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Rapid-Forming and Self-Healing Agarose-Based Hydrogels for Tissue Adhesives and Potential Wound Dressings Zhuo Zhang, Xiaolin Wang, Yitong Wang, and Jingcheng Hao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01764 • Publication Date (Web): 16 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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Rapid-Forming and Self-Healing Agarose-Based Hydrogels for Tissue Adhesives and Potential Wound Dressings Zhuo Zhang, Xiaolin Wang, Yitong Wang, and Jingcheng Hao* Key Laboratory of Colloid and Interface Chemistry & Key Laboratory of Special Aggregated Materials (Shandong University), Ministry of Education, Jinan 250100, P. R. China

Corresponding author: E-mail: [email protected] Phone: 0531-88366074 // Fax: 0531-88364750

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ABSTRACT To meet the progressive requirements of advanced engineering materials with superior physicochemical performances, self-healing and injectable hydrogels (AD hydrogels) based on agarose with pH-response were prepared through dynamic covalent Schiff-base linkages by simply mixing nontoxic agarose-ethylenediamine conjugate (AG-NH2) and dialdehyde-functionalized polyethylene glycol (DF-PEG) solutions. The self-healing and injectable capabilities of the hydrogels without any external stimulus are ascribed to dynamic covalent Schiff-base linkages between the aldehyde groups of DF-PEG and amine groups on AG-NH2 backbone. It is demonstrated that the AD hydrogels possess interconnected porous morphologies, rapid gelation time, excellent deformability and good mechanical strength. The incorporated Schiff’s base imparts the hydrogels to the remarkable tissue adhesiveness. In vivo hemostatic tests on rabbit liver demonstrates that the hydrogels are able to stanch the severe trauma effectively. Compared with the conventional gauze treatment, the total amount of bleeding sharply declined to be (0.19 ± 0.03) g and hemostasis time strikingly shorter than 10 s after treating with AD hydrogels. To sum up, the self-healing ability, cytocompatibility, and adhesion characteristic of the pH-responsive hydrogels make them promising candidates for long-lived wound dressings in critical situations.


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1. INTRODUCTION Many efforts have been made to fabricate wound dressing that perfectly meets the critical requirements of rapid wound closure and infection prevention.1,2 To date, various materials have been applied for wound dressings including rubber, foam, electrospun nanofiber, membrane and hydrogel, etc.3-6 Among different dressing materials, hydrogels reveal excellent characteristics including maintain a moist environment at the wound bed, absorb a degree of wound exudates and soothe the wound surface.7-9 Nevertheless, the simplicity of the conventional hydrogels which are deficient in dynamic and structural complexities has severely precluded their applications in various fields.10 Traditional hydrogel dressings with weak mechanical strength may suffer deterioration even damage permanently due to the external mechanical force after applying to the irregular wound sites. As a result, they exhibit a worse curative effect and a shorter lifespan.8,11 In order to solve this issue, Yang et al. employed 4-arm-PEG derivatives to construct three kinds of in situ antimicrobial hydrogels based on Schiff-base crosslinking incorporated vancomycin as an antimicrobial agent.12 The hydrogels can be promising candidates as wound dressings due to their excellent mechanical properties which can improve tissue adhesiveness and bacteria-sensitivity. The currenting challenges of hydrogels are lack of self-healing and injectable properties, which may impede their practical applications. Accordingly, it is a crucial goal to develop advanced hydrogels with self-healing and injectable capabilities as long-lived hemostats which exhibit merits of filling wounds with various shapes and in situ loading drugs. Recently,

advanced

engineering

hydrogels

that

possess

the

improved

physicochemical performances have been fulfilled by designs at the molecular scale and control over multiscale architecture.10 Inspired by self-healing feature of



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organisms such as human skin to automatically recover from injuries,13-15 self-healing hydrogels as promising candidates to spontaneously heal themselves upon mechanical damage and regain their original functionalities have been exploited on the basis of the concept of constitutional dynamic chemistry (CDC).16 Owing to the subtle combination of stability and reversibility, dynamic covalent bond such as Schiff’s base and acylhydrazone bond presents great advantages in constructing self-healing hydrogels.17,18 Previous works proved that Schiff’s base structure is frequently incorporated in networks as pH-responsive linker to form hydrogels with versatility.18-20

Carboxymethyl

chitosan

(CMC)

cross-linked

with

benzaldehyde-terminated 4-arm polyethylene glycol (PEG-BA) can form a strong hydrogel with rapidly self-healing performance based on dynamic Schiff-base linkages. Besides the excellent cytocompatibility, the hydrogels also show the improved hemostatic effect for a rabbit liver incision compared with sterile gauze, which can be utilized as potential hemostatic dressings for wound healing.21 Herein, a rapid-forming agarose-based injectable hydrogels with automatically self-healing property were constructed through the dynamic covalent Schiff-base linkages.

The

agarose-ethylenediamine

conjugate

(AG-NH2)

and

dialdehyde-functionalized PEG (DF-PEG) were synthesized, and then the AG-NH2/ DF-PEG hydrogels (AD hydrogels) can be rapidly generated within 15 s by simply mixing both solutions. The resultant hydrogels with interconnected porous structures displayed pH-responsive sol-gel transition behavior, excellent deformability and good mechanical strength. Compared with other agarose-based hydrogels, the AD hydrogels not only exhibit good biocompatibility, but also integrate well with tissue because of the fast reaction between amine groups of tissue proteins and remnant benzaldehyde groups of DF-PEG. In hemorrhaging rabbit liver model, the hydrogels


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showed superior hemostatic properties with sharply decreasing blood loss and hemostasis time. Owing to these favorable capabilities, the AD hydrogels can serve as the potential wound dressing materials for hemostasis and wound healing applications. 2. EXPERIMENTAL SECTION Chemical and Materials. PEG (MW 2000, N/A), 4-formylbenzoic acid (97%), N,N’-dicyclohexylcarbodiimide (DCC, 99%), 4-(dimethylamino) pyridine (DMAP, 99%), 1,1-carbonyldiimidazole (CDI, 98%), rhodamine B (N/A), methylene blue (N/A) were purchased from J&K Scientific Ltd and used directly. Agarose (AG, type II-A, Medium EEO, Sigma-Aldrich) was used as purchased. The solvents such as tetrahydrofuran (THF), diethyl ether, dimethyl sulfoxide (DMSO) of analytical grade were purchased from local vendors and used without further purification. Measurements and Characterization. 1HNMR spectra were recorded on Bruker Avance 400 spectrometer equipped with pulse-field gradient module (Z axis) using a 5 mm BBO probe at 400.13 MHz. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded with a KBr pellet method using a VERTEX-70/70v FT-IR spectrometer (Bruker Optics, Germany). Elemental analysis was performed using an organic elemental analyzer (Vario EI III). X-ray photoelectron spectroscopy (XPS) experiments were carried out on an X-ray photoelectron spectrometer (ESCALAB 250) with a monochromatized Al K X-ray source (1486.71 eV). The cross-section morphologies of lyophilized hydrogels with the gold coating were obtained by scanning electron microscopy (SEM, Zeiss G300). Wide-angle X-ray diffraction (WAXD) experiments were performed using a Rigaku D/Max 2200PC diffractometer



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equipped with a graphite monochromator and Cu Kradiation ( = 0.15418 nm) at ambient temperature. The 2 ranged from 10° to 30° for WAXD patterns. Rheological tests were carried out on a Haake RheoStress 6000 rheometer with a cone-plate sensor system (C35/1° Ti L07116, diameter: 35 mm, core angle: 1°). Synthesis of DF-PEG2000 and AG-NH2. The difunctionalized PEG was obtained by esterification of two hydroxyl groups terminated PEG with 4-formylbenzoic acid according to the previous report.20 Briefly, PEG2000 (1.63 g, 0.815 mmol), 4-formylbenzoic acid (0.49 g, 3.26 mmol) and DMAP (0.025 g, 0.205 mmol) were dissolved in anhydrous THF (50 mL) and then the solution was cooled to 0 °C. DCC (0.84 g, 4.075 mmol) were dissolved in anhydrous THF (2 mL) and added to the mixture solution dropwise with vigorous stirring under a nitrogen atmosphere. The final mixture was stirred at 25 °C for 20 h. Then the white precipitate was removed by filtration. The product was precipitated with (200 mL) diethyl ether and recrystallization three times. The final product was dried under vacuum and obtained as a white powder (1.42 g, 77%). 1H NMR (CDCl3, δ, ppm): 10.11 (s, 2H, CHO), 8.22 (d, J =8.3 Hz, 4H, CHCCHO), 7.96 (d, J = 8.3 Hz, 4H, CHCHCCHO), 4.53-4.51 (m, 4H, COOCH2), 3.86-3.65 (m, 166-168H, OCH2CH2O). IR (KBr): ν (cm-1) = 3433, 2887, 1967, 1717, 1468, 1352, 1281, 1112, 957, 843. Ethylenediamine

was

covalently

crosslinked

to

agarose

using

1,1-carbonyldiimidazole (CDI) activation method according to the previous report.22 To activate the hydroxyl groups of agarose, CDI (6.36 g, 0.039 mmol) was first dissolved in 40 mL DMSO and then added to the pre-dissolved agarose solution (100


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mg, 60 mL). The mixture was stirred at the room temperature for 2 h. Subsequently, excessive amounts of ethylenediamine (10 mL) was added to the solution and stirred for 24 h at the room temperature. The resulting mixture was dialyzed against deionized water for 5 days with a dialysis membrane (MWCO: 8000-14000 Da). The final product of AG-NH2 as a white solid was obtained by freeze-drying. The degree of substitution (DS) of the AG-NH2 was confirmed from the conventional elemental analysis and expressed by the molar percentage of ethylenediamine introduced to the hydroxyl groups on agarose (N: 9.67%, C: 38.90%, H: 5.928%). Preparation of AD Hydrogel. Typically, precursor solution 1 was prepared by dissolving AG-NH2 (100 mg) into 8 mL deionized water. Precursor solution 2 was obtained by dissolving DF-PEG (300 mg) into 2 mL deionized water. Then the precursor solution 2 was added to the solution 1 dropwise under votex and eventually homogeneous hydrogel precursor was fabricated. The AD hydrogels with various concentrations of AG-NH2 (c) and mass ratios of DF-PEG:AG-NH2 (r) were prepared in the same process. The AG hydrogels (c = 1 wt%) were prepared by dissolving agarose (100 mg) into 10 mL deionized water. The preparation procedures of AG-PEG hydrogels are same as those of AD hydrogels, except for the replacement AG-NH2 with agarose. In addition, the gelation time of hydrogels with different concentrations was also investigated by vial-tilting method.23 pH-Response Experiments. Schiff base is a kind of dynamic cross-linkers which can be destroyed by weak acidity and regenerated in neutral or alkaline conditions.19 Concentrated HCl aqueous solution (20L, 3 mol/L) was added to the model



hydrogel (r = 3, c = 1 wt%, 0.5 mL) under vortex to decompose the networks incorporated Schiff’s base. Subsequently, concentrated NaOH aqueous solution (20 L, 3 mol/L) was added to the sol system to neutralize acid and recover the gel networks. Rheological Characterization. To determine the rheological properties of the hydrogels, stress sweep tests were performed firstly to determine the linear viscoelastic region and investigated the mechanical strength of the hydrogels at different concentrations under a fixed oscillatory frequency of 1 Hz. The native AG hydrogel and AG-PEG hydrogel were treated as control. Oscillatory shear tests were carried out under an appropriate stress selected from the linear viscoelastic region with the frequency ranging from 0.01 to 10 Hz. To investigate the recovery properties of the model gels in response to applied shear forces, initially, stress sweep test of the self-healed AD hydrogel (r = 3, c = 1 wt%) was performed once again to investigate mechanical strength change after healing. Additionally, alternate step stress sweep tests were carried out to monitor the self-healing process of the AD hydrogel under a fixed oscillatory frequency of 1 Hz. Oscillatory shear stress (γ) were changed from small stress (γ = 1 Pa) to subsequent large stress (γ= 2000 Pa, which is bigger than upper limit value of linear viscoelastic region) with 100 s for each strain interval. Characterization of Self-Healing Property and Injectability of Hydrogels. Initially, the whole AD hydrogel (r = 3 and c = 1 wt%) was cut into halves, and the two gels were combined into an integral hydrogel. After being incubated in the container for 10 min at ambient condition, the self-healed hydrogel was immersed in


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PBS buffer solution for 2 h to monitor its healing condition. Then four pieces of AD hydrogels with different color were alternate merged into a united hydrogel at ambient condition to observe its recovery process. To manifest the injectability, AD hydrogels (r = 3 and c = 1 wt%) stained by rhodamine B were put into a 20 G needle and then injected into a glass slide to observe clearly. Two bulks AD hydrogels stained by rhodamine B and methylene blue were put into two needles and separately injected into a 5 mL beaker. The hydrogel pieces integrated into a whole entity in the bottom of the beaker for 1 min at room temperature which could be grabbed and hold by a tweezer. Adhesive Strength Test. To investigate the adhesive strength between the hydrogel and tissue, torsion test and bursting pressure test were performed. Porcine skin was selected as adherend due to its biological similarity to human dermis.24,25 The AD hydrogel (r = 3 and c = 1 wt%) was formed in situ on the prepared porcine skin without further purification act as experimental tissue substrates to mimic real clinical condition. After standing for 5 min, torsion force was applied on the hydrogels to investigate their adhesive strength on the tissue qualitatively. Furthermore, the bursting pressure test was carried out according to methods described by previous methods to evaluate adhesive strength quantitatively.26,27 The skin was punched a 3 mm circular hole with a scalpel and fixed on the experimental device. After the formation of AD hydrogels in situ on the hole for 5 min, the bursting pressure was measured by a digital manometer with continually injecting air using a syringe at 10 mL/min. The pressure at which it began to decrease was considered the bursting



pressure. The AG hydrogel and AG-PEG hydrogel formed on the hole were treated as control. All measurements were repeated six times. Values were shown as mean ± standard deviation (n = 6). In Vitro Cytotoxicity Assay. The cytotoxicity test was performed according to quantitative MTT cytotoxicity assay using human umbilical vein endothelial cells (HUVEC cells) and was evaluated by contacting extracts of the hydrogels.28-30 The hydrogel extracts were prepared by adding 20 g AD hydrogel fragments (r = 3 and c = 1 wt%) to 20 mL DMEM culture medium and being immersed at 37 °C for 12 h (extracts 1) or 24 h (extracts 2). After this period, the residual AD hydrogels were discarded. 80% extract was obtained by diluting the 100% extract with DMEM culture medium. Typically, HUVEC cells were suspended in cell culture medium and seeded into 96-well plates at a density of 2 × 103 cells per well, following by being incubated for 24 h at 37 °C in 5% CO2 to obtain a monolayer of cells. Then, the cells were treated with hydrogel extracts (1 and 2) at different concentrations for an additional time (24 or 48 h). Then, all the sample solutions were extracted after incubation and the cells were further incubated for 4 h after being supplemented with 20 L of 0.5 mg/mL of MTT solution. Finally, the culture medium was replaced with 200 L of DMSO to dissolve the formed formazan pigment. An absorbance of the DMSO solution at 570 nm was detected by a microplate reader (Synergy HT) in a 96-well culture plate. The relative cell viability was calculated as the ratio between the mean absorbance value of the sample and that of cells cultured in the pure DMEM. Cytotoxicity test was tested 3 times for each culture. Values were shown as mean ±


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standard deviation (n = 3). The level of significance was set as P < 0.05. In Vivo Hemostatic Ability of AD Hydrogel. In the rabbit liver wound healing experiments, a total of 4 white New Zealand rabbits (2 kg, random distribution of male and female) were used. After wiping ear with 75% ethanol, rabbit was anesthetized by intravenous injection of sodium pentobarbital (3 wt%, 4 mL) and followed by immobilizing on a surgical corkboard. Then, the liver of the rabbit was exposed by abdominal incision and the pre-weighted sterile gauze was placed beneath the target liver to absorb blood. An incision of 1 cm in length was made with a surgical scalpel on the liver lobe. Upon bleeding, bleeding site was immediately applied on 2 mL AD hydrogel, or a piece of sterile gauze with compression for hemostasis or no treatment. No treatment after the bleeding was considered as a negative control and sterile gauze was treated as a positive control. Hemostasis time was recorded and blood loss was estimated by weighing the sterile gauze placed under the bleeding liver lobe during the operative period. Following the hemostasis, liver tissue around wound site was obtained after dehydrating completely in paraformaldehyde solutions and stained with hematoxylin-eosin (H&E) for observation using light microscopy. All of the results were shown as mean ± standard deviation (n = 4). This animal study was performed at Shandong University Laboratory Animal Center and all experiments in this study were performed in the light of relevant laws and institutional guidelines. 3. RESULTS AND DISCUSSION Synthesis and Characterization of AD Hydrogels. Successful wound care with



sophisticated biochemical processes requires an ideal wound healing environment.31,32 Accordingly, versatile wound dressings should be designed to satisfy the requirements of complicated healing process. Herein, a fast-forming adhesive hydrogel dressing was designed, which was incorporated pH-responsive dynamic Schiff-base linkages to endow the hydrogel self-healing and injectable capabilities (Scheme 1). The Schiff’s base could be regarded as a quasi-covalent linkage due to the intrinsic dynamic equilibrium.19 Owing to the more favorable stability of aromatic Schiff’s base than the aliphatic counterparts,20,33 DF-PEG and AG-NH2 as gelators were synthesized in this work.

Scheme 1. Schematic illustration (A to D) and preparation (E) of self-healing AD hydrogels incorporated Schiff -base linkages. DF-PEG was prepared by facile esterification reaction between PEG and 4-formylbenzoic acid using DCC as dehydrating agent and DMAP as catalyst (Scheme S1A in Supporting Information (SI)). Form the 1H NMR spectrum of DF-PEG (Figure S1), new peaks belonging to the aldehyde (10.10 ppm) were clearly


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identified. The integration ratios among 8.22, 7.96 (benzene ring), 4.52 (ester methylene), and 3.65 (ether methylene) were 4:4:4:168, close to the theoretical value 4/4/4/172, manifest the successful synthesis of DF-PEG. From FT-IR spectra, the peak appeared at 1717 cm-1 was attributed to the stretching vibration of C=O in the aldehyde and ester carbonyls which can further illustrate the successful synthesis of DF-PEG (Figure S2). Ethylenediamine was covalently crosslinked to hydroxyl groups of agarose molecules via 1,1-carbonyldiimidazole (CDI) activation (Scheme S1B). By changing the amount of CDI added initially, the extent of substitution can be varied. As shown in Figure S3, the modification of AG can be confirmed by the signals at 1702 and 1270 cm-1. Absorbance peak at 1702 cm-1 manifests the combination of carbonyl group and amino group, and the signal at 1270 cm-1 indicates a C–O group in ester bond. Additionally, the two components of N 1s peak of AG-NH2 from XPS correspond to -NH- at 399.7 eV and -NH2 at 401.3 eV (Figure 1A). Both FT-IR and XPS spectra confirm the successful modification of ethylenediamine on the agarose molecule backbone. Because of the large molecular weight of AG-NH2, excessively thick AG-NH2 solution and tough characterization of amino, the degree of substitution value of AG-NH2 in 60% (Table S1) was estimated by conventional elemental analysis, rather than 1HNMR. The AD hydrogels can be easily prepared by homogeneously mixing AG-NH2 solution with DF-PEG solution at ambient temperature. This facile preparation process meets the requirements of wound dressings for severe wounds and urgent



conditions.12 In the spectrum of lyophilized AD hydrogel, the signal at 1717 cm-1 for the stretching vibration of C=O from DF-PEG entirely disappeared, while a new peak at 1640 cm-1 for stretching vibration of C=N from Schiff’s base appeared, representing the formation of Schiff’s base in the hydrogel networks (Figure 1B). Schiff-base linkages can be utilized as good stimuli-responsive linkers due to their sensitivity to pH and reversibility. In aqueous solution, the Schiff’s base can decompose at low pH and reform after acid being neutralized. The hydrogels incorporated Schiff-base linkages can progressively disintegrate in acidic condition and regenerate in neutral or alkaline environment. When the hydrogels were added the concentrated HCl aqueous solution (20 L, 3 mol/L), the AD hydrogels with various concentration of AG-NH2 (c) and mass ratios of DF-PEG:AG-NH2 (r) (r = 3 and c = 1 wt%) transferred to be solution within 2 min due to the decomposition of Schiff’s base in the networks (Figure S4). To neutralize the acid, NaOH aqueous solution (20 L, 3 mol/L) was added, and the hydrogels can rapidly reform within 60 s, indicating the reversibility of the hydrogel networks. Consequently, the pH-responsive hydrogels can achieve controlled-release of encapsulated cargos (i.e. cells and drugs) under different conditions. The gelation time of hydrogel dressings is quite vital for blood coagulation and rapid hemostasis in critical conditions.34 As shown in Figure 1C, the gelation time of AG hydrogels, AG-PEG hydrogels and AD hydrogels were estimated by using vial-tilting method. The gelation time of AD hydrogels is quite faster than those of control groups (AG and AG-PEG hydrogels). Upregulating the amount from 4 wt% to


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6 wt% decreased the gelation times to be about 10-15 s, and all the gelation time meets the requirement of fast hemostasis. Moreover, rapid gelation can avoid hydrogel precursor solutions or cargos diffusing away from the wound site to favor the applications in vivo.21 Excessively rapid gelation process might bring about undesired heterogeneous mixing of components and deficient therapy. To make a compromise of rapid hemostasis and homogenous composition, the 1 wt% AG-NH2 was adopted for further studies. As shown in Figure 1D, interconnected porous structures with uniform pore sizes ranging from 5 to 28m were revealed by SEM observations, which can maintain a moist environment at the wound bed and it is beneficial for wound healing.34 The XRD spectrum in Figure S5 further demonstrated the amorphous structures of hydrogels.

A

B -NH(399.7)

Intensity (a.u.)

-NH2 (401.3)

1717 CHO+COOR DF-PEG

1702 C-N

AG-NH2

Xerogel 1640 CH=N 408

405

402

399

396

C

2000

1500

1000

500

Wavenumber (cm-1)

Binding Energy (eV)

Gelation Time (s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80

60 s

60 s

60

40

15 s

20

0

AG c=1

AG-PEG r=3 c=1

AD r=3 c=1

10 s

10 s

AD r=3 c = 1.2

AD r=3 c = 1.5

Figure 1. (A) XPS spectrum for N 1S signal of the AG-NH2. (B) FT-IR spectra of DF-PEG, AG-NH2 and xerogels (r = 3 and c = 1 wt%). (C) The gelation time of



various hydrogels. (D) SEM images of AD hydrogel (r = 3 and c = 1 wt%). The mechanical strength and viscoelasticity of the hydrogel dressings play a vital role in protecting the wound from further severe trauma.11,35 Rheological measurements were carried out to estimate the mechanical properties of the hydrogels. The storage modulus (G′) and the yield stress (τ) from stress sweep measurements can evaluate the mechanical strength of hydrogels. G′ represents the plateau value in the stress sweep and the τ indicates the critical shear stress when G′ decreases suddenly.36,37 By comparing the AD hydrogels with the control groups (AG and AG-PEG hydrogels), one can see that the τ value (~ 2000 Pa) of AD hydrogels is quite larger than those of control groups (126 and 315 Pa), suggesting that AD hydrogels can bear a larger deformation under external stress due to the existence of dynamic Schiff-base linkages (Figure 2A).35 The AG hydrogels were fragile and stiff, contrary to the soft and stretchable properties of AD hydrogels macroscopically (Figure S6). The hydrogels with various c and r were also investigated. As shown in Figure S7A, when r was fixed at 3:1, the G′ increases obviously from 98 to ~2400 Pa with the c increase from 0.6 to 1.2 wt%, which should contribute to the denser structure packing with the increase of the polymer concentration. Compared with hydrogel (c = 1 wt%) with high mechanical strength, the hydrogels (c = 0.6 wt%) was too weak to support a well shape (Figure S8). When c was fixed at 1 wt%, the G′ apparently increases from 520 to ~1700 Pa with the R decrease from 3:2 to 6:1, which could contribute to the formation of more Schiff-base linkages and a denser polymer network packing with the increase of the DF-PEG concentration (Figure S7B). As the oscillatory shear tests


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revealed (Figure S9), G′ is consistently greater than G′′ on multiples of 10-100 after equilibrium, demonstrating the formation of solid-like hydrogels with the dense cross-linked networks and the elastic dominant behavior of AD hydrogels. Self-Healing Performance of AD Hydrogels. Smart hydrogel dressings possessed self-healing property could restore their original functionalities and maintain intact structures after external damage, contributing to prolonged lifetime and preferable curative effect. Macroscopic self-healing experiments were performed to visually evaluate the self-healing ability of AD hydrogels (r = 3 and c = 1 wt%). In order to test the self-healing properties of the hydrogels, two hydrogel samples stained with rhodamine B and methylene blue respectively were cut into two equal pieces for examining to form the integral hydrogels (Figure 3A). Without any external intervention, the different pieces of the hydrogels were jointed as a whole entity after 5 min. When lifted the reconstructed hydrogel, it could sustain integrity under gravity. The boundaries between the adjacent pieces turned obscure after healing at ambient temperature for 1 h. Another test for the self-healing properties of the hydrogels were performed, 4 pieces of broken hydrogel samples with alternate colors were contacted to form an intact hydrogel, which further prove the self-healing capability of AD hydrogels (Figure 3B). All these observations clearly indicated that the formation of



dynamic covalent Schiff-base linkages at the fracture interfaces of hydrogels rather than the simple adhesion. The dynamic covalent Schiff-base interactions between amine groups from AG-NH2 and benzaldehyde groups from DF-PEG exist cleavage and regeneration, which play a vital role in the rapid and autonomous self-healing capability of the hydrogels. To further testify the self-healing property, rheological characterizations were performed systematically. As the stress sweep test showed (Figure 2B), the G′ curve and the G″ curve remained constant and G′ was larger than G″ under the stress from 1 to ~1600 Pa, indicating that the formation of a dense and stiff cross-linked network remained intact without damage. When the G′ curve intersected with the G′′ curve at the stress of ~1800 Pa defined as critical point, gel state transformed into quasi-liquid, revealing the beginning of destruction for hydrogel networks. With a larger stress than this critical point, the G′ value exhibited a dramatic decline and was lower than the G′′ value, suggesting the thorough collapse of the hydrogel networks.38 The rheological recovery properties of the hydrogels were studied under secondary stress sweep and alternate step stress sweep experiments. The stress sweep test of the AD hydrogels (r = 3 and c = 1 wt%) was performed once again after the 5 min of the first test (Figure 2C). One can see that the G′ curve of self-healed hydrogels had no significant difference with that of the original hydrogels.


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These results confirmed the mechanical strength of the hydrogel suffers little damage after recovery. Based on the stress sweep results, alternate step stress sweep tests were performed subsequently to further validate the self-healing property of the AD hydrogels (Figure 2D). As the oscillatory shear stress stepped from 1 to 2000 Pa for 100 s, the G′ and the complex viscosity (│*│) overlapped. When the high stress (2000 Pa) was withdrew and a low stress (1 Pa) was applied, both G′ and │*│recovered to their original values within a few seconds. All these results clearly demonstrate that the AD hydrogels possess a rapid self-healing ability since the broken Schiff-base linkages reformed due to their reversible properties. 4

A

B

10

3

10

3

G', G'' (Pa)

10

2

1

10

AG, c = 1 AG-PEG, r = 3, c = 1 AD, r = 3, c = 1

0

1

10

1760 Pa 0

G' G''

10

-1

-1

10

2

10

-1

0

10

10

1

10

2

10

3

10

10

4

10

0

1

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Biomacromolecules

100

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Figure 2. (A) G′ as a function of the oscillatory stress for hydrogels of AG, AG-PEG



and AG-NH2/DF-PEG. (B) G′ and G′′ as a function of the oscillatory stress for AD hydrogels (r = 3 and c = 1 wt%). (C) The stress sweep tests for original and self-healed AD hydrogels (r = 3 and c = 1 wt%). (D) Cyclic G′ and │*│ values of AD hydrogel (r = 3 and c = 1 wt%) as alternate step stress switched from small stress (1 Pa) to large stress (2000 Pa) with a fixed 100 s interval. Injectability Test. As demonstrated by rheological data, the self-healing hydrogels flowed like liquid when applied with high shear stress, the G′ recovery rapidly when stress was removed. The unique property was harnessed to enable injection, which could supply more homogeneous distributions of cargos ex vivo and better controllable placement of hydrogels at the target site in vivo without cargos loss.39-41 In order to testify the injectability, the AD hydrogels which stained with rhodamine B was loaded into syringe and squeezed through 20-gauge needle on a glass slide in a line without clogging, exhibiting near-instantaneous reassembly (Figure 3C). Two AD hydrogel samples with different colors (one of them was stained with rhodamine B) were put into two needles and separately injected into a 5 mL beaker (Figure 3D). After 1 min, the mixed gels could be grabbed and hold by a tweezer with an intact structure. In the process of injection, the dynamic Schiff-base linkages dissociated under pressure and the hydrogel networks deformed. After squeezing, the smashed pieces reformed to an integrated hydrogel again due to the reconnection of Schiff-base linkages. Hence, the self-healing hydrogels with injectability can be the promising candidate for cargo carrier in a minimally invasive manner.


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Biomacromolecules

Figure 3. The self-healing process of AD hydrogels (r = 3 and c = 1 wt%). Two (A) or four (B) pieces of AD hydrogels with different colors were connected together alternately and jointed tightly as a whole entity after 5 min. The boundaries between the adjacent pieces turned obscure after healing for 1 h. The injectable process of the self-healing AD hydrogel (r = 3 and c = 1 wt%). (C) The AD hydrogels squeezed through 20G needle without clogging. (D) Two AD hydrogel samples with different colors (one of them was stained with rhodamine B were respectively injected into a beaker and recombined after 1 min. Adhesive Property of AD Hydrogels. Upon hemostasis applications, the hydrogel dressings with excellent adhesion ability can completely seal injuries to prevent bacterial infection and control bleeding.42,43 To evaluate the potential applications of the AD hydrogels for wound occlusion, torsion tests on porcine skin were firstly performed (Figure 4A).25 The AG, AG-PEG and AD hydrogels were prepared in situ on the surface of porcine skin, respectively. After vigorously shaking, the AD hydrogels could stick to the skin without dropping contrast to the compared experiments of AG and AG-PEG hydrogels (video 1 in the SI). When applied the torsion stress, the AD hydrogels could still adhere to the skin and remain intact, revealing their adhesive strength and deformability on the skin (video 2 in the SI).


Biomacromolecules

The hydrogels were applied on the incision of the skin as sealants to further test their bursting pressure (Figure S10).27 The bursting pressure of the AD hydrogels far exceeds those of control groups (AG and AG-PEG hydrogels) and even bigger than the arterial blood pressure (120 mmHg), indicating the capability of the AD hydrogels to stanch in wound healing (Figure 4C). As to the adhesive mechanism for the hydrogels, the AG and AG-PEG hydrogels noncovalently stick to surrounding tissues through hydrogen bonds, leading to their weak adhesive strength. For the AD hydrogels, both hydrogen bonds and Schiff-base linkages between residual aldehyde in hydrogel and amino in tissues improve the adhesion. Besides, π-π stacking interactions formed between the benzene ring of the aromatic Schiff’s base in the hydrogel network and the protein of the surrounding tissues could also enhance adhesive capability of hydrogel (Figure 4B).12,44 Accordingly, incorporating Schiff-base linkages into the hydrogel networks could enhance the adhesive strength between hydrogel and tissues, which improves the effect of wound healing.

C Bursting pressure (mmHg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 4. (A) Photographs of the AD hydrogels stained with rhodamine B adhered to porcine skin (a1) under torsion in different directions (a2), the AD hydrogel could


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stick to the skin without separation (a3). (B) Schematic illustration of adhesive mechanisms of AD hydrogels. (C) Results of bursting pressure tests for different hydrogels. (Values were shown as mean ± standard deviation, n = 6). Cytotoxicity Analysis. The assessment of cytotoxicity is critical to hydrogels applied for wound dressings. The components of AD hydrogels are DF-PEG and AG-NH2, which have been proved to be biocompatible.45,46 The biocompatibility of the AD hydrogel has been checked using MTT assay through the hydrogel extracts method, as shown in Figure 5. The hydrogel extracts were prepared by adding AD hydrogel fragments to DMEM culture medium and being immersed at 37 °C for 12 h (extracts 1) or 24 h (extracts 2). After incubating in hydrogel extracts (1 or 2) with different concentrations (80% and 100%) for 24 h (Figure 5A) and 48 h (Figure 5B), the cell viability of HUVEC cells is still larger than 80%, demonstrating that the AD hydrogels possess low cytotoxicity on HUVEC cells. Extract 1 Extract 2

100 80 60 40 20 0

B Cell Viability (%)

A Cell Viability (%)

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Biomacromolecules

Extract 1 Extract 2

100 80 60 40 20 0

80 %

Concentration

100 %

80 %

100 %

Concentration

Figure 5. Cytotoxicity of AD hydrogels on HUVEC cells through the extract method after incubation for (A) 24 h and (B) 48 h. (Values were shown as mean ± standard deviation, n = 3) In Vivo Hemostatic Ability Test. Minimizing trauma bleeding and hemostasis time are imperative for wound dressings to assure optimal effects and limit morbidity.



The hemostatic effect of the AD hydrogels was evaluated in rabbit liver hemorrhaging model to imitate the severe trauma (Figure 6A).47 After applying AD hydrogels (Figure 6B), sterile gauze, or no treatment at the hemorrhaging site, the amount of blood loss and hemostasis time were assessed. The total amount of bleeding after applying the AD hydrogels (r = 3 and c = 1 wt%) was 0.19 ± 0.03 g, whereas those of the gauze-treated and negative control were 0.71 ± 0.09 g and 1.17 ± 0.13 g, respectively (Figure 6F). The average hemostasis time was less than 10 s for the AD hydrogels, which was much shorter than those of gauze (72 ± 11 s) and negative control group (175 ± 22 s) (Figure 6G). All these results showed that the amount of bleeding was sharply declined and hemostasis time was obviously shortened after treated with AD hydrogels, indicating that the AD hydrogel can act as improved hemostatic materials in contrast to the sterile gauze. Compared with the other similar kind of dressing materials, the AD hydrogels also exhibit superiorities in effective hemostasis.21 Moreover, as the histological assessments shown, there is a narrow gap in the wound surface of the negative control group (Figure 6C), compared with a wide gap of the positive control group (Figure 6D). Meanwhile, a trauma with accumulation of blood cells and plates was observed clearly in hydrogel group (Figure 6E).


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G

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F Total Blood Loss (g)

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Biomacromolecules

0.8

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Figure 6. Snapshots of rabbit liver without injury (A); the wound site applied with AD hydrogels (B). H&E stained tissues with (C) no treatment (negative control group), D) sterile gauze (positive control group), and (E) AD hydrogel (r = 3 and c = 1 wt%). The total amount of bleeding (F) and hemostasis time (G) of wound site under different treatments. (Values were shown as mean ± standard deviation, n = 4) One can conclude that the incision would close gradually in natural hemostasis process leading to a narrow gap, while the gap would be broadened with press of sterile gauze. Owing the swelling property and porous structures, the hydrogels could promote accumulation of red blood cells and platelets by absorbing the contaminated



exudates of the wounds,48 resulting in the capture of the overflowing bleeding from the gaping wound. These characteristics endow AD hydrogels with remarkable hemostatic capability. 4. CONCLUSIONS In summary, self-healing and injectable hydrogels with pH-response were successfully developed by simply mixing the AG-NH2 and DF-PEG solutions based on the dynamic Schiff’s base. The dynamic equilibrium of Schiff-base linkages in networks of hydrogels can form and disintegrate easily under ambient condition, which impart the hydrogels with self-healing and injectable characteristics, as confirmed by the macroscopic self-healing tests and rheological recovery tests. The hydrogels possess the interconnected porous structures, rapid gelation time, excellent deformability and good mechanical strength, which are beneficial to hemostasis and wound healing applications. The AD hydrogels exhibit improved tissue adhesiveness due to the additional interactions between aromatic Schiff’s base and tissue protein. The AD hydrogels also show good cytocompatibility towards HUVEC cells and remarkable hemostatic capability after being applied to a rabbit liver incision immediately, in contrast to the sterile gauze. We believe that the injectable, pH-responsive, and self-healing AD hydrogels with superior tissue adhesiveness can be a potential candidate for the critical conditions acted as a rapidly hemostatic material. ■ ASSOCIATED CONTENT Supporting Information Synthesis of DF-PEG and AG-NH2, characterizations of DF-PEG and AG-NH2, pH sensitivity of the AD hydrogels, additional data on characterizations of AD hydrogels


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containing rheological measurements, macroscopic deformation tests, and schematic for bursting pressure test. The Supporting Information is available free of charge on the ACS Publications website at DOI:. ■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Phone: +86-531-88366074; Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This work was funded by the National Natural Science Foundation of China (Grant No. 21420102006). ■ REFERENCES 1. Boateng, J. S.; Matthews, K. H.; Stevens, H. N.; Eccleston, G. M. Wound healing dressings and drug delivery systems: A review. J. Pharm. Sci. 2008, 97, 2892-2923. 2. Ninan, N.; Forget, A.; Shastri, V. P.; Voelcker, N. H.; Blencowe, A. Antibacterial and anti-inflammatory pH-responsive tannic acid carboxylated agarose composite hydrogels for wound healing. ACS Appl. Mater. Interfaces 2016, 8, 28511-28521. 3. Xu, R.; Luo, G.; Xia, H.; He, W.; Zhao, J.; Liu, B.; Tan, J.; Zhou, J.; Liu, D.; Wang, Y.; Yao, Z.; Zhan, R.; Yang, S.; Wu, J. Novel bilayer wound dressing composed of silicone rubber with particular micropores enhanced wound re-epithelialization and contraction. Biomaterials 2015, 40, 1-11. 4. Rieger, K. A.; Birch, N. P.; Schiffman, J. D. Designing electrospun nanofiber mats to promote wound healing – a review. J. Mater. Chem. B 2013, 1, 4531-4541. 5. Xi, Y.; Dong, H.; Sun, K.; Liu, H.; Liu, R.; Qin, Y.; Hu, Z.; Zhao, Y.; Nie, F.; Wang,



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Table of Contents:

Entry: Rapid-Forming and Self-healing Agarose-Based Hydrogels with pH-response were constructed by dynamic covalent Schiff-base linkages through simply mixing nontoxic

agarose-ethylenediamine

conjugate

(AG-NH2)

and

dialdehyde-

functionalized PEG (DF-PEG) solutions. The incorporated Schiff’s base imparts the hydrogels to the remarkable self-healing capability, injectability and tissue adhesiveness. The hydrogels also show the effective hemostatic effect for a rabbit liver incision, which can be utilized as potential hemostatic dressings.

Title: Rapid-Forming and Self-Healing Agarose-Based Hydrogels for Tissue Adhesives and Potential Wound Dressings Z. Zhang, X. Wang, Y. Wang, Prof. J. Hao

Keywords: agarose-based hydrogel, self-healing property, tissue adhesion, effective hemostasis, wound dressing


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Biomacromolecules

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