Article pubs.acs.org/Macromolecules
Cobalt Cross-Linked Redox-Responsive PEG Hydrogels: From Viscoelastic Liquids to Elastic Solids Seraphine V. Wegner,*,†,‡,§ Franziska C. Schenk,†,‡ Sina Witzel,‡ Friedrich Bialas,‡ and Joachim P. Spatz†,‡ †
Department of New Materials and Biosystems, Max Planck Institute for Intelligent Systems, Heisenbergstr. 3, D-70569 Stuttgart, Germany ‡ Department of Biophysical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, D-69120 Heidelberg, Germany § Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany S Supporting Information *
ABSTRACT: We describe cobalt cross-linked redox-responsive 4-arm histidine-modified PEG (4A-PEG-His) hydrogels, which can be switched from self-healing viscoelastic liquids to form stable elastic solids through a simple oxidation step from Co2+ to Co3+. The dramatic change in gel properties is quantified in rheological measurements and is associated with the altered ligand exchange rate of the cross-linking cobalt ions. While Co2+ forms kinetically labile coordination bonds with low thermodynamic stability, Co3+ forms kinetically inert and highly stable coordination bonds. Unlike the Co2+ cross-linked hydrogels, the Co3+ cross-linked hydrogels do not dissolve in buffer and swell overtime, where they remain intact longer with increasing gel connectivity, increasing polymer concentration and decreasing temperature. Remarkably, these gels can even resist the strong chelator EDTA and withstand both low and high pH due to the low ligand exchange rates in the primary coordination sphere. Overall, the Co2+/3+ redox pair provides an attractive platform to produce redox-responsive materials with big deviations in mechanical and chemical properties.
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INTRODUCTION Materials that alter their physical properties (dimensions, shape, viscosity, swelling, self-healing, mechanical stability, host molecule release, etc.) in response to external stimuli in their environment are of great interest in sensing, health, construction, and electronics.1−4 To this end, metal coordination bonds are an attractive handle to obtain materials that respond to stimuli such as light, pH, temperature, mechanical forces, redox potential, and chemicals because depending on the metal ion, its oxidation state, and ligands, coordination bonds can range from strong, irreversible, and static to weak, labile, and dynamic.5−8 Copper, iron, and cobalt ions in particular have been used to obtain redox-responsive materials as these metals possess ions in two stable and accessible oxidation states with different chemical properties and are readily available.9−12 Meyer and co-workers integrated Fe2+/3+ and Cu+/2+ into preformed gel networks as additional crosslinkers and reported the reversible switching between a soft and a hard state by oxidation and reduction of the metal center, respectively.11,12 The gel−sol transition can be reversibly controlled with light in iron cross-linked poly(acrylic acid)13 and alginate14 hydrogels by taking advantage of the photoreduction of Fe3+ to Fe2+ and the oxidation of Fe2+ in air. More defined metal cross-linked hydrogels have been formed with polymers that contain bipyridyl chelating groups and have been cross-linked with Fe2+, Co2+/3+, and Cu+/2+ to obtain both temperature and redox-responsive materials.10,15,16 In all these examples, the predictable and altered coordination bond © XXXX American Chemical Society
properties in different oxidation states form the basis for these stimuli-responsive and redox-active materials. In nature, metal ions are also integrated into biogenic materials to increase hardness, toughness, adhesion, and selfhealing of biopolymers.17 For instance, in the biting tools of arthropods and annelids metal ions such as Zn2+ and Cu2+ are enriched at the edges, where the metal ions cross-link histidine and glycine rich proteins to increase hardness and stiffness in these unmineralized structures.18,19 Likewise, the byssal threads, which connect marine mussels to rocks, consist of specialized collagen proteins (PreCol proteins), which bind Zn2+ and Cu2+ through histidine rich domains.20,21 Remarkably, these byssal threads resist to regular wave impacts and can recover their mechanical properties in an acellular manner even when stressed beyond their yield points and have self-healing properties. Here, the metal coordination bonds act as reversible sacrificial bonds that re-form after rapture to restore mechanical strength.22 Biomimetic polymers that have histidine and imidazole side chains have been prepared to understand the metal-ion fortification and self-healing observed in biogenic materials.23,24 Exemplarily, a biomimetic histidine-modified 4arm poly(ethylene glycol) (4A-PEG-His), inspired by the histidine rich domains of PreCol proteins in mussels, can be cross-linked with divalent metal ions (Ni2+, Co2+, Cu2+, and Received: March 21, 2016 Revised: May 30, 2016
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DOI: 10.1021/acs.macromol.6b00574 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. (A) Cross-linking of the 4A-PEG-His with Co2+ or Co3+ results in hydrogels with very different physical properties due to the different coordination bond properties. In the Co2+-mediated cross-linking the coordination bonds have low thermodynamic stability and are kinetically labile, which results in a viscoelastic self-healing liquid. On the other hand, in the Co3+-mediated cross-linking, the coordination bonds are thermodynamically very stable and kinetically inert, which results in a form-stable elastic solid. The properties of the hydrogel can be switched from liquid to solid by oxidation of the Co2+ centers to Co3+. (B) Different ratios of cobalt ions to histidine end-group ratios (1:2 and 1:3 result) in different hydrogel connectivity. In the 1:2 gels on average 2 arms are connected to each other by a cobalt ion, while in the 1:3 gels 3 arms are connected at each cobalt ion. Consequently, at the same 4A-PEG-His concentration the 1:2 gels have a higher cross-linking density while while the 1:3 gels have a higher connectivity.
Zn2+) to form self-healing hydrogels.24 These gels are rheologically viscoelastic liquids, and the gel relaxation dynamics and self-healing rates correlate with the coordination bond dissociation rate constants for the different divalent metals rather than their bond strengths. Complementarily, the dynamic re-formation of metal ligand bonds by themselves25−29 or under light30,31 has been the basis of design for many synthetic self-healing materials. In this work we present a redox-responsive cobalt crosslinked PEG coordination hydrogel with well-defined coordination chemistry that can be switched from a viscoelastic liquid to an elastic solid by oxidizing Co2+ ions to Co3+ ions. Converting the cross-linking cobalt(II) ion to cobalt(III) ions drastically alters the gel properties as both thermodynamic stability and ligand exchange kinetics of the complexes are changed dramatically. First of all, as a d6 ion cobalt(III) typically forms complexes with much higher formation constants than the d7 ion cobalt(II).32,33 For example, formation constants of the ethylenediamine (en) complexes [Co(en)3]2+ and [Co(en)3]3+ are 1013 and 1048 and formation constants of the ethylenediaminetetraacetic acid (EDTA) complexes [Co(EDTA)]2− and [Co(EDTA)]− are 1016 and 1040, respectively.34 Second, cobalt(III) complexes are kinetically inert toward ligand exchange in their primary coordination sphere, while cobalt(II) complexes rapidly undergo ligand exchange reactions. For instance, while the hexaaqua complex of Co2+ has an exchange rate of 3.2 × 106 s−1 and the hexaaqua complexes of Fe2+/3+, Cu2+, Ni2+, and Zn2+ have exchange rates in the rage of 1.6 × 102−4.4 × 109 s−1, the water exchange rate in hexacoordinated Co3+ complex is about 10−6 s−1.35 This dramatic kinetic inertness of cobalt(III) has been exploited in bioinorganic chemistry to trap intermediates in metalloenzymes,36 to design redox-active metal based drugs,37 to stably immobilize proteins,38−40 and to cross-linking proteins.41 So far, the Co2+/3+ redox pair has only been used to cross-link bipyridyl-branched polyoxazoline polymers to form hydrogels, and it was reported that the slow ligand exchange of cobalt(III)
results in increased chemical resistance of these hydrogel.10 Here, we expand this concept and investigate in detail the differences of Co2+ and Co3+ cross-linked hydrogels. Toward this end, we use the bioinspired 4A-PEG-His polymer reported by Fullenkamp et al.24 but differently from Fullenkamp et al., who used labile cross-linking ions (Co2+, Zn2+, Cu2+, and Ni2+), we oxidize the Co2+ centers to Co3+ to obtain kinetically inert and thermodynamically very stable cross-linking. The altered oxidation state of cobalt has direct consequences for the mechanical and chemical properties of the hydrogel. The cobalt(II) cross-linking is dynamic and undergoes fast ligand exchange, and the bond strength is relatively weak, which results in a viscoelastic liquid with self-healing properties as reported. On the other hand, cobalt(III) cross-linking is kinetically inert and forms very stable bonds that are comparable to covalent bonds and results in an elastic solid. We quantify for the first time the change in rheological properties due the cobalt oxidation. Furthermore, we can alter the connectivity in the 4A-PEG-His cobalt(III) hydrogels through the well-defined cobalt coordination chemistry, and we investigated the parameters that influence gel properties in gel swelling studies. A key difference of the Co3+ mediated crosslinking is its inertness to ligand exchange and we probe the resistance of these gels toward the strong chelators EDTA as well as acidic and basic pH and reducing agents. Overall, we would like to establish the cobalt(II)/cobalt(III) redox couple as the basis for redox-responsive materials and believe that this concept can be extended to metal centers in synthetic and biogenic materials to alter their properties in a simple redox step.
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EXPERIMENTAL SECTION
General Procedure for the Hydrogel Preparation. 4A-PEGHis (Mw = 10 000 g/mol) and mPEG-His (Mw = 2000 g/mol) are synthesized as reported.24 Co2+ cross-linked hydrogels are prepared by mixing 4A-PEG-His (final concentration 50−150 mg/mL) with CoCl2 (1:2 or 1:3 cobalt:end-group ratio, 1 M CoCl2 stock solution) in 100 B
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Figure 2. Rheological characterization of 4A-PEG-His Co2+ and Co3+ coordination gels with (A) 1:2 and (B) 1:3 cobalt to end-group ratios. The storage (G′) and the loss (G″) moduli are shown as solid and open symbols, respectively. (C) The Young’s moduli of Co3+ cross-linked 4A-PEG-His hydrogels given in kPa. The values are the average of three independent experiments, and the errors are gives as the standard deviation.
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mM HEPES (pH 7) and adjusting the pH to 7 with 1 M NaOH as described in the literature.24 The 250 μL gels are oxidized with a solution of 20 μL of 1 M HEPES (pH 7) containing 3 equiv of H2O2 for each equivalent of Co2+ and oxidized to homogeneity overnight. Further, the 4A-PEG-His hydrogels are lyophilized for FTIR measurements. Optimization of Cobalt(II) Oxidation. Solutions of mPEG-His (150 mg/mL, Mw = 2000 g/mol) in 100 mM HEPES (pH 7) are incubated with CoCl2 (1:2 or 1:3 cobalt:end-group ratio) and treated with 0−3 equiv of H2O2 to Co2+. The absorbance at 550 nm is monitored over 7 h at room temperature. To obtain UV−vis spectra, 12 mM mPEG-His is mixed with either 6 or 4 mM CoCl2 (1:2 or 1:3 cobalt:end-group ratio) in 100 mM HEPES (pH 7) and incubated for 3 h after the addition of 2 equiv of H2O2. For the 1H NMR studies all solutions are prepared in D2O and 30 mM mPEG-His is mixed with 10 mM CoCl2 (1:3 cobalt:end-group ratio) in 100 mM HEPES, and the pH is adjusted to 7 with NaOH. For the oxidation 3 equiv of H2O2 is added, and the solution is incubated for at least 3 h. 1H NMR are measured on a 400 MHz instrument. Characterization of Hydrogel Swelling. 250 μL of 100 mM HEPES (pH 7) is added to 250 μL of cobalt(III) 4A-PEG-His hydrogels at 50−150 mg/mL with 1:2 or 1:3 cobalt:end-group ratios gels at 37, 25, and 4 °C. The swelling ratio of the gels is defined as the weight of the swollen gel divided by the dry weight of the 4A-PEG-His, which is calculated from the polymer concentration used. To access the chemical stability, 250 μL of 100 mg/mL 4A-PEG-His hydrogels with 1:2 cobalt to end-group ratios is incubated with 500 μL of 10 or 50 mM EDTA or ascorbic acid in 100 mM HEPES buffer at pH 7 or with 100 mM HEPES solutions with pH 2 or 10. The weight of the swollen gel is measured regularly after decantation of the excess solution, and after each measurement fresh solution with the reactants is added. Rheological Characterization. Frequency sweeps are performed on a Malvern Kinexus Pro rheometer equipped with an upper geometry probe head with a diameter of 0.80 cm and analyzed using RSpace software. Measurements are performed at 25 °C on unswollen gels with a frequency range from 0.01 to 16 Hz (100 to 0.1 rad/s) at 5% strain. These settings are adjusted to confirm that the hydrogels are measured in the linear viscoelastic range. The Young’s moduli are calculated as follows: Young’s modulus, E = 2G(1 + v), where G is the storage modulus and the average Poisson’s ratio for hydrogels is v = 0.5.42
RESULTS AND DISCUSSION The 4A-PEG-His inspired from the mussel PreCol protein is synthesized, and Co2+ hydrogels are formed as described in the literature.24 The Co2+ hydrogels are yellow, viscoelastic liquids, self-healing, and not form-stable gels as reported. When the cross-linking Co2+ ions are oxidized with hydrogen peroxide to obtain the corresponding Co3+ hydrogel, the gel properties change dramatically. The Co3+ hydrogels are red, form-stable, brittle, and solid (Figure 1A). This dramatic difference in gel properties is a direct reflection of the difference in ligand exchange rates of cobalt(II), which forms transient bonds and cobalt(III), which forms inert bonds. The bidentate histidine ligand at the end of each 4A-PEGHis arm allows to form hydrogels with well-defined connectivity (Figure 1A). Cobalt(II) as a d7 ion can form both tetra-, penta-, and hexacoordinated complexes, but cobalt(III) as a d6 ion has a very strong preference to form octahedral low-spin complexes.43 The Co2+ cross-linked 4APEG-His gels reported by Fullenkamp et al. use a cobalt:endgroup ratio of 1:2 so that each Co2+ connects 2-arms where 4 coordination sites are occupied by the histidine ligands on average.24 Bearing in mind the differences in coordination preference of Co2+ and Co3+, we used cobalt:end-group ratios of both 1:2 and 1:3 to obtain a mean coordination number of four and six with the histidine ligands, respectively (Figure 1B). The two different cobalt to end-group ratios result in different connectivities in the hydrogels; while in the 1:3 gels 3 arms are joined at each cobalt center on average, in the 1:2 hydrogels only 2 arms are connected at each cobalt center on average. Thus, at the same 4A-PEG-His concentration, the 1:3 gels have a higher connectivity, while the 1:2 gels have a higher crosslinking density. Besides the cobalt to end-group ratios also the total concentration of the polymer (4A-PEG-His) has an important effect of the gel properties as it controls the crosslinking density. To optimize the oxidation step from cobalt(II) to cobalt(III), we synthesized a single arm histidine-modified PEG (mPEGHis) and formed the corresponding cobalt(II) coordination complexes with 1:2 and 1:3 cobalt:end-group ratios. We added 0−3 equiv of hydrogen peroxide per Co2+ to the solutions and C
DOI: 10.1021/acs.macromol.6b00574 Macromolecules XXXX, XXX, XXX−XXX
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Figure 3. Swelling ratios of 4A-PEG-His Co3+ hydrogels over time with cobalt to end-group ratios of 1:2 and 1:3 at 4A-PEG-His concentrations of 50, 100, and 150 mg/mL at (A) 4, (B) 25, and (C) 37 °C. The values are the average of three independent experiments, and the error bars are the standard deviation.
The oxidation of the cobalt center from +2 to +3 changes the hydrogel’s physical properties, which is quantified by oscillatory rheometry (Figure 2A,B). The cobalt(II) gels are viscoelastic liquids (G″ > G′) with a crossover frequency of G′ (storage modulus) and G″ (loss modulus) at about 100 rad/s for 150 mg/mL 4A-PEG-His for both 1:2 and 1:3 cobalt:end-group ratios, which is in agreement with the curves reported by Fullenkamp et al.24 On the other hand, upon oxidation of the cross-linking Co2+ ions to Co3+ these gels became predominantly elastic solids (G′ is invariant of frequency and G′ > G″), and there is no crossover frequency in the measured range. The crossover frequency of G′ and G″ is an indicator of the materials transition from a predominantly elastic solid to a viscoelastic liquid and is inversely correlated with material relaxation and hence the coordination bond exchange dynamics. For the Co2+ and the by Fullenkamp et al. reported Ni2+, Zn2+, and Cu2+ gels the crossover frequencies are between 0.1 and 100 rad/s at 25 °C,24 while for the Co3+ gels the crossover frequency is outside the measurement window and expected to be at much lower frequencies. The crossover frequencies correlate with the ligand exchange rate constants for these metals, which are reported to be Zn2+ > Cu2+ > Co2+ > Ni2+ ≫ Co3+.35 For the unswollen cobalt(III) coordination gels, which are elastic solids, the Young’s moduli are calculated and these depend on both the 4A-PEG-His concentration and the connectivity of the gel (Figure 2C and Figure S5). The Young’s modulus increases linearly with the 4A-PEG-His concentration, which is expected as the Young’s modulus is directly related to the number of cross-links per unit volume in the hydrogel. Additionally, the 1:2 hydrogels are slightly softer than the 1:3 hydrogels. Here, we conclude that higher connectivity provided by the 1:3 complexes results in increased stability and stiffness of the hydrogels.
followed the oxidation by monitoring the absorption of the formed Co3+ center. These measurements demonstrate that 1 equiv of H2O2 is sufficient to oxidize the cobalt centers, but the oxidation reaction takes over 5 h and that the oxidation is completed within 2 h with 2 equiv of H2O2 in solution (Figure S1). However, in the hydrogels, where diffusion is hindered, we used 3 equiv of H2O2, and we observed that the gels only oxidized homogeneously overnight. The UV−vis spectra of solutions with 1:2 and 1:3 cobalt(III) to mPEG-His ratios give insight into the coordination environment of the cobalt(III) centers (Figure S2). Both the 1:2 and 1:3 complexes show an absorption maximum at ca. 500 nm, but the molar extinction coefficients calculated for the cobalt centers differ and are 37.8 and 54.7 M−1 cm−1, respectively. This result demonstrates that the cobalt(III) ions are in different coordination environments for these two samples, which is expected as on average 4 and 6 of the coordination site should be occupied by the mPEG-His in the 1:2 and 1:3 complex. Further, evidence for the cobalt coordination and the altered oxidation state come from IR and 1H NMR measurements. In the IR spectrum of 4A-PEGHis the band around 1670 cm−1, which is assigned to CN and CC bonds of the imidazole ring and the CO bond, shifts in partially to 1635 cm−1 upon Co2+ or Co3+ coordination (Figure S3). This shift to lower wavenumbers is expected and has been observed in numerous coordination complexes.44 Also in the 1H NMR, paramagnetic peak broadening is observed upon coordination of Co2+ for the aromatic protons in the imidazole ring, and this broadening is reversed when the Co2+ is oxidized to Co3+, which indicates the formation of a diamagnetic complex (Figure S4). In addition, it should be noted that the overall 1H NMR does not change with after the oxidation step, which indicates that the oxidation step does not harm the mPEG-His polymer. D
DOI: 10.1021/acs.macromol.6b00574 Macromolecules XXXX, XXX, XXX−XXX
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gel up to 4 days, and only after 6 days the gels started to dissolve in the EDTA solution (Figure 4). This is a remarkable
The swelling of metal cross-linked hydrogels reflect the bond inertness in the gels and are used to evaluate the effect of the 4A-PEG-His concentration, cross-linking, and temperature in the cobalt(III) hydrogels. The oxidation alters the gel behavior in buffer dramatically; while the cobalt(III) hydrogels swell in buffer over a long period of time, the cobalt(II) hydrogels dissolve completely in buffer within a few minutes. Therefore, swelling ratios for cobalt(II) hydrogels are not meaningful to measure. We also observe that cobalt(III) cross-linked gels get slightly softer overtime as they swell (Figure S6), which is explained by the decreasing cross-linking density upon water uptake. We measured the swelling ratios of gels at different temperatures (4, 25, and 37 °C) for 4A-PEG-His concentrations of 50−150 mg/mL and with cobalt:end-group ratios of 1:2 and 1:3 (Figure 3). In the swelling ratio measurements, an increase in value represents uptake of more water into gel network, while decreasing swelling ratios show that the gel is dissolving as His ligands are replaced with water ligands at the cobalt(III) centers. We observed that the total concentration of 4A-PEG-His and cobalt(III) is one of the determining factors of that influences swelling behavior. In general, the gels remain intact longer at higher the total 4A-PEG-His concentration. For examples at 37 °C gels with cobalt:end-group ratios of 1:2 keep swelling at 150 mg/mL 4A-PEG-His for 10 days, up to 5 days at 100 mg/mL 4A-PEG-His, and dissolve after a few hours at 50 mg/mL 4A-PEG-His (Figure 3C). The second factor that effected the gel swelling is the cobalt:end-group ratio with the 1:3 gels remaining intact longer than the 1:2 gels at the same 4A-PEG-His concentration. For example in Figure 3A, 50 mg/mL gels with 1:3 ratio keep swelling at 4 °C over the observed 10 days while 50 mg/mL gels with a 1:2 ratio dissolve within a few hours. Similarly, at 37 °C, 1:3 gels degrade slower than the 1:2 gels at 100 mg/mL 4APEG-His. It is noticeable that the 1:2 gels dissolved faster than the 1:3 gels despite a higher cross-linking density. This shows that the higher connectivity in the 1:3 gels contributes more to the stability than the higher cross-linking density in the 1:2 gels. A striking point here is that the increase in swelling ratios is almost the same for both 1:3 and 1:2 gels at the same total 4APEG-His concentration showing similar water uptake dynamics regardless of the gel connectivity and cross-linking density as long as the gels stay intact. As shown in Figure 3, temperature is another important factor that influences the cobalt(III) hydrogel integrity. In general, the hydrogels dissolve quicker with increasing temperature, which can easily be attributed to the thermally accelerated His ligand exchange with water at the Co3+ centers. For example, 50 mg/mL 4A-PEG-His gels with a 1:3 cobalt to end-group ratio swell at 4 °C over 10 days, dissolve gradually over 10 days at 25 °C, and dissolve within 1 day at 37 °C. Gels at 100 mg/mL with a 1:2 cobalt to end-group rations keep swelling for 10 days at 4 and 25 °C but start to dissolve at 37 °C after 5 days. Additionally, the water uptake into the system is not altered by temperature, and the swelling ratios over time are the same at all temperature as long as the gels do not start to dissolve. The cobalt(III) centers do not just undergo very slow ligand exchange with water but also with other ligands. Therefore, the cobalt(III) hydrogels can also withstand chelating conditions to some extent. To test this, we incubated 100 mg/mL gels with 1:2 cobalt:end-group ratio with 10 or 50 mM of the strong chelator EDTA and measured the swelling ratios. Remarkably, the hydrogels showed the same swelling behavior as the control
Figure 4. Chemical resistance of cobalt(III) cross-linked 4A-PEG-His hydrogels (100 mg/mL 4A-PEG-His hydrogels with 1:2 cobalt to endgroup ratio). The Co3+ hydrogels are incubated either in 100 mM HEPES at pH 2, 7, or 10, in 100 mM HEPES with 10 or 50 mM EDTA, or in 100 mM HEPES with 10 or 50 mM ascorbic acid. The values are the average of three independent experiments, and the error bars are the standard deviation.
resistance toward chelators, as metal cross-linked coordination gels typically dissolve when treated with chelators that have stronger binding than the coordinating group of the polymer. Most coordination gels are also sensitive to pH as the protonation of ligands at low pH and the competitive binding of OH− groups to metal centers at high pH can weaken or destroy the metal cross-linking. This behavior has also been observed with the Ni2+ cross-linked 4A-PEG-His coordination gels, which do not form at pH lower than 6 and higher than 9.24 Contrastingly, when we incubate formed Co3+ cross-linked 4APEG-His gels (100 mg/mL, 1:2 cobalt to end-group ratio) with solutions at pH 2 and 10, they display the same swelling behavior as a gel at pH 7 and the gels remain intact for at least 6 days in solution (Figure 4). Complementarily, we also evaluated if the swelling in buffer at pH 2 or 10 or in 50 mM EDTA has an effect on the rheological properties (Figure S6). The results show that after 1 day of incubation the Young’s moduli remain the same for all hydrogels at all pHs (2, 7, and 10) but that the incubation with 50 mM EDTA causes a slight decrease in stiffness. A key the reasons to use cobalt as a redox active center is its reversibility. To test if this is indeed the case, we incubated the 100 mg/mL gels with 1:2 cobalt:end-group cobalt(III) hydrogels with 10 or 50 mM ascorbic acid as a reducing agent and again measured their swelling ratios (Figure 4). The cobalt(III) gels changed color from red to yellow over time and dissolved over the course of 2 days. This shows that the cobalt(III) centers in the gel can be reversibly reduced back to cobalt(II) and alter the gel properties.
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CONCLUSIONS In this work, we demonstrate how the dramatically different coordination bonds formed by Co2+ and Co3+ can influence both the mechanical and the chemical properties in coordination hydrogels. The Co2+ cross-linking provides a transient linkage between polymers and therefore results in a viscoelastic liquid with self-healing kinetics that correlate with the ligand exchange rate as also revealed by Fullenkamp et al.24 Here we demonstrate that the Co3+ cross-linking provides permanent linkage between the polymers, which is comparable to a covalent bond. Consequently, the resulting hydrogel is an E
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(3) Tsitsilianis, C. Responsive reversible hydrogels from associative ‘‘smart’’ macromolecules. Soft Matter 2010, 6, 2372−2388. (4) Ahn, S.-K.; Kasi, R. M.; Kim, S.-C.; Sharma, N.; Zhou, Y. Stimuliresponsive polymer gels. Soft Matter 2008, 4, 1151−1157. (5) Whittell, G. R.; Hager, M. D.; Schubert, U. S.; Manners, I. Functional soft materials from metallopolymers and metallosupramolecular polymers. Nat. Mater. 2011, 10, 176−188. (6) Piepenbrock, M.-O. M.; Lloyd, G. O.; Clarke, N.; Steed, J. W. Metal- and Anion-Binding Supramolecular Gels. Chem. Rev. 2010, 110, 1960−2004. (7) Sui, X.; Feng, X.; Hempenius, M. A.; Vancso, G. J. Redox active gels: synthesis, structures and applications. J. Mater. Chem. B 2013, 1, 1658−1672. (8) Sun, Z.; Lv, F.; Cao, L.; Liu, L.; Zhang, Y. MultistimuliResponsive, Moldable Supramolecular Hydrogels Cross- Linked by Ultrafast Complexation of Metal Ions and Biopolymers. Angew. Chem. 2015, 127, 8055−8059. (9) Giammanco, G. E.; Sosnofsky, C. T.; Ostrowski, A. D. LightResponsive Iron(III)−Polysaccharide Coordination Hydrogels for Controlled Delivery. ACS Appl. Mater. Interfaces 2015, 7, 3068−3076. (10) Chujo, Y.; Sada, K.; Saegusa, T. Cobalt(III) Bipyridyl-Branched Polyoxazoline Complex as a Thermally and Redox Reversible Hydrogel. Macromolecules 1993, 26, 6320−6323. (11) Auletta, J. T.; LeDonne, G. J.; Gronborg, K. C.; Ladd, C. D.; Liu, H.; Clark, W. W.; Meyer, T. Y. Stimuli-Responsive Iron-Cross-Linked Hydrogels That Undergo Redox-Driven Switching between Hard and Soft States. Macromolecules 2015, 48, 1736−1747. (12) Harris, R. D.; Auletta, J. T.; Motlagh, S. A. M.; Lawless, M. J.; Perri, N. M.; Saxena, S.; Weiland, L. M.; Waldeck, D. H.; Clark, W. W.; Meyer, T. Y. Chemical and Electrochemical Manipulation of Mechanical Properties in Stimuli-Responsive Copper-Cross-Linked Hydrogels. ACS Macro Lett. 2013, 2, 1095−1099. (13) Peng, F.; Li, G.; Liu, X.; Wu, S.; Tong, Z. Redox-Responsive Gel-Sol/Sol-Gel Transition in Poly(acrylic acid) Aqueous Solution Containing Fe(III) Ions Switched by Light. J. Am. Chem. Soc. 2008, 130, 16166−16167. (14) Narayanan, R. P.; Melman, G.; Letourneau, N. J.; Mendelson, N. L.; Melman, A. Photodegradable Iron(III) Cross-Linked Alginate Gels. Biomacromolecules 2012, 13, 2465−2471. (15) Kawano, S.-I.; Fujita, N.; Shinkai, S. A Coordination Gelator That Shows a Reversible Chromatic Change and Sol-Gel PhaseTransition Behavior upon Oxidative/Reductive Stimuli. J. Am. Chem. Soc. 2004, 126, 8592−8593. (16) Chujo, Y.; Sada, K.; Saegusa, T. Iron(II) Bipyridyl-Branched Polyoxazoline Complex as a Thermally Reversible Hydrogel. Macromolecules 1993, 26, 6315−6319. (17) Degtyar, E.; Harrington, M. J.; Politi, Y.; Fratzl, P. The Mechanical Role of Metal Ions in Biogenic Protein-Based Materials. Angew. Chem., Int. Ed. 2014, 53, 12026−12044. (18) Lichtenegger, H. C.; Schoberl, T.; Bartl, M. H.; Waite, H.; Stucky, G. D. High abrasion resistance with sparse mineralization: Copper biomineral in worm jaws. Science 2002, 298, 389−392. (19) 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, 3219− 3225. (20) Vaccaro, E.; Waite, J. H. Yield and Post-Yield Behavior of Mussel Byssal Thread: A Self-Healing Biomolecular Material. Biomacromolecules 2001, 2, 906−911. (21) Harrington, M. J.; Waite, J. H. Holdfast heroics: comparing the molecular and mechanical properties of Mytilus californianus byssal threads. J. Exp. Biol. 2007, 210, 4307−4318. (22) Schmitt, C. N. Z.; Politi, Y.; Reinecke, A.; Harrington, M. J. Role of Sacrificial Protein-Metal Bond Exchange in Mussel Byssal Thread Self-Healing. Biomacromolecules 2015, 16, 2852−2861. (23) Srivastava, A.; Holten-Andersen, N.; Stucky, G. D.; Waite, J. H. Ragworm Jaw-Inspired Metal Ion Cross-Linking for Improved Mechanical Properties of Polymer Blends. Biomacromolecules 2008, 9, 2873−2880.
elastic solid, form-stable, not self-healing, brittle, and swell in buffer solutions for extended time before dissolving. The key difference in the mechanical behavior lies in the altered ligand exchange kinetics of Co2+ and Co3+ and also gives rise to a difference in chemical stability. While like most metal crosslinked coordination gels the Co2+ gels dissolve in solution and are sensitive to low and high pH and to chelators,24 the Co3+ cross-linked hydrogels can resist such chemical challenges due to their kinetic inertness. In general, the gels lose their integrity faster at higher temperatures as the ligand exchange reaction accelerates, with lower connectivity of the gel as fewer links have to be broken to devastate the network and at lower gel concentrations where the gel network is already highly hydrated. Metal ion cross-linking in nonmineralized proteinbased biomaterials is a general strategy to improve mechanical properties. In such materials divalent ions such as Zn2+, Cu2+, Ni2+, and Co2+ can be used quite interchangeably owing to their comparable coordination chemistry and ligand exchange kinetics. Here we depict a general strategy to transform these transient links into permanent ones and significantly alter mechanical properties of such metal cross-linked materials further, through a simple oxidation step from cobalt(II) to cobalt(III).
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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.6b00574. Oxidation kinetics of the mPEG-His-Co2+ complex, UV− vis and 1H NMR spectra of mPEG-His and its Co2+ and Co3+ complexes, IR spectra of 4A-PEG-His and its Co2+ and Co3+ complexes, and the Young’s moduli for Co3+ cross-linked hydrogels in relation to the 4A-PEG-His concentration and after swelling under different conditions (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel +49 6131 379135, e-mail
[email protected] (S.V.W.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Max-Planck Society for financial support. S.V.W. thanks the Baden-Württemberg Stiftung for a postdoctoral fellowship with the Eliteprogramm and the BMBF for funding her MaxSynBio research group (FKZ 031A359L). J.P.S. is the Weston visiting professor at the Weizmann Institute for Science. The research group is part of the excellence cluster CellNetworks at the University of Heidelberg.
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REFERENCES
(1) Liu, F.; Urban, M. W. Recent advances and challenges in designing stimuli-responsive polymers. Prog. Polym. Sci. 2010, 35, 3− 23. (2) Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Müller, M.; Ober, C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.; Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S. Emerging applications of stimuli-responsive polymer materials. Nat. Mater. 2010, 9, 101−113. F
DOI: 10.1021/acs.macromol.6b00574 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules (24) Fullenkamp, D. E.; He, L.; Barrett, D. G.; Burghardt, W. R.; Messersmith, P. B. Macromolecules 2013, 46, 1167−1174. (25) Bode, S.; Zedler, L.; Schacher, F. H.; Dietzek, B.; Schmitt, M.; Popp, J.; Hager, M. D.; Schubert, U. S. Self-Healing Polymer Coatings Based on Crosslinked Metallosupramolecular Copolymers. Adv. Mater. 2013, 25, 1634−1638. (26) Hong, G.; Zhang, H.; Lin, Y.; Chen, Y.; Xu, Y.; Weng, W.; Xia, H. Mechanoresponsive Healable Metallosupramolecular Polymers. Macromolecules 2013, 46, 8649−8656. (27) Mozhdehi, D.; Ayala, S.; Cromwell, O. R.; Guan, Z. Self-Healing Multiphase Polymers via Dynamic Metal−Ligand Interactions. J. Am. Chem. Soc. 2014, 136, 16128−16131. (28) Liang, H.; Zhang, Z.; Yuan, Q.; Liu, J. Self-healing metalcoordinated hydrogels using nucleotide ligands. Chem. Commun. 2015, 51, 15196−15199. (29) Foster, J. A.; Parker, R. M.; Belenguer, A. M.; Kishi, N.; Sutton, S.; Abell, C.; Nitschke, J. R. Differentially Addressable Cavities within Metal-Organic Cage-Cross-Linked Polymeric Hydrogels. J. Am. Chem. Soc. 2015, 137, 9722−9729. (30) Burnworth, M.; Tang, L.; Kumpfer, J. R.; Duncan, A. J.; Beyer, F. L.; Fiore, G. L.; Rowan, S. J.; Weder, C. Optically healable supramolecular polymers. Nature 2011, 472, 334−337. (31) Wang, Z.; Urban, M. W. Facile UV-healable polyethylenimine− copper (C2H5N−Cu) supramolecular polymer networks. Polym. Chem. 2013, 4, 4897−4901. (32) Bond, J.; Hobson, D. B. The stability constants of cobalt(III) chelates of polyaminopolycarboxylic acids. J. Chem. Soc. A 1969, 2155−2157. (33) Ogino, H.; Ogino, K. Redox potentials and related parameters of cobalt(III/II) complexes containing aminopolycarboxylates. Inorg. Chem. 1983, 22, 2208−2211. (34) Martell, A. E.; Smith, R. M. Standard Reference Database 46, 2001. (35) Helm, L.; Merbach, A. E. Inorganic and Bioinorganic Solvent Exchange Mechanisms. Chem. Rev. 2005, 105, 1923−1960. (36) Van Wart, H. E. Introduction of exchange-inert metal ions into enzymes. Methods Enzymol. 1988, 158, 95−110. (37) Graf, N.; Lippard, S. J. Redox activation of metal-based prodrugs as a strategy for drug delivery. Adv. Drug Delivery Rev. 2012, 64, 993− 1004. (38) Wegner, S. V.; Spatz, J. P. Cobalt(III) as a stable and inert mediator ion between NTA and His6-tagged proteins. Angew. Chem., Int. Ed. 2013, 52, 7593−7596. (39) Shao, S.; Geng, J.; Ah, Y. H.; Gogia, S.; Neelamegham, S.; Jacobs, A.; Lovell, J. F. Functionalization of cobalt porphyrinphospholipid bilayers with his-tagged ligands and antigens. Nat. Chem. 2015, 7, 438−446. (40) Wegner, S. V.; Schenk, F. C.; Spatz, J. P. Cobalt(III)-Mediated Permanent and Stable Immobilization of Histidine-Tagged Proteins on NTA-Functionalized Surfaces. Chem. - Eur. J. 2016, 22, 3156−3162. (41) Nguyen, D. T.; Cavazos, R. J.; Harris, A. N.; Petros, R. A. Werner Complexes Viewed Anew: Utilizing Cobalt Coordination Chemistry for ‘‘Traceless’’ Stimuli-Responsive Bioconjugation Involving Therapeutic Nanoparticles, Protein PEGylation, and Drug(Bio)polymer Conjugates. Comments Inorg. Chem. 2014, 34, 59−57. (42) Urayama, K.; Takigawa, T.; Masuda, T. Poisson’s Ratio of Poly(viny1 alcohol) Gels. Macromolecules 1993, 26, 3092−3096. (43) Maret, W.; Vallee, B. L. Cobalt as probe and label of proteins. Methods Enzymol. 1993, 226, 52−71. (44) Sarkar, B.; Wigfield, Y. The structure of copper(II)-histidine chelate. The question of the involvement of the imidazole group. J. Biol. Chem. 1967, 242, 5572−5577.
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DOI: 10.1021/acs.macromol.6b00574 Macromolecules XXXX, XXX, XXX−XXX
