Antimicrobial Property of Halogenated Catechols.

Bacterial infection associated with multidrug resistance (MDR) bacteria is increasingly becoming a significant public health risk. Herein, we synthesized a series of halogenated dopamine methacrylamide (DMA), which contains a catechol side chain modified with either chloro-, bromo-, or iodo-functional group. Catechol is a widely used adhesive moiety for designing bioadhesives and coating. However, the intrinsic antimicrobial property of catechol has not been demonstrated before. These halogenated DMA were incorporated into hydrogels, copolymers, and coatings and exhibited more than 99% killing efficiencies against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. More importantly, hydrogel containing chlorinated DMA demonstrated broad-spectrum antimicrobial activities towards multiple MDR bacteria, which included methicillin resistant S. aureus (MRSA), vancomycin resistant enterococci (VRE), multi antibiotics resistant Pseudomonas aeruginosa (PAER), multi antibiotics resistant Acinetobacter baumannii (AB) and carbapenem resistant Klebsiella pneumoniae (CRKP). These hydrogels also demonstrated the ability to kill bacteria in a biofilm while exhibiting low cytotoxic. Based on molecular docking and molecular dynamics simulation, Cl-functionalized catechol can potentially inhibit bacterial fatty acid synthesis at the enoyl-acyl carrier protein reductase (FabI) step. The combination of moisture-resistant adhesive property, inherent antimicrobial property, and the versatility of incorporating halogenated DMA into different polymeric materials greatly enhanced the potential for using these monomers for designing multifunctional bioadhesives and coatings.

Coacervation of Interfacial Adhesive Proteins for Initial Mussel Adhesion to a Wet Surface.

Coacervation of mussel adhesive proteins (MAPs) is proposed as a potential strategy that mussels may use during secretion due to their high concentration density, lack of dispersion into seawater, and low interfacial tension. Particularly, coacervations of interfacial MAPs, foot protein type-3 fast variant (fp-3F) and type-5 (fp-5), are important in the initial mussel adhesion process due to the relationship between the easy secretion/surface wetting properties of the coacervate and primer-like surface adhesive role of interfacial MAPs, which directly contact the marine surface. To the best of the authors' knowledge, this is the first report on coacervate formation of major recombinant interfacial MAPs with high charge densities and the highest 3,4-dihydroxyphenylalanine (Dopa) contents. Specifically, salt-induced coacervation of fp-3F is observed at low pH values corresponding to the acidified environment of the distal depression during mussel secretion. In addition, it shows enthalpy driven upper critical solution temperature behavior, possibly relying on bridging interactions between like-charged cationic fp-3Fs including salt-bridge and cation-π/π-π interactions in the presence of specific counterions, supported by Raman spectroscopy. It is believed that this study has broadened the scope of the understanding of coacervation of MAPs and may provide new insight for responsive biomaterial design.

Spatial distribution of proteins in the quagga mussel adhesive apparatus.

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious 'anchor' (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.

Green Synthesis of Silver Nanoparticles Stabilized with Mussel-Inspired Protein and Colorimetric Sensing of Lead(II) and Copper(II) Ions.

This articles reports a simple and green method for preparing uniform silver nanoparticles (AgNPs), for which self-polymerized 3,4-dihydroxy-l-phenylalanine (polyDOPA) is used as the reducing and stabilizing agent in aqueous media. The AgNPs functionalized by polyDOPA were analyzed by UV-Vis spectroscopy, high-resolution transmission electron microscopy (HR-TEM), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), Raman spectrophotometry, and X-ray diffraction (XRD) techniques. The results revealed that the polyDOPA-AgNPs with diameters of 25 nm were well dispersed due to the polyDOPA. It was noted that the polyDOPA-AgNPs showed selectivity for Pb2+ and Cu2+ detection with the detection limits for the two ions as low as 9.4 × 10-5 and 8.1 × 10-5 µM, respectively. Therefore, the polyDOPA-AgNPs can be applied to both Pb2+ and Cu2+ detection in real water samples. The proposed method will be useful for colorimetric detection of heavy metal ions in aqueous media.

In vivo residue-specific dopa-incorporated engineered mussel bioglue with enhanced adhesion and water resistance.

Misaminoacylation of 3,4-dihydroxyphenylalanine (Dopa) molecules to tRNA(Tyr) by endogenous tyrosyl-tRNA synthetase allowed the quantitative replacement of tyrosine residues with a yield of over 90 % by an in vivo residue-specific incorporation strategy, to create, for the first time, engineered mussel adhesive proteins (MAPs) in Escherichia coli with a very high Dopa content, close to that of natural MAPs. The Dopa-incorporated MAPs exhibited a superior surface adhesion and water resistance ability by assistance of Dopa-mediated interactions including the oxidative Dopa cross-linking, and furthermore, showed underwater adhesive properties comparable to those of natural MAPs. These results propose promising use of Dopa-incorporated engineered MAPs as bioglues or adhesive hydrogels for practical underwater applications.

Functional modification of mussel adhesive protein to control solubility and adhesion property.

Marine mussels produce strong underwater adhesives called mussel adhesive proteins (MAPs) that can adhere to a variety of surfaces under physiological conditions. Thus, MAPs have been investigated as a potentially sustainable alternative to conventional petrochemical-based adhesives. Recombinant MAPs would be promising for large-scale production and commercialization; however, MAPs are intrinsically adhesive, aggregative, and insoluble in water. In this study, we have developed a solubilization method for the control of MAP adhesion by fusion protein technique. Foot protein 1 (Fp1), a kind of MAP, was fused with the highly water-soluble protein, which is the C-terminal domain of ice-nucleation protein K (InaKC), separated by a protease cleaving site. The fusion protein exhibited low adhesion but high solubility and stability. Notably, Fp1 recovered its adhesive property after removal from the InaKC moiety by protease cleaving, which was evaluated and confirmed by the agglomeration of magnetite particles in water. The ability to control adhesion and agglomeration makes MAPs favorable prospects for bio-based adhesives.

Cell recognitive bioadhesive-based osteogenic barrier coating with localized delivery of bone morphogenetic protein-2 for accelerated guided bone regeneration.

Titanium mesh (Ti-mesh) for guided bone regeneration (GBR) approaches has been extensively considered to offer space maintenance in reconstructing the alveolar ridge within bone defects due to its superb mechanical properties and biocompatibility. However, soft tissue invasion across the pores of the Ti-mesh and intrinsically limited bioactivity of the titanium substrates often hinder satisfactory clinical outcomes in GBR treatments. Here, a cell recognitive osteogenic barrier coating was proposed using a bioengineered mussel adhesive protein (MAP) fused with Alg-Gly-Asp (RGD) peptide to achieve highly accelerated bone regeneration. The fusion bioadhesive MAP-RGD exhibited outstanding performance as a bioactive physical barrier that enabled effective cell occlusion and a prolonged, localized delivery of bone morphogenetic protein-2 (BMP-2). The MAP-RGD@BMP-2 coating promoted in vitro cellular behaviors and osteogenic commitments of mesenchymal stem cells (MSCs) via the synergistic crosstalk effects of the RGD peptide and BMP-2 in a surface-bound manner. The facile gluing of MAP-RGD@BMP-2 onto the Ti-mesh led to a distinguishable acceleration of the in vivo formation of new bone in terms of quantity and maturity in a rat calvarial defect. Hence, our protein-based cell recognitive osteogenic barrier coating can be an excellent therapeutic platform to improve the clinical predictability of GBR treatment.

Sutureless Transplantation of Amniotic Membrane Using a Visible Light-Curable Protein Bioadhesive for Ocular Surface Reconstruction.

The conjunctiva is a thin mucous membrane of the eye. Pterygium, a commonly appearing disease on the ocular surface, requires surgery to excise the conjunctiva to prevent visual deterioration. Recently, transplantation of the amniotic membrane (AM), which is the innermost membrane of the placenta, has been highlighted as an efficient method to cure conjunctiva defects because of its advantages of no side effects compared to mitomycin C treatment and not leaving additional scars on donor site compared to conjunctival autografting. However, to minimize additional damage to the ocular surface by suturing, AM transplantation (AMT) needs to be simplified by using a less invasive, time-saving method. In this work, a visible light-curable protein bioadhesive (named FixLight) for efficient sutureless AMT is applied. FixLight, which is based on bioengineered mussel adhesive protein (MAP), is easily applied between damaged ocular surfaces and transplanted AM, and rapidly cured by harmless blue light activation. Through in vivo evaluation using a rabbit model, the authors demonstrated that FixLight enabled facile, fast, and strong attachment of AM on sclera and promoted ocular surface reconstruction with good biocompatibility. Thus, FixLight can be successfully used as a promising clinical bioadhesive in opthalmological surgeries that require sutureless and rapid operation.

An injectable double cross-linked hydrogel adhesive inspired by synergistic effects of mussel foot proteins for biomedical application.

Hydrogel adhesives with high tissue adhesion, biodegradability and biocompatibility are benefit for promoting surgical procedures and minimizing the pain and post-surgical complications of patients. In this paper, an injectable mussel inspired double cross-linked hydrogel adhesive composed of thiolated mussel inspired chitosan (CSDS) and tetra-succinimidyl carbonate polyethylene glycol (PEG-4S) was designed and developed. CSDS was synthesized with thiol and catechol groups inspired by the synergistic effect of mussel foot proteins (mfps). The double cross-linked hydrogel was first formed by the addition of sodium periodate (or Fe3+) and then double cross-linked with PEG-4S. The results showed that the mechanical and adhesion properties of the double cross-linked hydrogels were significantly improved by the synergistic effects of the functional groups. And the prepared hydrogels showed good cytocompatibility which evaluated by determining the viability of L929 cells and human umbilical vein endothelial cells (HUVECs). Additionally, the biodegradability and biocompatibility in vivo were further confirmed by subcutaneous implantation in mice model, and the histological analysis results identified that the prepared hydrogels were in vivo biocompatible. This work presents an injectable mussel inspired double cross-linked hydrogels that can use as a potential hydrogel adhesive for biomedical application.

The position of lysine controls the catechol-mediated surface adhesion and cohesion in underwater mussel adhesion.

Intensive studies have found that 3,4-dihydroxyphenylalanine (Dopa) is one of the key molecules for underwater mussel adhesion. Although basic mechanisms of mussel adhesion have been elucidated, little is known about how mussels control the balance between surface adhesion and cohesion, which is critical for successful adhesion without peeling and/or tearing. In this work, we focused on lysine (Lys) molecules which are frequently flanked to Dopa residues in interfacial adhesive proteins, specifically their synergy and anti-synergy on surface adhesion and cohesion. Three model peptides were designed to characterize flanking Lys effects. Through nano-mechanistic analyses, we found that flanking Lys enhanced surface adhesion but disrupted Fe3+-mediated cohesion. Through nuclear magnetic resonance analyses and density functional theory calculations, we corroborated the synergetic effect on surface adhesion and anti-synergetic effect on cohesion. We also confirmed the consistency of flanking Lys effects in the actual protein system. Thus, we, for the first time, discovered that each Dopa molecule in interfacial adhesive proteins is participated in surface adhesion and cohesion differently through controlling the existence of flanking Lys. Our discovery enlightens how nature designs adhesive proteins through according roles of Dopa.

Comparison between Catechol- and Thiol-Based Adhesion Using Elastin-like Polypeptides.

Different chemistries have been utilized for adhesive materials to achieve adhesion in a humidified environment. l-3,4-dihydroxyphenylalanine (DOPA) found in marine mussel adhesive proteins has generated great interest because DOPA participates in multiple reaction mechanisms that confer the ability to adhere in wet conditions. However, the mussel adhesive complex also contains proteins with a relatively high thiol content, and these proteins can contribute to adhesion through the formation of disulfide bonds or interactions with DOPA. This work probes the individual contributions and interactions of DOPA and thiol chemistries to adhesion. To do so, we took advantage of the sequence flexibility in elastin-like polypeptides (ELPs) to create model proteins with highly similar sequences that are rich in either DOPA or thiol residues. The sequence similarity between the two ELP adhesives allowed us to focus on the differences between DOPA- and thiol-based adhesion. Curing kinetics in a wet setting, capability to recover from disturbance in the curing process, and cytocompatibility of the two adhesives were compared. Both chemistries resulted in cytocompatible materials. However, thiol chemistry had faster curing kinetics and higher adhesion strengths, whereas DOPA chemistry showed better recovery from disturbances during the curing process. By utilizing both DOPA- and thiol-based chemistry simultaneously and adding iron ions, we achieved fast curing kinetics, strong adhesion strengths, and good recovery from disturbances to curing. These insights into the contribution of these chemistries to adhesion provide important lessons for researchers designing adhesives that work in a humid environment.

Synthesis and characterization of incorporating mussel mimetic moieties into photoactive hydrogel adhesive.

Surgical adhesive is the optimal candidate for the replacement of traditional mechanical wound closure. In our paper, mussel adhesive proteins inspired hydrogel adhesive was prepared with 3, 4-dihydroxyphyenylanine acrylamide (DOPA-AA), poly (ethylene glycol) diacrylate (PEGDAA) and thiolated chitosan (CSS) by UV irradiation. DOPA-AA, containing catechol group and vinyl group, was successful synthesized and characterized by FTIR and 1HNMR. The gelation time, equilibrium water content, degradation, materials properties and adhesive strength of the hydrogels were studied. We found that the gelation time was retarded and the materials mechanical strength was decreased because of the inhibitory effect of catechol group. Equilibrium water content was slightly affect by the increasing concentration of DOPA-AA (1-5%). Nevertheless, catechol group can remarkably improve the adhesive properties because of the complex and durable interactions of the hydrogel, especially, the interaction between the thiol group of CSS and catechol group of DOPA-AA, which also greatly slowed down the degradation of the adhesive hydrogels. CSS and DOPA-AA was introduced to ensure the adhesive properties, DOPA-AA lend the adhesive nature to hydrogel and CSS can protect the catechol group from oxidation and enhance durable adhesion. Moreover, cytotoxicity of the resulting hydrogels showed that the L929 cell viability was weaken, it mostly probably induced by the catechol oxidation.

Sprayable Adhesive Nanotherapeutics: Mussel-Protein-Based Nanoparticles for Highly Efficient Locoregional Cancer Therapy.

Following surgical resection for primary treatment of solid tumors, systemic chemotherapy is commonly used to eliminate residual cancer cells to prevent tumor recurrence. However, its clinical outcome is often limited due to insufficient local accumulation and the systemic toxicity of anticancer drugs. Here, we propose a sprayable adhesive nanoparticle (NP)-based drug delivery system using a bioengineered mussel adhesive protein (MAP) for effective locoregional cancer therapy. The MAP NPs could be administered to target surfaces in a surface-independent manner through a simple and easy spray process by virtue of their unique adhesion ability and sufficient dispersion property. Doxorubicin (DOX)-loaded MAP NPs (MAP@DOX NPs) exhibited efficient cellular uptake, endolysosomal trafficking, and subsequent low pH microenvironment-induced DOX release in cancer cells. The locally sprayed MAP@DOX NPs showed a significant inhibition of tumor growth in vivo, resulting from the prolonged retention of the MAP@DOX NPs on the tumor surface. Thus, this adhesive MAP NP-based spray therapeutic system provides a promising approach for topical drug delivery in adjuvant cancer therapy.

Rapid in situ cross-linking of hydrogel adhesives based on thiol-grafted bio-inspired catechol-conjugated chitosan.

In this paper, a novel chitosan derivative, thiol-grafting bio-inspired catechol-conjugated chitosan was synthesized. The chemical structure of the synthesized catechol-conjugated chitosan was verified by 1H NMR, and its contents of thiol group and catechol group were determined by UV-vis spectrum. Four percent of catechol-conjugated chitosan aqueous solution could form hydrogels rapidly in situ in 1 min or less with the addition of sodium periodate. Rheological studies showed that the mechanical properties depend on the concentrations of catechol-conjugated chitosan and the molar ratio of sodium periodate to catechol groups. Additionally, the adhesive properties of the resulting adhesives were evaluated, and the adhesion strength of obtained adhesives was as high as 50 kPa because of the complex and multiple interactions, especially the anti-oxidation mechanism of thiol group. The in vitro cytotoxicity assays demonstrated an excellent biocompatibility of the catechol-conjugated chitosan hydrogels. Benefiting from the in situ fast cured, desired mechanical strength, biocompatibility and relatively high adhesion performance, these properties suggested that catechol-conjugated chitosan hydrogel adhesives have potential applications as tissue adhesive for soft tissues.

A bioinspired elastin-based protein for a cytocompatible underwater adhesive.

The development of adhesives that can be applied and create strong bonds underwater is a significant challenge for materials engineering. When the adhesive is intended for biomedical applications, further criteria, such as biocompatibility, must be met. Current biomedical adhesive technologies do not meet these needs. In response, we designed a bioinspired protein system that shows promise to achieve biocompatible underwater adhesion coupled with environmentally responsive behavior that is "smart" - that is, it can be tuned to suit a specific application. The material, ELY16, is constructed from an elastin-like polypeptide (ELP) that can be produced in high yields from Escherichia coli and can coacervate in response to environmental factors such as temperature, pH, and salinity. To confer wet adhesion, we utilized design principles from marine organisms such as mussels and sandcastle worms. When expressed, ELY16 is rich in tyrosine. Upon modification with the tyrosinase enzyme to form mELY16, the tyrosine residues are converted to 3,4-dihydroxyphenylalanine (DOPA). Both ELY16 and mELY16 exhibit cytocompatibility and significant dry adhesion strength (>2 MPa). Modification with DOPA increases protein adsorption to glass and provides moderate adhesion strength (∼240 kPa) in a highly humid environment. Furthermore, this ELP exhibits a tunable phase transition behavior that can be formulated to coacervate in physiological conditions and provides a convenient mechanism for application underwater. Finally, mELY16 possesses significantly higher adhesion strength in dry, humid, and underwater environments compared with a commercially available fibrin sealant. To our knowledge, mELY16 provides the strongest bonds of any rationally designed protein when used completely underwater, and its high yields make it more viable for commercial application compared to natural adhesive proteins. In conclusion, this ELP shows great potential to be a new "smart" underwater adhesive.

Diatom-Inspired Silica Nanostructure Coatings with Controllable Microroughness Using an Engineered Mussel Protein Glue to Accelerate Bone Growth on Titanium-Based Implants.

Silica nanoparticles (SiNPs) have been utilized to construct bioactive nanostructures comprising surface topographic features and bioactivity that enhances the activity of bone cells onto titanium-based implants. However, there have been no previous attempts to create microrough surfaces based on SiNP nanostructures even though microroughness is established as a characteristic that provides beneficial effects in improving the biomechanical interlocking of titanium implants. Herein, a protein-based SiNP coating is proposed as an osteopromotive surface functionalization approach to create microroughness on titanium implant surfaces. A bioengineered recombinant mussel adhesive protein fused with a silica-precipitating R5 peptide (R5-MAP) enables direct control of the microroughness of the surface through the multilayer assembly of SiNP nanostructures under mild conditions. The assembled SiNP nanostructure significantly enhances the in vitro osteogenic cellular behaviors of preosteoblasts in a roughness-dependent manner and promotes the in vivo bone tissue formation on a titanium implant within a calvarial defect site. Thus, the R5-MAP-based SiNP nanostructure assembly could be practically applied to accelerate bone-tissue growth to improve the stability and prolong the lifetime of medical implantable devices.

Mussel adhesion-employed water-immiscible fluid bioadhesive for urinary fistula sealing.

Urinary fistulas, abnormal openings of a urinary tract organ, are serious complications and conventional management strategies are not satisfactory. For more effective and non-invasive fistula repair, fluid tissue adhesives or sealants have been suggested. However, conventional products do not provide a suitable solution due to safety problems and poor underwater adhesion under physiological conditions. Herein, we proposed a unique water-immiscible mussel protein-based bioadhesive (WIMBA) exhibiting strong underwater adhesion which was employed by two adhesion strategies of marine organisms; 3,4-dihydroxy-l-phenylalanine (DOPA)-mediated strong adhesion and water-immiscible coacervation. The developed biocompatible WIMBA successfully sealed ex vivo urinary fistulas and provided good durability and high compliance. Thus, WIMBA could be used as a promising sealant for urinary fistula management with further expansion to diverse internal body applications.

Injectable dopamine-modified poly(ethylene glycol) nanocomposite hydrogel with enhanced adhesive property and bioactivity.

A synthetic mimic of mussel adhesive protein, dopamine-modified four-armed poly(ethylene glycol) (PEG-D4), was combined with a synthetic nanosilicate, Laponite (Na(0.7+)(Mg5.5Li0.3Si8)O20(OH)4)(0.7-)), to form an injectable naoncomposite tissue adhesive hydrogel. Incorporation of up to 2 wt % Laponite significantly reduced the cure time while enhancing the bulk mechanical and adhesive properties of the adhesive due to strong interfacial binding between dopamine and Laponite. The addition of Laponite did not alter the degradation rate and cytocompatibility of PEG-D4 adhesive. On the basis of subcutaneous implantation in rat, PEG-D4 nanocomposite hydrogels elicited minimal inflammatory response and exhibited an enhanced level of cellular infiltration as compared to Laponite-free samples. The addition of Laponite is potentially a simple and effective method for promoting bioactivity in a bioinert, synthetic PEG-based adhesive while simultaneously enhancing its mechanical and adhesive properties.