Responsive Double Network Hydrogels of Interpenetrating DNA and CB[8] Host–Guest Supramolecular Systems
A supramolecular double network hydrogel is presented by physical interpenetration of DNA and cucurbit[8]uril networks. In addition to exhibiting an increase in strength and thermal stability, the double network hydrogel possesses excellent properties such as stretchability, ductility, shear-thinning, and thixotropy. Moreover, it is enzymatically responsive to both nuclease and cellulase, as well as small molecules, showing great potential as a new soft material scaffold. Hybrid hydrogels are particularly important in materials science as they can be formulated to closely exhibit the combined physical properties of individual components and offer opportunities to selectively tailor material properties.1 One early and important example of this field is interpenetrating polymer networks (IPNs), which was introduced in the 1960s.2 Based on this concept, in 2003 Gong and co-workers developed materials called double network gels and reported an innovative and universal pathway to fabricate gels with extremely high mechanical performance which can be tailored to mimic a wide variety of load-bearing biological tissues, ranging from “soft” tissue such as muscle to firmer tissue such as bone.3 Generally, double networks consist of two individual chemically crosslinked networks with contrasting physical properties: one network as a rigid skeleton (the first network) and a second network as a soft and ductile substance.4 Interpenetration of these two networks with good homogeneity results in a non-linear increase in mechanical strength and resistance to fracture, often much greater than calculated values models would predict. The enhanced mechanical strength exhibited by such hydrogels is likely attributed to the stress transfer between the two interpenetrating networks, instead of the stress being confined to the smallest crosslink of a single component gel.5 However, current double network hydrogels are mostly based on fully or partially chemically crosslinked networks6 and the effect of replacing chemical crosslinks for physical crosslinks in such materials has not yet been studied. It is of great interest to examine the formation of double network hydrogels from non-covalent supramolecular systems as such materials will afford the ability to regulate crosslink dynamics and respond to a variety of external stimuli.7 Herein we present the first example of an entirely self-assembling and supramolecular double network hydrogel, which is formed from the combination of two distinctly different hydrogel systems: one through DNA hybridization,8 and the other by host–guest interactions of cucurbit[8]uril (CB[8]).9 Merging these two hydrogel formulations accesses a new material comprised of two interpenetrating networks that have no crosslinking interactions with each other. Unlike the formation process of chemically crosslinked double network hydrogels via a two-step polymerization, our supramolecular system can be fabricated by a simple “one-pot” mixing method attributed to the highly precise and specific recognition motifs. Figure 1 illustrates our strategy to which there are four fundamental components: DNA Y-scaffold, DNA linker, phenylalanine-functionalized carboxymethyl cellulose (CMC-phe) and CB[8]. The DNA Y-scaffold and DNA linker can distinctly bind each other on account of the precise hybridization of complementary DNA sequences to form the first crosslinked hydrogel network, referred to as DNA hydrogel.10 Alternatively, CMC-phe and CB[8] can also specifically recognize each other owing to the host–guest interaction between phenylalanine and CB[8] to form the second crosslinked hydrogel network, referred to hereon as CB[8] hydrogel.11 Upon combining these four components in phosphate-buffered saline (PBS) buffer (100 × 10−3 m, pH 7.4) via a “one-pot” mixing method, the corresponding units recognize one another, resulting in two interpenetrating supramolecularly assembled networks, namely a double network hydrogel. Notably, combining these two systems is of particular interest as they exhibit very different mechanical properties. For example, the DNA hydrogel has a very low tanδ (G″/G′) but is brittle, whereas the CB[8] hydrogel has a higher tan δ but is less susceptible to breakage. Our aim was to design a material of high modulus (on account of dual interpenetrating networks) whilst retaining or enhancing the most desirable properties of each single network. Furthermore, the resulting double network hydrogel would possess a full biodegradability profile as each single network can be selectively digested by specific enzymes, i.e., nuclease degradation of the DNA network and cellulase cleave of the CB[8] network. This allows for the selective degradation of one network whilst retaining the structure and mechanical property of the remaining single network. This prospect is particularly exciting as it potentially has application in enzymatically triggered response from a preselected network whilst retaining the hydrogel architecture and structure through the persistence of the remaining other.12 Each single and double network hydrogel formulation was prepared in PBS (100 × 10−3 m, pH 7.4) with a mass content of each single network of 1.5 wt%. As shown in Figure 2A, the freshly prepared CB[8] hydrogel is slightly turbid whereas the DNA hydrogel is optically transparent. The resultant double network hydrogel maintains an intermediate almost transparent state between the two corresponding independent networks. The differences in mechanical properties between the double network hydrogel and corresponding single network hydrogel formulations were also studied. As demonstrated in Figures 2B and S1 (Supporting Information), the separate addition of either CB[8] or CMC-phe components into the 1.5 wt% DNA hydrogel have little influence on the mechanical property of the DNA hydrogel. However, upon simultaneous addition of CB[8] and CMC-phe to the DNA hydrogel to form the double network hydrogel, the G′ increased from 80 ± 1.6 to 281 ± 2.4 Pa (also see Figure S2A, Supporting Information), which is higher than the sum of the two single network hydrogels (calculated double hydrogel, 230 ± 3.4 Pa). These results indicated that the two interpenetrating networks indeed increased the mechanical property of the double network hydrogel beyond the expected value; however, there is no increase in the order of magnitude of the modulus as previously demonstrated with chemically crosslinked hydrogels.[3] This difference can be attributed to the relatively weak interactions of supramolecular non-covalent crosslinking compared to stronger covalent conjugation, as the two networks experience stress, the physical crosslinks can be easily sheared. Interestingly, the internal supramolecular network structure appears responsive to increasing applied stretching frequency, similar to the CB[8] hydrogel and unlike the DNA hydrogel, implying the CB[8] hydrogel dominates the macroscopic structuring and viscoelastic response, Figure S2B (Supporting Information). A further study of the thermal behavior was also performed, during which G′ decreases with an increase in temperature and intersects with G″ at the gel–sol transition point. As shown in Figure 2C, the transition temperatures of the DNA hydrogel and the CB[8] hydrogel were 51 ± 0.5 °C and 46 ± 0.8 °C, respectively. However, the double network hydrogel impressively exhibited a higher gel–sol transition point at 62 ± 1.2 °C, again demonstrating that the two interpenetrating networks strengthened the thermal stability of the double network hydrogel. It was found that with an increase in mass content from 1.0 to 2.0 wt%, the G′ of double network hydrogel increased from 103.6 ± 1.6 Pa to 510.9 ± 4.0 Pa, and the value of tanδ decreased from 0.48 ± 0.015 to 0.38 ± 0.010 (Figure 2D), demonstrating that higher mass content affords higher G′ values, and therefore stronger double network hydrogels. These results also demonstrated that the double network hydrogel possesses an increased mechanical strength due to the interpenetrating interactions and it is very simple process to tune the mechanical strength by tailoring the mass contents. DNA hydrogels are crosslinked through hybridization of “sticky ends” between the DNA Y-scaffold and DNA linker. The rigid duplex structures and strong base-pair interactions result in the formation of a material that is relatively brittle. Upon compression, the hydrogels adhere to the testing parallel plate of the rheometer and after rheological analysis could be further stretched to 5233% (from 150 to 8000 μm) on retraction of the upper plate, Figure 2E. Specifically, the DNA hydrogels (1.5 wt%, 40 μL) exhibited a “narrow neck” during the stretching process followed by fracture (Figure S3, Supporting Information); moreover, they tended to lose their adherence to the top plate when stretched beyond 8 mm. Alternatively, CB[8] hydrogels (1.5 wt%, 40 μL) could readily be stretched to 8 mm without expressing a narrow neck (middle in Figure 2E) or as long as 2 cm when manipulated with tweezers before fracture (Figure S4, Supporting Information), on account of the flexible polymer chains and the reconfiguration of the CB[8] crosslinks offsetting any induced strain from the stretching process. Interestingly, the double network hydrogel (1.5 wt%, 40 μL) overcame the brittleness of the pure DNA hydrogel and demonstrated the good stretching properties inherent of the CB[8] hydrogel whilst maintaining shape persistence and rigidity of the DNA hydrogel, combined effects of the two interpenetrating non-covalent networks possessing dramatically different properties. Thus, the double network hydrogel exhibits truly emergent behavior upon deformation. This double network hydrogel could therefore be an ideal system to build a theoretical mechanics model to explain how force is transferred between rigid and flexible networks and this work is ongoing. We further investigated the stretching behavior of the double network hydrogel through manipulation with tweezers, Figure 2F, and found that the hydrogels can be stretched uniformly up to 2 cm without expressing a narrow neck, or as far as 3 cm without fracturing (Figure S5, Supporting Information) and the compressing/stretching process can be cycled for three times with good reversibility (Figure S6, Supporting Information). These observations also indicated that our double network hydrogel can overcome the brittleness of DNA hydrogels and retain the good ductility of CB[8] hydrogels.13 To investigate the combined behavior of the two interpenetrating networks, fluorescence labeling of the individual networks and confocal microscopy was undertaken. As shown in Figure 3A, pure DNA hydrogel networks were stained by the intercalating nucleic acid-specific dye GelGreen and exhibited uniform green color, indicating uniformity of the network. Alternatively, rhodamine isothiocyanate was chemically conjugated onto the CMC backbone. As shown in Figure 3B, the CB[8] hydrogel network exhibited red color with a few high density microdomains, which may be attributed to some small degree of folding of the CMC backbones or aggregation upon 2:1 complexation with CB[8]. The double network hydrogel appeared green, red, and orange under the green channel, red channel, and the merged channels respectively. As shown in Figure 3C (merged channels), the two network components are physically interpenetrating and bound within each other. This kind of binding behavior is the potential reason for observing strengthened mechanical properties and increased thermal stability as described in Figure 2. To further clarify the microstructure of the interpenetrating networks, scanning electron microscopy (SEM) was also used to investigate the detailed morphologies of lyophilized hydrogels. As shown in Figure 3, owing to the rigidity and helicity of double stranded DNA, the lyophilized DNA hydrogel (1.5 wt%) resulted in networks with smaller pores on the scale of tens of microns and thicker walls on the scale of several microns. On the contrary, the lyophilized CB[8] hydrogel (1.5 wt%) exhibited larger pores and thinner walls than that of the DNA hydrogel, likely attributed to the large flexibility of CMC polymers. Interestingly, the resultant double network hydrogel resulted in an intermediate morphology with a pore size similar to the DNA hydrogel and wall thickness close to the CB[8] hydrogel. This different morphology further verifies that the two network components are physically interpenetrating and bound within each other on the microscopic scale, altering the network structure, which in turn contributes to the overall properties on the macroscopic scale. As DNA hybridization and host–guest interactions between CB[8] and phenylalanine are both based on physical supramolecular interactions, the resultant double network hydrogel should exhibit an excellent shear-thinning behavior.14 This was first studied using steady shear rheology, Figure 4A. Upon increasing the shear rate from 0.001 to 0.01 s−1, the material expressed behavior of a Newtonian fluid as the dynamic viscosity remained constant. Beyond a shear rate of 0.01 s−1 the double hydrogel exhibited non-Newtonian shear thinning behavior and the viscosity dramatically decreased, a property related to plastic flow. The rheological properties of the double network hydrogel were further studied by oscillatory strain dependent rheology across an oscillatory strain range of 0.5–1000% at a fixed angular frequency of 1 Hz at 25 °C. As shown in Figure 4B, the G′ value rapidly decreased upon reaching 30% and had a crossing point with G″ at 300%, the gel-to-sol transition point, indicating a collapse of the gel state to a quasi-liquid sol state. It was observed that this gel–sol transition of the double network hydrogel was reversible and the quasi-liquid sol state can recover to its original value very rapidly when the strain was reverted from 1000% back to 1%, Figure 4C. Upon the application of a large amplitude oscillatory strain (1000%), the G′ value sharply decreased and became instantaneously lower than G″. The system persists at this quasi-liquid state under 1000% oscillation strain. When the applied strain was returned to 1%, both G′ and G″ were fully recovered without delay or fatigue and this process was recyclable. These results demonstrate that the double network hydrogels reported here have excellent shear thinning and rapid thixotropic properties, amenable to direct injection through a needle and syringe (Figure S7, Supporting Information). We further investigated the thixotropic behavior of the double network hydrogel as shown in Figure 4D and found that the shear stress increased with an increase of shear rate from 0.01 to 100 s−1 and vice versa, and the area below encompassed by the hysteresis loop was very small, indicating very little energy is required for structure breakdown. These results further verified that our double network hydrogel possesses both excellent thixotropic as well as shear-thinning properties, which may find great potential in injectable soft materials and drug delivery applications.15 Benefiting from the intrinsic properties of biopolymers such as DNA or cellulose, the resultant double network hydrogel exhibited dual-biodegradability profiles to nuclease and cellulase, respectively. As shown in Figure 5A, the double network hydrogel can be digested by EcoR I (restriction endonuclease with digestion site of GAATTC) at room temperature over 24 h, cleaving the DNA network component within the system and leaving the CB[8] hydrogel network alone with encapsulated DNA fragments. Alternatively, a similar effect can be achieved by addition of cellulase, which can specifically destroy the CB[8] hydrogel network, leaving the DNA hydrogel network component intact. Additionally, competitive substitution could also be used to selectively destroy one network. For example, adding free phenylalanine (in fivefold molar excess compared to CB[8]) into the double network hydrogel resulted in competition for the interaction between CB[8] and the grafted phenylalanine on the CMC backbone. If EcoR I and cellulase are both added into the double network hydrogel simultaneously, full degradation of the system can be realized, resulting in a one-way gel-to-sol transition. Furthermore, changes in the mechanical properties were observed by rheology. As shown in Figures 5B and S8 (Supporting Information), the G′ decreased from 276.8 ± 3.2 Pa to 160.2 ± 5.1 Pa after treatment by EcoR I for 24 h. Similarly, degradation by cellulase for 24 h caused the mechanical strength to decrease to 70.9 ± 2.3 Pa, which was exactly consistent with the value of the pure DNA hydrogel. Interestingly, competitive substitution by free phenylalanine also partially destroyed the CB[8] hydrogel network and lowered the mechanical strength to 106.6 ± 7.1 Pa. As expected, after incubation with EcoR I and cellulase for 24 h, the double network hydrogel was fully degraded and yielded a mixture that behaved as a solution whereby G′ tended to zero. These results demonstrated that our double network hydrogel possesses dual-enzymatic as well as small molecule responsiveness, enabling either selective partial or full degradation to meet different requirements for applications such as controlled drug delivery or orthogonal regulation of cell microenvironments.16 In summary, we demonstrated a responsive double network hydrogel entirely based on supramolecular interactions, i.e., DNA hybridization and host–guest recognition, which is distinctly different from previously reported chemically crosslinked double networks. Fluorescence labeling verified that the individual DNA and CB[8] networks were physically interpenetrating and bound within each other contributing to the increased mechanical properties and thermal stability. Furthermore, the interpenetrating behavior provides advantages for the double network hydrogel, i.e., excellent stretchability and ductility contributing resistance to fracture. Based on the dynamic and reversible supramolecular interactions, the double network hydrogel possesses shear-thinning and thixotropic properties, which show great potential as injectable soft materials for in vivo applications. Finally, on the account of the natural biodegradability of the DNA and cellulose backbones, the double network hydrogel is responsive to nuclease and cellulase as well as to small molecules. We believe that all of the advantages above make our supramolecular double network hydrogel an excellent new soft material scaffold that can be used in a wide variety of applications including dynamic surface coatings, controlled release and tissue engineering. C.L. and M.J.R. contributed equally to this work. The authors thank the National Basic Research Program of China (973 program, Grant No. 2013CB932803), the National Natural Science Foundation of China (Grant Nos. 91427302 and 21421064), the NSFC-DFG joint project TRR61, and Beijing Municipal Science and Technology Commission for financial support. M.J.R. thanks the University of Cambridge PhD Training Programme in Chemical Biology and Molecular Medicine. O.A.S. acknowledges an ERC Starting Investigator Grant (ASPiRe, 240629). Note: The figures were reset and the Acknowledgements appended on June 1, 2015, after initial publication online. 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