Article pubs.acs.org/Biomac
Mussel-Mimetic Protein-Based Adhesive Hydrogel Bum Jin Kim,† Dongyeop X. Oh,‡ Sangsik Kim,‡,§ Jeong Hyun Seo,∥ Dong Soo Hwang,*,†,‡,⊥ Admir Masic,# Dong Keun Han,▼ and Hyung Joon Cha*,†,‡,∥ †
School of Interdisciplinary Bioscience and Bioengineering, ‡Ocean Science and Technology Institute, §School of Environmental Science and Engineering, ∥Department of Chemical Engineering, and ⊥Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Korea # Department of Biomaterials, Max Planck Institute for Colloids and Interfaces, Potsdam 14424, Germany ▼ Center for Biomaterials, Korea Institute of Science and Technology, Seoul 136-791, Korea S Supporting Information *
ABSTRACT: Hydrogel systems based on cross-linked polymeric materials which could provide both adhesion and cohesion in wet environment have been considered as a promising formulation of tissue adhesives. Inspired by marine mussel adhesion, many researchers have tried to exploit the 3,4-dihydroxyphenylalanine (DOPA) molecule as a crosslinking mediator of synthetic polymer-based hydrogels which is known to be able to achieve cohesive hardening as well as adhesive bonding with diverse surfaces. Beside DOPA residue, composition of other amino acid residues and structure of mussel adhesive proteins (MAPs) have also been considered important elements for mussel adhesion. Herein, we represent a novel protein-based hydrogel system using DOPA-containing recombinant MAP. Gelation can be achieved using both oxdiation-induced DOPA quinone-mediated covalent and Fe3+-mediated coordinative noncovalent cross-linking. Fe3+-mediated hydrogels show deformable and self-healing viscoelastic behavior in rheological analysis, which is also well-reflected in bulk adhesion strength measurement. Quinone-mediated hydrogel has higher cohesive strength and can provide sufficient gelation time for easier handling. Collectively, our newly developed MAP hydrogel can potentially be used as tissue adhesive and sealant for future applications.
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INTRODUCTION The demand for soft tissue medical adhesives, which promote tissue regeneration and minimize surgery time by replacing common surgical procedures that can cause tissue damage, such as suturing and stapling, has been rising. However, conventional soft tissue adhesives do not fully satisfy the requirements to be classified as ideal medical adhesives.1 Cyanoacrylate and gelatinresorcinol-formaldehyde (GRF)-based adhesives can provide strong adhesion to tissue surfaces, but their usage is limited due to their chemical toxicity and heat generation during curing.2,3 Fibrin-based adhesives are biocompatible, exploiting the natural blood clotting process, but their applications are also limited due to poor adhesion strength.4,5 Alternative types of in situ cross-linkable polymer-based adhesives with less toxic crosslinkers have been developed to improve conventional tissue adhesives.6−11 Marine mussels tightly attach to wet surfaces, resisting mechanical stresses that arise from tough marine environments by secreting a proteinaceous thread-like adhesive aid, called byssus. Byssus is composed of 25−30 different types of mussel adhesive proteins (MAPs).12−15 3,4-Dihydroxy-phenylalanine (DOPA) residues, which are post-translationally modified amino acids, are found in all types of MAPs and play a key role in mussel adhesion by forming underwater adhesive bonds with diverse organic and inorganic surfaces and cohesive bonds © 2014 American Chemical Society
with all of the components in the byssal thread (such as neighboring DOPA residues, functional amine or thiol groups of neighboring amino acids, and multivalent metal ions).16,17 Fe3+-DOPA coordinative complexes were detected in the protective cuticle and bulk adhesive plaque of the byssus.18 In the protective cuticle, Fe3+ forms multiple bidentate complexes with DOPA of MAP type 1 (fp-1), which is the only known macromolecule in the cuticle to date.18 Especially, the Fe3+DOPA coordinative complex-mediated cross-link has attracted public attention due to its robust and reversible underwater bonding capability.19 Recent resonance Raman and surface force apparatus (SFA) studies suggested that the Fe3+-DOPA complexes in fp-1 are primarily responsible for the extraordinary mechanical properties of the protective cuticle, which exhibits the stiffness and hardness like epoxy and the high extensibility without catastrophic failure like rubber (ε ∼100%).19 Inspired by the catecholic cross-linking chemistry of DOPA, many studies have been attempted to develop hydrogel-based tissue adhesive. However, the application of natural MAPs was not feasible due to difficulties in directly obtaining sufficient Received: November 26, 2013 Revised: February 18, 2014 Published: March 20, 2014 1579
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DOPA:IO4−. For DOPA-Fe3+complexation, FeCl3 was added to the rfp-1 solution at a 3:1 molar ratio of DOPA:Fe3+, and the pH was increased using 1 N NaOH until the solution became dark pink. Absorbance Spectrophotometry Analysis. The stoichiometries of the DOPA-Fe3+ coordination and DOPA oxidation were monitored by a UV−visible spectrophotometer (Mecasys) using a quartz cuvette with a path length of 1 cm. A 10 mg/mL DOPA-containing rfp-1 solution was used for the analysis. The DOPA:Fe3+ and DOPA:IO4− ratios were applied as mentioned above. For Fe3+-mediated gelation, the pH was controlled using acetic acid or NaOH until the solution color changed. Resonance Raman Spectroscopy. For Raman spectroscopy analysis, a continuous laser beam was focused on the sample through a confocal Raman microscope (WITec) equipped with a piezo-scanner (PhysikInstrumente). A diode-pumped 785 nm near-infrared (NIR) laser excitation (Toptica Photonics AG) was used in combination with a 100 μm oil-immersed (Nikon; NA = 1.25) microscope objective. A laser power ranging between 15 and 30 mW was used for all measurements. The spectra from the 10 mg/mL rfp-1 solutions with Fe3+ for each buffer pH were acquired using an air-cooled CCD (Andor) behind a grating (300 g/mm) spectrograph (Princeton Instruments Inc.) with 6 cm−1 spectral resolution. The Raman spectra were processed and analyzed with Project Plus software (v 2.02; Witec). Rheological Analysis. The viscoelastic properties of the MAP hydrogels were measured using an Advanced Rheometric Expansion System (ARES) rheometer (Rheometric Science) with parallel plate geometry (25-mm-diameter rotating top plate). All tests were performed at 20 °C immediately after the gel sample was transferred onto the sample stage. Subsequent tests were conducted by monitoring the storage modulus (G′), loss modulus (G″), and loss tan δ parameter, which is calculated as G″ divided by G′. In the time sweep test, G′ was measured for ∼900 s at a fixed frequency of 10 Hz and a strain of 10% and normalized by each maximum value for facile comparison. Oscillatory shear testing of the gels as a function of frequency was performed at a constant 20% strain. Self-healing tests were performed by rupturing each gel to failure under increasing strain to 1000% at 80 Hz before monitoring G′ and G″ as functions of time. Bulk Adhesive Strength Measurement under Wet Conditions. Lap shear testing was performed to measure the bulk adhesive strength. To mimic the adhesive surfaces of actual skin tissues, porcine skin tissue surfaces (Stellen Medical) were used. The porcine skin tissue surfaces were cut into 10 mm × 10 mm squares and attached to aluminum fixtures using cyanoacrylate glue. Next, 50 wt % rfp-1 solution was applied to the surface of the porcine skin tissue, which was previously swelled in PBS; then, FeCl3 or NaIO4 was added, and another skin surface-attached aluminum fixture was placed on top of the original fixture. To induce in situ gelation of the Fe3+-mediated cross-linking gels, the samples were immersed in buffers with different pHs (sodium acetate buffer; pH 5.5, PBS; pH 7.4, and Tris buffer; pH 8.2) for 2 h. The quinone-mediated cross-linking samples were immersed in PBS for 2 h after being cured in air for various time periods (0, 5, and 30 min). The samples were lap shear-tested to failure on a universal testing machine (INSTRON) with a cross-head speed of 5 mm/min under ambient conditions. The wet adhesive strength was calculated from the measured maximum load and known area of adhesive overlap. To investigate the self-healing properties of the gels on a bulk scale, successive adhesive strength measurements were conducted six times. The separated adherents after the lap shear test were reassembled and immersed in PBS (pH 7.4) for 2 h. Then, the bulk adhesive strength of the reassembled adherents was measured in the same manner.
purified amounts from mussels. Thus, catechol-incorporated synthetic or natural polymers have been widely utilized as alternatives to fabricate hydrogel networks via catecholic crosslinking.20−34 Although these strategies have been assessed as facile and viable approaches to design mussel-inspired hydrogel systems, the structural and chemical differences with MAPs are neglected in some cases, such as for the use of synthetic polymers without any structures or any charges, e.g., DOPAgrafted polyethylene glycol (PEG-DOPA).20,22,26 From the structural point of view, two known fp-1 and type 2 (fp-2) MAPs, which are capable of forming the Fe 3+-DOPA coordinative complex, have their own solution secondary structures (polyproline type II helix and epidermal growth factor (EGF)-like, respectively).35,36 From the chemical point of view, most MAPs characterized to date are cationic polyelectrolytes which contain roughly equal amounts of both aromatic and cationic residues in their primary sequences. Recent reports have suggested cation−π bonding or π−π stacking as additional underwater adhesion mechanisms of MAPs with regard to their chemical difference.37−41 In this point of view, actual protein sequences could account for important parts of the mussel underwater adhesion. Therefore, we exploited, for the first time, a DOPA-containing recombinant MAP to establish a protein-based adhesive hydrogel system using noncovalent or covalent cross-linking network. With the help of a previously reported recombinant expression system to obtain sufficient amounts of MAPs,19,42−46 we could successfully prepare hydrogel formulations based on recombinant fp-1 (rfp-1) MAP, which is composed of 12 tandem repeats of the Mytilus fp-1 consensus decapeptide (AKPSYPPTYK), and realize a relatively strong adhesion strength in bulk scale. Escherichia coli mass-produced rfp-1 has shown its potential as an adhesive via nanomechanical SFA measurements.19
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MATERIALS AND METHODS
Preparation of DOPA-Containing MAP. rfp-1 MAP comprising 12 tandemly repeated decapeptides of fp-1 was produced using E. coli as previously reported.19,42−46 In brief, transformed E. coli cells were cultured in 10 L Luria−Bertani (LB) medium supplemented with 10 mg/mL kanamycin (Sigma) at 37 °C and 250 rpm. At optical density at 600 nm (OD600) of 0.6−0.8, 1 mM (final concentration) isopropylβ-D-thiogalactopyranoside (IPTG; Sigma) was added to the culture medium to induce rfp-1 expression, and cultured for 8 h at 37 °C and 250 rpm. After centrifuging culture broth at 18,000 g for 10 min at 4 °C, cell pellets containing rfp-1 were resuspended in 5 mL lysis buffer (10 mM Tris-HCl and 100 mM sodium phosphate; pH 8.0) per gram wet weight. Resuspended cells were lysed with constant cell-disruption systems (Constant Systems) at 20 kpsi. Lysates were centrifuged at 18,000 g for 20 min at 4 °C and the inclusion bodies containing expressed rfp-1 were collected for purification. The inclusion bodies were resuspended in 5% (v/v) acetic acid to extract rfp-1. The extraction solution was centrifuged at 18,000 g for 20 min at 4 °C, and the supernatant was collected, dialyzed in deionized water, and freezedried. To prepare DOPA-incorporated rfp-1, tyrosine residues of the produced rfp-1 were converted into DOPA by mushroom tyrosinase (Sigma); 1.5 mg/mL of rfp-1 solution in modification buffer (100 mM sodium phosphate dibasic, 20 mM boric acid, and 25 mL ascorbic acid; pH 6.8) was reacted with tyrosinase (100 μg/mL) for 1 h and dialyzed with 1% acetic acid. The modification yield was measured using the amino acid composition analysis method (Sykam) after HCl hydrolysis of the rfp-1, and confirmed repeatedly by Arnow colorimetric assay.47 Gelation of MAP. rfp-1 solution (20 wt %) in phosphate buffered saline (PBS; pH 7.4) was used for the gelation process. For covalent DOPA coupling, DOPA residues of rfp-1 were oxidized by NaIO4; NaIO4 was added to the rfp-1solution at a 2:1 molar ratio of
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RESULTS AND DISCUSSION Production of DOPA-Containing MAP. Among several types of previously reported recombinant MAPs,42−46 rfp-1 was considered the most appropriate candidate for hydrogel formulation which requires high contents of DOPA molecules and good solubility in aqueous buffer. We prepared highly 1580
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concentrated rfp-1 solution (>500 g/L in aqueous buffer) to form a hydrogel-based adhesive. To achieve gelation, ∼37% of the rfp-1 tyrosine residues were modified to DOPA molecules (∼7 mol % of total amino acids) using an in vitro mushroom tyrosinase enzymatic reaction (Figure 1). Because DOPA
the gelation induced by DOPA quinone-mediated coupling, a dark brown color (λmax ∼390 nm in Figure S1 in Supporting Information) was detected after treatment with NaIO4, indicating DOPA oxidation in rfp-1 to DOPA quinone. Color changes during Fe3+-mediated gelation of rfp-1 were also monitored according to pH variation (Figure S1 in Supporting Information); the gel was green under pH 4.5, purple at pH 5.5−6.9, and pink above pH 8.2, indicative of mono-, bis-, and tris-complexes of Fe3+-DOPA, respectively.48 Further evidence of the presence of Fe3+-DOPA complexes was gained from resonance Raman spectroscopy. The absorption of Fe3+-DOPA complexes in the visible spectral range can be exploited to produce resonance Raman spectra by applying a laser line that falls in the range of the characteristic absorption band. In this work, we excited the samples with a green laser (532 nm) and obtained Raman spectra that are characterized by typical Fe3+-DOPA coordination bands, namely, the Fe-catechol and catechol ring modes (Figure 3b). Notably, a remarkable similarity in spectral features was observed between the natural and recombinant fp-1 for all pH ranges. The presence of a band at 530 cm−1 is indicative of bidentate catechol-Fe complexes due to charge transfer (CT) interaction,49 whereas features at 587 and 636 cm−1 in the Fe− O stretches can indicate Fe3+-catechol complexes.48 As the pH changed, small differences in spectral features were observed; however, a significant increase in resonance signal intensity at higher pH values was observed, suggesting more tris Fe3+catecholate coordination.50 Slightly different with PEG-DOPA hydrogel cross-linked with Fe3+,24 the band at 530 cm−1 in the rfp-1 hydrogel with Fe3+ has similar band area to bands at 587 and 636 cm−1, implying better CT interaction in the rfp-1 hydrogel. Presumably, secondary structure (polyproline type II helix) in rfp-1 could contribute to enhancing the CT interaction. The chemical evidence obtained from Raman spectroscopy and the immediate color change observed upon the addition of Fe3+ ions are responsible for the stoichiometry of the Fe3+DOPA coordination. Many reports have shown that the pH can alter the catecholate coordination stoichiometry (mono-, bis-, or tris-) due to the deprotonation of catechol hydroxyl groups.26 Using spectrophotometry, the maximum absorbance of the tris- and bis-structures was observed to occur at approximately 500 nm (pink) and 548 nm (purple),
Figure 1. Amino acid composition analysis to quantify the modification yield of rfp-1. DOPA modification yield can be evaluated using calculated composition ratio of tyrosine and DOPA. Theoretical composition means a calculated composition based on rfp-1 sequence (AKPSYPPTYK)12. Experimental composition of all other amino acids showed a similar tendency with theoretical composition.
residues were created in the MAP, intermolecular networks of rfp-1 could be induced using two chemical approaches (Fe3+DOPA coordinative complexation and DOPA quinonemediated covalent coupling), thereby forming MAP-based hydrogels (Figure 2). Gelation of MAP and Chemical Identification of Its Cross-Linking. We achieved gelation of highly concentrated rfp-1 solution (>20 wt % in phosphate buffered saline (PBS), pH 7.4) by simply adding NaIO4 oxidant (DOPA:IO4− = 2:1) to induce covalent cross-linking via DOPA oxidation or by adding FeCl3 (DOPA:Fe3+ = 3:1) to form noncovalent crosslinking via Fe3+-DOPA coordinative complexation with pH elevation (pH > 8) (Figure 3a and Movie S1 in Supporting Information). In addition, characteristic color changes were observed during both covalent and noncovalent gelations; the color changes were quantitatively analyzed by UV−visible spectroscopy (Figure S1 in Supporting Information). During
Figure 2. Schematic representation of the MAP-based gelation process via Fe3+-DOPA coordination-mediated noncovalent cross-linking or DOPA quinone-mediated covalent cross-linking. 1581
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Figure 3. (a) Photographs of the gelation processes by the two cross-linking mechanisms. The bright brown color of the rfp-1 solution (top panel) changed to dark brown with the addition of oxidant NaIO4 (left middle panel), and gelation occurred within a few minutes (left bottom panel). Meanwhile, the rfp-1 solution changed to purple with the addition of FeCl3 (pH ∼6; right middle panel), and gelation occurred after the pH was elevated (pH > 8) by adding NaOH droplets (right bottom panel). (b) Raman spectroscopic analysis of Fe3+-DOPA coordinative complexes in the rfp-1 solution according to pH variation.
respectively.48 Interestingly, our results differ from a recent gelation study on cross-linking between PEG-DOPA and Fe3+ (mono- at pH < 5.6, bis- at pH 5.6−9.1, and tris- at pH > 9.1);26 the pH shift point from bis- to tris- in our results appears to correspond to the pH (8.2) of the natural sea environment. This finding could strengthen the argument for the putative mechanism of mussel byssal thread formation in the sea; fp-1 proteins secreted from the mussel feet contact enough of the environmental surroundings to form the cuticle layer of the byssus via Fe3+-DOPA tris-coordinative complexation. This interpretation is straightforward because the rfp-1 used in this study has partial amino acid sequences that are similar to those of natural fp-1. Rheological Behaviors of MAP-Based Hydrogels. To investigate the gelation behaviors of the rfp-1 solution mediated by the two different cross-linking mechanisms, bulk oscillatory rheology experiments were performed. First, the storage modulus (G′) of the rfp-1 solution after the addition of each type of cross-linking agent was monitored during the gelation period at constant strain and frequency (Figure 4a). The G′ value of the Fe3+-containing rfp-1 solution increased to approximately 75% of its maximum value immediately after the pH elevation caused by the addition of NaOH (aq). Meanwhile, it took approximately 5 min for the G′ value of the NaIO4-added rfp-1 solution to reach 75% of its maximum value, indicating that the gelation rate of Fe3+-DOPA coordinative cross-links is significantly higher than that of quinone-mediated covalent cross-links. These results suggest that the DOPA-Fe3+ coordinative interaction occurred more rapidly than the covalent interaction via DOPA oxidation, presumably due to its strong binding affinity (cumulative log Ks ∼40, where Ks is the equilibrium stability constant for Fe3+-DOPA complexes).26 Next, a frequency sweep test at constant strain was conducted to compare the mechanical properties of the quinone-mediated covalent and Fe3+-DOPA coordinative
noncovalent rfp-1 gels. The quinone-mediated covalent gel showed Hookean-like frequency-independent behavior with G′ ≫ loss modulus (G″), which indicates that its relaxation time (τ) is essentially infinite (Figure 4b). The frequency sweep of the Fe3+-mediated coordinative rfp-1gel at pH ∼9 showed Maxwell-like relaxation behavior with τ ∼5.88 s, in substantial agreement with a previous study of a PEG-DOPA hydrogel,26 and a steady state G′ (∼2000 Pa) similar to that of the quinonemediated covalent gel (Figure 4c). This behavior implies that at a basic pH (where the DOPA residues in the gel can be oxidized), the Fe3+-mediated gel was mainly cross-linked by bisor tris-catecholate coordination rather than by oxidative covalent bonds and that the gel, with its near-covalent G′ value, could also provide significant cohesive strength as a bulk adhesive. In addition, the relatively smaller τ of the Fe3+mediated noncovalent gel over the quinone-mediated covalent gel has important mechanical meaning for bulk adhesion; when pressed between rough-shaped adherents, the coordinative hydrogel with its small τ (i.e., high energy dissipation) can rapidly adapt to a deformed shape while the Hookean-like covalent gel may fracture. Apparently, our rheological analysis results seem to be similar to those of previous work on PEGDOPA hydrogel.26 However, we could identify different rheological behaviors in the case of the frequency sweep of Fe3+-mediated coordinative rfp-1 gel in low frequency ranges: the frequency range showing viscous behaviors (G′ ≪ G″) was much shorter than PEG-DOPA, resulting in smaller τ. This is probably because the random coiled structure of PEG could be readily deformed in low frequency ranges,51 while rfp-1 with stiff left-handed polyproline II helix structure might be hardly deformed.35 To evaluate the self-healing capabilities of the hydrogels after complete deformation, a time sweep test at constant frequency and strain was directly followed by a strain sweep test from 1% to 1000% at constant frequency. In the lower strain range with 1582
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Figure 4. Rheological behavior of the MAP-based hydrogels constructed via the two cross-linking mechanisms. (a) Time sweep analyses of both rfp1 gels. Frequency sweep analyses of the (b) quinone-mediated covalent rfp-1 gel and (c) Fe3+-mediated coordinative rfp-1 gel. (d) Self-healing behavior was identified in the Fe3+-mediated rfp-1 gel by strain sweep analysis followed by a time sweep analysis.
strain-independent loss tan δ, the covalent gel had an approximately 4.4-fold lower loss tan δ value than the coordinative gel, indicating that the covalent gel behaved more elastically than the coordinative gel (Figure 4d). The yield strain points, where the loss tan δ dramatically increased, were defined as 60% and 240% for the quinone- and Fe3+-mediated gels, respectively. The higher yield strain of the coordinative gel could potentially provide higher crack and fracture resistance when used as a bulk adhesive. In the time sweep test after the strain sweep, the loss tan δ value of the coordinative gel almost recovered to its original value and was 2-fold smaller than that of the covalent gel (Figure 4d). Therefore, it can be suggested that the Fe3+-mediated rfp-1 gel has self-healing property, which may be attributed to the reversible Fe3+-DOPA coordinative bonding.18,19,52 The self-healing property of the MAP-based hydrogel cross-linked via Fe3+ ions may be applicable in medical adhesives and implants, which require resistance against inevitable surgical damage or rough handling.53 Bulk Adhesive Strength of MAP-Based Hydrogels in Wet Condition using Porcine Skin. Finally, to evaluate the potential utilization of our newly developed MAP hydrogels as
medical adhesives, in vitro lap shear testing was performed using porcine skin tissue surfaces (Figures 5a,b). To our knowledge, the bulk adhesive strength of any polymeric hydrogels using the Fe3+-catecholate coordinative cross-linking mechanism has not yet been reported, potentially due to its high gelation rate. In our experiment, by varying the immersion buffer pH during the curing period (2 h) after the addition of Fe3+ ions to the rfp-1 solution, in situ gelation between the adhesive interfaces was achieved. As a result, the adhesive strength of the Fe3+-mediated noncovalent rfp-1 gel was observed to increase with increasing immersion buffer pH, measuring ∼130 kPa under basic (∼8.2) pH conditions (Figure 5c). The bulk adhesive strength of the quinone-mediated covalent rfp-1 gel was measured to be ∼40 kPa when the porcine skin adherents were immersed in water immediately after treatment with NaIO4 (Figure 5d). However, the strength of the gel dramatically increased as the curing time before water immersion increased, reaching ∼110 kPa with a relatively short curing time (5 min) before water immersion and increasing to ∼200 kPa with a 30 min curing time (Figure 5d). This result is consistent with the dynamic rheology experiments that the NaIO4-added rfp-1 solution needed at 1583
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temperature, and wet/dry) or adherent surface properties.20,23,27,54,55 Among the experimental adhesive strength values measured in wet environment with tissue surfaces,20,23,25,27 our MAP-based hydrogel adhesives showed better results than those of typical fibrin-based adhesive (∼10 kPa)23 and recently reported PEG-DOPA-based hydrogel systems (∼30 kPa).20,23,27 Interestingly, successive bulk adhesive strength measurements immediately after the lap shear test demonstrated that the Fe3+-mediated noncovalent rfp-1 gel retained its initial bulk adhesive strength for more than six cycles, while initial adhesive strength of the quinone-mediated covalent gel decreased gradually according to successive measurements and the quinone-mediated gel eventually lost its adhesion ability after four cycles (Figure 5e). These results might indicate that the Fe3+-mediated adhesive hydrogel also seems to have a selfhealing ability on the bulk scale. However, it might be necessary to perform further study and also careful interpretation of the self-healing behavior in bulk scale, due to complex influences of several interaction factors such as recently identified cation−π and π−π reversible underwater bonding in mussel adhesion.37−41 In the actual mussel adhesion process observed in nature, high levels of DOPA residues in type 5 (fp-5) and type 3 (fp-3) MAPs (28 mol % and 20 mol %, respectively), located in the adhesion plaque are involved in surface adhesion, and other types of MAPs, such as type 2 (fp-2) and type 4 (fp-4), located between the byssal thread and the adhesive plaque, might be more involved in cohesion. However, the relatively low amount (∼7 mol %) of DOPA residues (compared to natural MAPs) in our recombinant fp-1 should be involved in both adhesion and cohesion in the adhesive hydrogel system. Considering this fact, it may be necessary to use MAPs that have significantly higher DOPA contents and/or to induce cohesive functionality using other MAP types to develop strengthened mussel-mimetic medical adhesives in the future.
Figure 5. (a) Schematic illustration and (b) actual image of lap shear testing using porcine skin tissue surfaces. The adhesive strengths of (c) Fe3+-mediated rfp-1 gels according to immersion buffer pH and (d) quinone-mediated rfp-1 gels according to curing time before water immersion were measured using a universal INSTRON device. (e) Self-healing ability of the Fe3+- and quinone-mediated rfp-1 gel via successive bulk adhesive strength measurements in PBS (pH 7.4). The adhesive strength values were normalized by the initial adhesive strength value to simplify the comparison.
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CONCLUSIONS For the first time, gelation of DOPA-containing recombinant MAP was successfully achieved using both Fe3+-DOPA coordinative noncovalent cross-linking and DOPA quinonemediated covalent cross-linking. The mussel protein gels based on the two different cross-linking chemistries exhibited unique particular characteristics as bulk adhesives, although there was no notable difference in their bulk adhesive strengths. The Fe3+mediated MAP gel showed flexible viscoelastic behavior and a self-healing ability, which can be advantageous for adhesives used in surgical environments or requiring rough handling. The quinone-mediated MAP gel had a higher wet bulk adhesive strength due to its covalent cohesive bonding and was convenient for handling due to its gelation durability (in contrast to the rapidly formed Fe3+-mediated gel). Overall, our novel hydrogel system based on the MAP is potentially applicable as a medical adhesive and sealant.
least 5 min of curing time to be an adhesive gel with 75% of its maximum elastic modulus. Also, the higher adhesive strength value of quinone-mediated covalent gel compared to Fe3+mediated noncovalent gel might be due to the higher elastic modulus value which was identified by the rheological analysis (Figure 4). Both the quinone-mediated and the Fe3+-mediated rfp-1 gels mostly remained in both sides of the adherent surfaces after bulk adhesion tests (data not shown), implying that (1) cohesive failure mainly happened during the bulk adhesion test, (2) cohesive bonding by Fe3+-DOPA complexes or quinone-mediated covalent interactions might be the main factors for bulk adhesive strength in our system, and (3) adhesion between porcine skin and the cross-linked gels could be higher than the measured bulk adhesive strength of the gels. Adhesion cross-linkings between the rfp-1 gels and biological tissues can be responsible for not only DOPA chemistries such as quinone-mediated covalent cross-linkings with amine- or thiol groups of adjacent tissue surfaces,16,17 but also recently investigated cation−π or π−π interactions.37−41 Throughout many studies to measure bulk adhesive strength of DOPA-incorporated materials, the adhesive strength has been reported from tens of kPa to several MPa, depending on experimental conditions such as curing environments (time,
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ASSOCIATED CONTENT
S Supporting Information *
Absorbance spectra of the Fe3+-DOPA coordination-mediated and DOPA quinone-mediated rfp-1 gels measured by UV− visible spectroscopy (Figure S1) and movie about gelation process of the Fe3+-DOPA coordination-mediated rfp-1 gel (Movie S1). This material is available free of charge via the Internet at http://pubs.acs.org. 1584
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AUTHOR INFORMATION
Corresponding Authors
*(D.S.H.) E-mail:
[email protected]. *(H.J.C.) E-mail:
[email protected]. Present Address
(J.H.S.) School of Chemical Engineering, Yeungnam University, Gyeongsan 712−749, Korea Author Contributions
B.J.K., D.X.O., J.H.S., D.S.H., D.K.H., and H.J.C. designed the experiments. B.J.K., D.X.O., and S.K. performed the experiments. B.J.K., D.X.O., and A.M. analyzed the data. D.S.H. and H.J.C. supervised the research. B.J.K., D.X.O., J.H.S., D.S.H., and H.J.C. wrote the manuscript. H.J.C. is the principal investigator. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Marine Biomaterials Research Center grant from Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Korea (to H.J.C. and D.S.H.) and the Rising Star Program funded by POSTECH (to H.J.C.). D.X.O. was supported by the National Research Foundation grant (NRF-2013-Fostering Core Leaders of the Future Basic Science Program).
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dx.doi.org/10.1021/bm4017308 | Biomacromolecules 2014, 15, 1579−1585
