Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
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Internal Structure of Hyaluronic Acid Hydrogels Controlled by Iron(III) Ion−Catechol Complexation Jungju Ryu,†,‡ Sunhye Kim,† Inwook Oh,† Sota Kato,§ Takahiro Kosuge,§ Anna V. Sokolova,∥ Jeongwook Lee,† Hideyuki Otsuka,§ and Daewon Sohn*,† †
Department of Chemistry, Research Institute of Natural Sciences, Hanyang University, Seoul 04763, Korea Neutron Science Center, Korea Atomic Energy Research Institute, Daejeon 34057, Korea § Department of Chemical Science and Engineering, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan ∥ Australian Centre for Neutron Scattering, ANSTO, Lucas Heights, New South Wales 2234, Australia
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‡
S Supporting Information *
ABSTRACT: This study explores the internal structure of catechol-functionalized hyaluronic acid (HA−CA) hydrogels with different types of Fe3+-induced cross-linking. We previously reported HA−CA hydrogels cross-linked by covalent catechol coupling and by Fe3+−catechol coordination bonds. Here, we determine the internal structures of these gels using electron paramagnetic resonance, small-angle X-ray scattering, and neutron scattering. Phase-controllable structures were observed in relation to certain pH conditions and gelation pathways. First, we examined the structures of HA−CA gels developed from covalently cross-linked prenetworks, which allow additional Fe3+−catechol coordination bonds of mono, bis, and tris complexes depending on the pH condition. Second, we investigated the structural aspects of the gels preserved by Fe3+−catechol tris complexes, developed from both cross-linked prenetworks and un-cross-linked polymer solutions. The results show that the characteristics of the chains govern the network structures due to the changes in oxidation state of the functional groups, carboxylic acid and catechol, in the given environments. We also discuss the structural aspects, i.e., microphase separation, additional cross-linking within the restricted prenetworks, and locally stretched polymer chains. The observations here suggest that various structural characteristics can be considered to assist a number of different applications using biopolymers.
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environments.9,10 This remarkable feature has been demonstrated with catechol moieties that undergo reactions (oxidation and coordination) driven by Fe3+ ions, which is another component in mussel foot protein 1.9,10 These Fe3+induced catechol reactions have inspired researchers to develop biomedical devices and to impart useful functionalities such as self-healing, adhesion, tunable mechanical properties, and reinforced structures without mineralization.11−13 Thus, as part of the effort to realize novel hydrogels, catechol groups have been introduced on polymer chains, especially cationic biopolymer chitosan.14−16 In our previous work, catechol-modified hyaluronic acid (HA−CA) was successfully synthesized and cross-linked by using Fe3+-induced catechol reactions, i.e., catechol coupling reactions and Fe3+−catechol coordination bonds.17,18 The cross-linking varied with thermodynamic and kinetic conditions. Indeed, there were distinct differences in viscoelasticity, chemical structure, and surface morphology of the
INTRODUCTION Hyaluronic acid (HA), a high molecular weight glycosaminoglycan, is an important biopolymer that has a number of roles in living organisms. It is involved in biological functions such as tissue hydration, water transport, and rheological behavior in synovial fluid.1,2 HA has also been used to develop biomaterials for applications in biomedical and tissue engineering.3−5 Its desirable capabilities depend on its mechanical properties, which allow it to withstand specific loads and transmit them to biological surroundings. One approach to impart appropriate strength to polymer systems in general is to cross-link them. The use of cross-linked polymers has been reported in a variety of medical applications, including medical devices, bioscaffolds in tissue engineering, and drug delivery systems.5−8 We recently reported strategies that can create cross-links in HA chains through modification of 1,2-dihydroxybenzene (catechol), where dopamine is substituted for the carboxylic acid groups of the glucuronic acid units of HA monomers. Catechol is a well-known residue of 3,4-dihydroxylphenylalanine (DOPA) observed in mussel foot protein 1 and is responsible for instant responses as a shock absorber to protect interior areas in harsh © XXXX American Chemical Society
Received: April 30, 2019 Revised: August 8, 2019
A
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. Cross-linking of HA−CA based on Fe3+-induced catechol reactions: Fe3+−catechol coordination bonds and covalent catechol couplings. The gelation strategies were classified as instantaneous cross-linking (type I) and cross-linking with a reaction time gap of a precuring step (type II). The gels (1B) were preserved by Fe3+−catechol tris complexes formed from HA−CA solutions containing Fe3+ ions under pH 10−11. The prenetworks (M) provided various bond types in networks: 2A (pH 2−3), 2A′ (pH 4−5), 2B (pH 7−8), and 2C (pH 10−11). The phases and structures of polymer systems are not indicated here.
and evaluate the inhomogeneities that often exist in gel networks due to long-range polymer density differences. The observations reveal in-situ phenomena of the networks under various pH environments and demonstrate nanoscale behaviors of polymer networks displaying various structural features depending on gelation pathway. This study provides insight into the successful achievement of controllable networks that can be used in biological systems as original materials for drug delivery, bioscaffolds, and medical devices.
hydrogels depending on the type of cross-linking used in formulation.17 However, the structural cause of these properties has not been determined due to the obscured nanostructures of HA−CA hydrogels in the given environments. Here, we use small-angle scattering measurements to explore the internal structures of HA−CA hydrogels that have been cross-linked in specific ways. The HA−CA hydrogels are classified into two groups according to gelation process, i.e., cross-linking from polymer solutions and from prenetworks. The latter is cross-linked covalently by catechol coupling. In gels developed from prenetworks, the networks vary due to pH triggers, which change local environments in a polymer system and control the coordination states, such as the mono, bis, and tris complexes.19 The networks that develop from the solutions are composed of a single type of cross-linking with tris complexes that can be formed under a high pH. HA has a stiff backbone that favors an elongated conformation due to electrostatic repulsion of its carboxylic acid groups. The persistence lengths of HA have been reported to be 40−150 Å depending on ionic environment.20 HA’s solution properties have been studied by Horkay et al. with different salt concentrations and pH ranges that change interactions with neighboring HA molecules.2,21,22 Our study focused on the structural aspects of HA networks. First, we investigated the HA−CA hydrogels prepared from the prenetworks that allow phase-controllable structures by pH triggers. Second, the networks preserved by dual cross-linking (Fe3+−catechol tris complexes and covalent bonds of catechol coupling) were compared to the networks cross-linked by a single type of Fe3+−catechol tris complex. Small-angle scattering results of the HA−CA hydrogels analyze the local structures of polymers in given environments
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EXPERIMENTS
Samples. Catechol-modified hyaluronic acid (HA−CA) and HA− CA hydrogels were prepared as reported previously.17 Carboxylic acid groups of hyaluronic acid (HA, 200 kDa) were substituted with the amine groups of dopamine by a coupling reaction of 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC, Alfa Aesar) and 1-hydroxybenzotriazole hydrate (HOBt, SigmaAldrich, ≥97.0%).17,18 HA was dissolved in Dulbecco’s phosphate buffered saline (WELGENE, Korea), and then dopamine hydrochloride was added to the nitrogen-purged solution. The pH was maintained at 5.0−5.5 for 8 h. The solution was dialyzed (MWCO: 6000−8000 g/mol, SpectraPor) against a pH 5.0 aqueous solution for 2 days and then dialyzed again in deionized water purified in a Milli-Q (Millipore, Germany) system with a resistivity >18 MΩ cm for 1 day. Freeze-dried HA−CA was obtained with 37% substitution of catechol groups from the molar ratio of reactants of HA:EDC:HOBt:dopamine = 1:3:3:3. HA−CA chains with different substitution ratios were synthesized from the molar ratio modulation for 19.7%, 7.4%, and 3.7% substituted HA−CA. The catechol modification ratio of HA− CA was confirmed by 1H NMR (Bruker Advance III, 400 MHz). For the HA−CA hydrogels, the 37% substituted HA−CA was dissolved in deionized water at 3 wt %, and Fe3+ ions were added into the HA−CA solutions by preparation of an iron(III) chloride hexahydrate solution of 80 mM in 5% v/v acetic acid. The HA−CA solutions were treated with 100 μL of HA−CA homogeneously mixed B
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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stabilization for more than 1 h. The samples were measured at 25 ± 0.5 °C and maintained by using a circulation bath. The data were reduced on an absolute scale relative to the direct beam by using Mantid,23 and then solvent scattering was subtracted from that of the samples. The calculated contrast factor (ρHA−CA − ρD2O)2 was 1.50 × 10−11 Å−4, which did not vary with catechol modification ratio due to the similar neutron scattering length densities of the repeating units containing catechol and carboxylic acid. Scattering Intensity of Semidilute Solutions and Gels. The scattering intensity from polymer systems, gels, and semidilute solutions can be approximated with scattering functions that describe the contributions of spatial inhomogeneities, Istatic(q), and the concentration fluctuation of the polymer chains, Idyn(q): I(q) = Istatic(q) + Idyn(q).24,25 In solutions and gels that do not perturb the polymer chain conformation, Idyn(q) is given by the Ornstein− Zernike equation.23,24 The scattering intensity that emerged from the static inhomogeneities that is often observed in long-range scale is described with a Debye−Bueche term.24,26−28
with 11.7 μL of Fe3+ ions to give a 1:3 Fe3+:catechol molar ratio. The hydrogels were prepared with two different processes according to the presence of Fe3+-induced covalent cross-linking, as shown in Figure 1. First, the polymer solutions immediately produced networks with Fe3+−catechol tris complexes after addition of 20 μL of 1 M NaOH into the HA−CA solution, providing coordination bonds without preconstructed networks (type I). Second, pH-controlled phases were attained after a gelation time of 13 min, at which point Fe3+-induced covalent bonds were formed through the type II process, as shown in Figure 1. This gelation time was determined by an oscillatory time sweep process that provided steep increases in moduli at 825 s, showing elastic behavior.17 These prenetworks allowed pH-tunable phases in the range pH 2−11 with the addition of 10−20 μL of 1 M HCl or 1 M NaOH, as shown in 2A−2D of Figure 1. HA−CA solutions were additionally prepared by using polymer chains with catechol modification ratios of 3.7%, 7.4%, and 19.7%. The polymers were dissolved in deuterium oxide (D2O, Sigma-Aldrich, 99.9%) at a concentration of 1 wt %, followed by addition of Fe3+ ions and NaOH considering the same molar ratio of Fe3+:catechol of 1:3. Electron Paramagnetic Resonance (EPR). EPR measurements were performed on a JEOL JES-X320 X-band EPR spectrometer equipped with a JEOL variable temperature controller (JEOL ES13060DVT5). The spectra of samples were measured using a microwave power of 5−50 mW and a field modulation of 0.4 mT with a time constant of 0.03 s and a sweep rate of 0.25 mT/s at −150 °C. The samples were carefully inserted into EPR tubes with a diameter of 3.75 mm and a height of 43.5 mm. All measurements were performed within 20 min, from preparing the HA−CA gels to setting the EPR tubes into the EPR spectrometer. The detected signals were assessed qualitatively, considering the limitation of accurate weights of highly viscous samples and different amounts of the solvent water, which has a high dielectric loss. UV−Vis Spectrophotometry. UV−vis spectrophotometry was performed to monitor the oxidation of the Fe3+-induced HA−CA by using a UV−vis spectrometer (OPTIZEN 3220UV, Mecasys Co., Ltd.). The samples were loaded in 1 cm thick quartz cuvettes. The polymer solutions were prepared by dissolving 50 μm of 3 wt % HA− CA in 3 mL of 5% acetic acid containing iron cations (molar ratio of Fe3+ and catechol = 1:3). The high-pH samples were prepared by adding 1 M NaOH to the polymer solutions and to the aged solution. The polymer solutions were measured with the incubation time for oxidation of catechol groups. The formation of tris complexes was investigated in two different high-pH samples. Small-Angle X-ray Scattering (SAXS). SAXS experiments were performed at the Pohang Accelerator Laboratory in Korea to observe the internal structure of CA−HA hydrogel controlled by iron(III)− catechol complexation. The samples were measured on the 4C beamline with X-rays of 13.6 keV (resolution (ΔE/E): 2 × 10−4), and scattering patterns were obtained in the range of 0.005 Å−1 < q < 0.12 Å−1 (q = 4π sin(θ/2)/λ) at a distance of 5 m between the sample and the detector. Here, q, θ, and λ are the scattering vector, the scattering angle, and the wavelength, respectively. θ was calibrated with a standard sample of polyethylene-block-polybutadiene-block-polystyrene (SEBS) block copolymer. Scattering intensity was collected with a two-dimensional (2D) charge-coupled detector (Rayonix SX165, USA). Hydrogel samples with 2−3 mm thickness were loaded between Kapton windows. Empty cell scattering was subtracted from each data set. To avoid radiation damage, the time of each measurement did not exceed 120 s. Small-Angle Neutron Scattering (SANS). SANS was performed on the Bilby instrument23 at the Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology Organization (ANSTO), Australia. The instrument was used in time-of-flight mode. Wavelengths λ from 0.4 to 1.8 nm and Δλ/λ ≅ 10% were used for data reduction. The rear detector was placed 10 m from the sample. The top and bottom panels and left and right panels of the front detector were located 4 and 3 m from the sample, respectively. Then, 1 wt % HA−CA solutions were prepared in D2O followed by addition of Fe3+ ions and NaOH. The dispersed polymer solutions were loaded into Hellma quartz cells with a 2 mm thick path length after
I(q) =
Istatic(0) (1 +
ξ12q2)2
+
Idyn(0) 1 + ξ2 2q2
(1)
where ξ1 and ξ2 are the correlation lengths of two-phase structure with a sharp boundary27,28 and of polymer concentration fluctuations. In eq 1, the static term can be modified with the Guinier function which assumes the radius of gyration, Rg, as noninteracting domains of higher and lower monomer densities distributed in the networks.26 When scattering is observed in the range of qξ1 ≫ 1, a power law function is employed. The fractal structure arranged with noninteracting size, ξ, is expressed with eq 2, which provides parameters of fractal dimension, D, and correlation length, ξ.29 The equation is expressed with a Debye−Bueche form when D = 4. This model was applied to describe incomplete networks. I(0)
I(q) =
D /2
(1 + ( )ξ q ) D+1 3
2 2
(2)
In HA chains with semirigid character, the scattering function of eq 1 is modified with the second term of Idyn(q) ∼ q−1 that describes the local structure as rodlike chains.30 Considering the geometry of rodlike chains with a cross-sectional radius rc, the scattering function can be replaced with the equation25,31
I(q) =
Istatic(0) (1 +
ξ12q2)2
+
Idyn(0) 2 2 1/2
(1 + ξ2 q )
1 1 + rc 2q2
(3)
For the polymer gels and solutions of this study, the internal structures were interpreted on the basis of the concept of static and polymer concentration fluctuation contributions in scattering intensity by using the relation I(q) = Istatic(q) + Idyn(q). The scattering functions were adopted with proper modifications according to the characteristics of the polymer systems. Data analysis was performed using a package distributed by the National Institute of Standard Technology (NIST).32
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RESULTS AND DISCUSSION Cross-Linking of HA−CA Chains. HA−CA chains were cross-linked by Fe3+-induced catechol reactions, following the gelation strategies in Figure 1. Fe3+−catechol coordination bonds of tris complexes preserved the networks of 1B and 2C through different gelation paths (types I and II). The linkages arose instantaneously when the HA−CA solutions containing Fe3+ ions, 1A, were exposed to a high pH (type I). The solutions, 1A, could also develop another state, M, based on covalently cross-linked catechol groups, which were slowly generated from coupling reactions between the Fe3+-induced oxidative catechol group, o-quinone, and another catechol C
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules group.9 The gradual formation of o-quinone was observed by UV−vis spectrophotometry in Figure S1. The sample M has the absorption band at 410 nm which increases for the precuring time. The peak intensity increased over 13 min, and then it was saturated in a measurement time. As an intermediate state cross-linked partially by catechol coupling, the networks of M gave rise to prenetworks for further reactions to occur. This state was determined by a stage showing steep increases in elastic response (G′ > G″, where G′ and G″ are the storage and loss moduli, respectively) at around 13 min.17 These prenetworks could allow various structures (2A, 2B, and 2C) to be tuned by the pH, which may be involved in polymer conformational changes including coordination bonds between catechol groups and Fe3+ ions (type II). The respective samples consisted of different states of coordination bonds with mono, bis, and tris complexes, providing UV absorption peaks at 490, 520, and 714 nm, respectively, depending on the pH, as reported by our group and by other researchers.17,33 Effect of pH on Cross-Linking. For networks in a wide pH range of pH 2−11 (type II), the features of complexation, Fe(cat), Fe(cat)2, and Fe(cat)3, were estimated from the EPR results in Figure 2. The pH-tuned hydrogels showed two
groups. We focused on the representative signal of g = 1.003 to compare the intensity and integration of Fe3+ with those of the catechol group. Figure 2a shows the EPR spectra of the samples, indicating 2A at pH 2−3, 2A′ at pH 4−5, 2B at pH 7−8, and 2C at pH 10−11 through the type II process. By use of the distinct peaks at g = 4.2 and g = 1.003, the intensity and integration ratios for Fe3+/catechol are summarized in Figure 2b. The ratios from the tris complex of 2C were highest and then decreased toward 2B and 2A′ in the bis and mono complexes, respectively. Sample 1B also had a strong relative intensity at g = 4.2 in Figure S2. These results were consistent with earlier reports. Weisser et al. reported that the intensity at g = 4.2 could distinguish the spectral features among the coordination modes, suggesting that these differences in the spectra were attributed to differences from the completely rhombic high-spin Fe3+.35 The peak ratios of 2C were slightly higher than those of 1B, implying that 2C is composed of relatively well-organized tris-coordination bonds. In Figure S1, the UV−vis absorbance band at 480 nm coming from tris complexes was also slightly stronger in 2C. The results infer Fe3+-mediated polymer chains are stabilized with the time in the presence of Fe3+ ions. Fe3+ ions play dual roles being involved in both coordination and catechol coupling reactions. They accelerate the o-quinone formation at pH 3−4 (M), resulting in the stronger peak of 410 nm in 2C than that in 1B. Otherwise, Fe3+−catechol tris complexes are preferred over catechol coupling reactions in high pH because the metal coordination is instantaneous.19 As shown in Figure 2 and Figure S2, 2C and 1B provided distinct results from tris Fe3+−catechol complexes, displaying a strong signal at g = 4.2, which diminished in 2A′, 2B, and 1A. The morphologies of the various hydrogels were observed upon the pH-dependent cross-linking types, as shown in the optical microscope images (Olympus, BX51) of Figure 3. We dwell on the internal structures of the polymeric systems cross-linked in different environments. pH-Dependent Structure of HA−CA Hydrogels. Figure 4 shows the SAXS profiles of the HA−CA hydrogels prepared through the type II process. The obviously different scattering patterns exhibited phase-controllable structures in the pH range of 2−11. Polyelectrolytes generally lead to hierarchically ordered structures driven by interactions such as electrostatic and van der Waals forces.36 At low pH, the scattering profiles showed scattering peaks that indicated the presence of domains of localized chains with higher polymer concentrations. When charges were compensated, the attraction of the polymer chains became strong, resulting in development of regions with high polymer concentrations. In gels, macroscopic precipitation was not seen. Instead, the volume of the networks was favorably maintained due to the entropy balance of counterions from the neutralized ion−polymer regions.37 This process led to microphase separation, allowing for spatial order.37 HA is a weak polyelectrolyte in which anionic charged chains are neutralized for the following reasons: (1) carboxylic acid and catechol groups are deprotonated at low pH38 and (2) the gels contain Fe3+ ions at ∼8.4 mM. Thus, as a neutralized system at low pH, 2A showed the scattering feature of polymer-dense domains in the intermediate q range (0.01 Å−1 < q < 0.03 Å−1), where the estimated d-spacing (d = 2π/q) was 56.1 ± 0.1 nm at q = 0.0112 Å−1. The distance of 2A′ decreased to 29.1 ± 0.4 nm at q = 0.0216 Å−1, and scattering peaks were not found in 2B and 2C with higher pH.
Figure 2. EPR results of HA−CA hydrogels prepared by the type II process as a function of pH. (a) EPR spectra and (b) intensity ratios and integration ratios for 4.2 ≤ g ≤ 1.003, assigned to Fe3+ ions and catechol groups, respectively.
distinctly detectable components, a high-spin character of Fe3+ ions, and organic resonances derived from the catechol groups. The g value was undoubtedly attributable to the Fe3+ ions given its value of 4.183, which is ∼4.2 in the literature.34,35 The feature of g = 4.2 is found in high-spin Fe3+ with a completely rhombic environment.35 A value in the range of g = 1.3−0 was assigned to the organic resonance structure of the catechol D
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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Figure 3. Optical microscope images of the lyophilized hydrogels: (a) 2A, (b) 2B, (c) 2C, and (d) 1B.
gave rise to the radius of gyration, Rg, which increased from 27.5 ± 0.1 to 37.7 ± 0.1 nm during the time-evolution study. Thus, the results suggest that the neutralized chains could be entangled with domains in the intermediated region (R (= d/ 2) ∼ 19 and 28 nm) and generated large aggregations (Rg ∼ 28 and 38 nm) in the networks. The scattering intensity increased beyond the measured low-q range without scattering peaks in the sample stabilized for 2 h. A power law fitted the low-q region with a slope of −4, while the intensity of the high-q region was described with I(q) ∼ q−3. The results indicate that the ordered structure of the polymer-concentrated region changed to a surface fractal structure39 where the periodic domains were not valid. The spatial distances of the long-range inhomogeneities in regions dense and poor in polymers became longer. This scattering pattern provided the apparent correlation length, ξ = 11.5 ± 1.3 nm, by using eq 1 with the floating parameters of the fractal dimension. In 2A, the addition of protons induced structural changes of the networks based on microphase separation of the polymer chains, which formed polymer domains and aggregations. The networks of 2A originated from the intermediate sample of M, which was intended to have partial cross-linking, and were preserved by covalent cross-linking of catechol coupling, which reacted slowly. In pH 2−3, the chains are deprotonated, and mono complexes are not clearly shown because catechol and carboxylic acid have pKa values are 9.45 and 2.9, respectively.38 Thus, it is presumed that the phase separation continued to increase in size due to the pH-dependent behavior of HA−CA chains, which can aggregate in the precured networks. In particular, when Fe3+ ions are absent, the HA−CA chains show the proton-induced the correlation peak at 0.010 Å−1 (Figure S3), corresponding to the intensive scattering at q* = 0.011 Å−1 of the pattern of 2A. Figure 5a suggests the deprotonated chains that can stick each other give rise to fractal structure where the domains are merged. Otherwise, the covalent crosslinking mainly contributes to the decreases in size at the low-q region, q < 0.02 Å−1, which is described in next section. Therefore, the deprotonated chains have responsible for the microphase separation in 2A.
Figure 4. SAXS patterns of the intermediate networks of M and 1 h stabilized HA−CA hydrogels prepared with different pH conditions in the type II process and stabilized for 1 h.
For several samples, the scattering patterns deviated according to stabilization time. Figure 4 shows the results of gels stabilized for 1 h after pH treatment of sample M. However, it required more time to reach equilibrium, especially for samples at low pH. Figure 5a shows the evolution of scattering patterns over time after pH triggers for sample 2A. For the samples stabilized within 1 h, the profiles indicated an increase in domain size evaluated at scattering peaks in the intermediate q range. The intensity of these peaks decreased after incubation for 2 h. The domain sizes of the intermediate region were estimated at the broad peak positions which were determined by eq S1, as shown in Table S1. The distance d at q* increased from 36.7 to 56.1 nm from 20 min to 1 h, while parameter ξ2 was maintained at values of 20.1 and 19.3 nm, respectively. The scattering upturn at the lower q implied the presence of aggregated domains at larger distances. The distances in this regime were approximated with the Guinier function, I = exp(−q2Rg2/3), modified in the first term of eq 1.28 The results E
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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14.4 to 18.4 nm, as presented in Table S3. These results show the presence of large inhomogeneities that continued to increase in size, though the structural changes were not rapid compared to those of 2A. In Figure 5c, sample 2C presents a different scattering pattern that decreased monotonically with q. The scattering intensity had a power law fit of I(q) ∼ q−1 in the low-q region, and the intensity of the high-q region resulted in I(q) ∼ q−2 as features of polymer concentration fluctuations. The scattering patterns were analyzed using eq 1, which contained a power law term, due to the absence of data from the low-q region. The second term in provides the correlation length, ξ, from the polymer characteristics, found to be ξ = 6.8 ± 0.04 nm in the samples at 1 and 2 h. The values increased relative to that of the sample stabilized for 20 min and did not significantly vary after 1 h, as shown in Table S4. The scattering intensity in the high-q region depended on the q−2 behavior, which differed from the features observed in polyelectrolytes showing I(q) ∼ q−1 due to cross-linking via a two-step processes that introduces covalent bonds and in turn produce coordination bonds. With increases in the pH, the networks could be reorganized over the cross-links already formed, especially in the gel of 2C that contained additional anchoring regions where catechol moieties encounter Fe3+ ions at a ratio of 3:1. Thus, these structural changes could occur on relatively large length scales. The results demonstrate that the networks were reorganized with a parameter value of ξ = 6.8 nm and long-range distances showing stiff behavior due to pH triggers (pH = 10−11). The values of ξ increased relative to that of the sample stabilized for 20 min and did not significantly vary after 1 h, as shown in Table S4. Fe3+−catechol coordination bonds stabilize the catechol against the autoxidation. However, the coordination-based structure can also change with time through further oxidation of tris complexes. It has been reported that Fe3+ ions in tris complexes can be reduced to Fe2+, while the catechol is oxidized to semiquinone which react with oxygen to generate other radicals.40 Upon structural changes of polymer networks preserved by tris complexes, Takahara et al. reported the structure of catechol coordinative networks changed after 5 h.41 The network of 2A was almost constant in 2 h, showing stable structures against the further oxidation. Structure of HA−CA Hydrogels Using Reaction Time Gap. We compared the structures of HA−CA hydrogels prepared using reaction time gap, which provide the prenetworks of M. Figure 6 shows the time-dependent scattering patterns of M. The profiles of M exhibit an almost a single slope that is ambiguous to describe with two features for typical gel networks. The actual states of M demonstrate the partially cross-linked networks, which are considered as a fractal structure inside a finite correlation length. Thus, the patterns of M were analyzed with eq 2. We estimated two parameters of fractal dimension and correlation lengths from the scattering intensity that varies slightly with the curing time. As shown in Figure 6b and Table S4, the structure of M shifted to a mass fractal structure (2 < D < 3) from the swollen structure (D = 1.75), displaying the correlation lengths that gradually reduced to 15.8 nm from 20.1 nm, according to the time for further oxidative cross-linking. Consequently, the results indicate that the networks become dense with the oxidative cross-linking density. With regard to the networks developed from the precured M, we compared the 1 h stabilized samples of Figure 4. The
Figure 5. SAXS patterns of samples (a) 2A, (b) 2B, and (C) 2C stabilized for 20 min, 1 h, and 2 h. The lines are fitting results.
When the pH increased to 7−8, sample 2B showed scattering patterns that consisted of two features following I(q) ∼ q−4 on a large length scale at q < 0.02 Å−1 and I(q) ∼ q−2 on a small length scale at q > 0.02 Å−1, as shown in Figure 5b. The profiles could be analyzed by using eq 1, but the intensive scattering of the low-q region deviated from the fits, as shown in Figure S4. The scattering characteristics of the polymer-dense domains of microphase separation were significantly reduced, but the observed low-q features could be considered to be the result of noninteracting aggregation described with the Guinier function. In this pH range, the catechol groups participate in the formation of bis complexes with Fe3+ cations, while carboxylic acid groups are protonated.37 Thus, the coordinative catechol groups can lead to produce distinguished locals including further cross-linking in the prenetworks, M. The values of Rg increased over time after pH treatment via addition of NaOH, with Rg increasing from F
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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relation of Rg2 = 3ξ2 under the assumption that the polymer segments of the domains follow a Gaussian distribution.42 With increases in pH, 2B shows ξ = 10 nm, which decreased from that of the prenetworks. Another fitting model, the Guinier function, was applied to improve the fitting results of 2B that has intensive scattering at q < 0.02 Å−1. The results provided Rg = 14 nm, which was approximated to ξ = 8.9 nm. The results support the structure of 2B becomes dense with coordinative connections of bis complexes on the oxidative cross-linking networks. Thus, the additional cross-linking of bis complexes of 2B enhances the scattering intensity of the low-q region, implying the presence of dense domains developed on the covalent cross-linking of M. Otherwise, the high pH triggers organize the networks based on the extended chain behavior and stable tris complexes. 2C exhibited the monotonically decreasing slope of intensity with q−1 and the short lengths ξ = 6.8 nm, resulting in relatively well-distributed networks. We summarized the structural aspects of the hydrogels in Figure 7. The sample M shows a fractal structure (ξ = 20 nm) which provides an initial state to form networks depending on pH: 2A, 2B, and 2C. The polymer domains arise due to the microphase separation by deprotonation of the charged polymer chains in 2A, and the dense polymer regions with bis complexes appear in 2B. The correlation lengths approximated from the domain size of 2A and 2B decreased in the basic gels, 2C and 1B, which consists of tris complexes. The differences of correlation lengths between 2C and 1B describe that the precuring step provided the increases in ξ due to the additional connections associated with the covalently cross-linking regions. Structural Differences between 1B and 2C. Samples 1B and 2C were preserved by coordinative cross-linking of Fe3+− catechol tris complexes, a mussel-inspired reaction. The coordination bonding takes place instantaneously when the samples are exposed to a pH 10−11 environment. Meanwhile, under lower pH conditions, pH 4 in this study, the covalent bonds of Fe3+-induced catechol groups were generated. Here, the Fe3+ cations played the role of an oxidizing agent to produce o-quinone, which participated in coupling reactions with other catechol groups. This reaction was relatively slow. The instantaneous reaction of Fe3+−catechol tris coordination interrupted the formation of o-quinone in a pH 10−11 environment, but the mono complexes generated in pH 5 allowed cross-linking by catechol coupling reactions.17 Thus, the networks of 2C were formed with dual cross-links consisting of catechol-coupling covalent bonds and Fe3+− catechol tris complexes. In Figure 8a, the scattering intensity of the intermediate networks, M, increased drastically due to the curing of HA− CA in an aqueous solution, of which the SAXS pattern
Figure 6. SAXS patterns and fitting parameter values for fractal dimension and correlation lengths of M with time evolution.
scattering profiles were interpreted as shown in the previous section, considering the structures described with a mass fractal or a system containing noninteracting domains. The resulting parameter values of ξ and Rg are noted in Table 1. The Table 1. Correlation Lengths of Samples through Types I and II 2A ξ (nm)
a
22
M
2B
2C
1B′
1B
20
a
6.8
3.0
1.7
8.9 (10)
ξ was calculated by using the relation Rg2 = 3ξ2. The values of Rg are 38 and 14 nm in 2A and 2B, respectively.
a
domains of 1A (Rg = 38 nm) arise from the fractal structure with ξ = 20 nm of the precured M due to the microphase separation where the deprotonated chains can produce a correlation peak in Figure S3. For the consecutive comparisons, the correlation lengths were approximated with the
Figure 7. Structural aspects of (a−d) pH-dependent networks from type II process and (e) from type I. The arrows indicate correlation lengths, ξ. G
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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Figure 8b shows the scattering patterns of samples processed through type I. In pH 8−9, sample 1B′, which is not a gel due to the insufficient cross-linking of bis complexes, shows that the scattering intensity dramatically increased with the formation of inhomogeneous regions. In pH 10−11, the cross-links of sample 1B began with pH-triggered tris complexation from the HA−CA solution. The scattering pattern of 1B showed the typical behaviors of the two regimes of long-range inhomogeneities and polymer concentration fluctuations. The scattering intensity (q < 0.01 Å−1) associated with the inhomogeneities followed I(q) ∼ q−2 in 1B, while the scattering intensity of 1B′ shows I(q) ∼ q−3.5 in 1B′. The analysis using eq 1 provided ξ = 1.7 nm of 1B, which is shorter than ξ = 3.0 nm of 1B′. The results showed the gelation in high pH produces the relatively homogeneous structure. The scattering intensity of 1B decreased more steeply with a slope of −2.2 compared to that of 2C with a slope of −1.4 in the low-q region. In contrast, the intensity in the high-q region decreased monotonically with q−1, while the scattering intensity of 2C showed q−2 behavior. This implies that the gel networks depend on the structures formed in the previous stage as solutions or gels. In pH 10−11, the scattering pattern of HA−CA solution shows the decreases in intensity with q−1 that indicates crosssectional lengths of molecules at q > 0.1 Å−1 (Figure S3). The extended chains can influence on the network structure, as observed in 2C that follows I(q) ∼ q−1 at the low-q region. The extended chain behavior of 1B is shown at q > 0.02 Å−1. The results suggest that the prenetworks may provide the bulky regions which lead to the structure with larger correlation lengths (ξ = 6.8 nm) for the networks of 2C. In the case of 1B, the free chains are connected directly by coordinative crosslinks with tris complexes, and consequently, the shorter correlation lengths (ξ = 1.7 nm) were noted. However, the structures of both 2C and 1B are preserved by tris complexes which provide relatively larger correlation lengths compared to ξ = 0.83 nm HA−CA solution without cations in pH 10−11, as shown in Tables S4 and S6. The high-q feature of 1B indicated stiff chain behavior, as observed in anionic polysaccharides.22,25 For this plot, we employed eq 3, where the power law term fitted the excess scattering in the low-q region. The scattering intensity in the high-q region described a rodlike behavior with a crosssectional radius (rc), with values of ξ and rc at 3.0 ± 0.04 nm and 5.3 ± 2 Å, respectively, from eq 3, as shown in Table S7. These values were in good agreement with those reported for HA semidilute solutions by Horkay et al.22 In pH 10−11, the HA−CA solutions without metal ions provide the chain conformations with ξ = 1.25 nm and rc = 0.24 nm (Tables S6 and S7). The results showed that the polymer characteristic lengths of 1B arose from the stiff chains of HA−CA solutions and that the shorter correlation lengths differed from the longer distances of the chains in 2C networks. Semirigid Chains in HA−CA Solutions and Gels. Sample 1B appeared to contain hydrogel networks with the polymer characteristic of chain stiffness. The semirigid chains of 1B were evaluated with the persist length, lp, that participates in the infinite connection of the networks, compared with lp of HA−CA chains forming clusters. The solutions of HA−CA with different catechol modification ratios were prepared by dissolving polymers with a concentration of 1 wt %, followed by an addition of iron
Figure 8. SAXS patterns of 1B and 2C prepared through the different paths. (a) 2C developed through M which had preoxidation time in HA−CA solution. (b) 1B and 1B′ developed from HA−CA solution.
exhibited intensity depending on q−1, a behavior typically observed in stiff chains. These increases in scattering intensity are often accompanied by formation of inhomogeneities during the gelation process. The profile of M exhibited a steep decrease in scattering intensity with a slope close to −2. With increases in pH, the networks of 2C would be distorted strongly because three catechol functional groups from neighboring chains participate in the formation of a coordination bond. Furthermore, the polymer chains in 2C showed increases in the correlation lengths, associated with the local lengths of the covalently cross-linked area. This structural change was accompanied by rearrangement of the HA−CA chains, which tended to persist from the state of M cross-linked at pH 4, as shown by the scattering intensity that decreased monotonically with a slope of −1 in the low-q region. Thus, we anticipated that long-range inhomogeneities were organized with bundle-like aggregations in the measured q range of qξ1 ≫ 1. We examined the basic gels prepared from the prenetworks in pH 10−11, according to the molar ratios: 1:3 (2C), 1:6, and 1:12 of Fe3+:catechol. As shown in Figure S5 and Table S4, the samples with the ratios mismatching for tris complexes (1:6 and 1:12) exhibit that the intensity coming from large inhomogeneities increased at the low-q region, while the correlation lengths decreased. Therefore, the results support that tris complexes connect the extended chains associated with partially cross-linked regions and provide the less defective structure. H
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules chloride salts in a high-pH condition. These polymer chains formed clusters but not a gel state. The solutions were subjected to SANS measurements to investigate the propensity of characteristic lengths at catechol modification ratios of 3.7%, 7.4%, and 19.7%. In Figure 8a, the scattering intensity at low q characterized the clusters, indicating a mass fractal structure with I(q) ∼ q−2.5. The features in the high-q region demonstrated polymer concentration fluctuations for neutralized chains showing q−1 behavior. These two features were distinguished in the range of 0.01 Å−1 < q < 0.02 Å−1. Power law fitting was performed for the two regions of q with slopes −2.5 and −1. The results gave rise to a crossover point at qx = 0.014 Å−1 in the sample with a modification ratio of 3.7%, and the values of qx were slightly shifted toward qx = 0.013 Å−1 with increased modification ratio. The crossover points yielded information about aggregation generated by polymer chains that have a total size described with persistence length, lp, over a range of lp = 1/ qx, providing lp = 7.1 nm in the sample with the lowest modification ratio (3.7%) and lp = 7.7 nm in the other two samples (7.4% and 19.7%). These values were comparable to the results regarding the persistence lengths of hyaluronic acid in the literature.43,44 Thus, it was difficult to conclude that the estimated persistence lengths were correlated to the number of catechol groups. The results suggest that the intrinsic chain characteristics of hyaluronic acid are dominant factors contributing to the overall structure of the networks, though it is true that the catechol functional groups contributed to the formation of bonds at local points. We note that the scattering pattern in the gel state of 1B accounted for the stiff chain characteristics of HA−CA in Figure 9a. The persistence length was estimated at qx = 0.016 Å−1, where two power law fits intersected at a crossover point between the slope of −1 and the upturn of the scattering intensity from inhomogeneities, as shown in Figure 9b. This evaluation resulted in a persistence length of 6.2 nm. The obtained value of lp differed from the correlation lengths, which had a shorter length scale. This deviation implied that the concentration fluctuation modes were not isotropic to the chain axis.25 The shorter length of lp could be observed due to the polymer concentration that produces networks (3 wt %), as the observations of the concentration-dependent ξ and lp in gels45 and in semidilute polyelectrolyte solutions.46 These results showed that the structure of 1B was close to that of the initial solutions developing networks from concentrated solutions. For the relationship between an incipient gel and a completed network, Adolf and Martin suggested that the fully cured networks have some remnant of fractal structures of the incipient gel, bringing into question the homogeneous network assumption that describes distinguishable structure against the heterogeneous incipient gel.47 Our observations provide one of the possible cases that show the structural resemblance between the completed gels and the prenetworks, as the system connected with metal mediated catechol groups dangling on polymer chains. Thus, these observations suggested that the 1B networks were organized with persistence lengths of HA−CA chains, as shown in the simplified structure in Figure 10a, where the represented structure was associated with the long-range inhomogeneities in the microscales observed by small-angle scattering. The results also showed that the structure of 1B differed from that of 2C emerging from the prenetworks of M,
Figure 9. (a) SANS patterns of polymer solutions in terms of catechol modification ratio. The blue and purple patterns are scaled by 10 and 100 for clarity, respectively. (b) SAXS patterns of 1B and the data fits showing crossover point between two scattering features.
as presented in Figure 10b, suggesting the initial states before gelation governed the final gel structures.
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CONCLUSION HA, an important biopolymer, was cross-linked by introducing catechol moieties that were modified on the chain. Based on Fe3+-induced catechol reactions, various types of networks were presented. In this study, their internal structures were explored to improve our understanding of networks originating from different gelation strategies and to support the development of desirable biomaterials. Catechol moieties can take part in two different reactions in the presence of Fe3+ cations, leading to covalent bonds and to coordination bonds. These reactions take place competitively; i.e., covalent bonds appear slowly in a low-pH range, whereas coordination bonds are formed instantaneously in a high-pH range. With the reaction time gap between them, the two different gelation processes were considered to prepare various hydrogels. From the aged solutions, phase-controllable hydrogels were obtained by pH triggers (type II). Another method was gelation by instantaneous coordination bonds at high pH (type I). First, we probed the phase-controlled structures over wide pH ranges. EPR results proved the formation of coordination bonds, specifically the tris complex, which was responsible for the cross-linking induced by high-pH triggers. SAXS measurements revealed the structural changes associated with pH-dependent phase behavior. Second, hydrogels I
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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Figure 10. Representative scheme of HA−CA networks (1B and 2C) cross-linked in pH 10−11. The tris complexes are generated through different pathways: (a) instantaneous cross-linking from solutions and (b) cross-linking using a reaction time gap for the prenetworks.
the results indicate that formation of clusters was associated with semirigid chain segments at a size range corresponding to the q values of 0.013−0.014 Å−1. The persistence lengths in 1B were also noted to have similar values in the measurements of the solutions. This indicates hydrogel structures were derived from polymer chains that respond in given environments. When the gels were prepared through the pathway of type II, they did not exhibit stiff chain behavior. For tris complexation of 2C, three catechol groups from neighboring chains were necessary to form a cross-linking point. Therefore, the structures were associated with rearrangements from the prenetworks, M, as shown by the two features I(q) ∼ q−1 and I(q) ∼ q−2 in long-range and short-range scales, respectively, in sample 2C. Consequently, the distinct structural differences between 1B and 2C implied that the gelation pathway determined the hydrogel structures.47 The findings of various structures of HA−CA hydrogels suggest that the networks were controlled by gelation environment, which affects the properties of the polymer chains. In other studies of polymer gels using catechol groups in a mussel-inspired gelation process identical to that of 1B, catechol-functionalized neutral chains of polystyrene exhibited scattering intensity with q−2 behavior.52 In polyelectrolyte gels, stretched polymer chains were observed in chitosan gels and polyacrylate gels.50,51 Microphase separation has been reported in other polyelectrolyte gels in poor solvents depending on pH and salt concentration.53,54 In this study, catechol-modified HA polymers produced various structures based on pH and gelation pathway. The results allowed determination of the structures expected in the given environments, and they improved our knowledge of phenomena in polymer systems, which should be understood to achieve innovative applications in biomaterials and bioengineering fields.8,55,56
prepared through different gelation pathways were compared. The small-angle scattering results of HA−CA gels and solutions revealed how the networks are influenced by the gelation processes of types I and II. The nature of the stiff chain of HA−CA in a solution, I(q) ∼ q−1, was observed in the gel of 1B, and the dual-cross-linked gels of 2C gave rise to the high-q features originating from the prenetwork structure developed in the aged solutions. The hydrogel structures were governed by the given pH triggers that change conformational characteristics of the HA− CA chains and cross-linking types. With regard to the pHdependent chain conformations, similar to the results of 1B, the locally stretched chains have been observed in other polysaccharide polymer gels and solutions.22,25,48,49 This rigid chain behavior was also correlated to the results of polyelectrolyte gels synthesized by means of radical polymerization of monomers in a salt solution.50,51 Thus, our observations indicate that the gel networks depend on the characteristics of polymer chains under the given circumstances but not on the cross-linking type regardless of whether gelation occurs due to polymers or monomers, i.e., connections of polymer chains and growth by radical polymerization. With the pH triggers, the magnitudes of the protonation of the functional groups carboxylic acid and catechol were regulated. Thus, deprotonated polymer chains induced microphase separation in the networks, as with sample 2A, and the protonated catechol groups participated in coordination bonds, of which the states also depended on the pH. The pH-dependent cross-linking types contribute to form various networks. The state of M cross-linked partially with covalent bonds, providing prenetworks for further connections showing a fractal structure with I(q) ∼ q−2. We also observed that the coordination bonds of tris complexes support the relatively well-distributed networks compared with that of bis complexes, on the basis of the results of two groups: 2B, 2C and 1B′, 1B. The structures of HA−CA gels were determined by gelation pathway. When the hydrogels were established directly from polymer solutions, the persistence lengths created the network structures. With regard to the influences of catechol groups on persistence lengths, HA−CA solutions were investigated. There was no obvious propensity in modification ratio, but
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.9b00889. UV−vis spectrophotometry, EPR spectra of sample 1B, SAXS patterns of HA−CA solutions without Fe3+ ions, SAXS patterns and fitting results for 2B, SAXS patterns J
DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
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of the samples developed from M with different molar ratio of Fe3+ ions, tables of the fitting parameter values of the examined samples, NMR spectra of CA−HA (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hideyuki Otsuka: 0000-0002-1512-671X Daewon Sohn: 0000-0002-7200-9683 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (2017M3A9G8084539). The authors acknowledge the support of the Pohang Accelerator Laboratory (PAL) in providing the X-ray beamline. We are grateful to the ANSTO, ACNS, Australia, for access to the BILBY instrument.
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ABBREVIATIONS EPR, electron paramagnetic resonance; HA, hyaluronic acid; HA−CA, catechol-modified hyaluronic acid; SANS, smallangle neutron scattering; SAXS, small-angle X-ray scattering.
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DOI: 10.1021/acs.macromol.9b00889 Macromolecules XXXX, XXX, XXX−XXX
