situ synthesized chimera molecules (bio-ATTECs) can degrade the mitochondrion in live tumor cells and subsequently induce autophagic cell death, which has been further demonstrated by lung metastasis melanoma murine models. To the best of our knowledge, this is the first bioorthogonal activated MADTAC in live cells for inducing autophagic tumor cell death, which may promote the development of cell-specific MADTACs for precision therapeutics by avoiding undesired side effects.

Boosting Endogenous Copper(I) for Biologically Safe and Efficient Bioorthogonal Catalysis via Self-Adaptive Metal-Organic Frameworks.

As one of the most typical bioorthogonal reactions, the Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition (CuAAC) reaction has received worldwide attention in intracellular transformation of prodrugs due to its high efficiency and selectivity. However, the exogenous Cu catalysts may disturb Cu homeostasis and cause side effects to normal tissues. What is more, the intratumoral Cu(I) is insufficient to efficiently catalyze the intracellular CuAAC reaction due to oncogene-induced labile Cu(I) deficiency. Herein, in order to boost the endogenous Cu(I) level for intracellular drug synthesis through the bioorthogonal reaction, a self-adaptive bioorthogonal catalysis system was constructed by encapsulating prodrugs and sodium ascorbate within adenosine triphosphate aptamer-functionalized metal-organic framework nanoparticles. The system presents specificity to tumor cells and does not require exogenous Cu catalysts, thereby leading to high anti-tumor efficacy and minimal side effects both in vitro and in vivo. This work will open up a new opportunity for developing biosafe and high-performance bioorthogonal catalysis systems.

Dimensionality Engineering of Single-Atom Nanozyme for Efficient Peroxidase-Mimicking.

In nature, enzymatic reactions occur in well-functioning catalytic pockets, where substrates bind and react by properly arranging the catalytic sites and amino acids in a three-dimensional (3D) space. Single-atom nanozymes (SAzymes) are a new type of nanozymes with active sites similar to those of natural metalloenzymes. However, the catalytic centers in current SAzymes are two-dimensional (2D) architectures and the lack of collaborative substrate-binding features limits their catalytic activity. Herein, we report a dimensionality engineering strategy to convert conventional 2D Fe-N-4 centers into 3D structures by integrating oxidized sulfur functionalities onto the carbon plane. Our results suggest that oxidized sulfur functionalities could serve as binding sites for assisting substrate orientation and facilitating the desorption of H2O, resulting in an outstanding specific activity of up to 119.77 U mg-1, which is 6.8 times higher than that of conventional FeN4C SAzymes. This study paves the way for the rational design of highly active single-atom nanozymes.

Bioorthogonal-Activated In Situ Vaccine Mediated by a COF-Based Catalytic Platform for Potent Cancer Immunotherapy.

Personalized tumor vaccines have become a promising modality for cancer immunotherapy. However, in situ personalized tumor vaccines generated from immunogenic cancer cell death (ICD) and adjuvants are mired by toxic side effects and unsatisfactory efficiency. Herein, by functionalizing the reticular structure to optimize the catalytic activity of the materials, a series of biocompatible covalent organic framework (COF)-based catalysts have been designed and screened for establishing a bioorthogonal-activated in situ cancer vaccine in an efficient and safe way. Especially, pro-doxorubicin (pro-DOX) could be bioorthogonally activated in situ by the COF-based Fe(II) catalysts, which elicited ICD and released tumor-associated antigens (TAAs). This in situ prodrug activation strategy could minimize drug side effects and maximize treatment effects. More importantly, the system could also catalytically activate pro-imiquimod (pro-IMQ, a TLR7/8 immune agonist), which served as an adjuvant to amplify the antitumor immunity. Notably, this bioorthogonal-activated in situ cancer vaccine not only facilitated a strong antitumor immune response but also prevented the dose-dependent side effects of chemotherapeutic drugs, including systemic inflammation caused by the random distribution of adjuvants. To the best of our knowledge, it is the first time to devise an efficient catalytic platform for generating an in situ bioorthogonal-activated cancer vaccine, which would provide a paradigm for achieving secure and robust immunotherapy.

Transforming Intratumor Bacteria into Immunopotentiators to Reverse Cold Tumors for Enhanced Immuno-chemodynamic Therapy of Triple-Negative Breast Cancer.

Immunotherapy of triple-negative breast cancer (TNBC) has an unsatisfactory therapeutic outcome due to an immunologically "cold" microenvironment. Fusobacterium nucleatum (F. nucleatum) was found to be colonized in triple-negative breast tumors and was responsible for the immunosuppressive tumor microenvironment and tumor metastasis. Herein, we constructed a bacteria-derived outer membrane vesicle (OMV)-coated nanoplatform that precisely targeted tumor tissues for dual killing of F. nucleatum and cancer cells, thus transforming intratumor bacteria into immunopotentiators in immunotherapy of TNBC. The as-prepared nanoparticles efficiently induced immunogenic cell death through a Fenton-like reaction, resulting in enhanced immunogenicity. Meanwhile, intratumoral F. nucleatum was killed by metronidazole, resulting in the release of pathogen-associated molecular patterns (PAMPs). PAMPs cooperated with OMVs further facilitated the maturation of dendritic cells and subsequent T-cell infiltration. As a result, the "kill two birds with one stone" strategy warmed up the cold tumor environment, maximized the antitumor immune response, and achieved efficient therapy of TNBC as well as metastasis prevention. Overall, this strategy based on a microecology distinction in tumor and normal tissue as well as microbiome-induced reversal of cold tumors provides new insight into the precise and efficient immune therapy of TNBC.

Bioorthogonal Disruption of Pyroptosis Checkpoint for High-Efficiency Pyroptosis Cancer Therapy.

Pyroptosis is an inflammatory form of programmed cell death that holds great promise in cancer therapy. However, autophagy as the crucial pyroptosis checkpoint and the self-protective mechanism of cancer cells significantly weakens the therapeutic efficiency. Here, a bioorthogonal pyroptosis nanoregulator is constructed to induce pyroptosis and disrupt the checkpoint, enabling high-efficiency pyroptosis cancer therapy. The nanoregulator allows the in situ synthesis and accumulation of the photosensitizer PpIX in the mitochondria of cancer cells to directly produce mitochondrial ROS, thus triggering pyroptosis. Meanwhile, the in situ generated autophagy inhibitor via palladium-catalyzed bioorthogonal chemistry can disrupt the pyroptosis checkpoint to boost the pyroptosis efficacy. With the biomimetic cancer cell membrane coating, this platform for modulating pyroptosis presents specificity to cancer cells and poses no harm to normal tissue, resulting in a highly efficient and safe antitumor treatment. To our knowledge, this is the first report on a disrupting intrinsic protective mechanism of cancer cells for tumor pyroptosis therapy. This work highlights that autophagy as a checkpoint plays a key regulative role in pyroptosis therapy, which would motivate the future design of therapeutic regimens.

Integrating Incompatible Nanozyme-Catalyzed Reactions for Diabetic Wound Healing.

Multi-nanozymes are widely applied in disease treatment, biosensing, and other fields. However, most current multi-nanozyme systems exhibit only moderate activity since reaction microenvironments of different nanozyme are often distinct or even incompatible. Conventional assemble strategies are inapplicable for designing multi-nanozymes consisting of incompatible nanozymes. Herein, a versatile fiber-based compartmentalization strategy is developed to construct multi-nanozyme system capable of simultaneously performing incompatible reactions. In this system, the incompatible nanozymes are spatially distributed in distinct compartmentalized fibers, where different microenvironments can be tailored by controlling the doping reagent, endowing each nanozymes with the preferential microenvironments to exhibit their highest activity. As a proof of concept, pH-incompatible peroxidase-like and catalase-like catalytic reactions are tested to verify the feasibility of this strategy. By doping with benzoic acid in the desired location, the two pH-incompatible nanozymes can work simultaneously without interference. Further, it is demonstrated that the oxygen supply and antimicrobial power of the integrated platform can be applied for accelerating diabetic wound healing. It is hoped that this work provides a way to integrate incompatible nanozyme and broadens the application potential of multi-nanozymes.

A Homing Missile-Like Nanotherapeutic with Single-Atom Catalytic Sites for In Situ Elimination of Intracellular Bacterial Pathogens.

Intracellular bacterial pathogens hiding in host cells tolerate the innate immune system and high-dose antibiotics, resulting in recurrent infections that are difficult to treat. Herein, a homing missile-like nanotherapeutic (FeSAs@Sa.M) composed of a single-atom iron nanozyme (FeSAs) core coated with infected macrophage membrane (Sa.M) is developed for in situ elimination of intracellular methicillin-resistant S. aureus (MRSA). Mechanically, the FeSAs@Sa.M initially binds to the extracellular MRSA via the bacterial recognition ability of the Sa.M component. Subsequently, the FeSAs@Sa.M can be transported to the intracellular MRSA-located regions in the host cell like a homing missile under the guidance of the extracellular MRSA to which it is attached, generating highly toxic reactive oxygen species (ROS) for intracellular MRSA killing via the enzymatic activities of the FeSAs core. The FeSAs@Sa.M is far superior to FeSAs in killing intracellular MRSA, proposing a feasible strategy for treating intracellular infections by in situ generating ROS in bacterial residing regions.

Nanozyme-Based Supramolecular Self-Assembly As an Artificial Host Defense System For Treatment of Bacterial Infections.

The proper functioning of host defense system (HDS) is the key to combating bacterial infection in biological organisms. However, the delicate HDS may be dysfunctional or dysregulated, resulting in persistent infection, tissue damage, or delayed wound healing. Herein, a powerful artificial "host defense system" (aHDS) is designed and constructed for treatment of bacterial infections. First, the aHDS can quickly trap the bacteria by electrostatic interactions. Next, the system can be stimulated to produce large amounts of cytotoxic reactive oxygen species (ROS) and exert strong antibacterial effects, which can further regulate the immune microenvironment, leading to macrophage polarization from M0 to pro-inflammatory phenotype (M1) for synergistic bacteria killing. At the later stages, the system can exhibit excellent antioxidant enzyme-like activities to reprogram the M1 macrophage to anti-inflammatory phenotype (M2) for accelerating wound healing. This powerful aHDS can effectively combat the bacteria and avoid excessive inflammatory responses for the treatment of bacteria-infected wounds.

Lactate-Responsive Gene Editing to Synergistically Enhance Macrophage-Mediated Cancer Immunotherapy.

Combination therapies involving metabolic regulation and immune checkpoint blockade are considered an encouraging new strategy for cancer therapy. However, the effective utilization of combination therapies for activating tumor-associated macrophages (TAMs) remains challenging. Herein, a lactate-catalyzed chemodynamic approach to activate the therapeutic genome editing of signal-regulatory protein α (SIRPα) to reprogram TAMs and improve cancer immunotherapy is proposed. This system is constructed by encapsulating lactate oxidase (LOx) and clustered regularly interspaced short palindromic repeat-mediated SIRPα genome-editing plasmids in a metal-organic framework (MOF). The genome-editing system is released and activated by acidic pyruvate, which is produced by the LOx-catalyzed oxidation of lactate. The synergy between lactate exhaustion and SIRPα signal blockade can enhance the phagocytic ability of TAMs and promote the repolarization of TAMs to the antitumorigenic M1 phenotype. Lactate exhaustion-induced CD47-SIRPα blockade efficiently improves macrophage antitumor immune responses and effectively reverses the immunosuppressive tumor microenvironment to inhibit tumor growth, as demonstrated by in vitro and in vivo studies. This study provides a facile strategy for engineering TAMs in situ by combining CRISPR-mediated SIRPα knockout with lactate exhaustion for effective immunotherapy.

Target-Specific Bioorthogonal Reactions for Precise Biomedical Applications.

Bioorthogonal chemistry is a promising toolbox for dissecting biological processes in the native environment. Recently, bioorthogonal reactions have attracted considerable attention in the medical field for treating diseases, since this approach may lead to improved drug efficacy and reduced side effects via in situ drug synthesis. For precise biomedical applications, it is a prerequisite that the reactions should occur in the right locations and on the appropriate therapeutic targets. In this minireview, we highlight the design and development of targeted bioorthogonal reactions for precise medical treatment. First, we compile recent strategies for achieving target-specific bioorthogonal reactions. Further, we emphasize their application for the precise treatment of different therapeutic targets. Finally, a perspective is provided on the challenges and future directions of this emerging field for safe, efficient, and translatable disease treatment.

Chirality-Dependent Reprogramming of Macrophages by Chiral Nanozymes.

It is known that extracellular free radical reactive oxygen species (ROS) rather than intracellular ROS plays a non-substitutable role in regulation of tumor-suppressing (M1) tumor-associated macrophages (TAMs) polarization. However, most therapeutic nanoplatforms mainly provide intracellular ROS and exhibit insufficient accumulation near TAMs, which strongly limits the macrophage-based immunotherapeutic effects. Here we design and synthesize chiral MoS2 /CoS2 nanozymes with peroxidase (POD)-like and catalase (CAT)-like activities to efficiently modulate TAMs polarization and reverse tumor immunosuppression by harnessing their chirality-specific interactions with biological systems. MoS2 /CoS2 nanoparticles coordinated with d-chirality (d-NPs, right-handed) show improved pharmacokinetics with longer circulating half-life and higher tumor accumulation compared with their l (left-handed)- and dl (racemate)-counterparts. Further, d-NPs can escape from macrophage uptake in the tumor microenvironment (TME) with the aid of cell-unpreferred opposite chirality and act as extracellular hydroxyl radicals (⋅OH) and oxygen (O2 ) generators to efficiently repolarize TAMs into M1 phenotype. On the contrary, l-NPs showed high cellular uptake due to chirality-driven homologous adhesion between l-NPs and macrophage membrane, leading to limited M1 polarization performance. As the first example for developing chiral nanozymes as extracellular-localized ROS generators to reprogram TAMs for cancer immunotherapy, this study opens an avenue for applications of chiral nanozymes in immunomodulation.