Reinforcing DNA Supramolecular Hydrogel with Polymeric Multiple-Unit Linker
2022; Chinese Chemical Society; Volume: 5; Issue: 2 Linguagem: Inglês
10.31635/ccschem.022.202101523
ISSN2096-5745
AutoresYujie Li, Yuqiao Ding, Bo Yang, Tianyang Cao, Jiang‐Fei Xu, Yuanchen Dong, Quan Chen, Lijin Xu, Dongsheng Liu,
Tópico(s)Dendrimers and Hyperbranched Polymers
ResumoOpen AccessCCS ChemistryRESEARCH ARTICLE18 Mar 2022Reinforcing DNA Supramolecular Hydrogel with Polymeric Multiple-Unit Linker Yujie Li, Yuqiao Ding, Bo Yang, Tianyang Cao, Jiangfei Xu, Yuanchen Dong, Quan Chen, Lijin Xu and Dongsheng Liu Yujie Li Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084 , Yuqiao Ding Department of Chemistry, Renmin University of China, Beijing 100872 , Bo Yang Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084 , Tianyang Cao Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084 , Jiangfei Xu Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 , Yuanchen Dong CAS Key Laboratory of Colloid, Interface and Chemical Thermodynamics, Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Quan Chen State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 , Lijin Xu Department of Chemistry, Renmin University of China, Beijing 100872 and Dongsheng Liu *Corresponding author: E-mail Address: [email protected] Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084 https://doi.org/10.31635/ccschem.022.202101523 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail While supramolecular hydrogels have received growing interest due to their unique dynamic features, their relatively weak mechanical properties have largely limited their biomedical applications. In this study, we propose and demonstrate a strategy to reinforce the mechanical properties of supramolecular hydrogel by introducing polymeric multiple-unit linker (PMUL), which incorporates multiple supramolecular units into a polymeric backbone to crosslink supramolecular hydrogel. We demonstrated that PMUL can effectively improve the kinetic stability of supramolecular crosslinkers through multiple-unit interaction in a DNA supramolecular hydrogel model system, thus leading to higher mechanical strength. Meanwhile, the dynamic features of the supramolecular hydrogels have been well preserved, including shear-thinning, self-healing properties, and reversible thermal responsiveness. This strategy offers a simple but effective way for mechanical reinforcement of supramolecular hydrogels to construct novel biomaterials. Download figure Download PowerPoint Introduction Extracellular matrix (ECM) is a fascinating natural material, which can provide both mechanical support and abundant dynamic properties for cells. This seemingly contradictory combination between mechanical strength and dynamics is crucial to realize a lot of fundamental biological functions, which have been explained by the synergy of multiple noncovalent interactions among the hierarchical components of the ECM.1 Constructing such delicate structures to realize the marvelous mechanical properties of natural ECM in synthetic systems is of great interest in the field of biomaterials2 but remains extremely challenging. During recent decades, hydrogel materials, including both covalent and supramolecular systems, have been developed with the expectation to mimic ECM functions for cell biology.3 Covalently crosslinked hydrogels are well-known for their good mechanical strength, but lack of dynamic features. On the other hand, supramolecular hydrogel formed by self-assembly through noncovalent interactions, which is the same as ECM, is considered one of the most promising materials.4,5 The transient and reversible nature of noncovalent interaction endows supramolecular hydrogel with self-healing, shear-thinning properties and shape-adaptivity, which facilitate extraordinary dynamic features.6,7 However, the high association and dissociation rate of traditional single noncovalent interaction leads to the poor mechanical performance of supramolecular hydrogels,8 which limits their engineering applications. Therefore, reinforcing supramolecular hydrogel to increase its mechanical strength without compromising its dynamic features is extremely attractive and essential to construct novel biomaterials in many biomedical applications. In the past few decades, considerable effort has been made to enhance the mechanical strength of supramolecular hydrogels.9,10 A classical strategy is to increase the supramolecular crosslinking density with higher concentrations or ratios of the crosslinking moieties.11,12 However, high crosslinking density inherently leads to compact network with low porosity, poor permeability, and high viscosity, which limit the applications of hydrogels in biomedical fields. Introduction of covalent crosslinkers into the supramolecular network has also been proven to effectively reinforce supramolecular hydrogels.13,14 Nevertheless, the self-healing and other dynamic behaviors are impaired due to the irreversibility of covalent crosslinkers. Furthermore, cooperatively combining multiple supramolecular interactions to increase system complexity is also an effective method to enhance the mechanical performance. For example, nanocomposites (such as carbon nanomaterials, metal nanostructures, and nanofibers)15,16 or multiple orthogonal supramolecular crosslinking reactions17–19 can be incorporated into supramolecular hydrogels and serve as extra multivalent crosslinking sites. However, it should be noted that the change of composition may cause potential toxic effects and restrict their clinical applications. Recently, controlling the kinetics of supramolecular crosslinking interaction has been proposed for mechanical reinforcement of supramolecular hydrogels, which brought a promising potential to overcome the above challenge.10 It is known that the exchange kinetics of the supramolecular crosslinkers plays a dominant role in determining the bulk mechanical behaviors of supramolecular gels,20,21 and slower exchange rates of supramolecular interaction can lead to stronger materials.22 Therefore, improving the kinetic stability of supramolecular crosslinking interaction is a more intrinsic method to reinforce the hydrogels without direct impacts on their dynamic behaviors, which has been demonstrated in the lately reported Kinetically Interlocking Multi-Unit (KIMU) strategy.23,24 KIMU strategy can be described as multiple supramolecular half units covalently linked by polymeric chains that inhibit the synchronous and cascade dissociation of different units and thus greatly decrease the overall dissociation rates. Therefore, KIMU offers us an effective way to improve the kinetic stability of overall supramolecular interactions with a single supramolecular unit remaining unchanged, so stable supramolecular polymers can be prepared, even under very diluted conditions. Furthermore, as a model of the KIMU effect, supramolecular hydrogels self-assembled by DNA building blocks have been reported to exhibit relatively stronger mechanical strength.25,26 This unique mechanical performance of DNA supramolecular hydrogel is attributed to the slow kinetics of dehybridization of double-stranded DNA crosslinker, which also endows the hydrogel with multiple responsiveness and dynamic behaviors.27,28 Inspired by KIMU strategy and the synergy of multiple noncovalent interactions in natural ECM, we hypothesized that further incorporating multiple supramolecular units into a polymeric backbone with careful design to crosslink hydrogel could improve the mechanical strength of bulk material. We anticipated that through rational design, the composition, supramolecular crosslinking density, and dynamic behavior of the supramolecular hydrogels would be well preserved. In this study, we demonstrated a mechanical reinforcement strategy by introducing polymeric multiple-unit linker (PMUL) into a DNA supramolecular hydrogel model system self-assembled by DNA Y-scaffold and linker. The introduced PMUL improved the overall kinetic stability with enhanced mechanical strength of DNA supramolecular crosslinking due to the synergetic effect of multivalent interactions. Meanwhile, the dynamic features of the DNA supramolecular hydrogels could also be well preserved. This combination of mechanical strength and dynamic behaviors, as well as good biocompatibility, makes this mechanically reinforced DNA supramolecular (MRDS) hydrogel a good candidate for cell culture scaffolding and tissue engineering. Experimental Methods Synthesis of the PMUL The overview of the synthesis of the PMUL is presented in Supporting Information Figure S2. The long chain ssDNA was synthesized via the rolling circle amplification (RCA) reaction. Firstly, 5′-phosphorylated linear templates were cyclized using ssDNA CircLigase. To degrade non-circularized templates, the resulting mixture was further treated with exonuclease I and exonuclease III. The circular templates were then purified by filtration with 10 kDa cutoff. For the RCA reaction, circularized template was mixed with primer, bovine serum albumin (BSA), dNTPs, inorganic pyrophosphatase, and phi29 DNA polymerase in phi29 DNA polymerase buffer. The mixture was kept for 4 h at 30 °C to produce long ssDNA. It is worth mentioning that pyrophosphatase should be added during the RCA reaction to avoid the formation of DNA/MgP2O7 nanoflower structures.29 Upon hybridizing with the complementary strand, the PMUL was obtained. Details of the experimental procedures can be found in the Supporting Information. Preparation of MRDS hydrogel Stoichiometric amounts of DNA strands of the Y-scaffold (Y1, Y2, and Y3) were lyophilized and then phosphate-buffered saline (PBS) buffer (pH 7.4) was added to give a final concentration of 500 μM for each DNA strand. A certain amount of long chain ssDNA was mixed with complementary strands and stoichiometric amounts of DNA strands of the linker (L1 and L2), where the amount of complementary strands is equal to the repeated fragments of long-chain ssDNA. The percentages of linker replaced by PMUL in terms of the molar quantity of repeating units were controlled as 0%, 2.5%, 5%, 7.5%, and 10% to prepare different samples. The mixture was freeze-dried and added with PBS buffer (pH 7.4) to give a final concentration of 750 μM for each linker strand. The two resulting mixtures were both heated to 95 °C for 5 min and subsequently cooled to room temperature in 2 h. Then equivoluminal Y-scaffold and linker solutions were mixed. The solutions lost their fluidity and formed supramolecular hydrogels within a minute. Other experimental procedures, including rheological tests, three-dimensional (3D) cell culture, and a cell viability test are outlined in the Supporting Information. Results and Discussion System design and formation of MRDS hydrogels Currently developed supramolecular hydrogels as ECM mimics for cell culture had a broad range of mechanical strength to meet the requirement of different occasions30 ( Supporting Information Figure S1). In this study, we chose a typical KIMU DNA supramolecular (DS) hydrogel as a model system, which was self-assembled from DNA linker and DNA Y-scaffold building blocks as illustrated in Scheme 1. DNA linker was a linear DNA duplex containing 12 nt single-stranded DNA (ssDNA) sticky ends (A domain, red), while Y-scaffold was a three-armed DNA structure with three sticky ends (CA domain, complementary to A, red) complementary to A domain (detailed sequences are listed in Supporting Information Table S1). According to a previous study,25 a DNA supramolecular hydrogel would be formed upon mixing Y-scaffold and linker via DNA complementary base-pairing between the A and CA domain. Scheme 1 | Schematic representation of DS and MRDS hydrogel networks formation. PMUL incorporating multiple supramolecular units into a polymeric backbone is introduced to prepare MRDS hydrogel with higher kinetic stability of overall supramolecular crosslinking interaction. Download figure Download PowerPoint To enhance the mechanical strength of DS hydrogel, we further introduced PMUL which contained a periodic single-stranded crosslinking region (A domain, red) and a double-stranded spacer region (B/CB domain, complementary to B, blue). Multiple Y-scaffold could be further crosslinked by an additional covalent-like network through the polymeric chains of PMUL into kinetically trapped and interlocked states. Due to the slower exchange kinetics of supramolecular crosslinkers after the introduction of the PMUL (Scheme 1), we anticipated that the resulting MRDS hydrogel would exhibit higher mechanical strength, without sacrificing the dynamic nature of DS hydrogel. Therefore, the MRDS hydrogel would preserve the previously described characteristics of the DS hydrogel, such as injectability, self-healing ability, reversible thermal responsiveness, and good biocompatibility. PMUL was prepared as depicted in Supporting Information Figure S2, which was composed of a long ssDNA and the partially complementary strand CB. The long-chain ssDNA was prepared by RCA reaction,31 which is an isothermal enzymatic process that produces long chain ssDNA with repeating sequences. A circular template containing two CA-CB sequence domains was initially designed and prepared as shown in Supporting Information Figure S3a. Under room temperature, long-chain ssDNA consisting of A–B repeating units was synthesized under the catalysis of the phi29 enzyme ( Supporting Information Figure S3b). The PMUL was prepared by hybridizing the long-chain ssDNA with the complementary short strand CB ( Supporting Information Figure S4). To prepare MRDS hydrogel, a certain amount of linker in DS hydrogel was replaced by PMUL in terms of equivalent repeating units (A+B domain) to crosslink Y-scaffold completely. It should be noted that while the molar ratio was kept constant, only a small change of solid content (0.003%) was achieved when 10% linker was replaced, which could be neglected. To examine the influence of PMUL on the mechanical performance of the hydrogels, a series of MRDS hydrogels was prepared by varying the molar percentages of introduced PMUL (PPMUL), which were denoted as MRDS-PPMUL hydrogels. In our experiments, the highest PPMUL was set to 10% to avoid the potential change of the molecular network. Figure 1a showed that free-standing MRDS-10 hydrogel was relatively strong, retaining their shapes on tweezers. Figure 1 | (a) Representative MRDS-10 hydrogel stained with GelRed (red) free-standing at the bottom of tube and retaining its shape on tweezers. Scale bar, 10 mm. (b) Rheological time-sweep tests of DS hydrogel and MRDS hydrogels with a fixed strain of 1% and frequency of 1 Hz at 25 °C. (c) Rheological strain-sweep tests of DS hydrogel and MRDS hydrogels with a fixed frequency of 1 Hz at 25 °C. (d) Overview of oscillatory rheological properties of DS hydrogel and MRDS hydrogels (ω = 1 Hz, γ = 1%, 25 °C). **p < 0.01 (Student's t-test). Download figure Download PowerPoint Mechanical properties To investigate the mechanical reinforcement effect, rheological studies were applied to measure the mechanical strength of the hydrogels. The time-sweep test was first carried out with fixed strain of 1% and frequency of 1 Hz at 25 °C. As illustrated in Figure 1b, both DS hydrogel and MRDS hydrogels with different PPMUL exhibited stable solid-like behavior with the value of the storage modulus (G′) much higher than that of the loss modulus (G″). The hydrogels also exhibited stress relaxation when a strain of 10% was applied ( Supporting Information Figure S5). Strain amplitude sweep was then applied to the hydrogels at a fixed frequency of 1 Hz. As shown in Figure 1c, all samples exhibited linear viscoelastic responses at lower values of the applied strain (0.1–10%) where G′ and G″ remained almost constant. As the strain increased, the value of G′ rapidly decreased, and the value of G″ value increased. At a critical strain, the two curves intersected, and hydrogels collapsed into a sol state with broken network structures. The critical strain values of all samples were in the range of 40–60% without significant difference, indicating similar network structures. To reveal the mechanical reinforcement effect of introducing PMUL, the G′ taken at frequency (ω) = 1 Hz and strain (γ) = 1% was used as a measure of hydrogel mechanical strength and summarized in Figure 1d. The G′ of original DS hydrogel without PMUL was 1008 Pa. The presence of only 2.5% PMUL significantly enhanced the strength to 1500 Pa. Increasing PPMUL to 5%, the G′ value continued to increase to 1920 Pa. The G′ value was further improved with the higher PPMUL. In the samples tested, MRDS-10 hydrogel showed the highest reinforcement effect with a threefold increase of G′ (2810 Pa) than that of DS hydrogel. Therefore, the mechanical strength of DNA supramolecular hydrogels could be adjusted by simply changing PPMUL. Nevertheless, tan δ (defined as = G″/G′), which is a metric of hydrogel viscoelasticity, showed no significant difference. These results demonstrate the significant reinforcement of DNA supramolecular hydrogels with the strategy of introducing PMUL. This mechanical reinforcement can be explained by the synergistic effect of the following aspects. The multiple DNA supramolecular half-units were covalently connected in PMUL, which effectively trapped short DNA building blocks. According to the KIMU effect, the DNA supramolecular crosslinkers were anticipated to have a higher energetic barrier for dissociation and a slower exchange rate. This suggested that the supramolecular crosslinkers could achieve higher kinetic stability, which could lead to higher mechanical strength of the DS hydrogel (Scheme 1). Furthermore, the covalent connection of the supramolecular units decreased the amount of nick in the double-stranded DNA backbone. Thus, the rigidity of network backbone was increased, which also contributed to the mechanical reinforcement effect. Meanwhile, the polymeric chain also enhanced long-range interaction and the degree of entanglement, which further improved the mechanical strength of DNA supramolecular hydrogel. Thermorheological behavior To investigate the mechanism of mechanical reinforcement, thermorheological studies were performed on the hydrogels. The temperature-dependent mechanical strengths of all samples were first tested with temperature-sweep tests. As shown in Figure 2a, the G′ of the hydrogels continuously decreased while G″ slightly increased along with increasing temperature. Then the intersection of the G′ and G″ profile occurred, which corresponded to the temperature at which the crosslinking region of the A-CA region dissociated. We observed that all samples exhibited a close crossover point between 52 and 57 °C despite different mechanical moduli. This result indicates that the introduction of PMUL does not significantly affect the Gibbs free energy of association (ΔG) of each single crosslinking unit, which supports the principle of the mechanical reinforcement strategy. Figure 2 | (a) Temperature-ramp rheological test results of DS and MRDS hydrogels in the temperature range of 10–65 °C at a fixed frequency (1 Hz) and strain (1%). (b) Cyclic temperature-sweep measurement of MRDS-10 hydrogel with applied temperature being alternated between 25 and 60 °C at a fixed frequency (1 Hz) and strain (1%). (c–e) Master curves of frequency dependence of the storage modulus G′ and loss modulus G″ for (c) DS, (d) MRDS-5 and (e) MRDS-10 hydrogels obtained by time–temperature superposition shifts. (f) Arrhenius plot for the shift factors of DS, MRDS-5, and MRDS-10 hydrogels, respectively. Download figure Download PowerPoint To further study the thermoreversibility, the cyclic temperature-sweep measurement between 25 and 60 °C was conducted on MRDS-10 hydrogel. As presented in Figure 2b, when the temperature increased to 60 °C, a gel-to-sol transition occurred (G″ > G′). When cooling the hydrogels down, the mechanical moduli recovered to their initiate values and displayed a cooling curve that almost coincided with the heating curve ( Supporting Information Figure S6), indicating that the MRDS-10 hydrogels responded to temperature in a totally reversible way. This process could be cycled many times, demonstrating the good thermoreversibility of the hydrogel. Furthermore, frequency-sweep tests were applied to DS, MRDS-5, and MRDS-10 hydrogels at a fixed strain of 1% from 10 to 40 °C ( Supporting Information Figure S7). Based on the discussion above, A-CA hybridization was the dominant factor for the mechanical properties in this temperature range. According to the time-temperature superposition (tTs) principle, the frequency-dependent G′ and G″ profiles at different temperatures can be reduced to a single master curve by a horizontal shift factor (aT), which reflects the activation energy of the relaxation process. It should be noticed that the DNA crosslinker gradually dissociated with increasing temperature and could not simply be investigated by classical tTs, which was generally suitable for the constant network systems. Therefore, the deviation of the mechanical moduli caused by the change of the number of crosslinking points with temperature has been corrected by a previously reported protocol.32 Briefly, in addition to aT, another vertical shift factor (δbT = δT/Tr, where δ is an extra vertical shift factor, T and Tr were the experimental temperature and reference temperature, respectively) related to change of crosslinking density with temperature was also introduced to superimpose G′ and G″. As shown in Figures 2c–2e, master curves of G′ and G″ for DS, MRDS-5, and MRDS-10 hydrogels were constructed at a reference temperature of 25 °C. Over the entire frequency range of each sample, the value of G′ was obviously higher than that of G″, and G′ had a plateau regime at moderate frequencies. The temperature dependence of aT could be derived from the time-temperature superposition. The apparent activation energy (Ea), which is a measure of energy barrier that should be overcome to dissociate supramolecular crosslinkers, could be obtained from the linear fit of ln aT versus 1/T according to the Arrhenius equation ( Supporting Information Note S1). As shown in Figure 2f, Ea of DS hydrogel was calculated to be 111.06 kJ mol−1. After incorporation of 5% and 10% PMUL, the Ea of the hydrogels was increased to 177.86 and 233.70 kJ mol−1, respectively. This result indicated that the supramolecular interactions of crosslinkers in MRDS-5 and MRDS-10 hydrogels displayed higher energetic barriers and correspondingly slower association-dissociation kinetics than DS hydrogel. These results proved that PMUL could trap the DNA crosslinkers to a kinetically interlocked state through the synergetic effect of multiple-unit interaction without changing single interaction and thus reinforced the mechanical properties of DNA supramolecular hydrogels. Dynamic properties While our reinforcement strategy has been demonstrated to improve mechanical strength effectively, the MRDS hydrogel can also maintain the intrinsic dynamic properties of the supramolecular hydrogel. Therefore, we also compared the dynamic behaviors of both DS and MRDS hydrogels, including shear-thinning, self-healing ability, and thermal responsiveness. Here, MRDS-2.5 and MRDS-10 hydrogels were used as typical models to reveal these dynamic natures of MRDS hydrogels. To assess the shear-thinning of the hydrogels, steady-shear and step-shear measurements were performed on DS, MRDS-2.5, and MRDS-10 hydrogels. As shown in Figure 3a, the viscosity of MRDS-2.5 and MRDS-10 hydrogels decreased by more than three orders of magnitude over shear rates from 0.1 to 100 s−1, indicating its highly shear-thinning behavior similar to DS hydrogel. When we toggled shear rate between 1 and 100 s−1, the viscosity of MRDS-2.5 and MRDS-10 hydrogels decreased to 3–4 Pa·s at high shear rate, and quickly recovered the original viscosity at low shear rate (Figure 3b). This process could be cycled several times. This phenomenon indicated good possible injectability of the hydrogels, and therefore we applied MSDR-10 hydrogel to pass through 29-gauge needles. As illustrated in the inset of Figure 3a, the hydrogel was injected from the syringe needle smoothly, and a stable hydrogel fiber was instantly formed upon extrusion. These results demonstrate the good dynamic properties of the MRDS hydrogels which make them promising materials for biomedical applications. Figure 3 | (a) Steady-shear rheological measurements of DS, MRDS-2.5, and MRDS-10 hydrogels demonstrating highly shear-thinning behavior. The inset image shows the digital photograph of MRDS-10 hydrogel passing through 29-gauge needles without clogging. Scale bar, 5 mm. (b) Step-shear measurements of DS, MRDS-2.5, and MRDS-10 hydrogels with low shear rate (1 s−1, nonshaded areas) and high shear rate (100 s−1, shaded areas). (c) Step-strain measurements of DS, MRDS-2.5, and MRDS-10 hydrogels undergoing cyclic deformation of low strain (1%, nonshaded areas) and high strain (100%, shaded areas) at 1 Hz. (d) Rheological results of G′ and G″ for MRDS-10 hydrogels before vertically cutting the hydrogel in two pieces and after self-healing process. The inset image shows a representative self-healing process including cutting the hydrogel in two pieces and putting in contact for self-healing for 1 h at room temperature. (e) Thermo-plasticity of MRDS-10 hydrogel: the hydrogel fragments could be reshaped as a disc after heating and remolding. Download figure Download PowerPoint In addition to their shear-thinning behavior, MRDS hydrogels also exhibited excellent self-healing properties. As shown in Figure 3c, step-strain measurements were performed on DS, MRDS-2.5, and MRDS-10 hydrogels. Under high-magnitude strain (γ = 100%), the hydrogels collapsed to a quasi-liquid state (G″ > G′) within seconds, indicating the disruption of the DNA supramolecular crosslinking. When low magnitude strain (γ = 1%) was applied, both G′ and G″ fully recovered to their original value immediately, suggesting the reconstruction of the DNA supramolecular crosslinking network. This rapid and complete recovery of mechanical properties could also be repeated over several cycles. Combined with the rheological-strain sweep results, we concluded that the hydrogels were indeed crosslinked by supramolecular interactions. Then, a self-healing experiment for MRDS-10 hydrogel was conducted. As shown in the inset images of Figure 3d, two disc-shaped MRDS-10 hydrogels visualized by FAM (green) and ROX (red) were vertically cut into two pieces separately, and the two half pieces were placed together. We observed that the two half discs immediately stuck to each other at the cutoff surfaces when they made contact, and after one hour incubation, the boundary between the two pieces became obscure, demonstrating that the hydrogels completely self-healed. To further investigate the self-healing process quantitatively, MRDS-10 hydrogels were horizontally or vertically cut and self-healed, and rheological tests were applied to compare the mechanical strength of healed hydrogels with the precutting samples. As shown in Figure 3d and Supporting Information Figure S8, the G′ and G″ values of self-healed MRDS hydrogels were almost the same as those of original hydrogel regardless of cutting directions, indicating the complete recovery of the network structure of the hydrogels. These results demonstrate that the MRDS hydrogels possess excellent self-healing properties. Taking advantage of the good thermal-responsiveness and thermo-reversibility as discussed above, thermo-plasticity of the MRDS hydrogels was then explored.33,34 At high temperature, the hydrogels switch to a quasi-li
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