In Vivo Redox-Responsive SolâGel ... - ACS Publications

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In Vivo Redox-Responsive Sol−Gel/Gel−Sol Transition of Star Block Copolymer Solution Based on Ionic Cross-Linking Yoshiyuki Nakagawa,† Seiichi Ohta,‡ Akira Sugahara,† Masashi Okubo,† Atsuo Yamada,† and Taichi Ito*,†,‡ †

Department of Chemical System Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Center for Disease Biology and Integrative Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

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S Supporting Information *

ABSTRACT: Redox-responsive hydrogels have the potential for application in various fields, including biomedical science. We have developed a redox-responsive star block copolymer hydrogel based on iron ion cross-linking. The addition of the ferric ion (Fe3+) induced gelation of the star block copolymer solution within a few seconds, whereas the addition of the ferrous ion (Fe2+) did not. The resulting hydrogels cross-linked using Fe3+ showed storage moduli (G′) of 26−1400 Pa and were stable under physiological conditions for as long as 1 month. The cross-linking between the star arms produced by the addition of Fe3+ enabled a fast, redox-responsive sol−gel/gel−sol transition. Furthermore, the hydrogel showed excellent injectability and biocompatibility in vivo, resulting in a rapid sol−gel/gel−sol transition in subcutaneous tissues in response to redox stimuli, such as the administration of ascorbic acid or hydrogen peroxide.

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INTRODUCTION Various “smart” polymer materials that can respond to specific stimuli, such as pH, temperature, chemical substances, light, electrical fields, or a redox environment, have been investigated because of their diverse range of potential applications in health care, sensing, electronics, coatings, or textiles.1−5 In particular, redox-responsive hydrogels that exhibit a reversible sol−gel transition,6,7 changes in mechanical properties,8−10 or switchable swelling states11,12 in response to a surrounding redox environment have received increasing attention. Since the redox stimuli can be induced either chemically or electrochemically, these redox-responsive hydrogels have potential for a wide variety of applications. For example, they can be used in controlled drug release, where drugs are released in response to reducing agents, such as glutathione.13 They can also be applied as sensors to detect oxidizing or reducing agents.14,15 They have been further investigated for electrical actuators, where reversible switching of mechanical strength or the swelling state of the gels controlled by electrochemically applied redox stimuli is used as a tool for actuation.11,12 In these redox-responsive hydrogels, the formation and disappearance of cross-linking points are controlled by a redox environment through several approaches, such as changes in thiol/disulfide equilibria,13,16 hydrogen bonding,17 or host− guest interaction.6,18 Another interesting approach is to use the coordination of metallic ion cross-linkers to ligands. The stability constant of the metal−ligand complex varies with the oxidation state of the metallic ions, which can be used for redox-responsive coordinate bond formation. The versatility of this approach has enabled the development of a variety of © XXXX American Chemical Society

redox-responsive hydrogels using various ion cross-linkers.7−10,12,19−26 Copper,9,12 iron,7,8,10,21−26 and cobalt ions19,20 have been used in particular because these ions can transition between two stable oxidation states. The biomedical field is one of the most fascinating and challenging areas to investigate the application of such redoxresponsive hydrogels based on ionic cross-linking. When considering biomedical applications, the ferric ion (Fe3+) is a promising cross-linker. Iron plays an essential role in living systems through its involvement in oxygen transport, electron transfer, and catalytic action,27,28 which makes its use in biomaterials attractive. In fact, iron has several clinical applications, such as in hemostatic pharmaceuticals,29,30 hyperphosphatemia drugs,31 and iron-deficiency anemia drugs.32 The promise of iron as a cross-linker has also been demonstrated in investigations of the clinical use of hydrogels cross-linked with Fe3+.33 In previous studies, redox-responsive hydrogels based on Fe coordination have been investigated using polymers containing abundant ligand groups, including alginate,23,25,26 hyaluronan,23 pectin,23 chitin,22 glucomannan derivatives,21 poly(acrylic acid) (PAA),7,8,10,24 and polyphosphazene.12 However, the redox-responsive sol−gel/gel−sol transition of aqueous polymer systems based on Fe coordination has not been achieved in vivo so far. This is because of the difficulty in achieving the in situ formation of homogeneous hydrogels through coordination with free Fe3+ in Received: May 17, 2017 Revised: July 6, 2017

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DOI: 10.1021/acs.macromol.7b01020 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules living systems. In fact, direct addition of free Fe3+ often results in the precipitation of the polymers because intramolecular cross-linking, rather than intermolecular cross-linking, frequently occurs during the rapid contact with Fe3+.34 Therefore, strategic molecular design of the precursor polymer would be required to avoid this precipitation. Our group has previously reported the first example of a totally synthetic polymer that can form homogeneous hydrogels through direct addition of free calcium ions (Ca2+).35 That polymer was a star block copolymer with a dendritic polyester (DPE) core, a poly((oligoethylene glycol) methyl ether acrylate) (polyOEGA) inner layer, and a PAA outer layer (DPE-g-OEGA-b-AA). Prevention of the intramolecular crosslinking of the PAA outer layer by steric hindrance of the hydrophilic inner layer was suggested to be a key factor in the successful gelation. We expect that the same mechanism is also applicable to Fe3+, which would lead to the development of an in situ gellable and redox-responsive hydrogel. Since Fe3+ forms a more stable complex with carboxylate ligands through pseudocovalent bonds compared with Ca2+,36,37 the use of Fe3+ could reinforce the stiffness and stability of the hydrogels. In addition, Fe3+ would behave as a redox-responsive crosslinker. In the current study, the in situ formation of a star block copolymer hydrogel induced by direct addition of Fe3+ was investigated (Figure 1a). The mechanical strength and degradation kinetics of the obtained hydrogel were evaluated. The redox-responsiveness of this hydrogel, based on the

transition of iron ions between two oxidation states, was evaluated (Figure 1b). Finally, the redox-responsive sol−gel/ gel−sol transition was examined in vivo through subcutaneous administration in mice.

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EXPERIMENTAL SECTION

Materials. DPE (Boltorn H20) was kindly provided by Perstorp Japan Co., Ltd. (Tokyo, Japan). Oligo(ethylene glycol) methyl ether acrylate (OEGA; AM-90G) was kindly gifted by Shin-Nakamura Chemical Co., Ltd. (Wakayama, Japan). Iron(III) chloride hexahydrate (FeCl3·6H2O), iron(II) chloride tetrahydrate (FeCl2·4H2O), tert-butyl acrylate (tBA), hydrogen peroxide (H2O2), L(+)-ascorbic acid, and 10% formalin solution were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Chelate resin (DIAION CR20) was kindly provided by Mitsubishi Chemical Corporation (Tokyo, Japan). Ethylenediamine-N,N′-disuccinic acid trihydrate (EDDS) was kindly provided by Chubu Chelest Co. Ltd. (Osaka, Japan). Dialysis membranes (Spectra/Por, Molecular weight cutoff (MWCO) = 6−8 kDa) were purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA). Ultrafiltration membranes (NTU-2120, MWCO = 20 kDa) were kindly provided by Nitto Denko Corporation (Osaka, Japan). Synthesis and Characterization of the Star Block Copolymer (DPE-g-OEGA-b-AA). The star block copolymer (DPE-g-OEGA-bAA) was synthesized and characterized according to the procedure described in our previous publication.35 Details are shown in the Supporting Information. Inverted Vial Tests. The star block copolymer was placed in individual glass vials and dissolved in 100 μL of saline (20 wt %). The polymer solutions were then treated using 100 μL of ferric chloride (FeCl3) or ferrous chloride (FeCl2) at varying concentrations in saline, and the resulting mixtures were shaken gently at room temperature. After 5 s, the vials were turned upside down to determine whether gelation had occurred or not. Rheological Measurements. Rheological measurements were conducted with a rheometer (MCR301; Anton Paar, Graz, Austria) using a parallel-plate geometry (PP25; diameter = 25 mm, gap = 1.0 mm) for gel samples and a cone−plate geometry (CP50-1-27711; diameter = 50 mm, cone angle = 0.976°) for sol samples. The frequency dependencies of the storage modulus G′, loss modulus G″, and phase angle values of all of the samples were measured at a strain value of 5%. Dynamic strain sweep tests were performed for all of the samples from 0.1 to 10% at 1 Hz to confirm that this strain was within the linear viscoelastic regime. Spectroscopic Studies of the Iron−Carboxylate Complex. Formation of the complex between iron ions and carboxylate of the star block copolymer was analyzed using FT-IR and Mössbauer spectroscopy. In order to avoid the effect of air oxidation of the ferrous ions, all the samples were prepared under a nitrogen atmosphere using a glovebox. Saline was degassed through three freeze−pump−thaw cycles in advance and used as solvent. For the FT-IR measurements, 100 μL of the 20 wt % star block copolymer solution and 100 μL of the 200 mM iron chloride (FeCl3 or FeCl2) solution were mixed in a vial, and the resulting mixtures were lyophilized. The freeze-dried samples were subject to FT-IR measurements following the method mentioned in the Supporting Information. For Mössbauer spectroscopy, 500 μL of the 20 wt % star block copolymer solution and 500 μL of the 1.0 M FeCl2 solution were mixed in a vial. In the case of the hydrogel sample preparation, the resulting mixture was exposed to air to allow for oxidation of Fe2+ overnight. In case of the sol sample preparation, 1.6 M ascorbic acid was added to the resulting mixture in order to prevent the oxidation of Fe2+ during the sample preparation process. Note that for the Mössbauer measurements the Fe3+-cross-linked hydrogel was prepared through air oxidation of Fe2+ for a better signal-to-noise ratio. Both samples were lyophilized, and the lyophilized samples were sealed within a plastic bag and used for Mössbauer measurements. The 57Fe Mössbauer spectra were collected using a MFD-500A spectrometer

Figure 1. (a) Schematic illustration of the in situ formation of the star block copolymer hydrogel through the addition of FeCl3. The star block copolymer is composed of a dendritic polyester core and double hydrophilic block copolymer arms. (b) Schematic illustration of the redox-responsive sol-to-gel or gel-to-sol transition of the star block copolymer based on the transition of iron ions between two oxidation states. B


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(Topologic Systems Inc., Kanagawa, Japan) equipped with a 57Co gamma-ray source. The spectra were calibrated using the six lines of αFe, the center of which was taken as zero isomer shift. Mössbauer spectra were fitted using a MossWinn 3.0 program. Measurement of the Degradation Kinetics of the Hydrogels. The degradation over time of the star block copolymer hydrogel crosslinked using Fe3+ was measured gravimetrically. Hydrogel disks (0.25 mL) were prepared by mixing equal volumes of the star block copolymer solution (10, 20, or 40 wt %) and a FeCl3 solution. The concentration ratio of Fe3+ to carboxylate ([Fe3+]/[COO−]) was fixed at 0.5. The hydrogel disks were then incubated at 37 °C in 10.0 mL of phosphate buffered saline (PBS). The weight of the hydrogel disks was measured at each time point using an electric balance and then normalized by the initial weight. Cell Viability Assay. In vitro cell viability in the presence of the mixtures of the star block copolymer and iron ions was determined using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) assay (Cell Counting Kit-8; Dojindo Laboratories, Kumamoto, Japan) with NIH/3T3 mouse fibroblast cells (Riken Cell Bank, Tsukuba, Japan). Fibroblast cells were grown and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum at 37 °C in 5% CO2. Fibroblast cells were seeded in a 24-well plate at an initial density of 50 000 cells/well and incubated at 37 °C in 5% CO2 overnight. The medium was replaced with fresh medium, and then Transwell cell culture inserts (cat# 3422; Corning Incorporated, Corning, NY, USA) loaded with the mixture of the star block copolymer and iron ions (50 μL) were inserted into each well. Polymer concentration was varied from 5 to 20 wt %, whereas the concentration ratio of Fe3+/2+ to carboxylate ([Fe3+/2+]/[COO−]) was fixed at 0.5. The WST-8 assay was performed 24 h after the treatment using the mixture. A tetrazolium salt (WST-8) solution (50 μL) was added to each well, and then the resulting mixtures were incubated at 37 °C for 1 h. The absorbance of each well was measured at 450 nm using a plate reader (2030 ARVO V3; PerkinElmer, Waltham, MA, USA). The absorbance values were normalized relative to control wells where cells were incubated with the media in the absence of any test material. Subcutaneous Administration of the Materials. These experiments were performed at the Animal Research Section, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo. The Animal Care Committee of the University of Tokyo approved all of the procedures performed in this study before it began. ICR mice (4 weeks old, male) weighing 25 g were purchased from CLEA Japan, Inc. (Tokyo, Japan) and housed in groups in a 6 am−6 pm light−dark cycle. The star block copolymer was pretreated using an ethylene oxide sterilization process and then dissolved in saline at a concentration of 20 wt %. The other reagents, including 200 mM FeCl3, 200 mM FeCl2, 800 mM ascorbic acid, and 1.0 M H2O2, were prepared using saline as a solvent and sterilized using filtration (0.20 μm). Anesthesia was induced using isoflurane inhalation. The mixture of the star block copolymer solution (0.25 mL) and the FeCl3 solution (0.25 mL) was then administered subcutaneously into the posterodorsal wall using a dual syringe applicator with an 18-gauge needle (Terumo, Tokyo, Japan), followed by the administration of ascorbic acid solution (0.25 mL) into the same place using a syringe with a 25gauge needle. In addition, a mixture of the star block copolymer solution (0.25 mL) and the FeCl2 solution (0.25 mL) was prepared in advance and administered subcutaneously in the same way using a syringe with a 25-gauge needle, followed by administration of H2O2 solution (50 μL) into the same place using a syringe with a 25-gauge needle. The mice were sacrificed 1 day after the injections, and then the presence of any residue was evaluated. The subcutaneous tissues around the injection sites were sampled, fixed in a 10% formalin solution, and processed for histology (hematoxylin−eosin (HE)stained slides) using standard techniques.

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RESULTS AND DISCUSSION Gelation of the Star Block Copolymer Solution Induced by the Ferric Ion. The star block copolymer (DPE-g-OEGA-b-AA) was synthesized through a core-first approach, in accordance with our previous work.35 Star arms were grafted from the DPE core by sequential atom transfer radical polymerization (ATRP) of OEGA and tBA, followed by the deprotection of tert-butyl groups to obtain the star block copolymer. The average number of the star arms was calculated to be approximately nine, as discussed in our previous report.35 The degree of polymerization of the polyOEGA block and the PAA block was determined to be 18 and 27, respectively, from the 1H NMR spectra. The molecular weight distributions of the star polymers, as determined using gel permeation chromatography (GPC), are also shown in Figure S1. The gelation ability of the star block copolymer solution, following the addition of free Fe3+, was examined using an inverted vial test. The star block copolymer solution in saline (100 μL) was prepared in each vial, followed by the addition of FeCl3 solution in saline (100 μL). The final polymer concentration was 10 wt %, whereas the Fe3+ concentration was varied from 0 to 100 mM. After a gentle shaking of the mixtures, the vials were turned upside down to determine whether gelation had occurred or not. For comparison, the same experiments were performed using FeCl2 solution instead of FeCl3 solution. The star block copolymer solution (10 wt %) demonstrated gelation within 5 s when the concentration of Fe3+ exceeded a certain threshold (Figure 2a). The threshold concentration was 32 mM, where the concentration ratio of Fe3+ to carboxylate ([Fe3+]/[COO−]) was 0.16. However, in the presence of Fe2+ instead of Fe3+, the star block copolymer solution (10 wt %) remained as a free-flowing solution even when the concentration ratio of Fe2+ to carboxylate ([Fe2+]/[COO−]) reached 0.5 (Figure 2b). The same trends were observed when the

Figure 2. Results of the inverted vial tests. FeCl3 (a) or FeCl2 (b) solution was mixed with the star block copolymer solution in a vial, which was then turned upside down to check the gelation. Polymer concentration was set at 10 wt %, while iron ion concentration was varied from [Fe3+] or [Fe2+]/[COO−] = 0 to 0.5. C


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Macromolecules concentration of the star block copolymer was 5 or 20 wt % (Figure S2). The above results represent the first reported example of the in situ formation of totally synthetic hydrogels induced by the direct addition of free Fe3+. Since a PAA solution has been reported to cause precipitation when the [Fe3+]/[COO−] value reaches 0.025,38 the interaction of the PAA blocks of the star block copolymer with Fe3+ seems to be completely different compared with that of native PAA. The prevention of intramolecular cross-linking of the outer PAA blocks by the steric hindrance of the polyOEGA inner blocks can be attributed to the successful in situ formation of the homogeneous hydrogels, as in the case of Ca2+.35 Because of the precipitation problem, complex procedures have been conducted in previous work to obtain Fe3+-cross-linked hydrogels. For example, Yokoi et al.38 used a two-step procedure to prepare homogeneous PAA hydrogels crosslinked with Fe3+. They prevented the formation of large aggregates by adding free Fe3+ into a diluted PAA solution (≪5 wt %) dropwise, where the mixture was stirred vigorously. The dispersion was then heated to evaporate water, resulting in homogeneous hydrogels. Recently, Peng et al.7 used a Fe3+− citrate complex instead of free Fe3+ to slow down the rate of PAA−Fe3+ complex formation, leading to homogeneous PAA hydrogels. In the present work, gelation of the star block copolymer solution can be induced by simple addition of free Fe3+, and thus such complex procedures are not required. This feature enables the injection of Fe3+-cross-linked hydrogels into target sites, which broadens the range of their applications. Furthermore, the observed difference in the gelation ability between the mixtures with added Fe3+ and Fe2+ suggests the potential of the star block copolymer as a redox-responsive material. The different gelation ability of these hydrogels is because of the variation of the strength of cross-links formed by these iron ions. Henderson et al. concluded that the strength of ionic cross-links was mainly dominated by the stability constant of the metal−carboxylate complex.36 Considering that the stability constant log K1 of Fe3+−citrate and Fe2+−citrate complexes is 11.85 and 3.2, respectively,25 we can infer that the stability constant of the star block copolymer−Fe3+ complex is much higher than that of the star block copolymer−Fe2+ complex. This hypothesis is examined spectroscopically in the following section. Chemical Structure of the Iron−Carboxylate Complex. The structure of the complex formed within the mixture of the star block copolymer and iron ions was characterized spectroscopically using FT-IR (Figure 3a) and Mössbauer analyses (Figure 3b). The FT-IR spectrum of the star block copolymer showed three characteristic peaks from PAA at 1638, 1570, and 1408 cm−1. The peak at 1638 cm−1 (dashed arrow) reflects ν(CO) of the protonated carboxylate groups, while the other two peaks at 1570 and 1408 cm−1 (solid arrows) are assigned to νas(O− C−O) and νs(O−C−O) of the deprotonated carboxylate groups, respectively. We then recorded the FT-IR spectrum of the mixture of the star block copolymer and Fe3+. The peak at 1638 cm−1 disappeared, and the two peaks reflecting νas(O− C−O) and νs(O−C−O) (solid arrows) shifted to higher wavenumbers: 1594 and 1422 cm−1, respectively. In contrast, in the FT-IR spectrum of the mixture of the star block copolymer and Fe2+, the two peaks at 1570 and 1408 cm−1 disappeared, while the peak at 1638 cm−1 (dashed arrow) became larger.

Figure 3. (a) FT-IR spectra and (b) Mössbauer spectra of the star block copolymers and their mixtures with Fe3+ or Fe2+. The dashed arrows in (a) indicate the characteristic peaks reflecting ν(CO) of the carboxylates, and the solid arrows indicate the characteristic peaks reflecting νas(O−C−O) and νs(O−C−O) of the carboxylates.

From the observed shifts and shape changes in the FT-IR spectra, the coordination state of Fe3+ and Fe2+ with the star block copolymer can be estimated to be bidendate bridging and monodendate coordination, respectively, according to previous reports39,40 as follows. There are three possible modes of coordination: monodendate coordination (Figure S3a), bidenD


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Figure 4. (a) Frequency dependencies of the storage modulus G′ (closed symbols) and loss modulus G″ (open symbols) of the 10 wt % star block copolymer in the absence (circles) or presence (squares) of Fe3+ (Representative data). (b) G′ of the star block copolymer hydrogels cross-linked using Fe3+, measured at 1 Hz. Data are averages ± standard deviations (N = 4). The insets are images of the obtained hydrogels. [Fe3+]/[COO−] was fixed at 0.5.

block copolymer hydrogel cross-linked using Fe3+. In the following experiments, we fixed [Fe3+/2+]/[COO−] at 0.5 assuming that this amount of iron ions is sufficient against the amount of carboxylate groups. We measured the frequency (ω) dependences of storage modulus (G′) and loss modulus (G″) of the star block copolymer solution (10 wt %) in the absence or presence of Fe3+ (Figure 4a). The star block copolymer solution in the absence of Fe3+ showed the characteristic features of a viscoelastic liquid; G′ and G″ scaled with ω2 and ω, respectively. In the presence of Fe3+, on the other hand, the values of both G′ and G″ were frequency-independent across a wide range of frequencies, and G′ was larger than G″, indicating that this sample behaved as a viscoelastic solid. These results confirmed the successful gelation of the star block copolymer solution induced by the addition of Fe3+. Figure 4b further illustrates that Fe3+-cross-linked hydrogels can be formed with a wide range of polymer concentrations (5−20 wt %). The G′ (at 1 Hz) value increased with increasing polymer concentrations, from 26 Pa at 5 wt % to 1400 Pa at 20 wt %. These results suggest that the mechanical strength of the star block copolymer hydrogels can be tuned through their polymer concentration. The above results suggest a higher cross-linking density in the Fe3+-cross-linked hydrogel compared with the Ca2+-crosslinked hydrogel in our previous work.35 When Ca2+ was used as a cross-linker,35 a highly concentrated polymer solution (20 wt %) was required to prepare the hydrogel. In addition, the resulting hydrogel was considered to be a low cross-linkingdensity gel; G′ and G″ of the Ca2+-cross-linked hydrogel largely scaled with ωn, which is a characteristic behavior of cross-linked polymers at the gel point.42 In the present work, the formation of the Fe3+-cross-linked hydrogels at a wide range of polymer concentrations was demonstrated, and a high cross-linking density was suggested from the frequency independence of G′ and G″. These results arise from the higher stability constant of the Fe 3+ −carboxylate complex. It is known that the coordination of Fe3+ with carboxylate is much more stable than that of Ca2+ because of pseudocovalent bond formation.37 In the case of ionic cross-linking, the higher stability of the complex is reported to relate to a higher bond lifetime,36 therefore leading to a higher cross-linking density. Fe3+-cross-linking is also expected to affect the degradation kinetics of the star block copolymer hydrogel under

date chelating (Figure S3b), and bidendate bridging (Figure S3c). The monodendate structure has distinct double CO and single C−O bonds, while the bidendate structures, either chelating or bridging, have equivalent CO bonds. The bidendate structures are further classified into two modes: bidendate chelating where the ligands act as a chelating ligand of one metal and bidendate bridging where the ligands act as a cross-linker of two metals. Deacon and Philips39 reported an empirical rule to identify the mode of coordination from the gap in the wavenumbers between the peaks of νas(O−C−O) and νs(O−C−O), defined as Δν. If Δν of the complex is much smaller than that of the native carboxylate, the complex has the bidendate chelating structure. On the other hand, if the Δν of the complex is similar to that of the native carboxylate, the complex has the bidendate bridging structure. According to this rule, the possible coordination mode of Fe3+ with the star block copolymer can be estimated to be the bidendate bridging structure because the Δν of the resulting mixture (Δν = 172 cm−1) is similar to that of the native star block copolymer (Δν = 162 cm−1). In contrast, the star block copolymer and Fe2+ appeared to form a monodendate structure, as evidenced by the disappearance of the two peaks attributed to νas(O−C−O) and νs(O−C−O). Generally, a complex with a bidendate structure has a much higher stability constant compared with that with a monodendate structure.41 Therefore, we consider that this difference in complex structure is the reason for the difference in the gelation behavior between mixtures containing Fe3+ and Fe2+. Mössbauer analyses of the iron-cross-linked star block copolymer hydrogel and the iron-coordinated star block copolymer solution further confirmed the oxidation state of the iron ions. The Mössbauer spectrum of the former sample in the dried state showed an isomer shift (IS) value of 0.377 mm s−1 and small quadrupole splitting, which is a typical behavior of high-spin Fe3+. However, the Mössbauer spectrum of the latter sample in the dried state showed an IS value of 1.05 mm s−1 and large quadrupole splitting, indicating that this sample contains high-spin Fe2+. Therefore, the high-spin Fe3+ and the high-spin Fe2+ were responsible for the respective formation of the above-mentioned complexes. Mechanical Strength and Degradation Kinetics of the Star Block Copolymer Hydrogels Cross-Linked Using the Ferric Ion. Dynamic viscoelastic measurements were conducted to evaluate the mechanical properties of the star E


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block copolymer hydrogel presented here showed much higher stability against degradation. We consider the reason to be the stability of the metal−polymer complex. As mentioned above, the stability of the Fe3+−carboxylate complex is much higher than that of the Ca2+−carboxylate complex.37 We speculate that this stable complex formation prevented the release of Fe3+, which resulted in the longer degradation time of the hydrogels through maintenance of the cross-linking points. This prolonged degradation time would be beneficial for the future biomedical application of the star block copolymer hydrogel. Cytocompatibility of the Star Block Copolymer− Fe2+/3+. The cytocompatibility of the Fe3+-cross-linked and Fe2+-coordinated star block copolymer was evaluated through a cell viability assay using fibroblast cells (NIH/3T3). In order to avoid physical damage to the cells induced by contact with the hydrogels, we applied the materials via Transwell culture inserts, which have permeable membrane supports on the bottom. The cell viability was evaluated 1 day after the treatment with the materials, using a WST-8 assay. Regardless of the valence of the iron, the presence of the mixture of the 10 wt % star block copolymer solution and the iron chloride solution (50 μL) did not affect the viability of the NIH/3T3 cells (Figure 6a). The results are independent of the polymer

physiological conditions, which is an important factor for biomedical applications. The star block copolymer hydrogels cross-linked with Fe3+ were incubated at 37 °C in PBS (pH 7.4), and the weight of the hydrogels was measured at each time point and then normalized by the initial weight (Figure 5).

Figure 5. Degradation kinetics of the star block copolymer hydrogel cross-linked with Fe3+ at 37 °C in PBS (pH 7.4). The polymer concentration was set at 5 wt % (open triangles), 10 wt % (closed squares), and 20 wt % (open circles). Relative mass is the ratio of the weight of the hydrogel at each time point to the initial weight, expressed as a percentage. Data are averages ± standard deviations (N = 4).

The polymer concentration of the hydrogels highly affected their degradation rate. Complete degradation of the hydrogels at the polymer concentrations of 5, 10, and 20 wt % was observed after 6 h, 7 day, and 1 month of incubation, respectively (Figure S4a), suggesting that the stability of the hydrogels, as well as their mechanical strength as mentioned above, can be controlled through polymer concentration. The polymer concentration of the hydrogels also affected the initial gel swelling, which can be observed as initial increase in the relative mass beyond 100% for the 10 and 20 wt % hydrogels (Figure S4b). Comparing 20 wt % with 10 wt %, the initial gel swelling was suppressed as the polymer concentration increased, which is because of the increase in cross-linking density. Interestingly, as for the 20 wt % hydrogel, slight increase in the relative mass from day 3 to day 12 was observed in Figure S4a. This increase might represent swelling of the hydrogel as a result of decrease in cross-linking density during the first 3 days. We speculate that during the first several days the hydrogel showed burst release of Fe3+ as well as the star block copolymer, resulting in the decrease in cross-linking density. The degradation behavior of the star block copolymer itself was also investigated. The star block copolymer was incubated at 37 °C in a variety of solvents, and changes in its molecular weight were measured using GPC. The star block copolymer showed no pronounced degradation under a wide range of pH values or even in the presence of lipase or serum (Figure S5). These results suggest that the degradation of the star block copolymer hydrogels cross-linked using Fe3+ is induced mainly by the loss of cross-linking points as a result of bound Fe3+ release, rather than the degradation of the star block copolymer itself. Compared with cross-linking using Ca2+, as in our previous work,35 in which the hydrogel was completely degraded within 5 h under physiological conditions, the Fe3+-cross-linked star

Figure 6. Viability of fibroblast cells (NIH/3T3) after treatment with (a) the mixture of the 10 wt % star block copolymer with FeCl3 or FeCl2 or (b) the mixture of the star block copolymer with FeCl3 at different polymer concentrations, measured using a WST-8 assay after 1 day of incubation. Data are averages ± standard deviations (N = 4).

concentration in the range of 5−20 wt % (Figure 6b). Taken together with the low cytotoxicity of the star block copolymer itself, as shown in our previous report,35 these results suggest the good cytocompatibility of the iron-associated material. Considering the positive in vitro results presented above, we fixed the polymer concentration at 10 wt % and moved onto further experiments. Redox-Responsiveness of the Star Block Copolymer− Fe2+/3+. The different gelation properties achieved using Fe3+ and Fe2+ (Figure 2) suggest the potential of the star block copolymer as a redox-responsive system, mediated by the transition of the iron ions between two oxidation states. We examined the redox-responsive sol-to-gel or gel-to-sol transition using an inverted vial test. Various kinds of oxidizing agents, such as sodium hypochlorite, K3[Fe(CN)6], or ammonium cerium nitrate, as well as reducing agents, including glutathione, dithiothreitol, or sodium erythorbate, have been used to change the oxidation state of iron ions.6,16,22 Among them, we chose H2O2 and ascorbic acid as oxidizing and reducing agents because both ascorbic acid and 3% w/v H2O2 are used as F


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Macromolecules pharmaceuticals43,44 and are expected to show good biocompatibility. The results of the evaluation of the redox responsivity of the star block copolymer hydrogel are shown in Figure 7. The 10

supported by a dynamic viscoelasticity measurement where G″ scaled with ω (Figure 7b-1). An oxidizing agent, H2O2, was then added to the solution to initiate the oxidation of Fe2+ to Fe3+ in the star block copolymer solution. As a result, the formation of a homogeneous hydrogel was observed within a few minutes (Figure 7a-2). The G′ and G″ of the hydrogel remained unchanged across a wide range of frequencies, and G′ was larger than G″, illustrating the characteristics of a viscoelastic solid (Figure 7b-2). Furthermore, this hydrogel changed back to the solution state within a few minutes through the addition of a reducing agent, ascorbic acid (Figure 7a-3). The G″ scaled with ω again in the rheological measurements (Figure 7b-3). The results shown in Figure 7 successfully demonstrate the redox-responsive, reversible sol−gel transition of the star block copolymer solution containing iron ions. This sol−gel/gel−sol transition was mediated by the oxidation/reduction of iron ions, which worked as a valence-sensitive cross-linker. The transition of the oxidation state of iron ions, induced by an oxidizing or reducing agent, is quite rapid; generally, H2O2 oxidization and ascorbic acid reduction of free Fe3+ are reported to be completed within 1 s.45,46 As a result of this fast reaction, the star block copolymer−Fe2+/3+ showed a rapid redoxresponsive sol−gel transition within a few minutes. This response is rapid enough for various applications and is advantageous for use in vivo, which is a dynamically changing environment. Although a redox-responsive, reversible sol−gel transition using iron ions has been investigated in previous studies, the resulting systems did not show such a rapid and clear response as that presented in the current study. In the case of PAA− Fe2+/3+ 7,24 or chitin−Fe2+/3+ systems,22 a much longer reaction time was required; the sol-to-gel transition was induced by the air oxidation of Fe2+ for several hours to several days or a gradual electro-oxidization of Fe2+ on the surface of an

Figure 7. (a) Results of the inverted vial tests and (b) frequency dependencies of G′ (closed symbols) and G″ (open symbols) of the 10 wt % star block copolymer in the presence of iron ions: (1: circles) the 10 wt % star block copolymer solution (400 μL) containing Fe2+; (2: squares) after treatment using 1.0 M H2O2 (40 μL); and (3: triangles) after an additional treatment using 1.6 M ascorbic acid (200 μL). Note that the G′ values of samples 1 and 3 are below the detection limit and therefore are not presented in this figure.

wt % star block copolymer solution containing Fe2+ behaved as a free-flowing solution (Figure 7a-1), which was further

Figure 8. Subcutaneous injection of the 10 wt % star block copolymer hydrogels cross-linked using Fe3+ (a) without or (b) with post-treatment using ascorbic acid solution or the 10 wt % star block copolymer solution containing Fe2+ (c) without or (d) with post-treatment using H2O2 solution and evaluation of the mice 1 day after the injection: (left) pictures of the mice right after the injection, (middle) pictures of the subcutaneous tissue of the mice, and (right) HE stained pictures of the injection sites on day 1 (10×). Scale bar = 250 μm. G


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Macromolecules electrode. This is because the immediate oxidation of Fe2+ through the addition of oxidizing agents causes precipitation of the polymers rather than formation of homogeneous hydrogels. However, in the case of our star block copolymer, the prevention of precipitation by the block copolymer arms composed of a hydrophilic, bulky poly(ethylene glycol) bottlebrush block and a negatively charged PAA block enabled sol-togel transition via the rapid oxidation of Fe2+, which resulted in a rapid redox response. In addition, the star-shaped structure of the precursor polymer is considered to be another key factor to achieve this fast response. Unlike linear polymers, the starshaped polymers are not highly entangled in solution.47 The dissolved star-shaped polymers are also considered to be well dispersed by hydration and charge repulsion. Therefore, the disentanglement of the polymer chains might not be the ratedetermining step, and these unique properties of the star block copolymer resulted in a rapid and reversible sol−gel transition using redox stimuli. In Vivo Redox-Responsiveness and Biocompatibility of the Star Block Copolymer−Fe2+/3+ . The redoxresponsive, reversible sol−gel transition of the star block copolymer−Fe2+/3+, as well as its injectability and cytocompatibility, motivated us to examine its performance in vivo. The redox-responsive gel−sol/sol−gel transition of the star block copolymer−Fe2+/3+ in vivo was examined via subcutaneous transplantation of the materials into mice. The 10 wt % star block copolymer hydrogel cross-linked using Fe3+ was administered into two mice using a double barrel syringe; the star block copolymer and Fe3+ were mixed at the injection sites. One mouse was subsequently subjected to subcutaneous administration of an ascorbic acid solution, while the other did not receive further treatment. In addition, a 10 wt % star block copolymer solution containing Fe2+ was administered to two mice. One of these two mice was further administered H2O2 subcutaneously, while the other one was not. One day after the injections, the residue of the hydrogel material was still observed at the injection site in the mouse treated using the star block copolymer with Fe3+ (Figure 8a), while no hydrogel was observed in the mouse further treated using ascorbic acid solution (Figure 8b). These results indicate the in situ formation of the star block copolymer hydrogel cross-linked using Fe3+ and the subsequent in vivo gel-to-sol transition in response to the reduction caused by the ascorbic acid. In contrast, no material was observed in the mouse treated using the star block copolymer solution containing Fe2+ (Figure 8c), while formation of the hydrogel material was found in the mouse further treated using H2O2 (Figure 8d), suggesting an in vivo sol-to-gel transition in response to the oxidation caused by the H2O2. In addition, no mice showed adverse effects. The inflammation around the materials or injection sites was extremely mild; although accumulation of neutrophils, which are inflammatory cells recruited at the early stage of inflammation, was found in the HE-stained pictures (Figure 8 and Figure S6), the observed infiltration of inflammatory cells was considered to be at the same level with that observed in biocompatible hyaluronan-based materials.48,49 The prevention of Fe3+-mediated polymer precipitation as a result of the unique structure of the precursor polymer allowed the in situ hydrogel administration. The rapid and clear gel− sol/sol−gel transition shown in the in vitro experiments above was also achieved in vivo. In addition, clearance of the material from the injection sites was highly affected by whether the material was in a gel or sol state. When the material was

oxidized to a gel state, it was still observed at the injection sites 1 day after administration. Note that the residual hydrogel was found even 1 week after the administration (data not shown). This is caused by the high stability of the hydrogel under physiological conditions, as indicated in Figure 5. In contrast, when the material was reduced to a sol state, it was completely cleared from the injection sites within 1 day. We speculate that this rapid clearance is attributable to the unique features of the precursor polymer. The excellent solubility and dispersibility of the precursor polymer in aqueous solution are likely to contribute to the rapid clearance of the Fe2+-coordinated star block copolymer through the systemic circulation or the lymphatic system. A similar rapid clearance of injected material from the subcutaneous tissue was also observed in the case of the Ca2+-cross-linked star block copolymer hydrogel we previously reported.35 Since the star block copolymer has a polyester core, scission of the star arms can potentially occur by hydrolysis of the star core through enzymatic degradation. Considering that the molecular weight of the star arms and cores are ∼10 and ∼3 kDa, the cleaved polymers would be able to be excreted from the kidney, molecular weight cutoff of which is reported to be ∼60 kDa.50,51 Considering its good injectability, fast gel−sol/sol−gel transition in response to redox stimuli, rapid clearance of the material in the sol state, and good biocompatibility, this material is expected to be useful for a variety of biomedical applications. For example, local drug delivery with the feature of on-demand drug release could be achieved using this material. This material could also be used as a “smart” scaffold in tissue engineering; the implanted material can be removed immediately at a desired time in a noninvasive manner. Biosensors or actuators that work inside living systems could be another potential application for this material. One concern may be the toxicity of iron ions in the star block copolymer−Fe2+/3+ system. It has been reported that iron produces toxicity as a result of its ability to catalyze the generation of reactive oxygen species, which could cause damage to cells or tissues.27,28 At least in the present research, the HE staining images shown in Figure 8 did not show any severe inflammation around the material in vivo, and therefore the toxic effects of the Fe3+ cross-linker are considered to be mild. This is caused by the gradual release of iron ions from the coordination sites, which prevents a rapid increase in local iron concentrations. The released iron ions are then considered to bind with extracellular transferrin and enter the systemic circulation, followed by recycling for use in the production of hemoglobin or myoglobin or in hepatocytes.27,28 In the current study, we have developed a redox-responsive star block copolymer hydrogel based on Fe-cross-linking, and demonstrated its successful in vivo sol−gel/gel−sol transition as a result of its well-defined in situ gelation and its good biocompatibility. Further investigations on controlling the physical properties and functions of hydrogels through the design of the precursor polymers are now underway in our laboratory. We hypothesize that the stiffness of hydrogels, as well as the viscosity of the precursor solution, can be tuned by controlling the structure of the star block copolymers. For example, higher gel stiffness is expected if we use longer PAA chains or stronger ligands instead of carboxyl groups. These explorations could further improve the utility of the Fe3+-crosslinked star block copolymer hydrogel for a wide variety of biomedical applications. H


Macromolecules

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CONCLUSIONS We have developed an in situ gellable, redox-responsive star block copolymer hydrogel based on Fe-cross-linking. As a precursor polymer, we used a star block copolymer with a DPE core and double hydrophilic block copolymer arms composed of polyOEGA blocks and PAA blocks, which we have reported previously. Addition of Fe3+ to the star block copolymer solution induced the in situ formation of homogeneous hydrogels while the addition of Fe2+ did not, suggesting a difference in the cross-linking abilities of Fe3+ and Fe2+. The storage moduli of the star block copolymer hydrogels crosslinked using Fe3+ were 26−1400 Pa, and they were stable in PBS (pH 7.4) for as long as 1 month. In addition, a mixture of the star block copolymer solution and an iron chloride solution showed low cytotoxicity regardless of the valence of the iron. Furthermore, a rapid, redox-responsive sol−gel/gel−sol transition of the star block copolymer−Fe2+/3+, based on the transition of iron ions between two oxidation states, was observed through an in vitro inverted vial test. Finally, the successful in vivo gel−sol/sol−gel transition of this material, as a result of its in situ gelation and good biocompatibility, was demonstrated through mouse subcutaneous transplantation. The current results provide a promising platform for the design of redox-responsive, injectable hydrogels with good biocompatibility, which are applicable to a variety of biomedical applications.

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REFERENCES

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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.7b01020. Experimental details, molecular weight distributions of the star polymers, additional data for the inverted vial tests, possible modes of coordination of iron ions with carboxylate groups, additional data for degradation kinetics of the materials, and enlarged images of the HE-stained pictures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (T.I.). ORCID

Atsuo Yamada: 0000-0002-7880-5701 Taichi Ito: 0000-0002-1589-8242 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We offer our deepest thanks to Perstorp Japan Co., Ltd.. for supplying DPE, Shin-Nakamura Chemical Co., Ltd., for supplying OEGA, Mitsubishi Chemical Corporation for supplying chelate resin, Chubu Chelest Co., Ltd., for supplying EDDS, and Nitto Denko Corporation for supplying the ultrafiltration membrane. We also thank Professor Tei/Chung and Associate Professor Sakai at The University of Tokyo for providing access to a rheometer. Y.N. appreciates a Research Fellowship from the Japan Society for the Promotion of Science (JSPS). This work was supported by a Grant-in-Aid for JSPS Research Fellow (No. 16J08368). I


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