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.

Near-Infrared Light-Activatable DNA Tentacles for Efficient Inhibition of Tumor Metastasis by Bio-Orthogonal Cell Assembly.

Tumor metastasis remains a major challenge in cancer management. Among various treatment strategies, immune cell-based cancer therapy holds a great potential for inhibiting metastasis. However, its wide application in cancer therapy is restricted by complex preparations, as well as inadequate homing and controllability. Herein, we present a groundbreaking approach for bioorthogonally manipulating tumor-NK (natural killer) cell assembly to inhibit tumor metastasis. Multiple dibenzocyclootyne (DBCO) groups decorated long single-stranded DNA were tail-modified on core-shell upconversion nanoparticles (CSUCNPs) and condensed by photosensitive chemical linker (PC-Linker) DNA to shield most of the DBCO groups. On the one hand, the light-triggered DNA scaffolds formed a cross-linked network by click chemistry, effectively impeding tumor cell migration. On the other hand, the efficient cellular assembly facilitated the effective communication between tumor cells and NK-92 cells, leading to enhanced immune response against tumors and further suppression of tumor metastasis. These features make our strategy highly applicable to a wide range of metastatic cancers.

Design, synthesis and bioactivity evaluation of self-assembled PROTACs based on multi-target kinase inhibitors.

Proteolysis targeting chimera (PROTAC) is a heterobifunctional molecule with enormous potential for its ability to overcome the limitations of traditional inhibitors. However, its inherent disadvantages have been increasingly revealed, such as poor cell permeability caused by large molecule weight. Herein, to overcome the inherent shortcomings, intracellular self-assembly was proposed based on bioorthogonal reaction and molecular fragments, affording a novel type of self-assembled PROTACs. Two types of precursors incorporated with tetrazine and norbornene as bioorthogonal groups were designed and synthesized, and they could subsequently be conjugated in cells to generate novel PROTACs. Fortunately, ultrafast HRMS and HPLC assays indicated that self-assembled PROTACs driven by the bio-orthogonal reaction were detected in living U87 cells. Biological evaluation suggested that the precursor molecule LN-1 could degrade PDGFR-ß protein in a concentration-dependent manner, while cancer cells were co-treated with another precursor molecule, TzB. Our findings verified the feasibility of a self-assembly strategy in future development of novel PROTACs.

In Situ Prodrug Activation by an Affibody-Ruthenium Catalyst Hybrid for HER2-Targeted Chemotherapy.

Transition-metal catalysts exhibit great potential as therapeutic agents to inhibit tumor growth. However, the precise delivery and in situ catalysis are challenging in catalytic medicine. Herein, we report an anti-HER2 affibody-ruthenium catalyst hybrid, named Ru-HER2 for selective and effective killing of cancer cells. Ru-HER2 binds to the HER2 receptor on a tumor cell and in situ catalyzes the activation of gemcitabine prodrug, resulting in enhanced selectivity in suppression of tumor growth and reduction of side effects. Immunoblotting reveals that Ru-HER2 in combination with gemcitabine prodrug can not only induce DNA damage, but also effectively block the HER2 signaling pathway in cancer cells. Therefore, the HER2-targeted chemotherapy exhibits substantially high anticancer activity toward HER2-positive cancer cells in vitro and in vivo. In a word, we report the first affibody-ruthenium catalyst hybrid and reveal its potential for effective HER2-targeted cancer chemotherapy.

Bio-clickable mussel-inspired peptides improve titanium-based material osseointegration synergistically with immunopolarization-regulation.

Upon the osteoporotic condition, sluggish osteogenesis, excessive bone resorption, and chronic inflammation make the osseointegration of bioinert titanium (Ti) implants with surrounding bone tissues difficult, often lead to prosthesis loosening, bone collapse, and implant failure. In this study, we firstly designed clickable mussel-inspired peptides (DOPA-N3) and grafted them onto the surfaces of Ti materials through robust catechol-TiO2 coordinative interactions. Then, two dibenzylcyclooctyne (DBCO)-capped bioactive peptides RGD and BMP-2 bioactive domain (BMP-2) were clicked onto the DOPA-N3-coated Ti material surfaces via bio-orthogonal reaction. We characterized the surface morphology and biocompatibility of the Ti substrates and optimized the osteogenic capacity of Ti surfaces through adjusting the ideal ratios of BMP-2/RGD at 3:1. In vitro, the dual-functionalized Ti substrates exhibited excellent promotion on adhesion and osteogenesis of mesenchymal stem cells (MSCs), and conspicuous immunopolarization-regulation to shift macrophages to alternative (M2) phenotypes and inhibit inflammation, as well as enhancement of osseointegration and mechanical stability in osteoporotic rats. In summary, our biomimetic surface modification strategy by bio-orthogonal reaction provided a convenient and feasible method to resolve the bioinertia and clinical complications of Ti-based implants, which was conducive to the long-term success of Ti implants, especially in the osteoporotic or inflammatory conditions.

Bioclickable Mussel-Derived Peptides With Immunoregulation for Osseointegration of PEEK.

Polyether ether ketone (PEEK)-based biomaterials have been widely used in the field of spine and joint surgery. However, lack of biological activity limits their further clinical application. In this study, we synthesized a bioclickable mussel-derived peptide Azide-DOPA4 as a PEEK surface coating modifier and further combined bone morphogenetic protein 2 functional peptides (BMP2p) with a dibenzylcyclooctyne (DBCO) motif through bio-orthogonal reactions to obtain DOPA4@BMP2p-PEEK. As expected, more BMP2p can be conjugated on PEEK after Azide-DOPA4 coating. The surface roughness and hydrophilicity of DOPA4@BMP2p-PEEK were obviously increased. Then, we optimized the osteogenic capacity of PEEK substrates. In vitro, compared with the BMP2p-coating PEEK material, DOPA4@BMP2p-PEEK showed significantly higher osteogenic induction capability of rat bone marrow mesenchymal stem cells. In vivo, we constructed a rat calvarial bone defect model and implanted PEEK materials with a differently modified surface. Micro-computed tomography scanning displayed that the DOPA4@BMP2p-PEEK implant group had significantly higher new bone volume and bone mineral density than the BMP2p-PEEK group. Histological staining of hard tissue further confirmed that the DOPA4@BMP2p-PEEK group revealed a better osseointegrative effect than the BMP2p-PEEK group. More importantly, we also found that DOPA4@BMP2p coating has a synergistic effect with induced Foxp3+ regulatory T (iTreg) cells to promote osteogenesis. In summary, with an easy-to-perform, two-step surface bioengineering approach, the DOPA4@BMP2p-PEEK material reported here displayed excellent biocompatibility and osteogenic functions. It will, moreover, offer insights to engineering surfaces of orthopedic implants.

Using bio-orthogonally catalyzed lethality strategy to generate mitochondria-targeting anti-tumor metallodrugs in vitro and in vivo.

Synthetic lethality was proposed nearly a century ago by geneticists and recently applied to develop precision anti-cancer therapies. To exploit the synthetic lethality concept in the design of chemical anti-cancer agents, we developed a bio-orthogonally catalyzed lethality (BCL) strategy to generate targeting anti-tumor metallodrugs both in vitro and in vivo. Metallodrug Ru-rhein was generated from two non-toxic species Ru-N3 and rhein-alkyne via exclusive endogenous copper-catalyzed azide alkyne cycloaddition (CuAAC) reaction without the need of an external copper catalyst. The non-toxic species Ru-arene complex Ru-N3 and rhein-alkyne were designed to perform this strategy, and the mitochondrial targeting product Ru-rhein was generated in high yield (>83%) and showed high anti-tumor efficacy in vitro. This BCL strategy achieved a remarkable tumor suppression effect on the tumor-bearing mice models. It is interesting that the combination of metal-arene complexes with rhein via CuAAC reaction could transform two non-toxic species into a targeting anti-cancer metallodrug both in vitro and in vivo, while the product Ru-rhein was non-toxic towards normal cells. This is the first example that exclusive endogenous copper was used to generate metal-based anti-cancer drugs for cancer treatment. The anti-cancer mechanism of Ru-rhein was studied and autophagy was induced by increased reactive oxygen species and mitochondrial damage. The generality of this BCL strategy was also studied and it could be extended to other metal complexes such as Os-arene and Ir-arene complexes. Compared with the traditional methods for cancer treatment, this work presented a new approach to generating targeting metallodrugs in vivo via the BCL strategy from non-toxic species in metal-based chemotherapy.

Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields Labeled RNA for Conformational Analysis and Imaging.

Chemical labeling of RNA by using chemoselective reactions that work under biologically benign conditions is increasingly becoming valuable in the in vitro and in vivo analysis of RNA. Here, we describe a modular RNA labeling method based on a posttranscriptional Suzuki-Miyaura coupling reaction, which works under mild conditions and enables the direct installation of various biophysical reporters and tags. This two-part procedure involves the incorporation of a halogen-modified UTP analog (5-iodouridine-5'-triphosphate) by a transcription reaction. Subsequent posttranscriptional coupling with boronic acid/ester substrates in the presence of a palladium catalyst provides access to RNA labeled with (a) fluorogenic environment-sensitive nucleosides for probing nucleic acid structure and recognition, (b) fluorescent probes for microscopy, and (3) affinity tags for pull-down and immunoassays. It is expected that this method could also become useful for imaging nascent RNA transcripts in cells if the nucleotide analog can be metabolically incorporated and coupled with reporters by metal-assisted cross-coupling reactions.

Design and Engineering of Metal Catalysts for Bio-orthogonal Catalysis in Living Systems.

In the past two decades, bio-orthogonal transformations mediated by biocompatible metal catalysts in living systems have shown enormous potential in both synthetic biology and medicinal chemistry. These metal-mediated bio-orthogonal reactions, many of which could not be accomplished by natural enzymes, have created more possibilities in organic chemistry and biological sciences. Despite all of the challenges for making those abiotic catalysts work in complicated biological environments, many catalytic systems working in living systems have been reported, mediating different transformations such as uncaging of fluorescent probes, pro-drug activation, glycan or protein activation, labeling of proteins or cell surfaces, and in situ drug synthesis. This review categorizes and summarizes the recent development of synthetic metal catalysts for bio-orthogonal reactions in living systems within two decades. Ranging from simple metal complexes and macromolecular-scaffold-based catalytic systems to heterogeneous nanomaterial-based systems, we show those catalysts of diverse nature and highlight the strategies for their design and engineering. We analyze and describe the structure-property relationship of those biocompatible metal catalysts and show the importance of structural diversification and optimization for their potential applications. A brief overview of metal-mediated bio-orthogonal reactions for biological applications is also given, and the challenges and opportunities of this field are discussed from a long-term perspective.

Bio-orthogonal click reaction-enabled highly specific in situ cellularization of tissue engineering scaffolds.

Tissue engineering generally utilizes natural or synthetic scaffolds to repair or replace damaged tissues. However, due to the lack of guidance of biological signals, most of the implanted scaffolds have always suffered from poor in vivo cellularization. Herein, we demonstrate a bio-orthogonal reaction-based strategy to realize in situ specific and fast cellularization of tissue engineering scaffold. DBCO-modified PCL-PEG (PCL-PEG-DBCO) polymer was synthesized and then fabricated into PCL-PEG-DBCO film through electrospinning. Meanwhile, azide-labeled macrophages (N3 (+) macrophages) were obtained through metabolic glycoengineering. Through a series of in vitro dynamic and in vivo characterization, DBCO-modified films were noted to dramatically increase the selective capture efficiency and survival rate of N3 (+) cells. Additionally, there is negligible influence of covalent conjugation on cell viability and proliferation, indicating the feasibility of the bio-orthogonal click reaction-based tissue engineering strategy. Overall, this work shows the advantages of an in situ bio-orthogonal click reaction in realizing highly specific, efficient, and long-lasting scaffold cellularization. We anticipate that this general strategy would be widely applicable and useful in tissue engineering and regenerative medicine in the near future.