Injectable, Self-Healing, and Multi-Responsive Hydrogels via Dynamic

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Injectable, Self-Healing, and Multi-Responsive Hydrogels via Dynamic Covalent Bond Formation between Benzoxaborole and Hydroxyl Groups Yangjun Chen, ZhengZhong Tan, Wenda Wang, Yi-Yang Peng, and Ravin Narain Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01652 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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Injectable, Self-Healing, and Multi-Responsive Hydrogels via Dynamic Covalent Bond Formation between Benzoxaborole and Hydroxyl Groups Yangjun Chen*†‡# , Zhengzhong Tan‡§#, Wenda Wang‡, Yi-Yang Peng‡ , Ravin Narain*‡ †School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China ‡Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2G6, Canada §Department of Polymeric Materials, School of Materials Science and Engineering, Tongji University, Shanghai 201804, China KEYWORDS. glycopolymer; self-healing; multi-responsive; hydrogel; boronic ester

ABSTRACT: Hydrogels that are injectable, self-healing and multi-responsive are becoming increasingly relevant for a wide range of applications. In this work, we have successfully developed a novel approach in the fabrication of smart hydrogels with all the above properties. A symmetrical ABA triblock copolymer was first synthesized via atom transfer radical polymerization with a temperature responsive middle block and two permanently hydrophilic glycopolymer chains on both ends. Hydrogels were subsequently constructed by mixing the triblock copolymer with another linear hydrophilic copolymer bearing benzoxaborole groups. The interactions of the benzoxaborole groups with the sugar hydroxyl groups allowed the formation of dynamic covalent bonds. The resulting hydrogels exhibited temperature, pH, and sugar responsiveness. Rheological studies confirmed that the mechanical property can be tuned by changing the pH as well as the galactose/benzoxaborole molar ratio. Furthermore, the hydrogels showed excellent self-healing ability and shearthinning performance due to the inherent well known dynamic covalent bonds of boronic esters. Therefore, these types of hydrogels can have excellent biomedical applications.

Introduction Hydrogels, which are crosslinked three-dimensional (3D) networks with high water content, have been shown to be extremely useful for a myriad of biomedical applications, including 3D cell culture, tissue engineering, and drug delivery.1-4 Stimuli-responsive hydrogels are characterized by their abilities to response to certain kinds of stimuli such as changes in temperature, pH, light, and many others.5 These properties can be exploited to achieve functions such as stimuli-triggered drug release, molecular recognition and shape memory.6-8 Hydrogels that are responsive to a single kind or two different kinds of stimuli have been widely introduced. However, there is a need of hydrogels with multi-responsiveness, considering the sophisticated physiological environment in human bodies where hydrogels are expected to exert different functions effectively when exposed to different cues.9 In general, stimuli-responsive hydrogels can be designed by introducing sensitive properties to either the polymer chain or the crosslinking sites. For example, poly(Nisopropylacrylamide) (PNIPAAm)10-12 has been widely reported towards the fabrication of temperature

responsive hydrogels due to its lower critical solution temperature (LCST) character. Recently, dynamic covalent bonds (DCBs) have received increasing attention in the fabrication of “smart” hydrogels as the reversibility of crosslinking sites can provide not only stimuliresponsiveness but also self-healing property.13, 14 Several typical DCBs include Schiff-base, hydrazone, disulfide bond, boronic ester, and Diels-Alder reaction.15 Hence, integrated design of the polymer chain and crosslinking sites can lead to formation of multi-responsive and simultaneously self-healing hydrogels. Boronic esters have been intensively studied for constructing self-healing hydrogels due to reversible complexation between boronic acids and 1,2-/1,3-diols.16 Moreover, the mechanical property of the hydrogels based on boronic esters is reportedly associated with several factors, such as pH of the environment, pKa of the boronic acids, and the molar ratio of the two functional groups.17, 18 Nevertheless, one limitation for their biomedical use is that most boronic acids (mainly phenylboronic acids, PBAs), which have pKa values in the range of 8-9, are not able to form boronic esters at physiological pH.19 For this

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reason, great efforts have been put on the development of arylboronic acids and analogs with lower pKa values which will allow for hydrogel formation at neutral pH. 20, 21 Benzoxaborole with a pKa value of ~ 7.2 becomes the preferred component of hydrogels to grant them with feasible use in physiological conditions.22, 23 Our group has exploited dynamic arylboronic esters based on benzoxaborole-diol complexation in several biomedical applications.24-27 Meanwhile, among a wide range of diolpossessing substances, glycopolymers, which can be obtained from natural sugar molecules and well-assessed to be biocompatible,28, 29 have several advantages over other diol-contained polymers like polyvinyl alcohol (PVA) and dopamine-based polymers. For example, glycopolymers can be easily functionalized by copolymerizing with other functional monomers;30 glycopolymers are chemically stable and can avoid the undesired oxidation problem which might be a puzzle for polyphenols.31 Therefore, dynamic arylboronic ester based on benzoxaborole-glycopolymer complexation shows promising potential for constructing hydrogels used especially in biomedical areas.

of hydrogels can be adjusted upon temperature change, which should be ascribed to the formation of microaggregates by the collapsed temperature responsive polymer chains. These excellent properties endow the hydrogel with great potential for biomedical applications such as tissue engineering and controlled drug delivery.

Benefiting from the inherent pH and diol responsiveness, hydrogels crosslinked by boronic esters can be further designed to be multi-responsive by incorporation of other sensitive properties. Our group has previously prepared hydrogels by mixing glycopolymers with statistical PNIPAAm-based copolymers which have pendent benzoxaborole groups.32 The hydrogel showed triple-responsiveness to changes in temperature, pH, and sugar concentration. However, the formation of the hydrogel was highly dependent on the temperatureresponsive property of the PNIPAAm part and the rheological characterization was insufficient. Recently, Zhang et al. has prepared hydrogels based on a catecholcontained polymer crosslinked by bis(phenylboronic acid carbamoyl) cystamine (BPBAC) which showed triple pH-, glucose-, and redox-responsiveness.33 However, the hydrophobic crosslinker BPBAC should be dissolved in DMSO during the gelation process, which might be an issue when applied in biomedical field. Therefore, careful molecular design should be taken into consideration when two or more elements are incorporated together in one system for preparing multi-responsive hydrogels.

Scheme 1. a) Schematic Illustration of Injectable, SelfHealing, and Multi-Responsive Hydrogel Crosslinked by Benzoxaborole-Galactose Complexation. b) Formation of Hydrogel by Mixing PLDL and PAB Solutions at pH 7.4

In the present work, a novel ABA-type triblock copolymer, which consists of temperature responsive middle block and two permanently hydrophilic glycopolymer chains on the two ends was synthesized via a two-step atom transfer radical polymerization (ATRP) method. Hydrogel was subsequently formed by mixing the triblock copolymer and a statistical hydrophilic copolymer containing pendent benzoxaborole groups. The rapid DCBs formation of the benzoxaborole groups with the sugar hydroxyl groups allow the crosslinking of the polymer chains (Scheme 1). Rheological tests were performed to investigate the mechanical property as well as the self-healing behavior of the hydrogels. Moreover, the triple-responsiveness to pH, sugar, and temperature were confirmed, respectively. Interestingly, the modulus values

Experimental Section Materials. Di(ethylene glycol) methyl ether methacrylate (DEGMA), acrylamide (AAm), copper(I) bromide (CuBr), bipyridine (Bpy), diethyl meso-2,5-dibromoadipate, rhodamine B (RhB), methylene blue (MB), and 4,4 ′ azobis(4-cyanovaleric acid) (ACVA) were purchased from Sigma-Aldrich. DEGMA was passed through an aluminum column to remove the inhibitors prior to use. 2Lactobioamidoethyl methacrylamide (LAEMA)34 and 5methacrylamido-1,2-benzoxaborole (MAABO)32 were synthesized according to our previous reports. Characterization. 1H NMR spectra were recorded on a Varian 500 MHz spectrometer. The number (Mn) average molecular weight and polydispersity index (PDI) of water soluble PAB copolymer was measured by Viscotek conventional gel permeation chromatography (GPC) system equipped with two WAT011545 Waters Ultrahydrogel linear columns using 0.5 M sodium acetate/0.5 M acetic acid buffer as eluent at a flow rate of 1.0 mL/min. The GPC was calibrated by monodisperse pullulan standards (Mw = 5900-404,000 g/mol).25 The molecular weight of PDEGMA was determined by Agilent 1100 HPLC equipped with Phenogel columns and UV-Vis and ELSD detectors using tetrahydrofuran (THF) as eluent at a flow rate of 1 mL/min. Monodispersed polystyrene was used to calibrated the system. Synthesis and Characterization of the copolymers. The detailed synthetic routes for the two copolymers were displayed in Scheme 2. The ABA-type triblock copolymer PLAEMA-bPDEGMA-b-PLAEMA (denoted as PLDL) was synthesized


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via two-step ATRP using a di-functional initiator. With a target degree of polymerization (DP) of 200, DEGMA (1.13 g, 6.00 mmol) and diethyl meso-2,5-dibromoadipate (10.8 mg, 30.0 μmol) were first dissolved in methanol, then purged with nitrogen for 30 minutes in an ice bath. After the addition of CuBr (8.60 mg, 60.0 μmol) and Bpy (18.7 mg, 120 μmol), the solution was purged for another 15 minutes. Then the ice bath was removed, and the reaction tube remained sealed at room temperature to react overnight. The reaction was stopped when the tube was exposed to air. The mixture was first dialyzed against methanol for 1 day and then against deionized water (DI water) for 2 days to remove the catalyst and impurities. Then the solution was lyophilized for 2 days. The viscous and transparent gel-like product obtained was BrPDEGMA-Br with a yield of ~ 80% and its chemical structure was characterized by 1H NMR.

Scheme 2. a) Synthetic Route of ABA-Type Triblock CoPolymer PLDL by Two-Step ATRP Method. b) Synthetic Route of PAB by Conventional Free-Radical Polymerization PLDL was then synthesized using Br-PDEGMA-Br as macro-initiator. Lyophilized Br-PDEGMA-Br (600 mg) was dissolved in 2 mL of cold DI water. Galactose-contained monomer LAEMA (982 mg, DP set as 150) was dissolved in 1 mL of DI water and then added into the Br-PDEGMA-Br solution. The mixed solution was purged with nitrogen for 30 minutes in an ice bath. CuBr (5.70 mg, 40.0 μmol) and Bpy (12.4 mg, 80.0 μmol) was dissolved in 0.5 mL of methanol and then injected into the above solution. The solution was further purged with nitrogen for 15 minutes. The reaction tube remained sealed and the reaction was conducted at first in ice bath for 6 h and afterwards at room temperature for 24 h. The reaction was terminated when the tube was exposed to air. The mixture was dialyzed against DI water for 2 days to remove unreacted monomers and catalysts. Then the solution was lyophilized for 2 days. The white viscous solid obtained was PLDL with a yield of ~50% and its chemical structure was characterized by 1H NMR. The transmittance of 2.5 mg/mL PLDL aqueous solution was measured using UV-vis spectrophotometer. The LCST of PLDL was determined as the temperature at which the most rapid transmittance change occurred. The benzoxaborole-contained copolymer P(AAm-stMAABO) (denoted as PAB) was synthesized by free radical

polymerization of the hydrophilic monomer (AAm, 95 mol%) and benzoxaborole-based monomer (MAABO, 5 mol%). Briefly, AAm (607 mg, 8.55 mmol), MAABO (97.6 mg, 450 μmol), and ACVA (8.40 mg, 30.0 μmol) were dissolved in a mixture of dimethyl formamide (DMF, 4 mL) and DI water (2 mL). The mixture was placed in a 50 mL tube and degassed by purging with nitrogen for 30 min. After that, the tube was placed in an oil bath preheated to 70 °C and the reaction was allowed to be kept overnight. The resulting copolymer was purified by precipitation into acetone and dialysis against DI water for 2 days. PAB was obtained after freeze-drying with a yield of 89% and its chemical structure was characterized by 1H NMR. Aqueous GPC was applied to measure the Mn and polydispersity index (PDI) according to our previous reports.25, 32 Preparation and Characterization of Hydrogels. PLDL and PAB were dissolved separately in phosphate buffered saline (PBS) at a constant concentration of 10 wt %. Sonication was adopted to facilitate the dissolution. The two solutions were then mixed and stirred to obtain hydrogels. The formation of hydrogels was confirmed by test tube inversion method. To investigate the influence of pH, hydrogels with different pH (7.4, 8.4, 9.4) were prepared with the same amount of PLDL and PAB. To investigate the effect of molar ratio of the two functional groups, hydrogels with three different weight ratios (2:3, 1:1, 3:2) of PLDL : PAB were prepared at the same pH of 7.4 and the resultant hydrogels were named correspondingly as LB23, LB11, and LB32. All the prepared hydrogels kept the same solid content of 10 wt %. The rheological experiments were conducted using an AR-G2 rheometer (TA instruments) with a 20 mm 2.008 cone plate geometry at room temperature. To investigate the influence of pH and molar ratio of the two functional groups on the mechanical properties of the hydrogels, frequency sweep tests were performed with the frequency ranging from 0.1 rad/s to 100 rad/s at a constant strain of 1%. Hydrogels with different pH (7.4, 8.4 and 9.4) and weight ratios (3:2, 1:1 and 2:3) of the two polymer components were put on tests. To study the self-healing property, strain sweep from 0.1% to 500% was performed on hydrogel formed at pH of 7.4 with weight ratio of 1:1. Then, a time sweep was performed immediately at a constant low strain of 1% to test the recovery of storage modulus (G') and loss modulus (G"). More vividly, two pieces of hydrogels were cut in half, reconnected together and then left for an hour for healing. The shear-thinning property was confirmed by measuring the viscosity of the hydrogel as a function of shear rate ranging from 0.1 to 100 /s. The injectability of the hydrogel was further confirmed using following steps. 400 mg of hydrogel was put in a syringe and then injected through a 21-G needle. For better observation, two hydrogels were previously stained with pink RhB and blue MB, respectively. The injected hydrogels in the vial were left overnight to investigate the self-healing ability. The pH responsiveness of the hydrogel was examined using process below. The hydrogel was previously stained with RhB dye for better imaging. A little amount of



hydrochloride solution (HCl, 1 M) was added to 600 mg of hydrogel in a vial and followed by shaking. After the gel turned into solution, proper amount of sodium hydroxide solution (NaOH, 1 M) was added, and followed by stirring to rebuild the gel. The steps mentioned above were then repeated twice and the pictures of the reversible gel-sol-gel transition were taken. The sugar responsiveness of the hydrogel was examined by immersing 300 mg of hydrogel in 3 mL of PBS with 100 mM of fructose. Another piece of hydrogel immersed in pure PBS was set as control. Pictures were taken right after the immersion and after the gel in fructose solution was totally dissociated overnight. The temperature responsiveness was firstly examined by immersing the hydrogel formed at room temperature into 37 C water bath for 1 min and pictures before and after immersion were recorded for comparison. To further study the influence of temperature on the mechanical properties of hydrogels, rheological temperature ramp test in the range of 10-40 C with a heating rate of 2 C/min was performed on LB11 hydrogel. Moreover, frequency sweeps of LB11 hydrogel at 25 and 37 C were conducted separately and the modulus values were compared.

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galactose groups locating in two sides of the polymer chain should facilitate sufficient as well as homogeneous crosslinking.18 The LCST of PLDL was confirmed to be ~26.5 C (Figure S1), which is a bit higher than the wellreported LCST of PDEGMA (~26 C).36 The LCST of PLDL can be further adjusted in a wide range of temperature by copolymerization of DEGMA with other monomers bearing oligo ethylene glycol residues (OEGMA).

Results and Discussion Synthesis and Characterization of Polymers with Galactose and Benzoxaborole Groups. Synthesis of ABA-type triblock PLDL copolymer. Temperature sensitive polymers with LCST feature have been widely used in constructing stimuli-responsive systems. PNIPAAm is the most studied LCST polymer whereas still suffers from the appreciable toxicity which hinders its approval from FDA. Polymers bearing oligo ethylene glycol side chains such as PDEGMA have emerged as an excellent alternative to PNIPAAm due to their remarkable biocompatibility and facile tunability of LCSTs in a wide range of temperature.35-37 Therefore, PDEGMA was applied in the present work as the middle block with LCST feature. PDEGMA was synthesized using a difunctional initiator and its chemical structure was confirmed by the 1H NMR spectrum (Figure 1a). GPC results indicated that PDEGMA had a Mn of 42.9 kDa and a low PDI of 1.14, which should be attributed to the wellcontrolled nature of ATRP. Therefore, the number of repeated units per PDEGMA was calculated according to the GPC result to be 228. Next, PDEGMA was used as the macro-initiator for the subsequent polymerization of LAEMA. Polymerization of sugar monomers without protection has been developed by Narain and Armes.38, 39 Our group has previously reported a series of biomedical applications using LAEMA-based glycopolymers, for example, cationic glycopolymers for gene delivery24, 40, 41 and thermosensitive nanogels for controlled drug release.42, 43 The successful polymerization of LAEMA was confirmed by the appearance of typical peaks for sugar moiety in the 1H NMR spectrum (Figure 1b). By comparing the typical peak integral ( 4.36, 4.51 ppm) for LAEMA with that for the polymer backbone ( 1.83 ppm), the DP of LAEMA was determined by comparing with that of DEGMA to be ~164, which is close to the feed ratio. Our strategy by designing ABA-type PLDL polymer with

Figure 1. 1H NMR spectra of a) di-functional macroinitiator Br-PDEGMA-Br, b) ABA triblock copolymer PLDL bearing galactose groups, and c) PAB containing benzoxaborole groups.


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Synthesis of PAB copolymer. Due to the hydrophobicity of MAABO monomer, simple co-polymerization with hydrophilic AAm was applied to obtain water soluble PAB. Characteristic peaks of AAm and MAABO can be clearly observed from the 1H NMR spectrum (Figure. 1c). The molar percent of benzoxaborole in PAB was determined to be 8.5%, which was calculated by comparing the typical peak integral ( 7.30-7.75 ppm) of MAABO with that ( 1.172.42 ppm) of the polymer backbone. Aqueous GPC results (Figure S2) showed that PAB possessed a Mn of 60.9 kDa and a PDI of 2.96. Preparation and Characterization of Hydrogels. Benzoxaborole has been shown by Hall and coworkers to have strong binding affinity to cis 1,2/1,3-diols of saccharides under physiological conditions.44, 45 Therefore, complexation between galactose groups of PLDL and benzoxaborole groups of PAB can lead to crosslinking and subsequent hydrogel formation. To prepare the hydrogels, PLDL and PAB polymers were separately dissolved in PBS, followed by mixing with pipetting. Test tube inversion

method was adopted to confirm the formation of hydrogels as shown in Scheme 1, which happened quickly within ca. three minutes. As the formation of boronic ester is highly pH-dependent, hydrogels formed at different pH were compared by rheological tests. As shown in Figure 2a and 2b, both the values of G' and G" increased with increasing pH, which should be ascribed to the enhanced binding affinity of benzoxaborole with galactose at higher pH. The molar ratio of the two functional groups, or weight ratio of the two polymers accordingly, can also influence the mechanical properties of hydrogels. Three hydrogels with different weight ratios (2:3, 1:1, 3:2) of PLDL:PAB were compared. As shown in Figure 2c and 2d, both G' and G" became higher with increasing PLDL percentage. This could be due to the relatively high steric hindrance of the LAEMA part. Not all diol groups had been involved in the reaction with benzoxaborole due to the steric hindrance. Therefore, increasing the galactose/benzoxaborole ratio would increase the number of boronic ester bonds formed, increase the crosslinking density and thus enhance the mechanical property of the hydrogels.

Figure 2. a) Frequency sweep of hydrogels formed at different pH. b) Storage modulus of hydrogels at different pH collected at frequency of 1 Hz. c) Frequency sweep of hydrogels with different PLDL:PAB weight ratios. d) Storage modulus of LB23, LB11, and LB32 collected at frequency of 1 Hz. Self-Healing and Injectable Property of Hydrogel. The self-healing property was tested by oscillatory rheological measurements on the hydrogel formed at physiological pH of 7.4 and with a galactose/benzoxaborole ratio of 1:1. The hydrogel was first disrupted by increasing the applied strain from 0.1% to 500% and then allowed to heal at a low strain of 1% with both G' and G" recorded with time. Figure

3a shows the change of G' and G" versus time and the corresponding strain variation is recorded in Figure 3b. Both G' and G" underwent a plateau under low strain and then dramatically decreased with the value of G" exceeding that of G', which implied that the hydrogel was dissociated into solution state. The critical strain value (when G'=G") required for gel failure was determined here to be 47.7%.



However, when the large strain was released and a small strain of 1% was applied, both G' and G" increased immediately together with the reversion of the values of G' and G", indicating the rapid reconstruction of hydrogel network. Moreover, the value of G' can recover fully to the original level within 300 s, revealing the efficient selfhealing process without the addition of any external stimuli. To get a more direct understanding of the selfhealing property, two round shaped hydrogels dyed with RhB and MB respectively were cut in half, and reconnected together (Figure 3c). Then, after less than one hour, they automatically rejoined as one whole piece and the contact

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line was blurred as the two parts became mixed as one, creating a mixed color near the original contact line. The shear-thinning property of hydrogel is critical for its injectability. The viscosity was recorded as a function of shear rate. As shown in Figure 3d, the viscosity decreased steadily with the increase of shear rate (from 0.1 to 100 /s), which indicated a shear-thinning behavior. More intuitively, the hydrogel can be easily injected using a 21G needle into a vial (Image inserted in Figure 3d). It’s worth mentioning that the two gels being injected into the vial was left overnight and they became one whole piece of gel with mixed purple color (Figure 3e), providing another proof for the excellent self-healing property.

Figure 3. a) Storage modulus and loss modulus value change in response to the imposed strain. b) The corresponding strain variation: Increasing first from 0.1% to 500% to break the gel and then dropping back to a small strain of 1% for gel recovery. c) Images showing the self-healing process by reconnecting two pieces of hydrogel together. d) Shear-thinning property of the hydrogel. e) Self-healing phenomenon of hydrogels after being injected. pH, Sugar, and Temperature Responsive Behaviors of hydrogel. The hydrogel by careful design of components was expected to have triple-responsiveness. As previously described, the reversible binding of boronic ester is highly pH-dependent. The molecular dissociation at low pH and reformation at high pH of benzoxaborole-galactose complexation would cause the gel-sol-gel transition macroscopically. With the addition of 1 M HCl into the hydrogel and following mild shaking, the hydrogel was quickly transformed into a liquid solution because of the dissociation of the crosslinking sites (Figure 4a). Then with the addition of 1 M NaOH to neutralize the acid and followed by stirring, the rapid sol to gel transition was observed. After another two repetitions of the addition of HCl and NaOH, the reversible gel-sol-gel transition was

still observed, each in a short period of time, which indicated the excellent pH responsiveness of the hydrogel.


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Figure 4. a) pH responsiveness shown by repeatedly adding HCl and NaOH solution. b) Sugar responsiveness shown by adding fructose. c) Temperature responsiveness by increasing temperature from room temperature to 37 C. Because of the reversibility of arylboronic ester, addition of saccharides containing diol groups can completely break benzoxaborole-galactose complexation and consequently induce gel dissociation. Fructose, which reportedly has high binding affinity with benzoxaborole,23 was adopted here to demonstrate the sugar responsiveness of the hydrogel. As shown in Figure 4b, the hydrogel immersed in the PBS containing 100 mM of fructose can be totally dissolved after one day. By contrast, the RhB-dyed hydrogel immersed in pure PBS as control stayed considerably stable though some extent of RhB dye was released as well. The temperature responsive property of the hydrogel was mainly attributed to the PDEGMA block with LCST feature. As shown in Figure 4c, when the temperature rose up from room temperature (~22 C) to body temperature of 37 C, the gel turned opaque and adopted a milky white appearance, which should be induced by formation of hydrophobic PDEGMA micro-aggregates above its LCST of 26.5 C.37, 46

Figure 5. a) Temperature ramp test in the range of 10-40 C with a heating rate of 2 C/min. b) Frequency sweeps of LB11 hydrogel at 25 and 37 C, respectively. To further investigate the influence of temperature on the mechanical property of hydrogel, temperature ramp test was conducted on LB 11 hydrogel in the range of 10-40 C. As shown in Figure 5a, both storage and loss modulus can be adjusted with temperature change. Though there was a gentle drop of modulus in the range of 20.3-26.8 C, steady rise of modulus can be clearly observed when temperature increased from 26.8 C to 40 C. The transition point appeared closely to the LCST of PLDL, indicating the modulus increase should be induced by the formation of micro-aggregates inside the hydrogel network. The collapsed PDEGMA chains above its LCST endow the hydrogel with second physical crosslinking. Frequency sweeps of hydrogels at 25 and 37 C were displayed in Figure 5b. The storage modulus (1 Hz, 1% strain) increased from 111.6 Pa at 25 C to 207.5 Pa at 37 C. Therefore, this kind of material design may provide a facial approach to prepare hydrogels with tunable mechanical properties. Conclusions In summary, we have successfully synthesized a novel ABA-type triblock copolymer (PLDL) with LCST feature and galactose pendent groups. Hydrogels can be constructed by mixing solutions of PLDL and another hydrophilic copolymer bearing benzoxaborole groups due to formation of dynamic benzoxaborole-galactose complexation. The resultant hydrogels exhibited triple pH-



, sugar-, and temperature-responsiveness. Rheological studies demonstrated that the mechanical property can be tuned by changing the pH as well as the galactose/benzoxaborole molar ratio. Moreover, the hydrogels showed excellent self-healing and injectable ability gifted by the innate reversibility of arylboronic ester bonding. Therefore, the injectable, multi-responsive, and self-healing hydrogels would have great potential in a variety of biomedical applications.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Temperature responsive turbidity change of the triblock copolymer PLDL; Aqueous GPC trace of PAB copolymer. (PDF)

AUTHOR INFORMATION Corresponding Author *Email: [email protected], *Email: [email protected]

ORCID Yangjun Chen: 0000-0002-7449-9348 Ravin Narain: 0000-0003-0947-9719 Yi-Yang Peng: 0000-0002-3363-4937

Author Contributions The manuscript was written through contributions of all authors. #These authors contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Foundation for Innovation (CFI). Prof. Hongbo Zeng is thanked for the help with the rheometer. Di Wu and Prof. Dennis G. Hall are thanked for the help with the synthesis of benzoxaborole monomer and useful discussion.

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