Vitrification cryopreservation of ligaments based on zwitterionic betaine

Ligament cryopreservation enables a prolonged shelf life of allogeneic ligament grafts,which is fundamentally important to ligament reconstruction.However,conventional cryopreservation techniques fail to eliminate the damage caused by ice crystal growth and the toxicity of cryopreservation agents(CPAs).Here,we report a novel CPA vitrification formulation primarily composed of betaine for ligament cryopreservation.Comprehensive optimization was conducted on the methods for vitrification and rewarming,as well as the loading and unloading conditions,based on the critical cooling rate(CCR),critical warming rate(CWR),and permeation properties of the CPA.Using biomechanical and histological level tests,we demonstrate the superior performance of our method in ligament cryopreservation.After 30 days of vitrification cryopreservation,parameters such as the Young's modulus,tensile stress,denaturation temperature,and glycosaminoglycans content of the ligament remained essentially unchanged.This work pioneers the application of ice-free cryopreservation for ligament and holds great potential for improving the long-term storage of ligament,providing valuable insights for future cryopreservation technique development.

深低温保存下高效抗冻多肽的合理设计和机理探讨

The development of effective antifreeze peptides to control ice growth has attracted a significant amount of attention yet still remains a great challenge.Here,we propose a novel design method based on in-depth investigation of repetitive motifs in various ice-binding proteins(IBPs)with evolution analysis.In this way,several peptides with notable antifreeze activity were developed.In particular,a designed antifreeze peptide named AVD exhibits ideal ice recrystallization inhibition(IRI),solubility,and biocompatibility,making it suitable for use as a cryoprotective agent(CPA).A mutation analysis and molecular dynamics(MD)simulations indicated that the Thr6 and Asn8 residues of the AVD peptide are fundamental to its ice-binding capacity,while the Ser18 residue can synergistically enhance their interaction with ice,revealing the antifreeze mechanism of AVD.Furthermore,to evaluate the cryoprotection potential of AVD,the peptide was successfully employed for the cryopreservation of various cells,which demonstrated significant post-freezing cell recovery.This work opens up a new avenue for designing antifreeze materials and provides peptide-based functional modules for synthetic biology.

DNA: From Carrier of Genetic Information to Polymeric Materials

Research has shown that the DNA molecule can not only store genetic information but also serve as a polymeric biomolecule for the fabrication of functional materials. The unique precise molecular recognition capability and sequence programmability, combined with its good biocompatibility and biodegradability, impart the DNA molecule considerable potential for use in the construction of multifunctional materials. Depending on the composition, DNA-based materials have been generally categorized into pure DNA materials that are entirely composed of DNA and hybrid DNA materials that are composed of DNA and other functional compositions. Recently, we have developed a series of DNA-based materials that can be applied in diagnosis and therapy, and this review summarizes the relative work. Although challenges still exist regarding the real applications of DNA-based materials such as the high cost of DNA, the difficulty in scale-up, and the low resistance to nuclease, we believe that these drawbacks will be overcome with the development of technology, and new opportunities will emerge in the fi eld of diagnosis and treatment.

A Programmable Hybrid DNA Nanogel for Enhanced Photodynamic Therapy of Hypoxic Glioma

Photodynamic therapy(PDT)is a promising cancer therapy due to the evident advantages of a rapid curative eff ect,minimal or non-invasiveness,and circumvention of drug resistance.However,the hydrophobicity of photosensitizers and the hypoxic tumor microenvironment in solid tumors reduce the therapeutic eff ect of PDT immensely.Herein,we construct a programmable hybrid mesoporous silica nanoparticle/DNA nanogel(H-DNA nanogel)for enhanced PDT.The H-DNA nanogel is constituted with a virus-like mesoporous silica nanoparticle(VMSN)as the core to provide an appropriate nano-interface and a self-assembly programmable DNA hydrogel layer based on rolling circle amplifi cation(RCA)as the shell.Two kinds of G-quadruplex structures inserted with a hemin and zinc phthalocyanine(ZnPc)photosensitizer are introduced into the H-DNA nanogel by base pairing.The two modules of G-quadruplex structure work as an oxygen supplement in the hypoxic tumor microenvironment and increase the yield of singlet oxygen,respectively.Our hybrid DNA nanogel system provides a modular platform for effi cient cancer PDT and has great potential in the broader biomedical fi eld.

Aptamer-Based DNA Materials for the Separation and Analysis of Biological Particles

DNA is a biological macromolecule that carries genetic information in organisms.It provides a series of predominant bio-logical advantages,such as sequence programmability,high biocompatibility,and low biotoxicity.As such,it is a unique polymer material that shows great potential for application in biological and medical fields.DNA aptamers are short DNA sequences with a specific ability of molecular recognition.With its discovery,the application prospect of DNA materials has broadened,especially for the separation and analysis of biological particles.In this review,the functions and characteristics of DNA aptamers are introduced,and the applications of DNA materials in cell/exosome separation and cancer detection are summarized.The application prospect and possible challenges of DNA materials are predicted.

AIE活性抗冻水凝胶可实现低温环境下多级信息的加密与解密

抗冻水凝胶可在低温环境下调控内部水分子的冻结状态,进而维持其优异特性(如智能响应性、离子运输能力等)以及拓展其在低温环境下的应用。本文开发了一系列具有AIE活性的抗冻水凝胶,其可用于低温环境下的信息加密/解密。根据水凝胶中甜菜碱浓度的不同使其具有可调的冻结温度(T_(f))。当温度高于/低于Tf时,编码于水凝胶内的AIE分子表现出不发光/发光行为,从而实现信息的加密/解密。此外,通过调控水凝胶的冷冻程序,或者在水凝胶中原位引入具有光热效应的硫化铜纳米粒子并结合一定的照射条件,可实现多级信息的读取,从而增强信息的安全性。最后,由于该水凝胶对外界温度波动表现出不可逆性,因此可作为无需外界供能的超低温防伪标签,用于实时可视化监测冷链运输过程中细胞的活性。

Light-Mediated Spatiotemporally Dynamic Assembly of DNA Nanostructures in Living Cells Regulates Autophagy

The assembly of exogenous artificial architectures inside cells can regulate a series of biological events,which heavily relies on the development of spatiotemporally controlled molecular assembly systems.We herein report a designer deoxyribonucleic acid(DNA)nanostructure that enables light-mediated spatiotemporally dynamic assembly in living cells and consequently achieves efficient regulation of cell autophagy.The DNA nanostructure was constructed from i-motif moiety-containing branched DNA,photocleavable bond-containing linker,and tumor cell-targeting aptamer.After cellular uptake mediated by aptamers,under the spatiotemporal control of both UV light and late endosomal/lysosomal acidic environments,disassembly/reassembly of DNA nanostructure occurred via two rationally designed routes,generating microsized DNA assembly.As a result,autophagy was significantly enhanced with the increase of DNA assembly size.The enhanced autophagy showed an impact on related biological effects.Our system is expected to be a powerful tool for the regulation of intracellular events and cellular behaviors.

Inhibition of Defect-Induced Ice Nucleation, Propagation, and Adhesion by Bioinspired Self-Healing Anti-Icing Coatings

Anti-icing coatings on outdoor infrastructures inevitably suffer from mechanical injuries in numerous icing scenarios such as hailstorms,sandstorms,impacts of foreign objects,and icing–deicing cycles.Herein,the mechanisms of surface-defect-induced icing are clarified.At the defects,water molecules exhibit stronger adsorption and the heat transfer rate increases,accelerating the condensation of water vapor as well as ice nucleation and propagation.Moreover,the ice–defect interlocking structure increases the ice adhesion strength.Thus,a self-healing(at−20℃)antifreeze-protein(AFP)-inspired anti-icing coating is developed.The coating is based on a design that mimics the ice-binding and non-ice-binding sites in AFPs.It enables the coating to markedly inhibit ice nucleation(nucleation temperature<−29.4℃),prevent ice propagation(propagation rate<0.00048 cm^(2)/s),and reduce ice adhesion on the surface(adhesion strength<38.9 kPa).More importantly,the coating can also autonomously self-heal at−20℃,as a result of multiple dynamic bonds in its structure,to inhibit defect-induced icing processes.The healed coating sustains high anti-icing and deicing performance even under various extreme conditions.This work reveals the in-depth mechanism of defect-induced ice formation as well as adhesion,and proposes a self-healing anti-icing coating for outdoor infrastructures.

DNA-based nanosystems to generate reactive oxygen species for nanomedicine

Reactive oxygen species(ROS)are essential in various pathological and physiological processes.Developing nanosystems that generate ROS in a controlled manner is of great interest for nanomedicine.DNA nanotechnology offers a promising approach to constructing programmable ROS-generating platforms.By incorporating photosensitizers or metal ions,DNA nanostructures can be designed to produce ROS in a spatially and temporally desired fashion.DNA-based ROS-generating nanosystems hold great potential in intracellular homeostasis regulation,drug release,and cancer therapy.This review summarizes recent advances in developing DNA-based ROS-generating nanosystems,highlights their emerging biomedical applications,and discusses the opportunities and challenges for further applications.DNA nanotechnology provides a versatile toolkit to construct biocompatible ROS-generating platforms for next-generation nanomedicines.

Advanced Biotechnology for Cell Cryopreservation

Cell cryopreservation has evolved as an important technology required for supporting various cell-based applications,such as stem cell therapy,tissue engineering,and assisted reproduction.Recent times have witnessed an increase in the clinical demand of these applications,requiring urgent improvements in cell cryopreservation.However,cryopreservation technology suff ers from the issues of low cryopreservation effi ciency and cryoprotectant(CPA)toxicity.Application of advanced biotechnology tools can signifi cantly improve post-thaw cell survival and reduce or even eliminate the use of organic solvent CPAs,thus promoting the development of cryopreservation.Herein,based on the diff erent cryopreservation mechanisms available,we provide an overview of the applications and achievements of various biotechnology tools used in cell cryopreservation,including trehalose delivery,hydrogel-based cell encapsulation technique,droplet-based cell printing,and nanowarming,and also discuss the associated challenges and perspectives for future development.

Exploring novel cell cryoprotectants based on neutral amino acids

Cryoprotectants play a key role in cell cryopreservation because they can reduce cryoinjuries to cells associated with ice formation.To meet the clinical requirements of cryopreserved cells,cryoprotectants should be biocompatible,highly efficient and easily removable from cryopreserved cells.However,integration of these properties into one cryoprotectant still remains challenging.Herein,three biocompatible neutral amino acids,includingβ-alanine,γ-aminobutyric acid andε-aminocaproic acid,are first reported to have the potential as such ideal cryoprotectants.The results demonstrate that they can inhibit ice formation and reduce osmotic stress to provide extracellular and intracellular protection,thereby ensuring high cryopreservation efficiency for both anuclear and nucleated cells.More importantly,due to the remarkable osmotic regulation ability,the neutral amino acids can be rapidly removed from cryopreserved cells via a one-step method without causing observable damage to cells,superior to the current state-of-the-art cryoprotectants—dimethyl sulfoxide and glycerol.This work provides a new perspective to develop novel cryoprotectants,which may have dramatic impacts on solvent-free cryopreservation technology to support the cell-based applications,such as cell therapy and tissue engineering,etc.

Engineering CRISPR/Cas-based nanosystems for therapeutics, diagnosis and bioimaging

CRISPR/Cas system has been utilized to rationally manipulate intracellular genes,and it has been engineered as versatile and efficient gene editing tools with precise site-specificity and excellent targeting ability for therapeutics,diagnostics,and bioimaging.Here,the evolution and application of CRISPR/Cas systems were sketched chronologically.Landmark works were exemplified to illustrate the design principles of CRISPR/Cas systems.Furthermore,the delivery vectors of CRISPR/Cas system especially DNA nanomaterials-based vectors were categorized and illuminated.DNA nanomaterials are suitable for CRISPR/Cas system delivery via base pairing due to its sequence programmability and biocompatibility.Then the applications of CRISPR/Cas in diagnosis and genomic imaging were highlighted.At the end of the review,the challenges and opportunities of CRISPR/Cas systems were deeply discussed.We envision that the grant advances on CRISPR/Cas systems will promote the development of interdisciplinary fields in chemistry,biology and medicine.

Construction of Polymeric DNA Network and Application for Cell Manipulation

Comprehensive Summary Deoxyribonucleic acid(DNA)is a biomacromolecule,as well as a polymeric material,whose sequences with different manipulative structures enable them to implement a series of functions,such as reorganization,target,and catalysis.Compared to existing traditional materials incapable of multifunctional integration,the polymeric DNA network is a form of material that can achieve functional integration while maintaining specific DNA properties.Furthermore,precise target enabled by DNA network is one of the most essential components of cellular manipulation.Hence,the DNA network is indispensable and irreplaceable to cell manipulation that it is a versatile tool for the understanding of basic laws of living life and treatments of diseases,such as cell isolation,cell delivery,and cell interference.Herein,the construction of polymeric DNA network is briefly introduced from the aspects of assembly modules,construction methods,and properties.

Construction of DNA aggregates in cell milieu for bio-interference

Precise assembly of biomolecules into functional aggregate structures represents a key characteristic of nature living cells,and is critical for the cellular processes.The construction of artificial aggregates in living cells by responding to intracel-lular specific stimuli has been applied in elucidating molecular mechanisms of naturally cellular processes and interfering with cellular processes,which is poten-tial and significant in biomedicine.DNA(deoxyribonucleic acid)features sequence programmability,precise assembly and versatility,and therefore is regarded as the potential candidate for constructing versatile functional aggregates in living cells.In this review,we summarize our recent efforts of employing DNA to in situ construct versatile aggregates in living cells via responding intracellular triggers,and the sub-sequent bio-interference of the DNA aggregates.Finally,we discuss the remaining challenges and opportunities in thefield,and envision that rational design and con-struction of versatile DNA aggregates in living system would be a promising solution for precision and personalized therapeutics.

Hybridization chain reaction-based DNA nanomaterials for biosensing,bioimaging and therapeutics

DNA nanomaterials hold great promise in biomedical fields due to its excellent sequence programmability,molecular recognition ability and biocompatibility.Hybridization chain reaction(HCR)is a simple and efficient isothermal enzyme-free amplification strategy of DNA,generating nicked double helices with repeated units.Through the design of HCR hairpins,multiple nanomaterials with desired functions are assembled by DNA,exhibiting great potential in biomedical applications.Herein,the recent progress of HCR-based DNA nanomaterials for biosensing,bioimaging and therapeutics are summarized.Representative works are exemplified to demonstrate how HCR-based DNA nanomaterials are designed and constructed.The challenges and prospects of the development of HCR-based DNA nanomaterials are discussed.We envision that rationally designing HCR-based DNA nanomaterials will facilitate the development of biomedical applications.

A DNA micro-complex containing polyaptamer for exosome separation and wound healing

Exosomes(EXOs)have showed great potential in regenerative medicine.The separation of EXOs from complex biological media is essential for the down-stream applications.Herein,we report a deoxyribonucleic acid(DNA)-based micro-complex(DMC)containing polyaptamers,which realized the specific separation of EXOs from cell culture media and the significant promotion of wound healing.The synthesis of DMCs was based on a biomineralization process via rolling circle amplification(RCA)under the catalysis of phi29 DNA polymerase.To endow DMCs with the ability to capture EXOs,the DNA template of RCA was integrated with complementary sequence of aptamer that specifically recognized the CD63 proteins on EXOs.The obtained DMCs contained polyaptamers that can specifically capture the EXOs in cell culture media.The EXOs-capturing DMCs were collected by centrifugation,achieving the separation of EXOs.Mesenchymal stem cell(MSC)-derived EXOs(MSC-EXOs)were separated by this DMC-based strategy,and the separated MSC-EXOs significantly enhanced the migration ability of cells.In particular,the significant therapeutic efficacy of the DMCs with MSC-EXOs was verified in full-thickness wound excision mouse models,in which the wounds completely healed in 10 days.We envision that this DMC-based separation strategy can be a promising route to promote the development of EXOs in biomedicine.

A Humidity-Powered Soft Robot with Fast Rolling Locomotion

A range of soft robotic systems have recently been developed that use soft,flexible materials and respond to environmental stimulus.The greatest challenge in their design is the integration of the actuator,energy sources,and body of robots while achieving fast locomotion and well-defined programmable trajectories.This work presents such a design that operates under constant conditions without the need for an externally modulated stimulus.By using a humidity-sensitive agarose film and overcoming the isotropic and random bending of the film,the robot,which we call the Hydrollbot,harnesses energy from evaporation for spontaneous and continuous fast self-rolling locomotion with a programmable trajectory in a constant-humidity environment.Moreover,the geometric parameters of the film were fine-tuned to maximize the rolling speed,and the optimised hydrollbot is capable of carrying a payload up to 100%of its own weight.The ability to self-propel fast under constant conditions with programmable trajectories will confer practical advantages to this robot in the applications for sensors,medical robots,actuation,etc.

Surface display of carbonic anhydrase on Escherichia coli for CO_(2) capture and mineralization

Mineralization catalyzed by carbonic anhydrase(CA)is one of the most promising technologies for capturing CO_(2).In this work,Escherichia coli BL21(DE3)was used as the host,and the N-terminus of ice nucleation protein(INPN)was used as the carrier protein.Different fusion patterns and vectors were used to construct CA surface display systems forα-carbonic anhydrase(HPCA)from Helicobacter pylori 26695 andα-carbonic anhydrase(SazCA)from Sulfurihydrogenibium azorense.The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity,while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system.The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA.Cell frac-tionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell’s outer membrane as a fusion protein.The enzyme activity of the surface display strain E-22b-I RL S(11.43 U⋅mL^(−1) OD 600−1)was significantly higher than that of the intracellular expression strain E-22b-S(8.355 U⋅mL^(−1) OD 600−1)under optimized induction conditions.Compared with free SazCA,E-22b-I RL S had higher thermal and pH stability.The long-term stability of SazCA was also significantly improved by surface display.When the engineered strain and free enzyme were used for CO_(2) mineralization,the amount of CaCO_(3) deposition catalyzed by the strain E-22b-I RL S on the surface(241 mg)was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S(173 mg).These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO_(2) capture and mineralization.

Tianjin Biosecurity Guidelines for codes of conduct for scientists:Promoting responsible sciences and strengthening biosecurity governance

Formulated and endorsed by the international scientific community,the Tianjin Biosecurity Guidelines are a set of ten guiding principles and standards of conduct designed to promote responsible sciences and strengthen biosecurity governance at national and institutional levels.It may be used to develop new or enhance,supplement,and update the existing codes of conduct adaptive to a specific context and responsive to the bio-risks arising from the rapid advances in biological sciences.

Construction and applications of DNA-based nanomaterials in cancer therapy

As a biologically active macromolecule, deoxyribonucleic acid(DNA) has the advantages of sequence programmability and structure controllability and can accurately transmit sequence information to specific biological functions. Facing the complex internal microenvironment and heterogeneity in tumor treatment, the construction and applications of DNA-based nanomaterials have become a focus point of research. In particular, the hybridization of DNA molecules with other materials endows DNA-based nanomaterials with multiple functions such as targeting, stimulus responsiveness and regulations of biological activities, making DNA nanostructures great potential in the treatment of major human diseases.In this review, the construction and characteristics of DNA-based nanomaterials are introduced. Then,the functions and applications of DNA-based nanomaterials in the delivery of chemotherapy drugs and gene drugs, stimulus-responsive release and regulation of cell homeostasis are reviewed. Finally, the future development and challenges of DNA-based nanomaterials are prospected. We envision that DNAbased nanomaterials can enrich the nanomaterial system by rational design and synthesis and address the growing demands on biological and biomedical applications in the real world.