Stimuli-responsive gels are intriguing materials for various applications ranging from flexible electronics, artificial muscles, actuators, and sensors to the mimicry of extracellular matrix for cell culture and tissue regeneration [1, 2]. These applications usually require the gels to exhibit responsiveness to multiple triggers such as acid/base, redox signals, enzymes, heat, and light [3-5]. The early generation of responsive gels is designed based on the inherent property of used polymers, i.e., temperature-sensitive poly(N-isopropyl acrylamide) and pH-sensitive poly(acrylic acid). To expand the stimuli-responsiveness of the gels, dynamic covalent linkages such as boronate esters, disulfide bonds, thioesters, hemiaminal, and imine bonds or reversible non-covalent host-guest pairs such as cyclodextrin/azobenzene were introduced to form polymer networks [6-11]. These gels could respond to various stimuli by specific material design, but usually require the pre-synthesis of macromolecular building blocks with chemical reactive ligands on the backbone or side chains. This involves high complexity in material synthesis, which limits the large scale and repeatable preparation of corresponding gels [12]. An alternative strategy is synthesis of low molecular weight gelators containing dynamic covalent connections that can form fibrous networks via supramolecular assembly [13-17]. These supramolecular gels can be facilely prepared, but usually only respond to a single or two stimuli. In addition, the non-covalent forces holding the supramolecular gel are relatively weak, and this may lead to low gel stability [18]. It is hypothesized that dynamic covalent linkages can be used to directly crosslink small molecules with multiple functional groups [19, 20], yielding a class of all-small-molecule dynamic covalent gels. Here, we report the preparation of such gels by direct gelation of natural occurring polyphenol tannic acid (TA) with a series of commercially available inorganic borate (1–4) or organic boronate (5–6) building blocks via reversible boronate ester bonds. Though the boronate esters with catechol, 1, 2 or 1, 3 diols were widely used as reversible linkages in responsive polymeric gels [21-26], the direct gelation of naturally occurring small molecules using this dynamic covalent chemistry is yet to be investigated. The formed gels are responsive to acid/base, oxidants or reducing chemicals depending on the chosen borate/boronate building blocks.

TA is a natural occurring hydrophilic polyphenol with a high density of pyrogallic acid or catechol groups on its structure. Its excellent aqueous solubility and multiple reactive catechol groups make it an ideal candidate in the preparation of all-small-molecule dynamic covalent hydrogels [27-30]. Here, commercially available inorganic borates including NaBO₂, Na₂B₄O₇, Na₂BO₃ and H₃BO₃/ NaOH were firstly used to crosslink the TA molecules in aqueous solution via boronate ester bonds (Fig. 1a). Take NaBO₂ for example, the inorganic borate could form hydrogels with TA within a wide concentration ranges for both NaBO₂ and TA (Fig. 1b). A yellow-colored and transparent hydrogel (Gel 1) was formed within minutes by gently mixing the two solutions together at ambient temperature. Rheology result in Fig. 1c showed that the storage modulus (G') of a representative gel is higher than the loss modulus (G") after 1200 s, which is usually considered as the gelation time. We then investigated the effects of NaBO₂ and TA concentrations on the gel stiffness and gelation time (Figs. S1a-d in Supporting information). When the NaBO₂ concentration increased from 0.3 mol/L to 0.4 mol/L, the storage modulus of hydrogel increased and the gelation time decreased. With a further increase in NaBO₂ concentration, a reverse trend was observed. Assuming there are five binding sites on each TA molecule, 0.4 mol/L NaBO₂ is theoretically to interact with 0.16 mol/L TA via the formation of catechol-borate diesters. Therefore, a maximum storage modulus and shortest gelation time were achieved at 0.4 mol/L NaBO₂. More borates in the system will lead to increased catechol-borate monoesters, and decreased crosslinking degree in the gel. On the other hand, when the TA concentration was increased from 0.13 mol/L to 0.22 mol/L, the storage modulus increased, while the gelation time decreased. This is due to increased content of catechol-borate diesters and hydrogen bonding interactions in the network at higher TA concentrations. Like most hydrogels, the yielding TA/NaBO₂ gel displayed shear-thinning (Fig. 1d), thixo-tropic (Fig. 1e), self-healing (Fig. 1f) properties, and could be processed to different shapes using specific molds (Fig. 1g).

We next investigated the mechanism of TA/NaBO₂ gelation in aqueous solution by solution ¹¹B NMR spectroscopy. The samples were prepared at TA to NaBO₂ molar ratios ranging from 0:1 to 0.26:1 in deuterated water at pH 5.5 (no gelation observed under this condition, pH 5.5 is the pH value for TA/NaBO₂ mixture in Gel 1), and added into Teflon tubes for ¹¹B NMR measurement. As shown in Fig. 2a, NaBO₂ alone showed a sharp peak at 20.1 ppm at pH 5.5, which is the characteristic peak for tetrahedral borate anion [31]. After the addition of TA molecules, a new peak at 8.7 ppm was observed, and this peak is assigned to the borate monoester formed between catechol and tetrahedral borate anion. With increasing molar ratio of TA to NaBO₂, the intensity for the peak at 8.7 ppm is increased, and another peak at 14.7 ppm assigned to the borate diesters is observed. The chemical shifts for catechol-borate monoesters and diesters are in accordance with the reference results [31, 32]. The catechol-borate diesters are the major driven force for gelation between TA and tetrahedral borate (Fig. 2b). Besides ¹¹B NMR, we also characterized the TA/NaBO₂ gel by a resonance Raman Spectroscopy. Compared to free TA or NaBO₂, a new peak was observed at 1412 cm^-1 which is assigned to catechol-borate esters in the formed gels (Fig. 2c). The responsive behaviors of Gel 1 to acid or base were then investigated. As shown in Fig. 2d and Fig. S2 (Supporting information), the formed TA/NaBO₂ gel was relatively stable at pH 5.0–6.0, but rapidly dissolved in the presence of HCl or NaOH. This can be explained by the cleavage of catechol-borate diesters in acidic environments and hydrolysis/oxidation of TA in basic conditions [33-35]. These speculations were confirmed by ¹¹B NMR results in Fig. 2d, where the catechol-borate diesters were disappeared in both cases.

According to the analysis above, tetrahedral borate is the key structure for TA/borate gelation. Therefore, other inorganic borates that could hydrolyze into tetrahedral borate have the potential of gelation with TA via the dynamic covalent chemistry. Na₂B₄O₇ is a naturally occurring inorganic borate can be hydrolyzed to trigonal and tetrahedral borates at an appropriate molar ratio of 1:1 in aqueous solution (Fig. 2e). As shown in Fig. 2f, TA and Na₂B₄O₇ also form a transparent and yellow-colored gel after gentle mixing (Gel 2). In the ¹¹B NMR spectrum (Fig. 2g), a peak of 9.7 ppm is observed in the Na₂B₄O₇ solution (0.4 mol/L, pH 9.3), this peak is the weighted average of trigonal-state (2.5 ppm) and tetrahedral-state (20.1 ppm) borates in solution, which exhibiting a fast exchange between the two states within the NMR time scale [31]. The content of tetrahedral borates in the Na₂B₄O₇ solution can be modulated by facilely changing the solution pH, and this property can be used to tune the gel state. As shown in Fig. 2h, the TA/Na₂B₄O₇ hydrogel is also pH-responsive and stable TA/Na₂B₄O₇ gels were formed within the pH range of 5.0-6.0. Besides Na₂B₄O₇, NaBO₃ and a mixture of H₃BO₃ and NaOH at a molar ratio of 1:1 can be used to prepare dynamic covalent hydrogels with TA (Gel 3 and Gel 4, Fig. S3 in Supporting information).

TA has been widely reported with antibacterial activity by destabilizing the bacteria membrane [36]. We further investigated the in vitro antibacterial activity of the prepared hydrogels. As shown in Figs. 3a and b, the natural polyphenol TA and all the TA/ borate hydrogels effectively inhibited the growth of E. coli, suggesting the potent antibacterial activity of TA and TA-containing gels. The borates NaBO₂ and Na₂B₄O₇ showed limited effect on the inhibition of E. coli growth. Interestingly, NaBO₃ effectively killed the bacteria at an equal borate concentration. This can be explained by the yielding of hydrogen peroxide during the hydrolysis of NaBO₃ in aqueous solution. The TA/NaBO₂ hydrogels could be easily coated on substrates such as culture plates and glass slides due to the catechol groups on the gel surface (Fig. 3c). The SEM-EDS analysis in Fig. 3d suggests the successful coating of Gel 1 on a glass slide. After gel coating, the substrate effectively killed E. coli expressing green fluorescence protein (GFP, Fig. 3e). These results together suggest that the TA/ borate gels have potent antibacterial activity.

We further expanded the gel responsiveness by using organic boronates to crosslink with TA. 1, 4-Phenylenebisboronic acid (PBBA) with two boronate groups in the structure is expected to form catechol-boronate diesters for gelation (Fig. 4a) [21, 22]. TA and PPBA formed stable gels within a high concentration ranges for both chemicals (Fig. 4b), and the yielding gel (Gel 5) is yellow-colored with G' higher than G'' in the rheology studies (Fig. 4c). PBBA showed a broad peak at 31.5 ppm in ¹¹B NMR, which is assigned to the trigonal-state of boronates on the chemical [37]. After the addition of TA, a new peak at 13.6 ppm is observed which is attributed to the formed catechol-boronate ester (Fig. 4d). The peak for catechol-boronate ester is disappeared in the presence of HCl, which is similar to the behavior of catechol-borate esters for Gel 1 and Gel 2 (Fig. 4e and Fig. S4 in Supporting information). Besides acid and base, the yielding gel is also responsive to oxidant chemicals such as H₂O₂, which is due to the oxidative degradation of PBBA into inorganic borates by H₂O₂ (Fig. 4e). This process is confirmed by the disappearance of the boronate ester peak in the ¹¹B NMR upon H₂O₂ addition (Fig. 4d). In comparison, the gels prepared by TA and NaBO₂ showed weak sensitivity to H₂O₂ under the same condition (Fig. 4e).

Finally, a commercially available organic boronate 4-mercap-tophenylboronic acid (MPBA) was used as the boronate building block to prepare the dynamic covalent gels. MPBA was oxidized into dimers in the presence of DMSO and successfully formed a transparent gel with TA (Fig. 5a). ¹¹B NMR spectra suggested the formation of catechol-boronate esters in the mixture (Fig. 5b). The formed gel (Gel 6) responsiveness to acid, base, H₂O₂ (Fig. 5c and Fig. S5 in Supporting information), which is similar to Gel 5 in Fig. 4. Interestingly, Gel 6 also degraded rapidly in the presence of DTT, which is distinct from Gel 5. The DTT responsiveness of Gel 6 is explained by the cleavage of disulfide in the gel structure into thiol groups, which destroyed the crosslinking networks in the gel.

In summary, we reported a series of all-small-molecule dynamic covalent gels with antibacterial properties by direct gelation between TA and inorganic borates or organic boronates. In comparison with the responsive polymeric gels that require the pre-synthesis of macromolecular building blocks with chemical reactive ligands, the gels reported in this study are formed by direct gelation between natural occurring polyphenol TA and commercially available borate compounds. Besides facile construction, the all-small-molecule dynamic covalent hydrogels also show predictable degradation property. The hydrogels could be completely degraded into the small molecules upon multistimuli-responsiveness accord-ing to the chosen building blocks. The prepared dynamic covalent gels offer high flexibility to tune the physicochemical properties and responsiveness for various applications.

This work is supported by the National Natural Science Foundation of China (No. 21725402) and the Shanghai Municipal Science and Technology Commission (No. 17XD1401600).

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.cclet.2019.07.013.