Phase Controllable Hyaluronic Acid Hydrogel with Iron(III) Ion

Sep 20, 2016 - Department of Polymer Chemistry, School of Chemical Sciences, North Maharashtra University, Jalgaon 425-001, Maharashtra, India. Macrom...
0 downloads 13 Views 8MB Size
Download PDF
Article pubs.acs.org/Macromolecules

Phase Controllable Hyaluronic Acid Hydrogel with Iron(III) Ion− Catechol Induced Dual Cross-Linking by Utilizing the Gap of Gelation Kinetics Jeongwook Lee,† Kyeol Chang,‡ Sunhye Kim,† Vikas Gite,§ Hoeil Chung,‡ and Daewon Sohn*,† †

Research Institute for Convergence of Basic Science and ‡Analytical Spectroscopy Lab, Department of Chemistry, Hanyang University, Seoul 133-791, Republic of Korea § Department of Polymer Chemistry, School of Chemical Sciences, North Maharashtra University, Jalgaon 425-001, Maharashtra, India S Supporting Information *

ABSTRACT: Metal complexation-based gelation imparts load-bearing hydrogels with striking properties like reversibility, self-healing, and mechanical tunability. Using a bio-inspired metal−catechol complex, these properties have been introduced to a variety of polymer hydrogels, except hyaluronic acid, which is widely used in biological applications. In this research, we developed two different hyaluronic acid (HA) hydrogels by regulating the gelation kinetics of Fe3+ and a catechol cross-linker, including Fe3+-induced covalent bonding and coordination bonding. Dual roles of Fe3+ in catechol-modified HA (HA-CA), Fe3+−catechol coordination, and catechol oxidation followed by a coupling reaction were selectively applied for different gelations. Phase-changeable HA-CA gel was attributed to dominant Fe3+−catechol coordination with immediate pH control. Alternatively, allowing a curing time to form catechol coupling bonds resulted in color-changeable HA-CA gels with pH control. The gel structure is then preserved by dual cross-linking through covalent catechol-coupling-based coordinate bonds and electrostatic interactions between Fe3+ and HA-CA. The hydrogels showed enhanced cohesiveness and shock-absorbing properties with increasing pH due to coordinate bonds inspired by marine mussel cuticles. The present gelation strategy is expected to expand the utility of HA hydrogels in biological applications, offering easy control over the phase, gel network, and viscoelastic properties.

1. INTRODUCTION Adaptation of mussels to tidal phenomena has given rise to byssal thread, a fibrous holdfast secreted from the mussel foot. The soft interior portion of the byssal thread is protected by an outer cuticle coating that is largely composed of mussel foot protein 1, which serves as a shock absorber and helps the mussel endure the harsh and turbulent intertidal zone environment.1 Mussel foot protein 1 contains a variety of 3,4dihydroxyphenylalanine (DOPA) residues and metal ions, especially Fe3+.2,3 The remarkable nature of this cuticle has inspired bioengineering investigations into biomimetic loadbearing materials.4 In molecular structures, including DOPA residues, Fe3+ ions play a dual role serving as an oxidizing agent as well as undergoing complexation with 1,2-dihydroxybenzene (catechol) pendant groups.5 Fe3+−catechol complexation (log k = 20 for the monocomplex) is instantaneous, and the degree of Fe3+−catechol coordination can be transformed between the mono, bis, and tris complexes by modulating the pH.6 The coordination complex-based gelation imparts load-bearing hydrogels with very useful properties such as reversible gelation, self-healing, adhesion, structural hardness with no mineraliza© XXXX American Chemical Society

tion, usability in water, and tunability of mechanical properties.7,8 Catechol oxidation by Fe3+ leads to irreversible catechol coupling reactions.9 Although catechol coupling is much slower than coordination complexation, the irreversible covalent bonds of the former contribute to a solid gel structure that is impervious to external stimuli. These two disparate gelation methods, in terms of quantitative and kinetic factors, have been applied to catechol-modified synthetic polymers (PEG, polystyrene) to endow a reversible phase transition and regulate the cross-linking density and mechanical properties.10,11 Recent research on mussel cuticle inspired hydrogels has been concentrated on chitosan, a natural polymer and linear amino polysaccharide considered to be a promising polymer for biological applications.12,13 However, due to the strong cationic surface charge of chitosan, which arises from its amine functionality, a high ratio of catechol substitution and a long gelation time are required to alleviate the repulsive interaction between the cation (Fe3+) and a cationic polymer (chitosan).14 Received: June 5, 2016 Revised: September 2, 2016

A

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 1. (a) Schematic process of the catechol-modified HA (HA-CA). (b) 1H NMR spectrum of HA-CA synthesized by EDC/HOBt coupling. The degree of substitution is determined by comparison of peak integrals in the 6.8−7.2 ppm (red) and 1.8−2.2 ppm (blue). EDC and 312 mg of HOBt dissolved in 4 mL of 1:1 H2O:DMSO were then added, followed by the addition of 429 mg of dopamine hydrochloride. The pH of the solution was maintained between 5.0 and 5.5 for 8 h by adding 1 M HCl and 1 M NaOH. The solution was then dialyzed (MWCO: 6000−8000 g/mol, SpectraPor) against a pH 5.0 aqueous solution adjusted with 1 M HCl for 2 days and dialyzed again with deionized water for 1 day to fully remove unreacted reagents and salts. The resultant solution was lyophilized for 2 days. The molar ratio of reactants was HA:EDC:HOBt:dopamine = 1:3:3:3. 19% and 27% substituted HA-CA were synthesized by modulating the molar ratio of reactants as follows: HA:EDC:HOBt:dopamine = 1:1:1:1 for 19% substituted HA-CA and 1:2:2:2 for 27% substituted HA-CA. The catechol modification ratio of HA-CA was determined by 1 H NMR (Varian Mercury, 400 MHz). The ratio of catechol conjugation in the HA backbone was calculated by comparison of the 6.8−7.2 and 1.8−2.2 ppm peaks. The zeta potential of HA-CA at 25 °C was measured using a Zetasizer Nano ZS Analyzer (Malvern Ltd., UK). An aqueous solution (1 mg/mL in deionized water) was used to determine the surface charge of HA-CA at various pH values. 2.3. Preparation of Fe3+-Induced HA-CA Hydrogel and pHInduced HA-CA Hydrogel. The lyophilized HA-CA (37% substitution) was utilized for all gel formation processes. The HACA was fully dissolved in deionized water at 3% (w/v). To study the effect of the Fe3+:catechol ratio upon gelation, Fe3+ solutions of different concentrations (40 mM, 1:3 ratio; 80 mM, 2:3 ratio; and 120 mM, 3:3 ratio) were prepared by adding iron(III) chloride hexahydrate to a 5% acetic acid aqueous solution (v/v). 100 μL of the HA-CA aqueous solution was dispensed into a Petri dish. 23.4 μL of the Fe3+ solution was then added to the HA-CA solution, and the resulting suspension was uniformly mixed. The phase transition HA-CA hydrogel was prepared by immediately increasing the pH of the mixture by adding 20 μL of 1 M NaOH. The reverse transition from gel to liquid was performed by adding 20 μL of 1 M HCl. Alternatively, the HA-CA hydrogel formation to yield a hydrogel stable over a wide pH region was prepared by allowing the mixture of HACA and Fe3+ solution to cure. The end of the gel formation was identified by inverting the gel at various times. After curing, 10 μL of 1 M HCl and 1 M NaOH were selectively added to the samples for pH control followed by sufficient mixing, which resulted in yellow (pH 2− 3), green (pH 4−5), dark green (pH 7−8), and dark red (pH 10−11) HA-CA hydrogels. The pH-induced HA-CA hydrogel was synthesized by mixing with 100 μL of 3 wt % HA-CA and an oxidizing agent,

Hyaluronic acid (HA), a ubiquitous glycosaminoglycan in extracellular matrix, is widely used in tissue engineering because of its high viscoelasticity and space-filling property.15 In addition, the anionic charge of HA can contribute to improved gelation via its attractive interaction with Fe3+. Despite considerable research to develop coordinative cross-linked gels, there have not been any studies regarding the use of HA in cuticle-inspired hydrogels, and coordination complex-based HA gelation is unknown. Accordingly, in the present work we developed two different types of cuticle-inspired HA hydrogels: one that undergoes reversible phase transitions with changing pH and another that maintains its gel properties over a wide pH range. The phase transition is attributed to the dominance of Fe3+−catechol coordination in the gelation process. The latter HA hydrogel is formed by dual cross-linking of covalent catechol coupling based on Fe3+−catechol coordination in which the anionic charge of HA enables efficient gelation. The two types of Fe3+-induced HA hydrogel are regulated by crosslinking kinetics with curing time. The resulting gels show entirely different structures and mechanical properties under varying pH, owing to the cohesive and shock-absorbing properties of coordinate bonds inspired by mussel cuticle.

2. MATERIALS AND METHODS 2.1. Materials. Hyaluronic acid (HA, 200 kDa) was purchased from Lifecore Biomedical (Chaska, MN). 1-(3-(Dimethylamino)propyl)ethylcarbodiimide hydrochloride (EDC) was obtained from Alfa Aesar. 1-Hydroxybenzotriazole hydrate (HOBt, ≥97.0%), dopamine hydrochloride, sodium periodate (NaIO4), and iron(III) chloride hexahydrate were purchased from Sigma-Aldrich. Dulbecco’s phosphate buffered saline (DPBS) was obtained from WELGENE (Daegu, Korea). Deionized water with resistance greater than 18.2 MΩ·cm was used. 2.2. Synthesis of Catechol-Modified HA. Catechol-modified hyaluronic acid (HA-CA) was synthesized by an EDC/HOBt coupling reaction, where the carboxylic acid groups of HA are substituted with the amine groups of dopamine.16 300 mg of HA was dissolved in 100 mL of DPBS, and then the solution was purged with nitrogen for 30 min. The solution pH was reduced to 5.5 using 1 M HCl. 426 mg of B

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 2. (a, c) Gelation tests with quantitative variation of HA-CA weight percent and molar ratio of Fe:catechol and (e) phase changeable gelation with pH control at 1:3. UV−vis spectra of different molar ratios of Fe3+:catechol (b, d) and (f) pH control at 1:3.

Figure 3. (a) pH-induced gelation tests of HA-CA with or without NaIO4 oxidizing agent and (b) gel formation with the inverse method. (c) UV− vis spectra of the pH-induced gelation. NaIO4 (catechol:IO4− molar ratio of 1:1), immediately followed by adding 20 μL of 1 M NaOH. 2.4. UV−Vis Spectrophotometry. To monitor the type of crosslinking, Fe3+-induced HA-CA gelation was analyzed using a UV−vis spectrophotometer (OPTIZEN 3220UV, Mecasys Co., Ltd.), and samples were contained in a 1 cm quartz cuvette. The experiment in which various Fe3+:catechol ratios were tested was conducted with a 5% acetic acid blank sample for comparison. In each test, a 50 μL

portion of 3 wt % HA-CA was thoroughly mixed with 11.7 μL of the Fe3+ solution in 3 mL of 5% acetic acid. The increase in coordination state with increasing pH was investigated by adding 1 M NaOH to the solution prepared with the 1:3 Fe3+:catechol ratio. The characterization of pH-induced HA-CA hydrogel was performed by mixing 3 wt % HA-CA (50 μL) and NaIO4 (catechol:IO4− molar ratio of 1:1). 2.5. Morphology of the Gels. The hydrogel morphology was investigated by scanning electron microscopy (SEM). SEM images of C

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 4. SEM images of (a) the mixed solution of Fe3+ and HA-CA at pH 3, (b) hydrogel after an increase to pH 10, and (c) pH-induced gelation with 1:1 (catechol:NaIO4) at pH 10.

37% based on 1H NMR spectroscopy (Figure 1b). The optimum gelation condition for the phase transition of the HA hydrogel was investigated by quantitatively varying the weight percent and thus the molar ratios of Fe3+ and catechol. The gelation from liquid to hydrogel was inspected when the pH 3.2 solution mixture of HA-CA and Fe3+ was changed to gel at pH 10−11 with increasing pH. The 3 wt % HA-CA aqueous solution was the minimum concentration studied to yield a hydrogel at pH 10−11; gelation failed for the 1 and 2 wt % solutions (Figure 2a). The variation in the catechol molar ratio with constant Fe3+ influenced the solution color, especially at a 1:3 (Fe:catechol) ratio. The clear green color indicates the formation of the monocomplex, specifically, the coordination complex of one catechol and one Fe3+. The state of this metal− catechol complex was verified by UV−vis spectroscopy. The broad absorption peak of the monocomplex (714 nm) appeared strong for the solution with the 1:3 Fe3+:catechol ratio and slightly strong for the 1:2 solution; it was uncertain whether this peak appeared for the 1:1 solution (Figure 2b).18 A narrow absorption peak at 400 nm appeared, attributed to oquinone, formed by the oxidation of catechol by Fe3+.19 However, the gap between 1:2 and 1:3 at 400 nm was much smaller than that of the monocomplex peak, demonstrating that the 1:3 ratio was the optimal condition among those studied in terms of the monocomplex formation in acidic conditions. The effect of the Fe3+ ion concentration in solutions with constant catechol concentration was studied; the green color observed for the 1:3 solution turned to a dark yellow with increasing Fe3+ (Figure 2c). In addition, increasing Fe3+ yielded greater increases in the o-quinone absorption peak than in the monocomplex (Figure 2d). These results prove that Fe3+ ions added within the Fe:catechol range of 1:3 to 3:3 played a larger role in catechol oxidation, reducing the amount of catechol groups available for forming bis and tris complexes under basic conditions. Thus, the 1:3 ratio is the most suitable for maximizing the amount of reduced catechol that can participate in coordination complexation. A liquid−gel phase transition by varying the pH was performed using the 1:3 solution. A maximum of three phase transition cycles was observed owing to progressive dilution by the solutions added to change the pH. The green solution observed at pH 3−4 gelled immediately under increasing pH; the initial solution changed completely to a dark red hydrogel at pH 10−11 and existed in an intermediate state between liquid and gel at pH 7−8 (Figure 2e). The hypsochromic shift of the absorption spectra in Figure 2f shows that the coordination state of Fe3+−catechol changed from the monocomplex (714 nm) to the tris complex (490 nm) via the bis complex (570 nm) with increasing pH.18 This proves that dominant tris coordination is required for complete

the hydrogel surface were collected on an SNE-4000 M instrument (SEC Co., South Korea) operated at 15 kV. Hydrogel samples were directly lyophilized after gel formation, and all samples were coated with gold. 2.6. Raman Spectroscopy. The wide area illumination (WAI) scheme used in this research was developed by Kaiser Optical Systems (Ann Arbor, MI).17 An excitation laser (785 nm, diode laser) was magnified to form a circular illumination area with a diameter of 6 mm (area 28.3 mm2) to cover a large sample area. The scattered radiation was then collected by an array of 50 optical fibers and delivered to a charge-coupled device detector (Kaiser Raman Rxn1). The hydrogel samples used for Raman spectroscopy were prepared in the same manner as for SEM analysis. A laser power of 20 mW and exposure time of 2 s were used for all experiments. 2.7. Rheology of the Gels. The mechanical properties of hydrogels were analyzed using a rotational rheometer (Gemini 150, Malvern Instruments, UK) with a 20 mm parallel plate. All tests with gels were performed after transferring a gel sample to the sample stage, except for the liquid-phase and oscillatory time sweep studies. A frequency sweep was performed over the 0.1−10 Hz region with 10% constant strain. Oscillatory time sweeps were performed at 1 Hz under 10% constant strain. Self-healing tests were conducted by a strain sweep under increasing strain from 0.1% to 70% and then allowed to heal under 0% strain for 50 s. The gel re-formation was identified by the time sweep after the recovery time span. All experiments were conducted at 25 °C.

3. RESULTS AND DISCUSSION 3.1. Phase Transition HA-CA Hydrogel. 3.1.1. Optimum Gelation Condition. To obtain catechol-modified HA (HACA), HA was substituted with dopamine, a catecholic monomer, by a coupling reaction between ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole hydrate (Figure 1a). The degree of catechol modification in the HA backbone was

Figure 5. Rheological properties of the phase transition HA-CA hydrogel with pH control at a 1:3 Fe3+:catechol molar ratio (G′: storage modulus; G″: loss modulus). D

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 6. (a) Schematic process of HA-CA gel formation over the entire pH range, including color changes. (b) Moduli study by oscillatory time sweep after curing of the 1:3 (Fe3+:catechol) solution. (c) Zeta-potential curves of HA-CA at various pH.

Figure 7. Time-dependent moduli at (a) only pH enhancement to pH 10, (b) pH 10 condition with NaIO4 (1:1 = catechol:NaIO4), and (c) pH 10 condition with Fe3+ (1:3 = Fe3+:catechol).

catechol oxidation occurred. However, the liquid phase did not gel. Previous gelation studies of catechol-conjugated polymers were performed at high pH conditions and also by oxidizing agents such as NaIO4 to promote the extent of catechol oxidation. HA-CA with a 1:1 ratio (catechol:NaIO4) changed into a gel at pH 10 (Figures 3a,b) along with the observation of a deep red color. The absorption peak (400 nm) in Figure 3c proves that o-quinone existed more in the gel structure (high pH, with NaIO4) than the liquid phase (high pH, without NaIO4). Thus, pH-induced oxidative cross-linking gelation was not possible without an oxidizing agent, although pH enhancement influences the catechol oxidation. 3.1.3. Gel Structure and Rheological Property. The phase transition by the dominant tris complex also influences the gel structure. The morphology of the hydrogel was analyzed by SEM, indicating that the smooth morphology of the initial 1:3 solution changed dramatically to a porous structure at pH 10 (Figure 4b). Instantaneous cohesion by dominant tris coordination induces inhomogeneity into the gel structure, producing denser, tris-complexed HA parts in the main gel network in contrast to the looser pore parts. Alternatively, the morphology of the pH-induced HA-CA gel with NaIO4 was an irregular beehive structure without pores on the surface. A strong oxidative cross-linking structure composed of covalent bonds is a key factor that leads to a dense gel structure. To elucidate the viscoelastic properties of the phase-changeable hydrogel, samples at various pH were measured with an

Figure 8. Raman spectra of HA-CA hydrogel formed by pH control after curing of the 1:3 (Fe:catechol) molar ratio.

gelation. Without sufficient catechol coupling reaction, Fe3+− catechol acts as the primary cross-linking agent. 3.1.2. pH-Induced Oxidative Cross-Linking. To investigate the effect of increasing pH, which is able to contribute to oxidative cross-linking, a gelation experiment in the absence of Fe3+ was conducted at pH 10. Figure 3a shows that the color of HA-CA at pH 10 changed to dark orange, which means E

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Figure 9. (a−e) SEM images of 1:3 (Fe:catechol) HA-CA hydrogels after curing. Morphology of (a) the gelled surface after 13 min, (b) yellow color, (c) green color, (d) dark green color, and (e) dark red color HA-CA hydrogels under pH treatment. (f) Schematic illustration of the variation of coordination states with pH control.

3.2. Preservation of HA-CA Gel Structure by Dual Cross-Linking. 3.2.1. Inducement of Catechol Coupling. To form a phase-changeable hydrogel, dual roles of Fe3+ ions, oxidizing agent and coordination agents, are given too much emphasis on participating in coordination. Fe3+−catechol coordination, an instantaneous reaction, prevents o-quinone from undergoing coupling with reduced catechol, which is much slower than the metal complexation. However, sustaining the degree of the monocomplex for a sufficient time for catechol coupling induces an enhanced covalent cross-linking ratio that contributes to maintenance of the gel structures from

oscillatory rheometer by a frequency sweep. The mixture of Fe3+ and HA-CA at pH 3 displayed viscous properties (G′ < G″) at low frequency due to a shortage of cross-linking (Figure 5). In addition, the crossover point of 6.2 Hz was only observed for the pH 3 solution, which behaved according to the Maxwell viscoelasticity model, in contrast with both the pH 7 and 10 solutions that exhibited elastic features (G′ > G″).20 The storage modulus was rapidly enhanced with increasing pH, from 7.2 Pa (pH 3) to 191.3 Pa (pH 10) via 24.9 Pa (pH 7) at the 1 Hz frequency point; this showed the strong contribution of dominant tris complexation to gelation. F

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

external stimuli. A 1:3 (Fe3+:catechol) solution was gradually gelled within 13 min of curing time (Figure 6a). An oscillatory time sweep of the 1:3 solution was measured to monitor the gelation process. The initial viscous response (G′ < G″) changed to an elastic response (G′ > G″) at a crossover point of 200 s (Figure 6b). The elastic property was continuously enhanced by increasing G′ without a specific plateau. The gelation time of 13 min obtained by the inversion method well matched the modulus results, which showed rapidly increasing G′ at 825 s. In addition, gradually increasing moduli after the gelation time demonstrated that the slow catechol coupling reaction was not fully complete. Additional covalent cross-links by unreacted o-quinone beyond the gelation time contribute to the improved storage modulus of nearly 1000 Pa at 2000 s, which is appropriate for applications demanding high mechanical strength. The storage modulus was also improved by changing the Fe3+:catechol molar ratio (Figure S1). Compared to 130 Pa for the 1:3 solution, the 2:3 solution showed an enhanced G′ value greater than 250 Pa, which attributes to an increase in the oxidative cross-linking by Fe3+induced oxidation. The gelation time could be reduced by increasing the catechol modification ratio in HA, which leads to more cross-linking (Table S1). 3.2.2. Attractive Interaction between HA and Fe3+. Alternatively, previous similar studies reported that catecholmodified chitosan was able to gel in acidic conditions (∼pH 3) given enough curing time for the Fe3+-induced coupling reaction.21 However, due to the strong cationic charges of chitosan and thus the repulsive interaction with Fe3+, a high ratio of catechol modification (∼70%) is required to reduce the gelation time. Compared to chitosan, the gelation time of the HA hydrogel was remarkably reduced despite the low substituted ratio of catechol: 18% catechol-substituted chitosan required 9 h for gelation whereas 19% catechol-substituted HA required 35 min (Table S1).14 Thus, catechol coupling and other factors have a strong effect on the gelation in the case of HA. The zeta-potential value of HA-CA indicates anionicity over nearly the entire pH region, even at the strongly acidic pH 2 (Figure 6c). The strong anionic property of HA is mainly due to the presence of carboxylate in the HA backbone.22 The carboxylate group in HA had been applied to HA gelation with Fe3+, which was cross-linked by electrostatic interactions between Fe3+ and carboxylate. Although the support of anionic carboxylate is partially applicable due to the reaction conditions at the 1:3 ratio with a pH of 3.21, which is less than the pKa of carboxylic acid (∼3.75), the negative zeta value still contributes to spontaneous gelation by attractive interaction. 3.2.3. Gel Formation with pH Control. On the basis of the covalent catechol cross-linking and attractive interaction, additional coordinate bonds arising from increasing pH lead to hydrogel formation throughout the whole pH region, with accompanying variation in color. The four colors of HA hydrogel at different pH represent coordination states of Fe3+ with the catechol moiety. As mentioned above in reference to Figure 2f, the HA hydrogels were green at pH 4−5, dark green at pH 7−8, and dark red at pH 10−11 (Figure 6a), with the colors respectively attributed to mono, bis, and tris coordination between Fe3+ and catechol. Compared to the liquid and intermediate states of the phase transition gel, maintenance of the hydrogel formation at pH 4−8 is possible with this gelation method; the dark green hydrogel can be especially applied in physiological conditions. In addition, hydrogel formation is maintained in strongly acidic conditions

Figure 10. Rheological properties of HA-CA hydrogels formed at pH 2.5, 4.5, 7.0, and 10.0 after curing as measured by frequency sweep characterization.

Figure 11. Rheological verification of the self-healing ability of Fe3+induced HA-CA hydrogels formed at pH (a) 2.5, (b) 4.5, (c) 7.0, and (d) 10.0 after curing. Gels were degraded under increasing oscillatory strain followed by a healing time of 50 s and then recovery monitoring by oscillatory time sweep.

G

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

uneven hydrogel surface resembling an irregular beehive (Figure 9b). Vacant spaces visible on the hydrogel surface were mainly due to the disassembly of Fe3+ from the monocomplex at pH 2−3, which leaves only the covalent cross-linking network.21 Increasing the pH caused the hydrogel to change color from yellow to green, accompanied by swelling of the gel structure following the network pattern of the yellow gel (Figure 9c). These changes were attributed to the introduction of the monocomplex and enhanced swelling capacity of HA with increasing pH.24 Alternatively, a series of pH increases transformed the flat gel network into a curled structure. Compared to the green gel, the gel network of the dark green color hydrogel was condensed into a straight directional gel structure (Figure 9d). The condensed gel structure became completely rolled at pH 10−11, leading to uplift of the gel network (Figure 9e). The gradual condensation of the gel structure with increasing pH results from reinforcement of the cohesion by an increase of the coordination state because the tris complex exhibits the strongest cohesive properties. 3.2.7. Rheological Property. The mechanical properties of the dual cross-linking hydrogels were investigated by frequency sweeping. The slope of the moduli decreased with increasing pH (Figure 10). This result was mainly attributed to variation of both of the additional gelation process with unreacted catechol and HA chain rearrangement at each pH condition. Unreacted catechol moieties in the hydrogel network induced extra coupling reactions as well as coordination with Fe3+, which is available for bonding due to the spare coordination sites in the mono and bis complexes compared to the tris complex. In addition, chain rearrangement of the high molecular weight HA demands substantial time to reach the minimum free energy. The process of rearrangement actively progresses at lower pH conditions with frequency sweep, especially at high frequency, compared to the dark red color gel that is already organized with strong tris coordination.25 Based on the comparison of the storage modulus, the improvement in mechanical strength was proportional to the increase in the coordination state. Compared to other gels, the dark red color gel shows unusual behavior that does not correspond to the typical Hookean elastic model or Newtonian behavior.19,26 This phenomenon arises from tris coordination, where energy from an applied load is better dissipated than in the cases of mono or bis coordination, as inspired by the shock-absorbing role of the cuticle of mussel byssus.1,20 3.2.8. Self-Healing Ability. The self-healing ability of Fe3+ induced HA-CA hydrogels were demonstrated by association of the oscillatory strain sweep and time sweep. The strain applied to the gels was enhanced from 0.1% to 70% strain and then allowed to recover under 0% strain for 50 s. In Figure 11, Fe3+induced hydrogels were degraded under increasing oscillatory strain, which induces moduli inversion at each crossover point. However, time sweep results indicate that Fe3+-induced hydrogels exhibit instantaneous healing and recover their original strength within 50 s. This self-healing behavior of the gel is attributed to coordinative interaction between Fe3+ and catechol groups. The coordinate bond plays a sacrificial role under high stress, while gels are degraded by shear strain. However, reversible and dynamic equilibrium of coordinate bonds readily reorganizes the gel network, leading to the rapid re-formation of gels.14,19

of pH 2−3, which are harsh conditions for gelation due to the dissociation of the coordination complexes. The absorption peak in Figure S2 shows that the monocomplex was disassembled with decreasing pH because of the reduced affinity between Fe3+ and catechol due to the full protonation of the catechol groups. Despite the absence of the coordination bonding, covalent cross-linking-based gelation enabled the formation of a gel, which was yellow due to the improved solubility of Fe3+ with reduced pH. 3.2.4. Monitoring of the Gelation Process. To investigate the effects and kinetics of oxidative cross-linking on gelation, time-dependent rheological behavior was monitored by oscillatory time sweep experiments. Figure 7a shows that the elastic property is slowly increasing, which results in retardation of the crossover point (777 s). The insufficient G′ value at 2000 s (118.3 Pa) and the small gap between G′ and G″ prove that oxidative cross-linking is partially processed, and o-quinone remains intact instead of the catechol coupling reaction. In contrast, pH-induced gel with oxidizing agent (Figure 7b) is analogous to the result of Figure 6b in terms of early crossover point (345 s) and high storage modulus (657.5 Pa) at 2000 s. However, the 1:3 (Fe3+:catechol) solution is cured by both monocomplex and oxidative cross-linking in comparison with only oxidative cross-linking in a pH-induced gelation with NaIO4. Because of the slow catechol coupling reaction, the crossover point of pH-induced gelation with NaIO4 is later than the 1:3 (Fe3+:catechol) solution. Compared to the former results, gelation with Fe3+ at pH 10 exhibits an elastic response from beginning to end. This behavior indicates that gelation by the tris complex is performed in a moment at high pH conditions. In addition, the G′ value was not enhanced with the time point because most Fe ions are involved in coordinative cross-linking. Consequently, the lack of Fe3+ as an oxidizing agent causes low oxidative cross-linking, which leads to a lower G′ value of Figure 7c in comparison to the higher G′ value of Figure 7b. 3.2.5. Raman Spectra. The resonance Raman spectral characteristics of Fe3+−catechol coordination in the HA hydrogel network were analyzed by wide Raman spectroscopy, performed using a near-infrared (785 nm) laser excitation source. Interaction between Fe3+ and phenolic oxygen atoms of catechol was represented by peaks near 531, 590, and 633 cm−1, whereas peaks near 1270, 1325, 1420, and 1483 cm−1 represented ring vibrations of Fe3+−catechol complexes (Figure 8).23 The peaks related to the Fe−O interaction and ring vibration were not observed at pH 2.5, analogous to the case of HA-CA without Fe, which supports the result of Figure S2. However, increasing the pH from 4.5 to 10 via pH 7 induced an increase in the intensity of the peaks corresponding to Fe3+− catechol coordination. The peak near 531 cm−1 indicates the charge transfer (CT) interaction of the bidentate chelation between Fe3+ and the catechol oxygen. The area ratio of the CT peak to other Fe−O interaction peaks (590, 633 cm−1) was 19% at pH 4.5, 25% at pH 7.0, and 31% at pH 10. The growth of this area ratio with increasing pH demonstrates an increase in the bidentate complexation, specifically an alteration in the Fe3+−catechol coordination in the HA hydrogel network from the mono to tris complex via the bis complex.19 3.2.6. Structural Cohesiveness. Changes in the hydrogel structure with alternating changes in pH were investigated by SEM. The smooth morphology of the 1:3 solution was maintained during gelation with increasing curing time (Figure 9a). However, HCl treatment to reduce the pH led to an H

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

cells, from microorganisms to mammals. Environ. Microbiol. 2001, 3 (2), 81−91. (7) Broomell, C. C.; Mattoni, M. A.; Zok, F. W.; Waite, J. H. Critical role of zinc in hardening of nereis jaws. J. Exp. Biol. 2006, 209 (16), 3219−3225. (8) Harrington, M. J.; Waite, J. H. pH-dependent locking of giant mesogens in fibers drawn from mussel byssal collagens. Biomacromolecules 2008, 9 (5), 1480−1486. (9) Smejkalova, D.; Conte, P.; Piccolo, A. Structural characterization of isomeric dimers from the oxidative oligomerization of catechol with a biomimetic catalyst. Biomacromolecules 2007, 8 (2), 737−743. (10) Menyo, M. S.; Hawker, C. J.; Waite, J. H. Versatile tuning of supramolecular hydrogels through metal complexation of oxidationresistant catechol-inspired ligands. Soft Matter 2013, 9 (43), 10314− 10323. (11) Xu, H.; Nishida, J.; Ma, W.; Wu, H.; Kobayashi, M.; Otsuka, H.; Takahara, A. Competition between oxidation and coordination in cross-linking of polystyrene copolymer containing catechol groups. ACS Macro Lett. 2012, 1 (4), 457−460. (12) Brzonova, I.; Steiner, W.; Zankel, A.; Nyanhongo, G. S.; Guebitz, G. M. Enzymatic synthesis of catechol and hydroxyl-carboxic acid functionalized chitosan microspheres for iron overload therapy. Eur. J. Pharm. Biopharm. 2011, 79 (2), 294−303. (13) Zvarec, O.; Purushotham, S.; Masic, A.; Ramanujan, R. V.; Miserez, A. Catechol-functionalized chitosan/iron oxide nanoparticle composite inspired by mussel thread coating and squid beak interfacial chemistry. Langmuir 2013, 29 (34), 10899−10906. (14) Yavvari, P. S.; Srivastava, A. Robust, self-healing hydrogels synthesised from catechol rich polymers. J. Mater. Chem. B 2015, 3 (5), 899−910. (15) Annabi, N.; Tamayol, A.; Uquillas, J. A.; Akbari, M.; Bertassoni, L. E.; Cha, C.; Camci-Unal, G.; Dokmeci, M. R.; Peppas, N. A.; Khademhosseini, A. 25th anniversary article: rational design and applications of hydrogels in regenerative medicine. Adv. Mater. 2014, 26 (1), 85−124. (16) Xu, X.; Jha, A. K.; Harrington, D. A.; Farach-Carson, M. C.; Jia, X. Q. Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. Soft Matter 2012, 8 (12), 3280−3294. (17) Kim, M.; Chung, H.; Woo, Y.; Kemper, M. New reliable Raman collection system using the wide area illumination (WAI) scheme combined with the synchronous intensity correction standard for the analysis of pharmaceutical tablets. Anal. Chim. Acta 2006, 579 (2), 209−216. (18) Sever, M. J.; Wilker, J. J. Visible absorption spectra of metalcatecholate and metal-tironate complexes. Dalton T 2004, 7, 1061− 1072. (19) Holten-Andersen, N.; Harrington, M. J.; Birkedal, H.; Lee, B. P.; Messersmith, P. B.; Lee, K. Y. C.; Waite, J. H. pH-induced metal-ligand cross-links inspired by mussel yield self-healing polymer networks with near-covalent elastic moduli. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (7), 2651−2655. (20) Barrett, D. G.; Fullenkamp, D. E.; He, L. H.; Holten-Andersen, N.; Lee, K. Y. C.; Messersmith, P. B. pH-based regulation of hydrogel mechanical properties through mussel-inspired chemistry and processing. Adv. Funct. Mater. 2013, 23 (9), 1111−1119. (21) Guo, Z. W.; Ni, K. F.; Wei, D. Z.; Ren, Y. H. Fe3+-induced oxidation and coordination cross-linking in catechol-chitosan hydrogels under acidic pH conditions. RSC Adv. 2015, 5 (47), 37377− 37384. (22) Ko, D. Y.; Moon, H. J.; Jeong, B. Temperature-sensitive polypeptide nanogels for intracellular delivery of a biomacromolecular drug. J. Mater. Chem. B 2015, 3 (17), 3525−3530. (23) Xu, H.; Nishida, J.; Wu, H.; Higaki, Y.; Otsuka, H.; Ohta, N.; Takahara, A. Structural effects of catechol-containing polystyrene gels based on a dual cross-linking approach. Soft Matter 2013, 9 (6), 1967− 1974. (24) Shah, C. B.; Barnett, S. M. Swelling behavior of hyaluronic-acid gels. J. Appl. Polym. Sci. 1992, 45 (2), 293−298.

4. CONCLUSIONS In summary, the present paper introduced two different types of HA-CA hydrogel formation: one including a phase transition from liquid to gel with changing pH, and another where the gel is maintained over a wide range of pH region, regulated by Fe3+-induced covalent and coordination bonding. Fe3+ plays dual roles in these gels, undergoing Fe3+−catechol coordination and catechol oxidation followed by a coupling reaction, which can be used selectively for the different gelation. The phasechangeable HA-CA gel is formed by dominant Fe3+−catechol coordination with immediate pH control. Alternatively, allowing time for curing to form covalent bonds results in color-changeable HA-CA gels with pH control, in which the gel structure is preserved by dual cross-linking with covalent catechol-coupling-based coordinate bonds and electrostatic interactions between Fe3+ and HA-CA. The hydrogels show enhanced cohesiveness and shock absorption with increasing pH due to increasing coordinate bonds. The present gelation strategy, which is inspired by the gelation of the mussel byssus cuticle, is expected to expand the utility of HA hydrogels in biological applications, offering easy control of the phase, gel network, and viscoelastic properties.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b01198. Figures S1−S3 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (D.S.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF no. 2015M2B2A9032029).

■

REFERENCES

(1) Harrington, M. J.; Masic, A.; Holten-Andersen, N.; Waite, J. H.; Fratzl, P. Iron-clad fibers: a metal-based biological strategy for hard flexible Coatings. Science 2010, 328 (5975), 216−220. (2) Sun, C. J.; Waite, J. H. Mapping chemical gradients within and along a fibrous structural tissue, mussel byssal threads. J. Biol. Chem. 2005, 280 (47), 39332−39336. (3) Holten-Andersen, N.; Mates, T. E.; Toprak, M. S.; Stucky, G. D.; Zok, F. W.; Waite, J. H. Metals and the integrity of a biological coating: the cuticle of mussel byssus. Langmuir 2009, 25 (6), 3323− 3326. (4) Holten-Andersen, N.; Jaishankar, A.; Harrington, M. J.; Fullenkamp, D. E.; DiMarco, G.; He, L. H.; McKinley, G. H.; Messersmith, P. B.; Lee, K. Y. C. Metal-coordination: using one of nature’s tricks to control soft material mechanics. J. Mater. Chem. B 2014, 2 (17), 2467−2472. (5) Fullenkamp, D. E.; Barrett, D. G.; Miller, D. R.; Kurutz, J. W.; Messersmith, P. B. pH-dependent cross-linking of catechols through oxidation via Fe3+ and potential implications for mussel adhesion. RSC Adv. 2014, 4 (48), 25127−25134. (6) Schweigert, N.; Zehnder, A. J. B.; Eggen, R. I. L. Chemical properties of catechols and their molecular modes of toxic action in I

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules (25) Hong, S.; Yang, K.; Kang, B.; Lee, C.; Song, I. T.; Byun, E.; Park, K. I.; Cho, S. W.; Lee, H. Hyaluronic acid catechol: a biopolymer exhibiting a pH-dependent adhesive or cohesive property for human neural stem cell engineering. Adv. Funct. Mater. 2013, 23 (14), 1774− 1780. (26) Lee, B. P.; Dalsin, J. L.; Messersmith, P. B. Synthesis and gelation of DOPA-modified poly(ethylene glycol) hydrogels. Biomacromolecules 2002, 3 (5), 1038−1047.

J

DOI: 10.1021/acs.macromol.6b01198 Macromolecules XXXX, XXX, XXX−XXX