Robust and Multifunctional Therapeutic Nanoparticles against Peritonitis-Induced Sepsis.

Apart from bacterial growth and endotoxin generation, the excessive production of reactive radicals linked with sepsis also has a substantial impact on triggering an inflammatory response and further treatment failure. Hence, the rational design and fabrication of robust and multifunctional nanoparticles (NPs) present a viable means of overcoming this dilemma. In this study, we used antibiotic polymyxin B (PMB) and antioxidant natural polyphenolic protocatechualdehyde (PCA) to construct robust and multifunctional NPs for sepsis treatment, leveraging the rich chemistries of PCA. The PMB release profile from the NPs demonstrated pH-responsive behavior, which allowed the NPs to exhibit effective bacterial killing and radical scavenging properties. Data from in vitro cells stimulated with H2O2 and lipopolysaccharide (LPS) showed the multifunctionalities of NPs, including intracellular reactive oxygen species (ROS) scavenging, elimination of the bacterial toxin LPS, inhibiting macrophage M1 polarization, and anti-inflammation capabilities. Additionally, in vivo studies further demonstrated that NPs could increase the effectiveness of sepsis treatment by lowering the bacterial survival ratio, the expression of the oxidative marker malondialdehyde (MDA), and the expression of inflammatory cytokine TNF-α. Overall, this work provides ideas of using those robust and multifunctional therapeutic NPs toward enhanced sepsis therapy efficiency.

Rapid Construction of 18F-Triazolyl-tetrazines through the Click Reaction.

Due to the fast reaction rate, 18F-labeled tetrazines have been widely applied in positron emission tomography (PET) imaging in cancer research and drug discovery. In this work, several functional 18F-triazolyl-tetrazines were rapidly obtained through an optimized copper-catalyzed alkyene-azide cycloaddition reaction system in >99% radiochemical conversions. Notably, the commonly used 18F-labeled azides were isolated through cartridges and directly used for cycloadditions, which greatly simplified the labeling procedure. The assembled triazolyl-tetrazines demonstrated high in vitro stability and reaction kinetics, exhibiting considerable potential for the development of PET agents.

Tetrazine-Isonitrile Bioorthogonal Fluorogenic Reactions Enable Multiplex Labeling and Wash-Free Bioimaging of Live Cells.

Developing fluorogenic probes for simultaneous live cell labeling of multiple targets is crucial for understanding complex cellular events. The emerging [4+1] cycloaddition between tetrazine and isonitriles holds promise as a bioorthogonal tool, yet existing tetrazine probes lack reactivity and fluorogenicity. Here, we present the development of a series of tetrazine-functionalized bioorthogonal probes. By incorporating pyrazole adducts into the fluorophore scaffolds, the post-reacted probes displayed remarkable fluorescence turn-on ratios, up to 3184-fold. Moreover, these modifications are generalizable to various fluorophores, enabling a broad emission range from 473 to 659 nm. Quantum chemical calculations further elucidate the turn-on mechanisms. These probes enable the simultaneous labeling of multiple targets in live cells, without the need for a washing step. Consequently, our findings pave the way for advanced multiplex imaging and detection techniques for cellular studies.

Versatile polyphenolic platforms in regulating cell biology.

Polyphenolic materials are a class of fascinating and versatile bioinspired materials for biointerfacial engineering. In particular, due to the presence of active chemical groups, a series of unique physicochemical properties become accessible and tunable of the as-prepared polyphenolic platforms, which could delicately regulate the cell activities via cell-material contact-dependent interactions. More interestingly, polyphenols could also affect the cell behaviors via cell-material contact-independent manner, which arise due to their intrinsically functional characteristics (e.g., antioxidant and photothermal behaviors). As such, a comprehensive understanding on the relationship between material properties and desired biomedical applications, as well as the underlying mechanism at the cellular and molecular level would provide material design principles and accelerate the lab-to-clinic translation of polyphenolic platforms. In this review, we firstly give a brief overview of cell hallmarks governed by surrounding cues, followed by the introduction of polyphenolic material engineering strategies. Subsequently, a detailed discussion on cell-polyphenols contact-dependent interfacial interaction and contact-independent interaction was also carefully provided. Lastly, their biomedical applications were elaborated. We believe that this review could provide guidances for the rational material design of multifunctional polyphenols and extend their application window.

Overcoming Spectral Dependence: A General Strategy for Developing Far-Red and Near-Infrared Ultra-Fluorogenic Tetrazine Bioorthogonal Probes.

Bioorthogonal fluorogenic dyes are indispensable tools in wash-free bioimaging of specific biological targets. However, the fluorogenicity of existing tetrazine-based bioorthogonal probes deteriorates as the emission wavelength shifts towards the NIR window, greatly limiting their applications in live cells and tissues. Herein, we report a generalizable molecular design strategy to construct ultra-fluorogenic dyes via a simple substitution at the meso-positions of various far-red and NIR fluorophores. Our probes demonstrate significant fluorescence turn-on ratios (102 -103 -fold) in the range 586-806 nm. These results will greatly expand the applications of bioorthogonal chemistry in NIR bioimaging and biosensing.

Concise Synthesis of Functionalized Cyclobutene Analogues for Bioorthogonal Tetrazine Ligation.

Novel bioorthogonal tools enable the development of new biomedical applications. Here we report the concise synthesis of a series of aryl-functionalized cyclobutene analogues using commercially available starting materials. Our study demonstrates that cyclobutene acts as a small, strained dienophile to generate stable substrates suitable for bioorthogonal tetrazine ligation.

A General Strategy to Design Highly Fluorogenic Far-Red and Near-Infrared Tetrazine Bioorthogonal Probes.

Highly fluorogenic tetrazine bioorthogonal probes emitting at near-infrared wavelengths are in strong demand for biomedical imaging applications. Herein, we have developed a strategy for forming a palette of novel Huaxi-Fluor probes in situ, whose fluorescence increases hundreds of times upon forming the bioorthogonal reaction product, pyridazine. The resulting probes show large Stokes shifts and high quantum yields. Manipulating the conjugate length and pull-push strength in the fluorophore skeleton allows the emission wavelength to be fine-tuned from 556 to 728 nm. The highly photo-stable and biocompatible probes are suitable for visualizing organelles in live cells without a washing step and for imaging of tumors in live small animals to depths of 500 µm by two-photon excitation.

Activation and Delivery of Tetrazine-Responsive Bioorthogonal Prodrugs.

Prodrugs, which remain inert until they are activated under appropriate conditions at the target site, have emerged as an attractive alternative to drugs that lack selectivity and show off-target effects. Prodrugs have traditionally been activated by enzymes, pH or other trigger factors associated with the disease. In recent years, bioorthogonal chemistry has allowed the creation of prodrugs that can be chemically activated with spatio-temporal precision. In particular, tetrazine-responsive bioorthogonal reactions can rapidly activate prodrugs with excellent biocompatibility. This review summarized the recent development of tetrazine bioorthogonal cleavage reaction and great promise for prodrug systems.

Advances in Tetrazine Bioorthogonal Chemistry Driven by the Synthesis of Novel Tetrazines and Dienophiles.

Bioorthogonal chemistry has found increased application in living systems over the past decade. In particular, tetrazine bioorthogonal chemistry has become a powerful tool for imaging, detection, and diagnostic purposes, as reflected in the increased number of examples reported in the literature. The popularity of tetrazine ligations are likely due to rapid and tunable kinetics, the existence of high quality fluorogenic probes, and the selectivity of reaction. In this Account, we summarize our recent efforts to advance tetrazine bioorthogonal chemistry through improvements in synthetic methodology, with an emphasis on developing new routes to tetrazines and expanding the range of useful dienophiles. These efforts have removed specific barriers that previously limited tetrazine ligations and have broadened their potential applications. Among other advances, this Account describes how our group discovered new methodology for tetrazine synthesis by developing a Lewis acid-promoted, one-pot method for generating diverse symmetric and asymmetric alkyl tetrazines with functional substituents in satisfactory yields. We attached these tetrazines to microelectrodes and succeeded in controlling tetrazine ligation by changing the redox state of the reactants. Using this electrochemical control process, we were able to modify an electrode surface with redox probes and enzymes in a site-selective fashion. This Account also describes how our group improved the ability of tetrazines to act as fluorogenic probes by developing a novel elimination-Heck cascade reaction to synthesize alkenyl tetrazine derivatives. In this approach, tetrazine was conjugated to fluorophores to produce strongly quenched probes that, after bioorthogonal reaction, are "turned on" to enhance fluorescence, in many cases by >100-fold. These probes have allowed no-wash fluorescence imaging in living cells and intact animals. Finally, this Account reviews our efforts to expand the range of dienophile substrates to make tetrazine bioorthogonal chemistry compatible with specific biochemical and biomedical applications. We found that methylcyclopropene is sufficiently stable and reactive in the biological milieu to act as an efficient dienophile. The small size of the reactive tag minimizes steric hindrance, allowing cyclopropene to serve as a metabolic reporter group to reveal biological dynamics and function. We also used norbornadiene derivatives as strained dienophiles to undergo tetrazine-mediated transfer (TMT) reactions involving tetrazine ligation followed by a retro-Diels-Alder process. This TMT reaction generates a pair of nonligating products. Using nucleic acid-templated chemistry, we have combined the TMT reaction with our fluorogenic tetrazine probes to detect endogenous oncogenic microRNA at picomolar concentrations. In a further display of dienophile versatility, we used a novel vinyl ether to cage a near-infrared fluorophore in a nonfluorescent form. Then we opened the cage in a "click to release" tetrazine bioorthogonal reaction, restoring the fluorescent form of the fluorophore. Combining this label with a corresponding nucleic acid probe allowed fluorogenic detection of target mRNA. In summary, this Account describes improvements in tetrazine and dienophile synthesis and application to advance tetrazine bioorthogonal chemistry. These advances have further enabled application of tetrazine ligation chemistry, not only in fundamental research but also in diagnostic studies.

Positron Emission Tomography Imaging of Platelet-Derived Growth Factor Receptor ß in Colorectal Tumor Xenograft Using Zirconium-89 Labeled Dimeric Affibody Molecule.

Platelet-derived growth factor receptor ß (PDGFRß) is overexpressed in a variety of malignant cancers, plays a critical role in tumor angiogenesis, and has been proven as a valuable target for cancer treatment. In this pilot study, a dimeric affibody molecule, ZPDGFRß, was prepared and radiolabeled with positron emission radionuclide zirconium-89 for PET imaging of colorectal tumors by targeting PDGFRß expression in vivo. The PDGFRß-binding capability of dimeric affibody was evaluated by flow cytometry, immunofluorescent staining, and whole-body optical imaging. Then, ZPDGFRß was conjugated with DFO-Bn-NCS and radiolabeled with 89Zr. Targeted binding capability of 89Zr-DFO-ZPDGFRß to PDGFRß expressing cells was investigated by cellular assay in vitro and microPET/CT imaging in vivo. Dimeric ZPDGFRß affibody had specifically higher binding capability with PDGFRß expressing pericytes rather than LS-174T cancer cells, and well colocalized with tumor neovasculature by flow cytometry and immunofluorescent assay. ZPDGFRß was successfully labeled with 89Zr by DFO chelating with yield of 94.1 ± 3.53%. 89Zr-DFO-ZPDGFRß indicated preserved specific binding ability with PDGFRß expressing cells and effective inhibiting capability to PDGF-ß ligands ( P < 0.05) in vitro. Biodistribution indicated that tumor uptake of 89Zr-DFO-ZPDGFRß reached the peak of 6.93 ± 0.64%ID/g, and the tumor-to-blood ratio was 5.5 ± 0.6 at 2 h post-injection. LS-174T xenografts were clearly visualized by microPET/CT imaging through 1 to 4 h post-injection of 89Zr-DFO-ZPDGFRß affibody conjugate. In conclusion, the 89Zr-DFO-ZPDGFRß conjugate demonstrated specific and high binding ability with colorectal tumor, which indicated its use as a potential radiopharmaceutical for diagnostic imaging of tumor associate vasculatures with PET/CT.

Organocatalytic and Scalable Syntheses of Unsymmetrical 1,2,4,5-Tetrazines by Thiol-Containing Promotors.

Despite the growing application of tetrazine bioorthogonal chemistry, it is still challenging to access tetrazines conveniently from easily available materials. Described here is the de novo formation of tetrazine from nitriles and hydrazine hydrate using a broad array of thiol-containing catalysts, including peptides. Using this facile methodology, the syntheses of 14 unsymmetric tetrazines, containing a range of reactive functional groups, on the gram scale were achieved with satisfactory yields. Using tetrazine methylphosphonate as a building block, a highly efficient Horner-Wadsworth-Emmons reaction was developed for further derivatization under mild reaction conditions. Tetrazine probes with diverse functions can be scalably produced in yields of 87-93 %. This methodology may facilitate the widespread application of tetrazine bioorthogonal chemistry.

Alkoxy Tetrazine Substitution at a Boron Center: A Strategy for Synthesizing Highly Fluorogenic Hydrophilic Probes.

Strongly fluorogenic boron dipyrromethene (BODIPY)-tetrazine probes have been obtained by introducing an alkoxy tetrazine fragment at the boron center. The fluorescence signal from these probes strongly increases by up to 225-fold after reaction with bioorthogonal coupling partners, and the hydrophilicity of probes is improved, such that they are suitable for live-cell imaging.

A Bioorthogonal Near-Infrared Fluorogenic Probe for mRNA Detection.

There is significant interest in developing methods that visualize and detect RNA. Bioorthogonal template-driven tetrazine ligations could be a powerful route to visualizing nucleic acids in native cells, yet past work has been limited with respect to the diversity of fluorogens that can be activated via a tetrazine reaction. Herein we report a novel bioorthogonal tetrazine uncaging reaction that harnesses tetrazine reactivity to unmask vinyl ether caged fluorophores spanning the visible spectrum, including a near-infrared (NIR)-emitting cyanine dye. Vinyl ether caged fluorophores and tetrazine partners are conjugated to high-affinity antisense nucleic acid probes, which show highly selective fluorogenic reactivity when annealed to their respective target RNA sequences. A target sequence in the 3' untranslated region of an expressed mRNA was detected in live cells employing appropriate nucleic acid probes bearing a tetrazine-reactive NIR fluorogen. Given the expansion of tetrazine fluorogenic chemistry to NIR dyes, we believe highly selective proximity-induced fluorogenic tetrazine reactions could find broad uses in illuminating endogenous biomolecules in cells and tissues.

Electrochemical Control of Rapid Bioorthogonal Tetrazine Ligations for Selective Functionalization of Microelectrodes.

We demonstrate that bioorthogonal tetrazine ligations can be utilized to rapidly modify electrode surfaces, both with redox probes and enzymes. Furthermore, we show that the redox-active nature of 1,2,4,5-tetrazines can be exploited to gain electrochemical control over surface modification. To our knowledge this is the first demonstration of controlling a tetrazine ligation by changing the redox state of one of the reactants. We utilize the redox-switchable feature of tetrazine ligations for the site-selective functionalization of a 10 µm spaced interdigitated array of microelectrodes. In addition, we were able to achieve potential controlled ligation of the redox enzyme horseradish peroxidase to a macroscopic planar electrode. The rapid kinetics, bioorthogonal reactivity, and electrochemical control provided by tetrazine ligations should lead to numerous applications related to electrode functionalization.

Bioorthogonal tetrazine-mediated transfer reactions facilitate reaction turnover in nucleic acid-templated detection of microRNA.

Tetrazine ligations have proven to be a powerful bioorthogonal technique for the detection of many labeled biomolecules, but the ligating nature of these reactions can limit reaction turnover in templated chemistry. We have developed a transfer reaction between 7-azabenzonorbornadiene derivatives and fluorogenic tetrazines that facilitates turnover amplification of the fluorogenic response in nucleic acid-templated reactions. Fluorogenic tetrazine-mediated transfer (TMT) reaction probes can be used to detect DNA and microRNA (miRNA) templates to 0.5 and 5 pM concentrations, respectively. The endogenous oncogenic miRNA target mir-21 could be detected in crude cell lysates and detected by imaging in live cells. Remarkably, the technique is also able to differentiate between miRNA templates bearing a single mismatch with high signal to background. We imagine that TMT reactions could find wide application for amplified fluorescent detection of clinically relevant nucleic acid templates.

In situ synthesis of alkenyl tetrazines for highly fluorogenic bioorthogonal live-cell imaging probes.

In spite of the wide application potential of 1,2,4,5-tetrazines, particularly in live-cell and in vivo imaging, a major limitation has been the lack of practical synthetic methods. Here we report the in situ synthesis of (E)-3-substituted 6-alkenyl-1,2,4,5-tetrazine derivatives through an elimination-Heck cascade reaction. By using this strategy, we provide 24 examples of π-conjugated tetrazine derivatives that can be conveniently prepared from tetrazine building blocks and related halides. These include tetrazine analogs of biological small molecules, highly conjugated buta-1,3-diene-substituted tetrazines, and a diverse array of fluorescent probes suitable for live-cell imaging. These highly conjugated probes show very strong fluorescence turn-on (up to 400-fold) when reacted with dienophiles such as cyclopropenes and trans-cyclooctenes, and we demonstrate their application for live-cell imaging. This work provides an efficient and practical synthetic methodology for tetrazine derivatives and will facilitate the application of conjugated tetrazines, particularly as fluorogenic probes for live-cell imaging.

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.

Waste Tea-Derived Theabrownins for Solar-Driven Steam Generation.

Solar-driven seawater desalination has been considered an effective and sustainable solution to mitigate the global freshwater crisis. However, the substantial cost associated with photothermal materials for evaporator fabrication still hinders large-scale manufacturing for practical applications. Herein, we successfully obtained high yields of theabrownins (TB), which were oxidation polymerization products of polyphenols from waste and inferior tea leaves using a liquid-state fermentation strategy. Subsequently, a series of photothermal complexes were prepared based on the metal-phenolic networks assembled from TB and metal ions (Fe(III), Cu(II), Ni(II), and Zn(II)). Also, the screened TB@Fe(III) complexes were directly coated on a hydrophilic poly(vinylidene fluoride) (PVDF) membrane to construct the solar evaporation device (TB@Fe(III)@PVDF), which not only demonstrated superior light absorption property and notable hydrophilicity but also achieved a high water evaporation rate of 1.59 kg m-2 h-1 and a steam generation efficiency of 90% under 1 sun irradiation. More importantly, its long-term stability and exceptionally low production cost enabled an important step toward the possibility of large-scale practical applications. We believe that this study holds the potential to pave the way for the development of sustainable and cost-effective photothermal materials, offering new avenues for utilization of agriculture resource waste and solar-driven water remediation.

A bioorthogonal cell sorting strategy for isolation of desired cell phenotypes.

Here we describe a cost-effective and simplified cell sorting method using tetrazine bioorthogonal chemistry. We successfully isolated SKOV3 cells from complex mixtures, demonstrating efficacy in separating mouse lymphocytes expressing interferon and HeLa cells expressing virally transduced green fluorescent protein post-infection.

UV absorption enhanced polydopamine coating.

Mussel-inspired polydopamine (PDA) coatings have gained significant attention in various fields, including biomedicine, energy, detection, and UV protection, owing to their versatile and promising properties. Among these properties, UV shielding stands out as a key feature of PDA coatings. Nevertheless, the current methods for tuning the UV-shielding properties of PDA coatings are quite limited, and only rely on thickness adjustment, which might involve additional issues like color and visible light transmittance to the coating layer. In this study, we propose a facile and modular approach to enhance the UV absorption of PDA coatings by incorporating thiol-heterocycle (TH) derivatives. Both pre- and post-modification strategies can effectively impede the formation of conjugated structures within PDA, leading to enhanced UV absorption within the PDA layers. More importantly, these strategies can improve the UV absorption of PDA coatings while reducing the visible light absorption. Furthermore, this method enabled efficient regulation of the UV absorption of PDA coatings by altering the ring type (benzene ring or pyridine ring) and substituent on the ring (methoxyl group or hydrogen atom). These PDA coatings with enhanced UV absorption demonstrate great promise for applications in UV protection, antibacterial activity, wound healing and dye degradation.