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Chitosan Based Water-Resistant Adhesive. Analogy to Mussel Glue Kazunori Yamada,† Tianhong Chen,‡ Guneet Kumar,| Oleg Vesnovsky,§ L. D. Timmie Topoleski,§ and Gregory F. Payne*,‡,| Department of Industrial Chemistry, College of Industrial Technology, Nihon University, 1-2-1 Izumi-cho, Narashino, Chiba 275-8575, Japan; Department of Chemical and Biochemical Engineering and Department of Mechanical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250; and Center for Agricultural Biotechnology, 5115 Plant Sciences Building, University of Maryland, College Park, Maryland 20742 Received February 7, 2000
Using analogies from nature, we investigated the possibility that tyrosinase-catalyzed reactions of 3,4dihydroxyphenethylamine (dopamine) could confer water-resistant adhesive properties to semidilute solutions of the polysaccharide chitosan. Rheological measurements showed that the tyrosinase-catalyzed, and subsequent uncatalyzed, reactions lead to substantial increases in the viscosity of the chitosan solutions. Samples from these high-viscosity modified-chitosans were spread onto dry glass slides, the slides were lapped and clipped together either in air or after being submerged in water, and the bound slides were held under water for several hours. Adhesive shear strengths of over 400 kPa were observed for these modified chitosan samples, while control chitosan solutions conferred no adhesive strength (i.e., the glass slides separated in the absence of measurable forces). High viscosities and water-resistant adhesive strengths were also observed when semidilute chitosan solutions were treated with the known cross-linking agent, glutaraldehyde. Further studies indicate a relationship between the increased viscosities and water-resistant adhesion. These results demonstrate that the renewable biopolymer chitosan can be converted into a waterresistant adhesive. Introduction Nature provides numerous examples of functional materials, and there is considerable interest in mimicking nature to generate high-performing, environmentally friendly materials. One of the best-studied examples is the water-resistant adhesive protein used by marine animals (e.g., mussels) to adhere to wet or submerged surfaces.1,2 The simplified schematic in Figure 1a shows that these animals are believed to use a polyphenolic adhesive protein. This protein consists of conserved decapeptide repeats that are rich in lysine, hydroxyproline, and dihydroxyphenylalanine (DOPA) residues.3 Commonly, it is believed that the animals use a catechol oxidase enzyme to convert these o-diphenolic residues to o-quinones that are reactive and undergo a variety of nonenzymatic reactions.2,4 The nonenzymatic reactions of o-quinone residues can lead to the cross-linking of secreted proteins and the formation of a protein gel5,6 that is believed to confer adhesive strength.7 Originally, it was believed that the o-quinone residues undergo cross-linking reactions with the -amino group of lysine residues. However, chemical * Corresponding author. Telephone: (301)-405-8389. Fax: (301)-3149075. E-mail:
[email protected]. † Nihon University. ‡ Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County. § Department of Mechanical Engineering, University of Maryland, Baltimore County. | University of Maryland, College Park.
characterization studies have been unable to detect such linkages,8 and more recent studies with synthetic proteins indicate that lysine residues are not required for gel formation.9 In fact, this latter study suggests that oxidation of DOPA residues is required for gel formation, but un-oxidized DOPA residues are required for water-resistant adhesion.9 Obviously, the complexity of quinone chemistry has confounded efforts to understand the mechanism of waterresistant adhesion.2 Relevant to the roles of phenols and quinones to adhesion, three additional facts are worth noting. First, phenolformaldehyde polymers are widely used for adhesives and these synthetic polymers have phenolic residues similar to those in the mussel’s polyphenolic adhesive protein. There has also been considerable study of plant polyphenols (e.g., tannins and lignins) as replacements for synthetic phenolic adhesives.10 Second, synthetic quinone-containing polymers are known to offer interesting adhesive properties.11 Finally, o-diphenols (e.g., DOPA residues) can undergo autoxidation reactions to o-quinones, making it difficult to ascertain whether a functional property results from the reduced or oxidized species. Despite the uncertainties in adhesion mechanism, there has been considerable effort to produce an adhesive protein for commercial purposes. Adhesive proteins have been extracted from mussels and studied for medical applications.12 However, it seems unlikely that extracted protein could be cost-
10.1021/bm0003009 CCC: $19.00 © 2000 American Chemical Society Published on Web 04/13/2000
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Figure 1. Schematic depictions of biological processes leading to (a) mussel glue adhesion and (b) cuticule sclerotization (hardening) and (c) the analogy used in this work to create a water-resistant adhesive from the polysaccharide chitosan.
effective for lower value adhesive applications. Biotechnological routes for mussel glue production have been investigated5,6 but these routes can be complicated by the need for posttranslational modifications to convert tyrosine to DOPA residues and possibly to convert proline to hydroxyproline residues. Synthetic methods for mussel glue formation have also been reported.13,14 Synthesis is complicated by the need to produce a high molecular weight protein (>100 kD) but is facilitated by the fact that the protein is composed of small repeating sequences. Irrespective of the production route, the sensitivity of the DOPA residues to oxidation can be problematic during processing. An interesting alternative approach for preparing adhesive proteins was reported by Kaleem et al.15 who chemically grafted phenolic moieties onto the protein gelatin to allow these modified proteins to undergo enzymatic oxidation and cross-linking. In our study, we are attempting to mimic the function of the adhesive protein using a different biological analogy and a different biopolymer. The biological analogy for our study is the process of cuticular sclerotization in insects (i.e., hardening of the insect shell). During sclerotization, Figure 1b shows that the enzyme tyrosinase is believed to oxidize low molecular weight sclerotizing precursors such as N-acetyldopamine.16-18 The o-quinones generated from this reaction undergo subsequent nonenzymatic cross-linking reactions with proteins and this “quinone tanning” yields a hardened outer integument. Waite19 suggested that terrestrial animals could use low molecular weight cross-linking compounds, while marine animals exploited cross-linking residues on high molecular weight compounds (e.g., proteins) to avoid the problem that secreted, water-soluble compounds can be readily lost in marine environments. Again, the complexity of quinone chemistry has precluded a full understanding of the sclerotization process. However, it appears that the most important cross-linking sites on the protein may
be histidine residues.20,21 Presumably, histidine residues are more reactive than the -amino group of lysine residues because the histidine residue has a lower pKa (6.0 for histidine versus 10.5 for lysine). By analogy to insect sclerotzation, Figure 1c shows that we are using tyrosinase to oxidize a low molecular weight compound, 3,4-dihydroxyphenethylamine (dopamine). Further, to facilitate quinone reactions, we are using a biopolymer that has amino residues with moderately low pKa’s. However, the biopolymer for our study is a polysaccharide and not a protein. Specifically, we are using the polysaccharide, chitosan, that has primary amino groups with pKa’s of about 6 to 6.5.22,23 In related work, Muzzarelli and coworkers24,25 chemically grafted phenolic residues onto chitosan and then used tyrosinase to “quinone-tan” their modified chitosan. These investigators provided evidence that their “quinone-tanned” chitosan had formed a cross-linked gel. In previous studies, we showed that tyrosinase can react with low molecular weight phenolic substrates to generate “quinone-tanned” chitosan and that these “quinone-tanned” chitosans also behave as gels.26 The goal of the present study is to determine whether “quinone-tanned” chitosans also offer water-resistant adhesive properties. Materials and Methods Chitosan, 3,4-dihydroxyphenethylamine (dopamine), and tyrosinase (EC1.14.18.1) from mushroom were obtained from Sigma Chemical Co. Tyrosinase was reported by the manufacturer to have a specific activity of 5350 U/mg. A chitosan solution of 1.6 w/v% was prepared by adding 1.6 g of chitosan to 100 mL of water and intermittently adding HCl to maintain the pH at 2-3. After the mixture was stirred for 24 h, undissolved material was removed from the chitosan solution by vacuum filtration. For individual experiments, the chitosan solution
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was diluted typically to 0.32 and 0.48 w/v% with deionized water. Assuming complete deacetylation, these chitosan concentrations correspond to molar concentrations of amino groups of 20 or 30 mM, respectively. Before conducting the enzymatic reaction, the pH of the chitosan solutions was adjusted to 5.8-6.0 using small amounts of 2 M NaOH solution. To initiate reaction, dopamine and tyrosinase were added to the chitosan solutions. As described in the text, the mixtures were allowed to react for varying times. In some cases, the steady shear viscosities of the reaction mixtures were measured using a Brookfield DV II+ viscometer with S25 and S34 spindles at a rotation speed of 1 rpm. To examine the adhesive properties of the reaction mixture, we selected glass microscope slides (Fisher Scientific) as the adherend. The glass slides were first cleaned by soaking for 24 h in a mixture of water, H2SO4, and K2CrO7 (10:5:1). After rinsing with deionized water, the glass slides were oven-dried at 60 °C. To study adhesion properties, approximately 50 mg of the reaction mixture was spread onto each face of two glass slides, and the faces were placed in contact with a 25 × 25 mm overlapping surface area. After the overlapping surfaces were pressed together, the samples were clipped together with two binder clips and immersed in water. The pair of clips applied a force of approximately 27 N to the sample. In this procedure, we observed that the majority of the reaction mixture applied to the slides was extruded when the surfaces were clipped. Weight measurements indicated that on the order of 6 mg of the reaction mixture remained between the slides after clipping the samples together, and this value could not be rigorously controlled. On the basis of the initial concentrations of material (chitosan and dopamine), we estimate that only about 30 µg of dry material remained between the glass slides. The adhesive layer thickness, as measured by a micrometer, was never observed to exceed 10 µm. In an alternative procedure for preparing samples, we spread the reaction mixture onto clean glass slides, immersed the slides in water, and then lapped and clipped the slides together while the slides were submerged. Shear strength was measured at room temperature with a hydraulic mechanical testing system (Bionix 858 Testing System, MTS, Minneapolis, MN). One end of the glass slide sample was attached to the load cell and the other end to the actuator of the mechanical testing system. The samples were loaded in tension until failure. The values of shear strength were calculated by dividing the force to separate the bonded glass slides by the overlapping surface area. Chemical analysis was performed using UV-visible spectrophotometry (Spectronic Genesys). Sample preparation and analytical procedures are briefly described in the text and further details have been reported eslewhere.26-28
Results and Discussion Chemical Analysis of “Quinone-Tanned” Chitosan. To provide chemical evidence for “quinone-tanning” we first used UV-visible spectrophotometry with reacting solutions. When 0.5 mM dopamine was reacted with tyrosinase, the solution was visually observed to change from colorless to a yellowish-red. Figure 2a shows changes in UV-visible absorbance of this solution. Initially the solution has absorbance characteristic of dopamine (λmax ) 280 nm) while a progressive increase in the absorbance at 300 and 475 nm is observed over time. These latter two peaks are characteristic of dopaminochromesan intramolecular cyclized product of the intermediate quinone.29,30 A definitive peak associated
Figure 2. Spectrophotometric evidence for “quinone-tanning” of chitosan: (a) UV-visible spectra for solutions containing dopamine (0.5 mM) and tyrosinase (10 U/mL); (b) UV-visible spectra for solutions containing dopamine (0.5 mM), chitosan (0.24 w/v %; 15 mM equivalent amino groups) and tyrosinase (10 U/mL); (c) UV-visible spectra for heterogeneously modified chitosan films (spectra e-h) prepared by incubating chitosan films with dopamine (6 mM) and tyrosinase (60 U/mL). Control chitosan films were incubated with either dopamine (spectra c) or tyrosinase (spectra d), while the control cellulose film (spectra b) was incubated with both dopamine and tyrosinase.
with the intermediate quinone, dopaminoquinone (λmax ) 390 nm), is not observed in Figure 2aspresumably due to its
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rapid conversion to dopaminochrome.29 The results in Figure 2a agree with those of previous researchers,29,30 who reported the following reaction sequence:
When chitosan (0.24 w/v %; 15 mM equivalent amino groups) was included in the reaction mixture with dopamine (0.5 mM) and tyrosinase, the solution was observed to change from colorless to a reddish-brown and ultimately to black. Figure 2b shows the UV-visible spectra of this solution during the course of the reaction. Initially, the UV-visible spectra is characteristic of dopamine. During the first couple hours of reaction, absorbance peaks at 300 and 475 nm are observed, suggesting some dopaminochrome formation. Upon further reaction, a broad absorbance peak is observed at about 360 nm and also broad absorbance is observed at wavelengths greater than 500 nm. The difference between parts a and b of Figure 2 suggests that the above reaction sequence is altered in the presence of chitosan. Presumably, chitosan’s amino groups can react with dopaminoquinone to form a Michael’s-type adduct in competition with the dopaminochrome-forming intramolecular reaction. The high chitosan concentrations used in this experiment and the low pKa of chitosan’s amino groups should favor this intermolecular reaction compared to the intramolecular dopaminochrome-forming reaction. The formation of Michael’s-type adducts between different quinones and amines has been reported to result in absorption peaks between 340 and 400 nm.11,13,31 This observation is consistent with our observed absorbance at 360 nm (Figure 2b) and suggests that dopaminoquinone may form a Michael’s-type adduct with chitosan. It is also possible that dopaminoquinone and/or dopaminochrome may undergo Schiff-base formation with chitosan’s amino groups. Additional support for a reaction between tyrosinasegenerated quinones and chitosan was provided by reacting dopamine with tyrosinase in the presence of chitosan and cellulose films. After reaction, these films were extensively washed, and their UV-visible spectra were measured as described previously.26-28 When chitosan films were incubated with both tyrosinase and dopamine, the films were observed to become brown. Spectra e through h of Figure 2c show that the UV-visible absorption of the modified chitosan films progressively increases with reaction time. Especially prominent in these spectra is the broad absorption peak between 360 and 380 nm. No browning or increase in UV-visible absorption was observed for control chitosan films incubated in the presence of either tyrosinase or dopamine (spectra c and d in Figure 2c). Also, spectra b of Figure 2c shows no increase in UV-visible absorption for a control cellulose film that was incubated with tyrosinase and dopamine. Cellulose is a useful control because the only structural difference between these polysaccharides is that cellulose has a hydroxyl group at the C-2 position of the repeating units while chitosan has amino groups at this position. The observed “tanning” for chitosan, but not
Figure 3. Increase in viscosity associated with the enzymatic conversion of dopamine and subsequent nonenzymatic reaction with chitosan. (a) Steady shear viscosities were measured for solutions containing various combinations of chitosan (0.32 w/v %; 20 mM equivalent amino groups), dopamine (10 mM), and tyrosinase (60 U/mL). (b) Steady shear viscosities were measured for solutions containing dopamine (10 mM), tyrosinase (60 U/mL), and varying levels of chitosan.
cellulose, suggests that chitosan’s amino groups are involved in the “tanning” reaction. In summary, the results in Figure 2 provide UV-visible evidence for the covalent modification of chitosan by tyrosinase and dopamine. The differences between chitosan and cellulose, and the observed absorption peak between 360 and 380 nm for the modified chitosan is consistent with a covalent modification through chitosan’s amino groups. Physical Analysis of “Quinone-Tanned” Chitosan. Initial studies were conducted to demonstrate that chitosan’s properties are altered by incubation with tyrosinase and dopamine. For this, we prepared semidilute solutions of chitosan (0.32 w/v%) and monitored viscosity as a function of incubation time. Figure 3a shows that the viscosity of a solution containing chitosan, dopamine, and tyrosinase increased beginning about 2 h after initiating the reaction. After 24 h of reaction the viscosity had increased 600-fold to 74 000 cP. No increase in viscosity was observed for
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Figure 4. Adhesive shear strength of various modified chitosan preparations. Reaction conditions for generating chitosan solutions/ gels are discussed in the text (CC ) chitosan concentration, CD ) dopamine concentration, CT ) tyrosinase activity, and t ) reaction time). After reaction, chitosan solutions/gels were spread onto clean, dry glass slides. These slides were either, lapped and clipped in air (designated “In air”), or submerged in water and lapped and clipped while submerged (designated “Under water”). All samples were held under water for 24 h after which they were removed from water, unclipped, and tested to determine the shear strength required to separate the glass slides. Mean and standard deviations for the shear strength measurements were obtained from four or more replicates except for the controls (reaction mixture lacking tyrosinase and the neutralized chitosan samples), which were obtained from three replicates.
controls containing chitosan and tyrosinase or controls containing dopamine and tyrosinase. For the control containing chitosan and dopamine, the viscosity was observed to increase after 20 hspresumably due to autoxidation of dopamine. Figure 3b shows that the viscosity increase is larger when higher concentrations of chitosan are reacted with tyrosinase and dopamine. Presumably, the high viscosities observed in Figure 3 are associated with the gelation of chitosan.26 Gelation could result if the dopamine reactions lead to the cross-linking of chitosan. The observation that dopamine-modified chitosan could not be dissolved under either acidic or basic conditions is consistent with a crosslinking of chitosan. To determine if tyrosinase-catalyzed and subsequent uncatalyzed reactions can confer water-resistant adhesive properties to chitosan, we performed the following experiment. Homogeneous solutions containing tyrosinase, dopamine, and chitosan were reacted for 24 h. After reaction, the material was spread onto two clean and dry glass slides. The glass slides were then lapped together, clipped, and immediately submerged in distilled water. After being submerged for 24 h, the glass slides were taken from the water, the clips were removed, and the shear force required to separate the glass slides was measured. The first entry in Figure 4 shows that 265 kPa of shear was required to separate the slides when the reaction mixture contained chitosan (0.32 w/v %), dopamine, and tyrosinase. For comparison, we performed studies with controls containing the following: chitosan; chitosan and tyrosinase; chitosan and dopamine; and tyrosinase and dopamine. In all these controls, the glass
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slides separated when the clips were removed, and thus these control formulations offered no adhesive strength under the conditions studied. If the control containing chitosan and dopamine (but not tyrosinase) was allowed to react for 72 (and not 24) h, 85 kPa of adhesive strength was observed as indicated in Figure 4. The adhesive strength in this control presumably results from the autoxidation of dopamine and subsequent reaction of the quinone with chitosan. To confirm the water-resistant adhesive property, we employed a second experimental procedure. In the second procedure, the reaction mixture was spread onto dry glass slides but the slides were submerged in water and then lapped and clipped together under water (as opposed to being lapped and clipped together in air). After the slides were submerged for 24 h, the shear force required to separate them was measured, and results from this procedure are shown as the darkened areas in Figure 4. Again, Figure 4 shows that when the reaction mixture contained chitosan (0.32 w/v%), dopamine, and tyrosinase, considerable shear (105 kPa) was required to separate the glass slides. As reported above, if the reaction mixture contained only two components (chitosan and tyrosinase; chitosan and dopamine; or tyrosinase and dopamine), the glass slides separated immediately after unclipping. The lack of adhesive strength for these controls is not surprising since these control reaction mixtures were dilute solutions which were likely to have been immediately washed from the glass slides upon submersion in water. If the reaction mixture containing chitosan and dopamine was incubated for 72 (and not 24) h, a gel-type material was formed and 70 kPa adhesive strength was observed as shown in Figure 4. Three additional results are shown in Figure 4. First, Figure 4 shows that adhesive strengths increased by 50% to over 400 kPa when chitosan concentrations were increased from 0.32 to 0.48 w/v%. Second, water-resistant adhesive characteristics could be obtained if chitosan was reacted with glutaraldehydesa better characterized cross-linking agent.32 Specifically, we incubated a 0.32% chitosan solution with a low level of glutaraldehyde (1 mM). After 24 h of reaction, we observed a high viscosity (86 000 cP) in the reaction mixture and measured substantial water-resistant adhesive strength (140-280 kPa depending on the experimental procedure). These results indicate that the tyrosinase reaction is not required to confer water-resistant adhesive behavior to semidilute chitosan solutions. Presumably, alternative procedures that yield chitosan gels can confer water-resistant adhesion. Finally, the last entries in Figure 4 show that chitosan gels formed by neutralization can yield waterresistant adhesive properties. Because of the difficulty of creating chitosan gels by neutralization of semidilute solutions, the procedures used in this experiment were somewhat different from those used for the other entries in Figure 4. Specifically, we spread chitosan solutions (pH ) 6) onto glass slides and immersed the slides into 1% NaOH for 5 h to create the neutralized chitosan gels. After gel formation, the slides were either lapped in the base solution or were removed from the base solution and then lapped in air. In both cases, after the slides were lapped and clipped, they were immersed in distilled water for 24 h.
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Figure 5. Increase in shear strength as a function of reaction time. The reaction solution contained chitosan (0.48 w/v %; 30 mM equivalent amino groups), dopamine (10 mM), and tyrosinase (60 U/mL). Samples were taken from the reaction mixture at various times and spread onto dry glass slides. Lapping and clipping of the glass slides was conducted either in air (designated “In air”), or after the slides were submerged in water (designated “Under water”). Mean and standard deviations for the shear strength measurements were obtained from five replicates, except for the initial samples and the final sample “In air”, which were obtained from three replicates.
The results in Figure 4 indicate that tyrosinase and dopamine can confer water-resistant adhesive properties to chitosan. The strengths reported in Figure 4 are about an order of magnitude lower than values reported for adhesive proteins.14 These differences may be due to differences in procedure or differences in materials. With respect to procedure, Yu and Deming14 used solutions containing high concentrations of adhesive protein (40 mg protein per 100 µL solvent), applied these solutions to dry adherends, and allowed bonding to occur in a temperature-controlled oven. In contrast, we used semidilute solutions (
