Synthetic biology-based bacterial extracellular vesicles displaying BMP-2 and CXCR4 to ameliorate osteoporosis.

Osteoporosis (OP) is a systematic bone disease characterized by low bone mass and fragile bone microarchitecture. Conventional treatment for OP has limited efficacy and long-term toxicity. Synthetic biology makes bacterial extracellular vesicle (BEVs)-based therapeutic strategies a promising alternative for the treatment of OP. Here, we constructed a recombinant probiotics Escherichia coli Nissle 1917-pET28a-ClyA-BMP-2-CXCR4 (ECN-pClyA-BMP-2-CXCR4), in which BMP-2 and CXCR4 were overexpressed in fusion with BEVs surface protein ClyA. Subsequently, we isolated engineered BEVs-BMP-2-CXCR4 (BEVs-BC) for OP therapy. The engineered BEVs-BC exhibited great bone targeting in vivo. In addition, BEVs-BC had good biocompatibility and remarkable ability to promote osteogenic differentiation of BMSCs. Finally, the synthetic biology-based BEVs-BC significantly prevented the OP in an ovariectomized (OVX) mouse model. In conclusion, we constructed BEVs-BC with both bone-targeting and bone-forming in one-step using synthetic biology, which provides an effective strategy for OP and has great potential for industrialization.

Advancing homogeneous networking principles for the development of fatigue-resistant, low-swelling and sprayable hydrogels for sealing wet, dynamic and concealed wounds in vivo.

Effective sealing of wet, dynamic and concealed wounds remains a formidable challenge in clinical practice. Sprayable hydrogel sealants are promising due to their ability to cover a wide area rapidly, but they face limitations in dynamic and moist environments. To address this issue, we have employed the principle of a homogeneous network to design a sprayable hydrogel sealant with enhanced fatigue resistance and reduced swelling. This network is formed by combining the spherical structure of lysozyme (LZM) with the orthotetrahedral structure of 4-arm-polyethylene glycol (4-arm-PEG). We have achieved exceptional sprayability by controlling the pH of the precursor solution. The homogeneous network, constructed through uniform cross-linking of amino groups in protein and 4-arm-PEG-NHS, provides the hydrogel with outstanding fatigue resistance, low swelling and sustained adhesion. In vitro testing demonstrated that it could endure 2000 cycles of underwater shearing, while in vivo experiments showed adhesion maintenance exceeding 24 h. Furthermore, the hydrogel excelled in sealing leaks and promoting ulcer healing in models including porcine cardiac hemorrhage, lung air leakage and rat oral ulcers, surpassing commonly used clinical materials. Therefore, our research presents an advanced biomaterial strategy with the potential to advance the clinical management of wet, dynamic and concealed wounds.

A paintable ophthalmic adhesive with customizable properties based on symmetrical/asymmetrical cross-linking.

In situ and efficient incision sealing for ophthalmic surgery remains unresolved. Current commercially available gel adhesives often suffer from unsuitable gelation time, difficulty in micro-delivery, and mismatched degradation period, leading to difficulties for application in ocular tissue areas. Herein, a novel hydrogel adhesive was developed based on the simultaneous crosslinking of poly(lysine) (PLL) and lysine (Lys) with an end-modified active ester multi-arm polyethylene glycol (PEG) via the amidation reaction, where the residual terminal active ester of PEG can also bond to amino groups on tissue to provide strong adhesion. Due to the different molecular structures around their amino groups, PLL and Lys can crosslink with 4-arm-PEG-NHS (active ester) respectively, to form symmetrical and asymmetrical crosslinking networks, which exhibit various mechanical properties. Therefore, just by adjusting PLL/Lys ratios, the PEG-PLL-Lys hydrogel can easily possess a suitable gelation time, appropriate mechanical properties and matched degradation rate. As a result, a paintable, readily accessible and biocompatible ophthalmic tissue adhesive (sealant) is prepared for sealing ocular tissue incision. Considering the simple strategy and outstanding performance, the PEG-PLL-Lys hydrogel is promising for clinical transformation.

Enlisting a Traditional Chinese Medicine to tune the gelation kinetics of a bioactive tissue adhesive for fast hemostasis or minimally invasive therapy.

Gelation kinetics is important in tailoring chemically crosslinked hydrogel-based injectable adhesives for different applications. However, the regulation of gelation rate is usually limited to varying the gel precursor and/or crosslinker concentration, which cannot reach a fine level and inevitably alters the physical properties of hydrogels. Amidation reactions are widely used to synthesize hydrogel adhesives. In this work, we propose a traditional Chinese medicine (Borax)-input strategy to tune the gelation rate of amidation reaction triggered systems. Borax provides an initial basic buffer environment to promote the deprotonation process of amino groups and accelerate this reaction. By using a tissue adhesive model PEG-lysozyme (PEG-LZM), the gelation time can be modulated from seconds to minutes with varying Borax concentrations, while the physical properties remain constant. Moreover, the antibacterial ability can be improved due to the bioactivity of Borax. The hydrogel precursors can be regulated to solidify instantly to close the bleeding wound at emergency. Meanwhile, they can also be customized to match the flowing time in the catheter, thereby facilitating minimally invasive tissue sealing. Because this method is easily operated, we envision Borax adjusted amidation-type hydrogel has a promising prospect in clinical application.

A reduced polydopamine nanoparticle-coupled sprayable PEG hydrogel adhesive with anti-infection activity for rapid wound sealing.

There is a growing demand to develop sprayable hydrogel adhesives with rapid-forming and antibacterial abilities to instantly seal open wounds and combat pathogen infection. Herein, we propose to design a polydopamine nanoparticle (PDA NP) coupled PEG hydrogel that can quickly solidify via an amidation reaction after spraying as well as tightly binding PDA NPs to deliver reactive oxygen species (ROS) and induce a photothermal effect for bactericidal activity, and provide a hydrophilic surface for antifouling activity. The molecular structure of the 4-arm-PEG-NHS precursor was regulated to increase its reactivity with 4-arm-PEG-NH2, which thus shortened the gelation time of the PEG adhesive to 1 s to allow a fast solidification after being sprayed. The PEG-NHS precursor also provided covalent binding with tissue and PDA NPs. The reduced PDA NPs have redox activity to convey electrons to oxygen to generate ROS (H2O2), thus endowing the hydrogel with ROS dependent antibacterial ability. Moreover, NIR irradiation can accelerate the ROS release because of the photothermal effect of PDA NPs. In vitro tests demonstrated that H2O2 and the NIR-photothermal effect synergistically induced a fast bacterial killing, and an in vivo anti-infection test also proved the effectiveness of PEG-PDA. The sprayable PEG-PDA hydrogel adhesive, with rapid-forming performance and a dual bactericidal mechanism, may be promising for sealing large-scale and acute wound sites or invisible bleeding sites, and protect them from pathogen infection.

Coupling PEG-LZM polymer networks with polyphenols yields suturable biohydrogels for tissue patching.

Poor mechanical performances severely limit the application of hydrogels in vivo; for example, it is difficult to perform a very common suturing operation on hydrogels during surgery. There is a growing demand to improve the mechanical properties of hydrogels for broadening their clinical applications. Natural polyphenols can match the potential toughening sites in our previously reported PEG-lysozyme (LZM) hydrogel because polyphenols have unique structural units including a hydroxyl group and an aromatic ring that can interact with PEG via hydrogen bonding and form hydrophobic interactions with LZM. By utilizing polyphenols as noncovalent crosslinkers, the resultant PEG-LZM-polyphenol hydrogel presents super toughness and high elasticity in comparison to pristine PEG-LZM with no obvious changes in the initial shape, and it can even withstand the high pressure from sutures. At the same time, the mechanical properties could be widely adjusted by varying the polyphenol concentration. Interestingly, the PEG-LZM-polyphenol hydrogel has a higher water content than other polyphenol-toughened hydrogels, which may better meet the clinical needs for hydrogel materials. Besides, the introduction of polyphenols endows the hydrogel with improved antibacterial and anti-inflammatory abilities. Finally, the PEG-LZM-polyphenol (tannic acid) hydrogel was demonstrated to successfully patch a rabbit myocardial defect by suturing for 4 weeks and improve the wound healing and heart function recovery compared to autologous muscle patches.

Redox-Channeling Polydopamine-Ferrocene (PDA-Fc) Coating To Confer Context-Dependent and Photothermal Antimicrobial Activities.

Microbial disinfection associated with medical device surfaces has been an increasing need, and surface modification strategies such as antibacterial coatings have gained great interest. Here, we report the development of polydopamine-ferrocene (PDA-Fc)-functionalized TiO2 nanorods (Ti-Nd-PDA-Fc) as a context-dependent antibacterial system on implant to combat bacterial infection and hinder biofilm formation. In this work, two synergistic antimicrobial mechanisms of the PDA-Fc coating are proposed. First, the PDA-Fc coating is redox-active and can be locally activated to release antibacterial reactive oxygen species (ROS), especially ·OH in response to the acidic microenvironment induced by bacteria colonization and host immune responses. The results demonstrate that redox-based antimicrobial activity of Ti-Nd-PDA-Fc offers antibacterial efficacy of over 95 and 92% against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), respectively. Second, the photothermal effect of PDA can enhance the antibacterial capability upon near-infrared (NIR) irradiation, with over 99% killing efficacy against MRSA and E. coli, and even suppress the formation of biofilm through both localized hyperthermia and enhanced ·OH generation. Additionally, Ti-Nd-PDA-Fc is biocompatible when tested with model pre-osteoblast MC-3T3 E1 cells and promotes cell adhesion and spreading presumably due to its nanotopographical features. The MRSA-infected wound model also indicates that Ti-Nd-PDA-Fc with NIR irradiation can effectively eliminate bacterial infection and suppress host inflammatory responses. We believe that this study demonstrates a simple means to create biocompatible redox-active coatings that confer context-dependent antibacterial activities to implant surfaces.

Tag-Free Site-Specific BMP-2 Immobilization with Long-Acting Bioactivities via a Simple Sugar-Lectin Interaction.

The construction of a biomaterial matrix with biological properties is of great importance to developing functional materials for clinical use. However, the site-specific immobilization of growth factors to endow materials with bioactivities has been a challenge to date. Considering the wide existence of glycosylation in mammalian proteins or recombinant proteins, we establish a bioaffinity-based protein immobilization strategy (bioanchoring method) utilizing the native sugar-lectin interaction between concanavalin A (Con A) and the oligosaccharide chain on glycosylated bone morphogenetic protein-2 (GBMP-2). The interaction realizes the site-specific immobilization of GBMP-2 to a substrate modified with Con A while preserving its bioactivity in a sustained and highly efficient way, as evidenced by its enhanced ability to induce osteodifferentiation compared with that of the soluble GBMP-2. Moreover, the surface with Con A-bioanchored GBMP-2 can be reused to stimulate multiple batches of C2C12 cells to differentiate almost to the same degree. Even after 4 month storage at 4 °C in phosphate-buffered saline (PBS), the Con A-bioanchored GBMP-2 still maintains the bioactivity to stimulate the differentiation of C2C12 cells. Furthermore, the ectopic ossification test proves the in vivo bioactivity of bioanchored GBMP-2. Overall, our results demonstrate that the tag-free and site (i.e., sugar chain)-specific protein immobilization strategy represents a simple and generic alternative, which is promising to apply for other glycoprotein immobilization and application. It should be noted that although the lectin we utilized can only bind to d-mannose/d-glucose, the diversity of the lectin family assures that a specific lectin could be offered for other sugar types, thus expanding the applicable scope further.

Role of polydopamine's redox-activity on its pro-oxidant, radical-scavenging, and antimicrobial activities.

Polydopamine (PDA) is a bioinspired material and coating that offers diverse functional activities (e.g., photothermal, antioxidant, and antimicrobial) for a broad range of applications. Although PDA is reported to be redox active, the association between PDA's redox state and its functional performance has been difficult to discern because of PDA's complex structure and limitations in methods to characterize redox-based functions. Here, we use an electrochemical reverse engineering approach to confirm that PDA is redox-active and can repeatedly accept and donate electrons. We observed that the electron-donating ability of PDA offers the detrimental pro-oxidant effect of donating electrons to O2 to generate reactive oxygen species (ROS) or, alternatively, the beneficial antioxidant effect of quenching oxidative free radicals. Importantly, PDA's electron-donating ability depends on its redox state and is strongly influenced by external factors including metal ion binding as well as near-infrared (NIR) irradiation. Furthermore, we demonstrated that PDA possesses redox state-dependent antimicrobial properties in vitro and in vivo. We envision that clarification of PDA's redox activity will enable better understanding of PDA's context-dependent properties (e.g., antioxidant and pro-oxidant) and provide new insights for further applications of PDA. STATEMENT OF SIGNIFICANCE: We believe this is the first report to characterize the redox activities of polydopamine (PDA) and to relate these redox activities to functional properties important for various proposed applications of PDA. We observed that polydopamine nanoparticles 1) are redox-active; 2) can repeatedly donate and accept electrons; 3) can accept electrons from reducing agents (e.g., ascorbate), donate electrons to O2 to generate ROS, and donate electrons to free radicals to quench them; 4) have redox state-dependent electron-donating abilities that are strongly influenced by metal ion binding as well as NIR irradiation; and 5) have redox state-dependent antimicrobial activities.

A PEG-Lysozyme hydrogel harvests multiple functions as a fit-to-shape tissue sealant for internal-use of body.

In situ formation of surgical sealants to stop internal fluids leakage is more attractive compared to the traditional suture or staple. However, commercial sealants have weak points in tissue adhesive, cell affinity, antibacterial etc., which make them remain suboptimal for internal use of body. It is required to develop multifunctional sealants that can meet clinical needs. Herein, a PEG-lysozyme (LZM) injectable sealant composed of 4-arm-PEG and lysozyme was developed. Lysozyme offers free amine groups to rapidly cross link with PEG. The hydrogel can tightly adhere to tissues and provide good mechanics to withstand high pressure. Moreover, lysozyme innately confers antibacterial and cell affinity on the hydrogel that are usually lacking in marketed sealants. The hydrogel is easily operated to seal gas or blood leakage in a rabbit trachea and artery defect. Moreover, it can close the transmural left ventricular wall defect on a beating heart. The traumatic organ functions completely recovered postoperatively. Considering the good biocompatibility and the simple fabrication process, the PEG-LZM hydrogel is promising to clinical transformation. More broadly, our work indicates that nature-occurring molecules are versatile building blocks for construction of materials and confer functions, which represents a simple tragedy to develop advanced functional biomaterials.

A turn-on near-infrared fluorescence probe with aggregation-induced emission based on dibenzo[a,c]phenazine for detection of superoxide anions and its application in cell imaging.

A new turn-on near-infrared fluorescence probe (BDP) based on dibenzo[a,c]phenazine for superoxide anion detection with aggregation-induced emission properties as well as a desirable large Stokes shift was designed and synthesized. After BDP reacted with superoxide, the initial diphenyl-phosphinyl groups of BDP were cleaved, resulting in the production of the pyridinium modified fluorophore (BD) with near-infrared emission. The fluorescent sensor BDP has a high selectivity for superoxide anions over some other intracellular ROSs, reductants, metal ions and amino acids. When HepG2 cells undergo apoptosis and inflammation, BDP is a good probe to keep track of the endogenous superoxide anion level by confocal laser scanning microscopic imaging.

Targetable N-annulated perylene-based colorimetric and ratiometric near-infrared fluorescent probes for the selective detection of hydrogen sulfide in mitochondria, lysosomes, and serum.

Hydrogen sulfide (H2S) serves an effective role in biological systems as the acknowledged third endogenous gasotransmitter, so it makes great sense to detect and analyze H2S sensitively and quantitatively in subcellular environments, such as in mitochondria and lysosomes where H2S is widespread and functions as the mediator. Considering the excellent photophysical properties and multiple modification sites, N-annulated perylene (NP) was firstly chosen as the fluorophore to design a series of colorimetric and ratiometric near-infrared (NIR) fluorescent probes for the sensitive and selective detection of H2S. The probes showed near-infrared fluorescence at 681 nm in the absence of H2S. But with the addition of H2S, the NIR fluorescence decreased sharply and a new fluorescence peak at approximately 481 nm dramatically increased in a short response time, which could be clearly observed using the naked eye. Their large ratiometric fluorescence changes (about 200 nm), excellent selectivity and stability would be helpful for its detection in biological systems, and the limit of detection of the probe was calculated down to 139 nM. The reaction mechanism was studied as well. The targetable probes (Mito-NPNM and Lyso-NPNM) were also successfully employed to detect endogenous H2S in the mitochondria and lysosomes of living cells respectively. Besides, these probes were successfully applied to quantify H2S at low concentrations in serum where H2S levels are of great significance as an important indicator of various diseases.

Self-assembly of dual drug-delivery coating for synergistic bone regeneration.

Bone regeneration for the treatment of bone diseases represents a major clinical need. Introducing recombinant human bone morphogenetic protein-2 (rhBMP-2) into biomaterials is an extensively used approach to induce osteogenic differentiation and accelerate bone regeneration. However, serious adverse events can occur in the event of an overdose of rhBMP-2. Dexamethasone (DEX) is a synthetic hydrophobic glucocorticoid, which can enhance rhBMP-2-induced osteogenic differentiation by binding to a glucocorticoid receptor intracellularly. In this study, we have developed a multilayered composite coating made of poly(l-lactide-co-glycolide) (PLGA) nanoparticles, heparin and chitosan to deliver DEX and rhBMP-2 dually. The coating can reserve DEX and rhBMP-2 using the building blocks of the PLGA nanoparticles and heparin. Sustained release of DEX and rhBMP-2 by this coating was achieved. Moreover, a flow cytometry assay suggests that the PLGA nanoparticles could be transported across the cell membrane and presumably could improve the intracellular delivery of DEX via cell internalization. The in vitro osteogenesis studies reveal that the dual drug-loaded coating has a synergistic osteogenic differentiation effect on C2C12 myoblasts, as indicated by the upregulation of the alkaline phosphatise activity and osteo-related gene expression. In addition, µCT and histological analysis of the in vivo experiments demonstrate that the dual drug-loaded coating induced more ectopic bone formation than the individual drug-loaded coating. Therefore, this study demonstrates that our coating system can reserve these two drugs and deliver them locally to cells with the ability to induce rapid osteogenic differentiation and bone regeneration synergistically. Compared to other reported DEX/rhBMP-2 delivery systems, our coating system represents a simple, safe and effective dual drug delivery alternative. Moreover, since a layer-by-layer strategy is easily applied onto varying substrates, our coating system can be combined with many commercially available or existing biomaterials to improve their osteogenetic performance.