Interplay between viscoelastic and chemical tunings in fatty-acid-based polyester adhesives: engineering biomass toward functionalized step-growth polymers and soft networks

This Article describes the synthesis and characterization of renewable self-adhesive coatings with tunable viscoelastic properties and equipped with well-defined amounts of carboxylic acid "sticker" groups with adhesion promoting characteristics. Hydroxyl-ended polyesters with various architectures (linear, branched) were synthesized by melt polycondensation of dimerized fatty acids and fatty diols and then cured with maleic anhydride-modified triglycerides (such as maleinized soybean oil) in the presence of the amidine catalyst 1,8-diazabicyclo[5.4.0]undec-7-ene. The curing reaction of alcoholysis has the dual effect of chain extending/cross-linking the base polymers via creation of polymeric half-esters linkages while introducing carboxylic acid functions within the gel structure. We demonstrated how the adhesion properties can be finely tuned from molecular design and formulation of the network precursors and how the rheology and functionality of the coatings influence the adhesive bond formation and development. These renewable polyester adhesives proved to be suitable materials for pressure-sensitive adhesives applications with respect to adhesion strength, viscoelasticity, and functionality. In addition, the environmental benefits of such materials are briefly discussed.     

Catechol-functionalized chitosan/pluronic hydrogels for tissue adhesives and hemostatic materials

Bioinspired from adhesion behaviors of mussels, injectable and thermosensitive chitosan/Pluronic composite hydrogels were synthesized for tissue adhesives and hemostatic materials. Chitosan conjugated with multiple catechol groups in the backbone was cross-linked with terminally thiolated Pluronic F-127 triblock copolymer to produce temperature-sensitive and adhesive sol-gel transition hydrogels. A blend mixture of the catechol-conjugated chitosan and the thiolated Pluronic F-127 was a viscous solution state at room temperature but became a cross-linked gel state with instantaneous solidification at the body temperature and physiological pH. The adhesive chitosan/Pluronic injectable hydrogels with remnant catechol groups showed strong adhesiveness to soft tissues and mucous layers and also demonstrated superior hemostatic properties. These chitosan/Pluronic hydrogels are expected to be usefully exploited for injectable drug delivery depots, tissue engineering hydrogels, tissue adhesives, and antibleeding materials.     

Marine adhesive proteins: natural composite thermosets

Marine environments are severely challenging for the performance and durability of synthetic adhesives. Factors commonly associated with adhesive failure are weak boundary layers (water, oxides), adhesive erosion and swelling. For many permanently attached marine organisms such as barnacles, mussels, oysters, etc., however, underwater adhesion is 'business-as-usual'. Knowledge about the chemistry and bioprocessing of these marine adhesives will provide profound insights for the evolution of a new generation of environmentally safe, water-resistant adhesives. Despite their apparent structural diversity, marine adhesives are essentially analogous to composite thermosets, that is, the adhesive consists of fibre, filler and catalyst molecules that are dispersed in a cross-linked resin rendering it resistant to heat and solvents. The fibres and fillers in these composites are variable. e.g. collagen, fibroin, chitin present as fibres, and sand, shell, air and water present as fillers. The precured resins of seven organisms including members of the Mollusca, Annelida, and Platyhelminthes have now been isolated and partially sequenced. These are proteins with basic isoelectric points, high levels of the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA), and an extended, flexible conformation. The DOPA functional group in particular is thought to play a key role in (a) the chemisorption of these polymers to surface underwater, and (b) covalent cross-linking or setting of the adhesive, the latter reaction catalysed by the enzyme catecholoxidase. Much more needs to be done to explore the details of the adhesive processing and delivery strategies used by these organisms.     

Environmental post-processing increases the adhesion strength of mussel byssus adhesive

Marine mussels (Mytilus trossulus) attach to a wide variety of surfaces underwater using a protein adhesive that is cured by the surrounding seawater environment. In this study, the influence of environmental post-processing on adhesion strength was investigated by aging adhesive plaques in a range of seawater pH conditions. Plaques took 8–12 days to achieve full strength at pH 8, nearly doubling in adhesion strength (+94%) and increasing the work required to dislodge (+59%). Holding plaques in low pH conditions prevented strengthening, causing the material to tear more frequently under tension. The timescale of strengthening is consistent with the conversion of DOPA to DOPA-quinone, a pH dependent process that promotes cross-linking between adhesive proteins. The precise arrangement of DOPA containing proteins away from the adhesive-substratum interface emphasizes the role that structural organization can have on function, an insight that could lead to the design of better synthetic adhesives and metal-coordinating hydrogels.     

Surface-enhanced Raman spectroscopy of DOPA-containing peptides related to adhesive protein of marine mussel, Mytilus edulis

The common blue marine mussel adheres to underwater surfaces using an adhesive protein (Mefp-1) extruded from its foot. This highly hydroxylated protein contains a number of unusual amino acids, including 3,4-dihydroxyphenylalanine (DOPA), which is thought to contribute to the crosslinking of the extruded threads and adhesion to the substratum. Mefp-1 adheres to a wide variety of surfaces and is ultimately biodegradable. In this study we use surface-enhanced Raman spectroscopy (SERS) to characterize the adsorption of DOPA-containing peptides on colloidal gold. The peptides are simplified fragments of the Mefp-1 consensus decapeptide repeat, Ala-Lys-Pro-Ser-Tyr-DHP-Hyp-Thr-DOPA-Lys. Our results show that the peptides TDeltaKA, PTDeltaKA, and PPTDeltaKA (where Delta represents DOPA) coordinate to the gold surface through the catechol oxygens of the DOPA residue and through primary amine groups. The diproline sequence introduces conformational constraints that influence the conformations of the adsorbed peptides. These findings lay the groundwork for developing synthetic adhesives for underwater and medical applications.     

Synthesis and gelation of DOPA-modified poly(ethylene glycol) hydrogels

3,4-Dihydroxyphenylalanine (DOPA) residues are known for their ability to impart adhesive and curing properties to mussel adhesive proteins. In this paper, we report the preparation of linear and branched DOPA-modified poly(ethylene glycol)s (PEG-DOPAs) containing one to four DOPA endgroups. Gel permeation chromatography-multiple-angle laser light scattering analysis of methoxy-PEG-DOPA in the presence of oxidizing reagents (sodium periodate, horseradish peroxidase, and mushroom tyrosinase) revealed the formation of oligomers of methoxy-PEG-DOPA, presumably resulting from oxidative polymerization of DOPA endgroups. In the case of PEG-DOPAs containing two or more DOPA endgroups, oxidative polymerization resulted in polymer network formation and rapid gelation. The amount of time required for gelation of aqueous PEG-DOPA solutions was found to be as little as 1 min and was dependent on the polymer architecture as well as the type and concentration of oxidizing reagent used. Analysis of reaction mixtures by UV-vis spectroscopy allowed the identification of reaction intermediates and the elucidation of reaction pathways. On the basis of the observed reaction intermediates, oxidation of the catechol side chain of DOPA resulted in the formation of highly reactive DOPA-quinone, which further reacted to form cross-linked products via one of several pathways, depending on the presence or absence of N-terminal protecting groups on the PEG-DOPA. N-Boc protected PEG-DOPA cross-linked via phenol coupling and quinone methide tanning pathways, whereas PEG-DOPA containing a free amino group cross-linked via a pathway that resembled melanogenesis. Similar differences were observed for the rate of gel formation as well as the molecular weight between cross-links ((-)M(c)), calculated using equilibrium swelling and the Flory-Rehner equation.     

Stickiness--some fundamentals of adhesion

We review some adhesion mechanisms that have been understoodin the field of synthetic adhesives, and more precisely foradhesives that adhere instantaneously (a property named tackiness)and whose adhesive strength usually depends on the applied pressure(pressure-sensitive adhesives). The discussion includes effectsof surface roughness, elasticity, cavitation, viscous and elasticfingering, substrate flexibility.     

3,4-dihydroxy-L-phenylalanine as a cell adhesion molecule in serum-free cell culture

In this article, we examined the feasibility of using 3,4‐dihydroxy‐L ‐phenylalanine (DOPA) as a cell adhesion molecule in serum‐free cultures of anchorage‐dependent mammalian cells. DOPA is a critical, functional element in mussel adhesive proteins and is known to bind strongly to various natural or synthetic materials. DOPA coating on culture plates was confirmed using X‐ray photoelectron spectroscopy and energy‐dispersive spectroscopy. Human dermal fibroblasts (HDFs) were cultured on DOPA‐coated, fibronectin‐coated, or no material‐coated culture plates in serum‐free medium. HDFs cultured on DOPA showed the highest cell adhesion ratio, spreading, and viability but the lowest apoptotic activity. Therefore, DOPA may be a useful cell‐adhesion molecule for serum‐free culture. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1055–1060, 2012     

Rheological study of genipin cross-linked chitosan hydrogels

This paper reports the rheological behavior of chitosan solutions that have been cross-linked with different amounts of genipin, at body temperature and physiological pH. The effect of the cross-linker loading on the rheological properties of hydrogels has been evaluated. The oscillatory time sweep method was used to monitor the dynamic viscoelastic parameters during in situ (i.e., in the rheometer) gelation experiments, enabling the determination of the gelation time. The stress and frequency sweeps were employed to measure G' of the cured hydrogels. It was found that the solutions of chitosan cross-linked with genipin, under physiological conditions, could form relatively strong elastic gels when compared to those of pure chitosan. Moreover, the gelation time obtained from the crossover of G' and G' was in excellent agreement with the value obtained from the Winter-Chambon criterion. A significant reduction on this parameter was achieved even at low genipin concentrations. This behavior suggests that these formulations are able to be produced in situ and thus constitute promising matrices for cells and bioactive molecule encapsulations.     

厚壳贻贝足丝黏附蛋白mfp-3的重组表达及黏附功能分析

厚壳贻贝（Mytilus coruscus）中富含各种黏附蛋白分子,其中贻贝足丝蛋白3（mussel foot protein-3, mfp-3）是贻贝用以与外界基质进行黏附的主要蛋白分子.贻贝足丝中天然的mfp-3的含量低,水溶性差,因此纯化困难.本文以厚壳贻贝足丝蛋白mfp-3的cDNA序列为目的基因,用PCR法扩增Mfp-3基因,并成功构建含有多聚组氨酸标签的重组mfp-3原核表达载体pET-21a/ Mfp-3.经IPTG（isopropylthio-β-D-galactoside）诱导表达出重组蛋白,利用亲和层析和反相高效液相色谱分离纯化,获得分子量为9.18 kD的重组蛋白.经酪氨酸酶催化、玻璃包被和石英晶体微天平（quartz crystal microbalance,QCM）分析.结果表明,重组厚壳贻贝mfp-3蛋白经酪氨酸酶催化后,L-3,4-二羟基苯丙氨酸(即多巴,L-3,4- dihydroxyphenylalanine, DOPA) 含量较高并且具有较好的黏附性能.上述研究为开发以mfp-3黏附蛋白为来源的生物粘合剂奠定了良好的基础.     

Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking

Poly(L-lysine)/hyaluronan (PLL/HA) films were chemically cross-linked with a water soluble carbodiimide (EDC) in combination with a N-hydroxysulfo-succinimide (NHS) to induce amide formation. Fourier transform infrared spectroscopy confirms the conversion of carboxylate and ammonium groups into amide bonds. Quartz crystal microbalance-dissipation reveals that the cross linking reaction is accompanied by a change in the viscoelastic properties of the films leading to more rigid films. After the cross-linking reaction, both positively and negatively ending films exhibit a negative zeta potential. It is shown by fluorescence recovery after photobleaching measured by confocal laser scanning microscopy that cross-linking dramatically reduces the diffusion of the PLL chains in the network. Cross linking also renders the films highly resistant to hyaluronidase, an enzyme that naturally degrades hyaluronan. Finally, the adhesion of chondrosarcoma cells on the films terminating either with PLL or HA is also investigated. Whereas the non cross-linked films are highly resistant to cell adhesion, the cells adhere and spread well on the cross-linked films.     

Mechanisms of temporary adhesion in benthic animals

Adhesive systems are ubiquitous in benthic animals and play a key role in diverse functions such as locomotion, food capture, mating, burrow building, and defence. For benthic animals that release adhesives, surface and material properties and external morphology have received little attention compared to the biochemical content of the adhesives. We address temporary adhesion of benthic animals from the following three structural levels: (a) the biochemical content of the adhesive secretions, (b) the micro‐ and mesoscopic surface geometry and material properties of the adhesive organs, and (c) the macroscopic external morphology of the adhesive organs. We show that temporary adhesion of benthic animals is affected by three structural levels: the adhesive secretions provide binding to the substratum at a molecular scale, whereas surface geometry and external morphology increase the contact area with the irregular and unpredictable profile of the substratum from micro‐ to macroscales. The biochemical content of the adhesive secretions differs between abiotic and biotic substrata. The biochemistry of the adhesives suitable for biotic substrata differentiates further according to whether adhesion must be activated quickly (e.g. as a defensive mechanism) or more slowly (e.g. during adhesion of parasites). De‐adhesion is controlled by additional secretions, enzymes, or mechanically. Due to deformability, the adhesive organs achieve intimate contact by adapting their surface profile to the roughness of the substratum. Surface projections, namely cilia, cuticular villi, papillae, and papulae increase the contact area or penetrate through the secreted adhesive to provide direct contact with the substratum. We expect that the same three structural levels investigated here will also affect the performance of artificial adhesive systems.     

Smart hydrogels for in situ generated implants

The objective of this study was to explore the use of reverse thermo-responsive (RTG) polymers for generating implants at their site of performance, following minimally invasive surgical procedures. Aiming at combining syringability and enhanced mechanical properties, a new family of injectable RTG-displaying polymers that exhibit improved mechanical properties was created, following two different strategies: (1) to synthesize high-molecular-weight polymers by covalenty joining poly(ethylene glycol) and poly(propylene glycol) chains using phosgene as the coupling molecule and (2) to cross-link poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblocks after end-capping them with triethoxysilane or methacrylate reactive groups. While the methacrylates cross-linked rapidly, the triethoxysilane groups enabled the system to cross-link gradually over time. The chain-extended PEO/PPO copolymers had molecular weights in the 39 000-54 000 interval and exhibited improved mechanical properties. Reverse thermo-responsive systems displaying gradually increasing mechanical properties were generated by cross-linking triethoxysilane-capped (EO)(99)-(PO)(67)-(EO)(99) (F127) triblocks. Over time, the ethoxysilane groups hydrolyzed and created silanol moieties that subsequently condensated. With the aim of further improving their mechanical behavior, F127 triblocks were reacted with methacryloyl chloride and the resulting dimethacrylate was subsequently cross-linked in an aqueous solution at 37 degrees C. The effect of the concentration of the F127 dimethacrylate on the mechanical properties and the porous structure of the cross-linked matrixes produced was assessed. Rheometric studies revealed that the cross-linked hydrogels attained remarkable mechanical properties and allowed the engineering of robust macroscopic constructs, such as large tubular structures. The microporosity of the matrixes produced was studied by scanning electron microscopy. Monolayered conduits as well as structures comprising two and three layers were engineered in vitro, and their compliance and burst strength were determined.     

Effect of dimethyladipimidate and dimethylsuberimidate on cell-cell adhesion in rat fibroblasts

In a study performed to identify the molecular mechanisms which regulate cell to cell adhesion and contact inhibition in neoplastic and syngeneic normal cells of the rat we have observed that the adhesive capacity depends on the reagents used, either EDTA or trypsin, to release the cells from monolayer. Taking profit of this last property and of the possibility of blocking free -NH2 groups on membrane proteins with specific cross-linking reagents "in vitro", we have studied in this work the behaviour of the proteins of the cell coat involved in cell to cell adhesion of rat fibroblasts FG/2. The cross-linking reagents used were dimethyladipimidate (DMA) and dimethylsuberimidate (DMS). The cells were exposed to the reagents at 0 degrees C for 30'. Cell to cell adhesion was measured by determining the percentage of single cells labeled with 3H-leucine, adhering to a confluent monolayer at different incubation times. The inhibitory effect on cell to cell adhesion brought about by cross-linking reagents indicates that a) EDTA-released cells are more sensitive to both imides than those released with trypsin, b) DMA is more effective on trypsin-released cells and c) DMS is more effective on EDTA-released cells. Therefore, we conclude that the inhibition of adhesion by reaction with the two cross-linking reagents is more likely due to a stiffening of the molecules of the cell coat involved in the adhesion, rather than to the modification of -NH2 residues which should specifically participate to adhesive process.     

The effect of filament shortening on the mechanical properties of gel-filtered actin

To address the claim that filaments polymerized from highly purified (gel-filtered) F-actin acquire the elastic properties of a solid attributable to chemical cross-linking, we measured the rheologic spectrum of the dynamic storage modulus, G', and loss modulus, G' from 5 x 10(-4) to 0.5 Hz for gel-filtered actin alone and in the presence of the actin shortening protein, gelsolin. We confirmed that gel-filtered filamentous actin is a highly elastic material as evidenced by a relatively frequency-independent G', which is consistent with either topologically constrained filaments or a chemically cross-linked gel. Introduction of gel-filtered actin oligomers, however, caused the behavior of gel-filtered actin to become more frequency-dependent and almost identical to that of non-gel-filtered actin, suggesting that the effect of gel filtration on the mechanical behavior of actin is topologic. This conclusion is further supported by the finding that shortening of the actin filaments by the addition of gelsolin at molar ratios to actin of from 1:8000 to 1:500 causes a gradual decrease in elasticity and increase in the amount of flow.     

The phylogeny and chemical diversity of quinone-tanned glues and varnishes

1. 3,4-Dihydroxyphenyl-L-alanine (DOPA)-containing proteins are widely distributed throughout the animal kingdom and appear to serve chiefly as waterproof adhesives and varnishes. 2. The unique chemical and physical stability of these adhesives and varnishes is imparted by quinone-tanning, an oxidative process that leads to the polymerization of DOPA-containing and other proteins. 3. Recent advances in the biochemistry of DOPA-containing proteins suggest that most consist of tandemly repeated sequence motifs. Each motif contains DOPA, a basic amino acid (usually lysine), and abundant glycine or proline. 4. The DOPA residues undergo catechol oxidase-catalyzed conversion to o-quinones at the onset of quinone-tanning. 5. The complexity of quinone chemistry is discussed with regard to quinone-tanning.     

Polypeptide cross-linking in chloroplast membranes

Washed chloroplast membranes from romaine lettuce leaves were treated with the cross-linking reagent dimethyladipimidate (DMA) for various periods of time and subsequently analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparative examination of the electrophoretic profiles from control and treated membranes revealed that the light-harvesting chlorophyll-protein complex (LHCPC) was readily cross-linked to yield “dimers” and “oligomers” of higher molecular weight. Two polypeptides, of 25 and 23 kilodaltons, previously identified as two subunits of the LHCPC, were the major cross-linked species; other peptides were also cross-linked, but to a much lesser extent. These results suggest that cross-linking of chloroplast membranes with DMA, under our conditions, occurs primarily among the components of the LHCPC. We also measured the photosystem II activity in control and DMA-treated chloroplasts and found no impairment of this photochemical activity in the cross-linked chloroplasts as compared with controls.     

Cytochemical Localization of Polyphenol Oxidase Activity in K2‐Bodies of Saprolegnia ferax Secondary Zoospores

Zoospores of the oomycete Saprolegnia ferax release adhesive material from K‐bodies at the onset of attachment to substrates. To understand more fully how K‐bodies function in adhesion, enzyme activity was investigated cytochemically in secondary zoospores. Presence of catalase, a marker enzyme for microbodies, was explored in the diaminobenzidine (DAB) reaction. Although pH 9.2 DAB‐staining characteristic of catalase activity was detected in the granular matrix regions of K‐bodies, reaction controls indicated that the reaction was due to oxidative enzyme activity other than catalase. Because polyphenol oxidase (PPO) is another metal‐containing enzyme capable of oxidizing DAB, activity of this enzyme was tested with a more specific substrate, dihydroxyphenylalanine (DOPA). In the DOPA procedure, reaction product was exclusively localized within K‐bodies, indicating the presence of PPO. Results with three methods of reaction controls (elimination of substrate, addition of a PPO enzyme inhibitor, and heat‐inactivation of enzymes) all supported the presence of PPO in K‐bodies. This study highlights potential roles for K‐body PPO in stabilization of adhesion bodies by: cross‐linking matrix phenolic proteins or glycoproteins as K‐bodies discharge adhesives onto substrates, or polymerizing phenolics protective against microbial attacks of the adhesion pad.     

Self-assembly and adhesion of DOPA-modified methacrylic triblock hydrogels

Marine mussels anchor to a variety of surfaces by secreting liquid proteins that harden and form water-resistant bonds to a variety of surfaces. Studies have revealed that these mussel adhesive proteins contain an unusual amino acid, 3,4-dihydroxy-L-phenylalanine (DOPA), which is believed to be responsible for the cohesive and adhesive properties of these proteins. To separate the cohesive and adhesive roles of DOPA, we incorporated DOPA into the midblock of poly(methyl methacrylate)-poly(methacrylic acid)-poly(methyl methacrylate) (PMMA-PMAA-PMMA) triblock copolymers. Self-assembled hydrogels were obtained by exposing triblock copolymer solutions in dimethyl sulfoxide to water vapor. As water diffused into the solution, the hydrophobic end blocks formed aggregates that were bridged by the water-soluble midblocks. Strong hydrogels were formed with polymer weight fractions between 0.01 and 0.4 and with shear moduli between 1 and 5 kPa. The adhesive properties of the hydrogels on TiO2 surfaces were investigated by indentation with a flat-ended cylindrical punch. At pH values of 6 and 7.4, the fully protonated DOPA groups were highly adhesive to the TiO2 surfaces, giving values of approximately equal to 2 J/m2 for the interfacial fracture energy, which we believe corresponds to the cohesive fracture energy of the hydrogel. At these pH values, the DOPA groups are hydrophobic and have a tendency to aggregate, so contact times of 10 or 20 min are required for these high values of the interfacial strength to be observed. At a pH of 10, the DOPA groups were hydrophilic and highly swellable, but less adhesive gels were formed. Oxidation of DOPA groups, a process that is greatly accelerated at a pH of 10, decreased the adhesive performance of the hydrogels even further.     

Novel Proteins Identified in the Insoluble Byssal Matrix of the Freshwater Zebra Mussel

The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion.