Bioorthogonal Engineered Virus‑Like Nanoparticles for Efficient Gene Therapy

Gene therapy offers potentially transformative strategies for major human diseases.However,one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue.Here,we report a novel virus-like nanoparticle,the bioorthgonal engineered viruslike recombinant biosome(reBiosome),for efficient gene therapies of cancer and inflammatory diseases.The mutant virus-like biosome(mBiosome)is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site,and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry.The results show that the reBiosome exhibits reduced virus-like immunogenicity,prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci(like tumor and arthritic tissue).Furthermore,reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems,respectively.In conclusion,this study develops a universal,safe and efficient platform for gene therapies for cancer and inflammatory diseases.

Bioorthogonal microglia-inspired mesenchymal stem cell bioengineering system creates livable niches for enhancing ischemic stroke recovery via the hormesis

Mesenchymal stem cells(MSCs)experience substantial viability issues in the stroke infarct region,limiting their therapeutic efficacy and clinical translation.High levels of deadly reactive oxygen radicals(ROS)and proinflammatory cytokines(PC)in the infarct milieu kill transplanted MSCs,whereas low levels of beneficial ROS and PC stimulate and improve engrafted MSCs’viability.Based on the intrinsic hormesis effects in cellular biology,we built a microglia-inspired MSC bioengineering system to transform detrimental high-level ROS and PC into vitality enhancers for strengthening MSC therapy.This system is achieved by bioorthogonally arming metabolic glycoengineered MSCs with microglial membrane-coated nanoparticles and an antioxidative extracellular protective layer.In this system,extracellular ROSscavenging and PC-absorbing layers effectively buffer the deleterious effects and establish a microlivable niche at the level of a single MSC for transplantation.Meanwhile,the infarct’s inanimate milieu is transformed at the tissue level into a new living niche to facilitate healing.The engineered MSCs achieved viability five times higher than natural MSCs at seven days after transplantation and exhibited a superior therapeutic effect for stroke recovery up to 28 days.This vitality-augmented system demonstrates the potential to accelerate the clinical translation of MSC treatment and boost stroke recovery.

Synthetic host-guest pairs as novel bioorthogonal tools for pre-targeting

Due to its simplicity, high efficiency, and chemo-selectivity, bioorthogonal chemistry has shown a great application potential in pre-targeting.Currently, four bioorthogonal pairs as targeting tools, including (strept)avidin/biotin, antibody/antigen, oligonucleotide hybridization and IEDDA tools, have been developed and applied in targeted delivery.Nevertheless, all of these tools still suffer from some limitations, such as difficult modification, biochemical fragility and larger molecular weight for biological association tools, as well as chemical instability for IEDDA tools.Synthetic host-guest pairs with relatively small molecular sizes not only possess strong chemical stability, but also have the features of fast conjugation rate, tunable binding affinity , easy modification, and high chemo-selectivity.Consequently, they can be used as a novel non-covalent bioorthogonal tool for pre-targeting.In order to further promote the development of host-guest pairs as novel bioorthogonal tools for pre-targeted delivery, we firstly calculate their conversion rate to make researcher aware of their unique advantages;next, we summarize the recent research progress in this area.The future perspectives and limitations of these unique tools will be discussed.This review will provide a systemic overview of the development of synthetic host-guest pairs as novel bioorthogonal tools for pre-targeting, and may serve as a “go for” resort for researchers who are interested in searching for new synthetic tools to improve pre-targeting.

Injectable bioorthogonal hydrogel (BIOGEL) accelerates tissue regeneration in degenerated intervertebral discs

Intervertebral disc(IVD)degeneration is a leading cause of back pain and precursor to more severe conditions,including disc herniation and spinal stenosis.While traditional growth factor therapies(e.g.,TGFβ)are effective at transiently reversing degenerated disc by stimulation of matrix synthesis,it is increasingly accepted that bioscaffolds are required for sustained,complete IVD regeneration.Current scaffolds(e.g.,metal/polymer composites,non-mammalian biopolymers)can be improved in one or more IVD regeneration demands:biodegradability,noninvasive injection,recapitulated healthy IVD biomechanics,predictable crosslinking,and matrix repair induction.To meet these demands,tetrazine-norbornene bioorthogonal ligation was combined with gelatin to create an injectable bioorthogonal hydrogel(BIOGEL).The liquid hydrogel precursors remain free-flowing across a wide range of temperatures and crosslink into a robust hydrogel after 5-10 min,allowing a human operator to easily inject the therapeutic constructs into degenerated IVD.Moreover,BIOGEL encapsulation of TGFβpotentiated histological repair(e.g.,tissue architecture and matrix synthesis)and functional recovery(e.g.,high water retention by promoting the matrix synthesis and reduced pain)in an in vivo rat IVD degeneration/nucleotomy model.This BIOGEL procedure readily integrates into existing nucleotomy procedures,indicating that clinical adoption should proceed with minimal difficulty.Since bioorthogonal crosslinking is essentially non-reactive towards biomolecules,our developed material platform can be extended to other payloads and degenerative injuries.

Click chemistry and drug delivery:A bird's-eye view

Click chemistry has been proven to be very useful in drug delivery.Due to the availability of a large number of click reactions with a various characteristics,selection of appropriate chemistry for a given application is often not a trivial task.This review is written for pharmaceutical researchers who are interested in click chemistry applications and yet may not be click chemistry experts.For this,the review gives an overview of available click reactions organized by application types.Further,the general understanding of click reactions being fast and high yielding sometimes overshadows the need to analyze reaction kinetics in assessing suitability of a given reaction for certain applications.For this,we highlight the need to analyze the relationship among reaction kinetics,concentration effects,and reaction time scales,knowing that lack of such analysis could easily lead to failures.Further,possible issues such as chemical stability with various click reagents are also discussed to aid experimental designs.Recent examples and extensive references are also provided to aid in-depth understanding of technical details.We hope this review will help those interested in using click chemistry in drug delivery to select the appropriate reactions/reagents and minimize the number of pitfalls.

Click and bioorthogonal hyaluronic acid hydrogels as an ultra-tunable platform for the investigation of cell-material interactions

The cellular microenvironment plays a major role in the biological functions of cells.Thus,biomaterials,especially hydrogels,which can be design to mimic the cellular microenvironment,are being increasingly used for cell encapsulation,delivery,and 3D culture,with the hope of controlling cell functions.Yet,much remains to be understood about the effects of cell-material interactions,and advanced synthetic strategies need to be developed to independently control the mechanical and biochemical properties of hydrogels.To address this challenge,we designed a new hyaluronic acid(HA)-based hydrogel platform using a click and bioorthogonal strain-promoted azide-alkyne cycloaddition(SPAAC)reaction.This approach facilitates the synthesis of hydrogels that are easy to synthesize and sterilize,have minimal swelling,are stable long term,and are cytocompatible.It provides bioorthogonal HA gels over an uncommonly large range of stiffness(0.5-45 kPa),all forming within 1-15 min.More importantly,our approach offers a versatile one-pot procedure to independently tune the hydrogel composition(e.g.,polymer and adhesive peptides).Using this platform,we investigate the independent effects of polymer type,stiffness,and adhesion on the secretory properties of human adipose-derived stromal cells(hASCs)and demonstrate that HA can enhance the secretion of immunomodulatory factors by hASCs.

Metabolic Labeling and Imaging of Cellular RNA via Bioorthogonal Cyclopropene−Tetrazine Ligation

RNA labeling is vital for the study of an RNA structure,cellular distribution,localization,and metabolism.Herein,we report N6 cyclopropane-modified adenosine(cpA)as a new analog for metabolic RNA labeling.We successfully applied inverse electrondemand Diels–Alder(iEDDA)chemistry to label cellular RNA with cpA.This labeling technique is practical and provides a new platform to study RNA roles in cells in a metal-free manner.This simple and robust assay represents a significant advancement in the profiling methods of the nascent transcriptome using chemical approaches.

Systematic investigation of bioorthogonal cellular DNA metabolic labeling in a photo-controlled manner

Bioorthogonal cleavage and ligation reactions together form one more integrated system about the repertoire of bioorthogonal chemistry,capacitating an array of thrilling new biological applications.The bond-cleavage type and position of biomolecular remain a great challenge,which determines the metabolic pathway of the targets in living systems.Herein we designed two linkages of methylene and carbonyl group attached the N-3 position of the 5-ethynyl-2’-deoxyuridine(EdU)base or the oxygen atom at deoxyribose 3’position to a photocaging group,which would be cleaved by irradiation with 365 nm ultraviolet light.EdU derivatives linked by methylene at the N-3 position had better photodecage efficiency and stability in the absence of light.This paper provides a strategy for studying the nucleoside metabolic pathways in cells,which can easily and conveniently evaluate the effect of the position and type of the linkages.The developed strategy affords a reference for controlling spatial and temporal metabolism of small-molecule drugs,allowing direct manipulation of intact cells under physiological conditions.

Proximity Chemistry in Living Systems

Enzyme-and catalyst-generated reactive species have been leveraged in the past decade to covalently label biomolecules within a short range of a defined site or space inside cells or at the cell–cell interface.Due to their high spatial resolution,such proximity labeling strategies have been coupled with various bioanalytical techniques for dissecting dynamic and complex biological processes.Here,we review the development of enzyme-and catalyst-triggered proximity chemistry and their applications to identifying protein interaction networks as well as cell–cell communications in living systems.

Matthew effect:General design strategy of ultra-fluorogenic nanoprobes with amplified dark–bright states in aggregates

Fluorescence imaging,a key technique in biological research,frequently utilizes fluorogenic probes for precise imaging in living systems.Tetrazine is an effective emission quencher in fluorogenic probe designs,which can be selectively damaged upon bioorthogonal click reactions,leading to considerable emission enhancement.Despite significant efforts to increase the emission enhancement ratio(I_(AC)/I_(BC))of tetrazine-functionalized fluorogenic probes,the influence of molecular aggregation on the emission properties has been largely overlooked in these probe designs.In this study,we reveal that an ultrahigh I_(AC)/I_(BC)can be realized in the aggregate system when tetrazine is paired with aggregation-induced emission(AIE)luminogens.Tetrazine amplifies its quenching efficiency upon aggregation and drastically reduce background emissions.Subsequent click reactions damage tetrazine and trigger significant AIE,leading to considerably enhanced I_(AC)/I_(BC).We further showcase the capability of these ultra-fluorogenic systems in selective imaging of multiple organelles in living cells.We term this unique fluorogenicity of AIE luminogen-quencher complexes with amplified dark-bright states as“Matthew effect”in aggregate emission,potentially providing a universal approach to attain ultrahigh I_(AC)/I_(BC)in diverse fluorogenic systems.

Rational integration of defense and repair synergy on PEEK osteoimplants via biomimetic peptide clicking strategy

Polyetheretherketone(PEEK)has been widely used as orthopedic and dental materials due to excellent mechanical and physicochemical tolerance.However,its biological inertness,poor osteoinduction,and weak antibacterial activity make the clinical applications in a dilemma.Inspired by the mussel adhesion mechanism,here we reported a biomimetic surface strategy for rational integration and optimization of anti-infectivity and osteo-inductivity onto PEEK surfaces using a mussel foot proteins(Mfps)-mimic peptide with clickable azido terminal.The peptide enables mussel-like adhesion on PEEK biomaterial surfaces,leaving azido groups for the further steps of biofunctionalizations.In this study,antimicrobial peptide(AMP)and osteogenic growth peptide(OGP)were bioorthogonally clicked on the azido-modified PEEK biomaterials to obtain a dual-effect of host defense and tissue repair.Since bioorthogonal clicking allows precise collocation between AMP and OGP through changing their feeding molar ratios,an optimal PEEK surface was finally obtained in this research,which could long-term inhibit bacterial growth,stabilize bone homeostasis and facilitate interfacial bone regeneration.In a word,this upgraded mussel surface strategy proposed in this study is promising for the surface bioengineering of inert medical implants,in particular,achieving rational integration of multiple biofunctions to match clinical requirements.

Click CAR-T cell engineering for robustly boosting cell immunotherapy in blood and subcutaneous xenograft tumor

The adoptive transfer of chimeric antigen receptor-T(CAR-T)cells has shown remarkable clinical responses in hematologic malignancies.However,unsatisfactory curative results and side effects for tumor treatment are still unsolved problems.Herein we develop a click CAR-T cell engineering strategy via cell glycometabolic labeling for robustly boosting their antitumor effects and safety in vivo.Briefly,paired chemical groups(N3/BCN)are separately incorporated into CAR-T cell and tumor via nondestructive intrinsic glycometabolism of exogenous Ac4GalNAz and Ac4ManNBCN,serving as an artificial ligand-receptor.Functional groups anchored on cell surface strengthen the interaction of CAR-T cell and tumor via bioorthogonal click chemistry,further enhancing specific recognition,migration and selective antitumor effects of CAR-T cells.In vivo,click CAR-T cell completely removes lymphoma cells and minimizes off-target toxicity via selective and efficient bioorthogonal targeting in blood cancer.Surprisingly,compared to unlabeled cells,artificial bioorthogonal targeting significantly promotes the accumulation,deep penetration and homing of CAR-T cells into tumor tissues,ultimately improving its curative effect for solid tumor.Click CAR-T cell engineering robustly boosts selective recognition and antitumor capabilities of CAR T cells in vitro and in vivo,thereby holding a great potential for effective clinical cell immunotherapy with avoiding adverse events in patients.