Duplicating Dynamic Strain-Stiffening Behavior and Nanomechanics

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Duplicating Dynamic Strain-Stiffening Behavior and Nanomechanics of Biological Tissues in a Synthetic Self-Healing Flexible Network Hydrogel Bin Yan,†,‡,⊥ Jun Huang,‡,⊥ Linbo Han,‡,⊥ Lu Gong,‡ Lin Li,‡ Jacob N. Israelachvili,§ and Hongbo Zeng*,‡ †

College of Light Industry, Textile & Food Engineering, Sichuan University, Chengdu 610065, China Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 1H9, Canada § Department of Chemical Engineering, Materials Department, Materials Research Laboratory, University of California, Santa Barbara, California 93106, United States ‡

S Supporting Information *

ABSTRACT: Biological tissues can accurately differentiate external mechanical stresses and actively select suitable strategies (e.g., reversible strain-stiffening, self-healing) to sustain or restore their integrity and related functionalities as required. Synthetic materials that can imitate the characteristics of biological tissues have a wide range of engineering and bioengineering applications. However, no success has been demonstrated to realize such strainstiffening behavior in synthetic networks, particularly using flexible polymers, which has remained a great challenge. Here, we present one such synthetic hydrogel material prepared from two flexible polymers (polyethylene glycol and branched polyethylenimine) that exhibits both strain-stiffening and self-healing capabilities. The developed synthetic hydrogel network not only mimics the main features of biological mechanically responsive systems but also autonomously self-heals after becoming damaged, thereby recovering its full capacity to perform its normal physiological functions. KEYWORDS: hydrogel, strain-stiffening, self-healing, dynamical covalent bonds, flexible network mediate filaments,12,13 and collagen,14,15 as well as synthetic protein mimics such as polyisocyanopeptides.16−19 However, this adaptive behavior is absent in a wide variety of synthetic materials such as synthetic flexible network hydrogels. Instead of strain-stiffening, most synthetic flexible hydrogels generally tend to soften under external stress.12,20−23 Meanwhile, this strain-stiffening strategy is not always effective, in particular, when the deformation induced by an external mechanical force surpasses the maximum strain of these systems, resulting in irreversible material rupture or damage. In the case of irreversible damage of materials, another strategyselfhealingcomes into effect: the biological system can autonomously heal and mend ruptures, cuts, and tears and at the same time recover their designated physiological functions.

oft biological systems such as blood vessels,1,2 mesentery tissue,3 lung parenchyma,4 cornea,5,6 and blood clots7 can adopt strategies to manipulate deformation under external mechanical stresses to maintain their integrity and properly perform their physiological functions. Most of these biological systems strain-stiffen (viz., they become stiffer as applied stress or strain on the systems increases),8−12 which is one of the many strategies employed by biological systems to effectively prevent damage when subjected to large deformations, especially when the deformation induced by an external mechanical force is below the maximum strain these systems can sustain. Although strain-stiffening behavior is commonly adopted by biological systems to enhance their chance of survival, achieving this particular mechanical response in synthetic materials has been very challenging: the main efforts have been directed at developing biomimetic materials with similar mechanical responses by utilizing semiflexible biopolymers with stiff, helical architectures, including a series of biological proteins, such as microtubules,8,9 actin,10,11 inter-

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© 2017 American Chemical Society

Received: July 19, 2017 Accepted: September 28, 2017 Published: September 28, 2017 11074

DOI: 10.1021/acsnano.7b05109 ACS Nano 2017, 11, 11074−11081

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Figure 1. Measured mechanical properties of polymer hydrogels as a function of polymer concentration and temperature. (A) Differential modulus K′ of polymer hydrogels vs stress under varying polymer concentration at 25 °C with fixed NH2/CHO ratio in all the hydrogels. (B) Single master curve was obtained after rescaling (normalizing) the data in (A) with the plateau modulus G0 and the critical stress σcrit. (C) Critical stress σcrit for polymer hydrogels with concentration of 5.55 and 5.0 wt % at different temperatures. (D) Temperature dependence of differential modulus K′ vs stress for polymer hydrogels at concentration of 5.55 and 5.0 wt %, respectively, with a fixed NH2/CHO ratio. (E) Single master curve obtained by rescaling the data in (D) with the plateau modulus G0 and the critical stress σcrit. (F) Critical stress σcrit of the polymer hydrogels with varying polymer concentration.

Despite recent progress in self-healing biomaterials,24−35 it remains a challenge to design a smart synthetic material from flexible synthetic polymers that has the ability to differentiate among different types of external mechanical stresses and adopt a strategy of simultaneous strain-stiffening and self-healing that recovers full functional integrity. Here, we present a facile fabrication of self-healing strainstiffening hydrogels by simply mixing two kinds of synthetic flexible polymers in physiological solution. The Schiff base reactions between the amino groups and aldehyde groups on the two polymers lead to a self-cross-linking hydrogel network. Notably, the resulting hydrogels exhibit obvious strainstiffening behavior when applying a stress above some critical value. This biomimetic stress-stiffening response can be easily tuned by varying the polymer concentrations and temperature, as well as by choosing cross-linkers of different chain lengths. In addition, such types of hydrogels are able to automatically (self)heal from undesired damages which readily restores 100% of their integrity. This self-healing ability is attributed to the reversible cross-linking interactions through dynamic Schiffbase bonds in the polymer networks.23,36 The reported strategy based on dynamic covalent bonds for achieving self-healing and strain-stiffening properties can be further applied to design and fabricate a wide range of nanomaterials (e.g., nanocomposites, hydrogels) with applications in tissue engineering, drug delivery, etc.

patible telechelic difunctional PEGs with two aldehyde end groups with molecular weights of 4 and 20 kDa, labeled P1 and P2, respectively, were synthesized by esterification of hydroxylterminated PEGs of different molecular weights with 4formylbenzoic acid. P1 and P2 have been determined to terminate with benzylaldehyde groups at both ends by Fourier infrared transorm spectrophotometry (FTIR), 1H NMR, and gel permeation chromatography (GPC) and are well-defined and retain the low polydispersity similar to that of their parental PEG counterparts (see Figures S1 and S2 in the Supporting Information (SI) for details of the polymer synthesis and characterizations). The hydrogels were formed at room temperature by mixing PBS solutions of the two polymers at suitable concentrations to yield a final polymer concentration in the hydrogel between 3.85 and 6.25 wt %. For all the PEI/PEG hydrogels studied, it was found that the gelation time is less than 10 min (see Figure S3 and the Hydrogel Formation section in the SI for details). To guarantee that the Schiff base reactions within the polymer network were fully achieved, the formed hydrogels were kept undisturbed for 1 h before measurements. One striking feature of the developed hydrogels is their strain-stiffening behavior, that is, a strong, well-defined, and nonlinear stress response displayed after some critical external stress. Such stiffening response is generally pervasive in soft biological tissues but is extremely rare in synthetic flexible polymer hydrogels. Rheology tests (see Figure S4) clearly demonstrate that the PEI/PEG hydrogels show mechanical responses in two distinct regimes: a low-stress linear and a highstress nonlinear regime. To better (and quantitatively) describe their mechanical behavior, the differential modulus, K′, was

RESULTS AND DISCUSSION The self-healing strain-stiffening hydrogels are polymer networks prepared from two hydrophilic polymers: polyethylene glycol (PEG) and branched polyethylenimine (PEI). Biocom11075

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Figure 2. (A) Normalized shear modulus for hydrogel samples P1 and P2 during oscillatory shear at ω = 10 rad/s as a function of the strain amplitude at 25 °C. The two hydrogels tested have the same polymer concentration and NH2/CHO ratio. (B) Dynamic strain amplitude cyclic test (γ = 1% or higher strains above σc) of the same P1 hydrogel tested in (A) at 25 °C with a frequency of ω = 10 rad/s, showing clear strainstiffening behaviors. (C) Dynamic strain amplitude cyclic test (γ = 1% or higher strains above σc) of the same P2 hydrogel tested in (A) at 25 °C with a frequency of 10 rad/s, again showing clear strain-stiffening behaviors. (D) Continuous strain amplitude sweep cyclic tests (γ sweeping from 0 to 650% and then relaxing back to 0 strain for 60 s) of the same P2 hydrogel tested above, showing that the strain-stiffening response of the prepared hydrogel is totally reversible and stable and does not break the hydrogel network when the applied strain is below γmax.

defined as the part derivative of the stress, σ, with respect to the strain, γ, K′ = ∂σ/∂γ, and was employed to differentiate the two regimes. In the low-stress regime, the plateau modulus G0 = K′, and in the high-stress “stiffening” regime (σ > σcrit), the modulus K′ increases with increasing stress σ, where σcrit is the critical stress at which nonlinear, but still reversible elastic, response starts. The hydrogels in this work were prepared by cross-linking PEI chains with PEG via Schiff base bonds where the ratio of amine to aldehyde groups from the two polymers is fixed while the influence of the total polymer concentration on the resulting hydrogels is investigated. Figure 1A shows that both the plateau modulus G0 and the critical stress σcrit of the PEI/ PEG hydrogels strongly depend on the polymer concentrations in the hydrogels. Decreasing the polymer concentrations from 6.25 to 3.85 wt % could lower G0 by 3 orders of magnitude. This strong polymer concentration-dependent mechanical behavior of the synthetic PEI/PEO hydrogels is similar to those observed with biopolymer gels based on actin,10 fibrin collagen,14 and ethylene-glycol-functionalized polyisocyanopeptides.18 The measured moduli of the synthetic hydrogels with different polymer concentrations were normalized by the plateau modulus, K′/G0, and plotted as a function of the normalized stress σ/σcrit (Figure 1B). Interestingly, the normalized data for all the PEI/PEG hydrogel samples overlap and follow a master curve, as shown in Figure 1B, demonstrating a correlation of (K′/G0) ∝ log(σ/σcrit) in the strain-stiffening regime. Figure 1C clearly shows that on decreasing the polymer concentrations from 6.25 to 3.85 wt %, the σcrit required to trigger strain-stiffening of the hydrogels decreases from ∼200 Pa to less than 10 Pa, which is the biologically accessible stress range that cells can apply.37 The strain-stiffening response of the PEI/PEG hydrogels to external stress is also sensitive to temperature change: Figure

1D−F shows that for the hydrogels with polymer concentrations of 5.55 and 5 wt %, increasing the temperature from 15 to 35 °C significantly lowers G0 and σcrit, whereas the plots of the normalized moduli (K′/G0) vs the normalized stress (σ/ σcrit) at different temperatures almost follow the same master curvedisplaying K′/G0 ∝ σ/σcrit in the strain-stiffening regime. Such mechanical behaviors are also observed in dynamic temperature sweep experiments (Figure S5), in which the elastic modulus G′ and loss modulus G″ were measured as a function of temperature from 0 to 50 °C at a heating rate of 5 °C/min. Apparently, as the temperature increases, both G′ and G″ decrease. The temperaturedependent mechanical behaviors are attributed to the dynamical characteristics of the Schiff base bonds involved in hydrogel formation. The weakened mechanical properties with increasing temperature are most likely due to thermal deterioration of the bonding strength of the Schiff base connections.38 Another approach to tune the mechanical behavior of the strain-stiffening hydrogels is to adjust the length between the network strands (lc). In the PEI/PEG hydrogel systems, lc is mainly determined by the chain length of the cross-linkers, that is, the contour length of the PEG chains used. It is widely accepted that varying the length of the cross-linker lc alters the onset of nonlinear strain-stiffening.35,36 Therefore, another cross-linker (P2) with longer chain length (molecular weight 20 kDa) was synthesized and used as the reference to investigate the effect of lc on the mechanical properties of the hydrogels. To rule out the effect of the concentration and crosslinking density, the comparison was made between hydrogels based on P1 and P2 with the same polymer concentration at a fixed NH2/CHO ratio. Representative mechanical responses of the hydrogel networks with applied strain are shown in Figure 2A for PEI/PEG hydrogels of P1 and P2 (5 wt % polymers). 11076

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Figure 3. Self-healing properties of hydrogels. (A) Strain sweep measurements of original and self-healed P1 hydrogels (polymer concentration = 5.55 wt %) at 25 °C (storage modulus G′ and loss modulus G″ as a function of the strain). (B) Surface forces apparatus results (force−distance profiles obtained from three sequential force measurements) show the force magnitudes and reversibility of the Schiff base bonds formed between the amine and benzylaldehyde groups of P1 and PEI 60K. Photographs (C−F) show self-healing evidence of P1 hydrogels: (C) original P1 hydrogel; (D) P1 hydrogel after it was cut into two pieces; (E) two cut pieces brought together, where the selfhealing process occurred instantaneously, and (F) healed hydrogel withstanding the stretching with tweezers.

one or some of the following: (i) The predicted γmax is the ideal case with all the PEG strands to be fully extended before the hydrogel network breaks down. In reality, this can barely occur as the cross-linkers of the hydrogel network are unable to reorganize into a fully extended trans-conformation before breaking down during straining. (ii) The approximation of l0 above might not be very precise as the PEG polymer strands in the hydrogel networks experience different interactions compared to those in the bulk solution. (iii) The Schiff base connections between PEG and PEI are dynamic and relatively easy to break during stretching, thereby limiting the extension of the PEG strands. To explore the reversibility of the strain-stiffening behavior, cyclic dynamic strain step tests were conducted by applying a low strain γ = 1% (lower than γcrit) and a high strain on hydrogels of P1 and P2. As shown in Figure 2B,C for the P1 and P2 hydrogels, their strain-stiffening behaviors are fully reversible: both hydrogels exhibit stiffening when subjected to high strains (between γcrit and γmax), whereas they restore the linear state (G′/G0 ∼ 1.0) when the applied strain is reduced to γ = 1%. However, employing a cross-linker with a shorter chain length, P1 hydrogel exhibits a more sensitive strain-stiffening behavior than P2 hydrogel. For instance, under the same applied strain of γ = 100% (higher than γcrit for both hydrogels), the P1 hydrogel demonstrates a strain-stiffening change of G′/ G0 ∼ 2 while its counterpart P2 hydrogel only shows G′/G0 ∼ 1.2. Another important observation was that the mechanical response of the PEI/PEG hydrogels is barely dependent on the strain history. Actually, this reproducible strain-stiffening behavior under repeated straining is critical for applications involving artificial extracellular matrices because most biological systems, including skin, blood clots, tendons, and ligaments, must withstand repeated stretches and strains with every motion of the body. Unlike most un-cross-linked biopolymer networks that strain-stiffen at successively higher strain upon repeated straining, the strain-stiffening properties of PEI/PEG hydrogel remain unchanged with subsequent cyclic strain

The G′ properties of both the P1 and P2 hydrogels exhibit similar dependence on the applied strain γ: G′ maintains almost constant (G′/G0 ∼ 1.0) when γ falls below a critical strain γcrit but increases rapidly when γ exceeds γcrit and continues increasing until it reaches a maximum strain where rupture of the hydrogel network occurs (Figure 2A). Both γcrit and γmax demonstrate a marked dependence on the chain length of the cross-linkers: Figure 2A shows that the hydrogel prepared with P2 (of longer lc) shows a higher γcrit for the onset of nonlinear strain-stiffening than does the P1 hydrogel; the P2 hydrogel can also withstand a higher maximum strain γmax. This unusual strain-stiffening behavior of the PEI/PEG hydrogels is believed to be attributed to the nonlinear stretching and finite extensibility of the PEG strands among the hydrogel networks.39,40 Thus, the maximum strain the hydrogel can withstand (γmax) is related to the maximum uniaxial extension ratio of PEG polymer chains (λmax) and can be roughly predicted using the following equations:40,41 λmax =

l0 =

( θ2 )

nl sin lmax = l0 l0

⎡ ⎤ lp 2lplmax ⎢1 − (1 − e−lmax / lp)⎥ lmax ⎣ ⎦

−1 γmax = λmax − λmax

(1)

(2) (3)

where lmax is the contour length of a fully extended polymer chain that can be estimated by the end-to-end distance of the chain, composed of n bonds of length l with an angle θ between neighboring bonds, extended to the all-trans conformation; l0 is the end-to-end length of the upstretched polymer chains and is determined by using the persistence length lp of PEG in water (reported to be ∼0.37 nm by Lee et al.41). Based on eqs 1−3, the predicted values of γmax for the P1 and P2 hydrogels are calculated to be 5.7 and 13.1, respectively, which are larger than the experimental values of 2.2 and 7.0. The difference between the experimental results and calculations is most likely due to 11077

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ACS Nano Scheme 1. Illustration of the Self-Healing Mechanism for the PEI/PEG Hydrogel Networka

a

The network is mainly based on dynamic covalent bonding between amine rich polymer (PEI) and dibenzylaldehyde-functionalized PEG.

sweeps alternating between γ = 0 and 650% (lower than γmax) at the same frequency. As shown in Figure 2D, the strain-stiffening behavior is fully reversible and repeatable, showing no hysteresis, as is evident in the strain-stiffening profile of the eighth cycle that exactly follows the same trace of the first cycle; that is, when subjected to a high strain of 650%, G′ and G″ of the P2 hydrogel increases by 1 order of magnitude, whereas once the strain returns to 0, G′ and G″ swiftly recover. Moreover, the onset of nonlinearity happens at the same γcrit through the eight strain sweep cycles (see Figure S5A for further details). However, it should be noted that for both the P1 and P2 hydrogels, once subjected to a strain higher than γmax, the reversible strain-stiffening property can be weakened or even eliminated. For example, for P1, Figure S5B shows that when P1 hydrogel is under cyclic dynamic strain step tests between γ = 1 and 250%, the hydrogel stiffens with its initial G′/G0 of ∼3.3 at γ = 250% but fails to withstand this high strain with time: with G′/G0 gradually dropping from 3.3 to 2.8 after ∼120 s. Thus far, we have demonstrated that the strain-stiffening behaviors of the hydrogels based on synthetic polymers are robust and can be suitably tuned and controlled by varying the chain length of the cross-links as well as the polymer concentration in the hydrogels and temperature. We now consider the outstanding self-healing properties of these PEI/PEG hydrogels. As shown in Figures 3A and S4, even when a very high strain of γ = 1000% (higher than γmax) is applied to induce breakage or detrimental damage to the P1 hydrogels of different polymer concentrations, these hydrogels can automatically and rapidly heal to fully restore their integrity and strain-stiffening response after they are allowed to relax back to a low strain of γ = 5%. This self-healing capability is further confirmed by repeated dynamic strain step tests (γ = 5

or 400%) on the P1 hydrogel (5 wt %), where the recovery of the mechanical properties of the tested hydrogel is complete and reproducible (see Figure S6A). To determine the mechanism of this excellent self-healing behavior, a surface forces apparatus (SFA) was employed to measure the interactions involved in the PEI/PEG hydrogels. Two comparative experiments were conducted to measure the Schiff base or hydrogen bonding interactions between P1 or PEG and PEI. Briefly, two mica surfaces were first coated with positively charged PEI, followed by injection of P1 or PEG solution between the coated mica surfaces. The whole system was equilibrated for 15 min before measurements (see Figures 3B and S6B). The SFA results indicate that two different kinds of reversible interaction mechanisms are involved in the overall self-healing process. First, the interaction between PEI and unmodified PEG is highly reversible but relatively weak, with a small adhesion of ∼0.7 mN/m (mJ/m2), which probably originates from van der Waals bonds between the polymer networks or hydrogen bonds between PEI and PEG (see Figure S6B for details). In contrast, a (second) strong and reversible interaction is found between PEI and P1 (i.e., benzylaldehydecapped PEG), as shown in Figure 3B. The adhesion reaches ∼12 mN/mabout 17 times stronger than that between PEI and unmodified PEG. Interestingly, this high adhesion is reversible, as confirmed by the SFA results in sequential approach−separation force measurements. Figure 3B shows that the adhesion remains almost the same with only ∼3 mN/ m different for three sequential measurements at the same interaction area, or “spot”, on the two surfaces. Considering the slight difference in the chemical structures between PEG and P1, we can attribute this significantly enhanced and reversible adhesion between P1 and PEI to the Schiff base bonds between benzylaldehyde and amine groups, which endows the PEI/PEG 11078

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was adopted here to enhance the yield in dibenzylaldehyde functionalization. A solution of 4-formylbenzoyl chloride (2.02 g, 1.2 mmol) and freshly distilled THF (30 mL) was slowly added to a mixture of PEG20K (2.5 g, 0.125 mmol), triethylamine (1.2 mmol), and DCM (30 mL) at room temperature under an argon atmosphere. Then, the reaction mixture was allowed to reflux under argon for 24 h. After being cooled to room temperature, the mixture was filtered and the filtrate was concentrated. The crude residue was precipitated out in diethyl ether and collected by filtration, which was further purified by repeated dissolution in DCM and precipitation in diethyl ether three times. The polymer P2 was obtained as a white solid after drying under vacuum overnight. Hydrogel Preparation. Branched polyethylenimine (60 kDa) and telechelic dibenzylaldehyde-functionalized poly(ethylene glycol)s (e.g., P1 and P2) were dissolved in PBS separately at a concentration of 10 wt %. If necessary, pH of the resultant stock solutions was adjusted to 7.4 with concentrated HCl. A typical hydrogel was prepared as follows: PEI solution (0.4 mL) was added to a homogeneous mixture of P1 solution (0.6 mL) and PBS buffer (0.8 mL) under vortex. After being mixed, the resultant solution was allowed to keep still and the gelation occurred in 30 s. All of the other gels were prepared using the same procedures except that the stock solutions of PEI and P1/P2 were mixed at different ratios. The gelation time was determined using a pipet method as described by Elisseef et al.42 Briefly, determined amount of P1 and PEI60K solutions was added and mixed in an Eppendorf tube. The whole mixture was repeatedly pipetted up and down until it was impossible to pipet anymore. The time at which the solutions could not be pipetted is defined as the gelation time. We found that the gelation time for all the PEG/PEI hydrogels reported in the article is below 10 min, and it decreased with increasing polymer concentration applied for hydrogel formation. Characterization Methods. Fourier Transform Infrared Spectrophotometry. The FTIR spectra were obtained on a Thermo Scientific Nicolet iS50 Fourier transform infrared spectrophotometer equipped with diamond crystal/built-in all-reflective diamond ATR. The spectra were recorded over a frequency range of 4000−400 cm−1 directly on polymer powders. Gel Permeation Chromatography. GPC measurements on polymers were carried out on a Waters system equipped with a refractive index detector (RI 2414) and a UV/vis 2489 detector. A Waters 510 liquid chromatography pump equipped with one (HR4E) Styragel column was used at 40 °C. THF was used as the eluent at an elution rate of 1 mL min−1, and polystyrene standards were used for calibration. Nuclear Magnetic Resonance. 1H NMR measurements were performed on a Varian-Inova (400 MHz) spectrometer at room temperature. Chemical shifts are reported in parts per million (δ) relative to TMS as the internal reference. The polymer samples were dissolved in CCl3D at a concentration of 5 mg/mL. Rheology Analysis. Rheological characterization was performed on a TA Instruments AR-G2 stress-controlled rheometer fitted with a Peltier stage using a 20 mm parallel-plate configuration. The hydrogel samples were equilibrated at the desired temperature for 1 h before measurements were performed. To test self-healing behaviors of these hydrogels, dynamical oscillatory strain amplitude sweep measurements were conducted at a frequency of 10 rad/s at different temperatures with the strain change from 0.1 to 1000% to achieve a strain failure, followed by a time-dependent modulus observation at 5% strain. The temperature dependences of G′ and G″ were measured with a frequency of 10 rad/s and a heating rate of 1 °C/min. The nonlinear regime was studied using a prestress protocol as described before,16 where the gel at a desired temperature was subjected to a constant prestress σ0 = 0.2−200 Pa with a small oscillatory stress superposed at a frequency of ω = 10−0.1 Hz. The superposed oscillatory stress was at least 10 times smaller than the applied prestress. The data in the paper were recorded at 10 rad/s. Surface Forces Apparatus. A SFA was used to quantitatively study the interaction forces between fresh cleaved mica surfaces across polymer solutions.43 Briefly, two back-silvered muscovite mica surfaces (thickness ∼5 μm, grade #1, S&J Trading, USA) were glued onto

hydrogels with excellent self-healing ability. Based on the rheology and SFA results above, therefore, the self-healing behavior of the PEI/PEG hydrogel is proposed to be mainly achieved based on a reversible interaction process as illustrated in Scheme 1, where the dynamic covalent bonding between amine rich polymer (PEI) and dibenzylaldehyde-functionalized PEG is the main driving mechanism. To further explore the self-healing mechanism(s) of the PEI/ PEG hydrogels, their macroscopic self-healing properties were studied and are demonstrated in Figure 3C−F and Movie S1 in the SI, which show that two divided hydrogel pieces can adhere to each other and automatically heal into one continuous piece upon contact. The healed hydrogel shows complete recovery of its mechanical properties as quantitatively demonstrated by the rheological tests (Figure 3A), which can apparently regain its full integrity even under the very harsh and damaging stretching by the tweezers (see Figure 3F).

CONCLUSIONS In summary, this work demonstrates a biomimetic self-healable strain-stiffening hydrogel that can be readily prepared from two flexible synthetic polymers (branched PEI and telechelic difunctional PEGs). The hydrogels show highly sensitive strain-stiffening behaviors that can be easily tuned by varying the polymer concentration in the hydrogels, cross-linker length, and temperature. The hydrogels can withstand repeated deformations and quickly recover their mechanical properties and structures via strain-stiffening when the applied strain is below γmax (that can be as high as 700%). When the applied strain is above γmaxcausing detrimental damage to the hydrogels, the hydrogels can self-heal to fully recover their integrity via the dynamical Schiff base bonds among the hydrogel network. The synthetic hydrogels developed in this work are highly biocompatible and can be readily modified with desirable functional groups via extra amine groups on the branched PEI component, thereby more closely mimicking the superior strain-stiffening performance of natural biopolymer hydrogel materials, and/or showing high flexibility for further functionalization for a wide variety of bioengineering applications. This work also provides insights into the development of advanced biomimetic hydrogel materials with strain-stiffening and self-healing capabilities. MATERIALS AND METHODS Materials. Tetrahydrofuran (THF) and dichloromethane (DCM) were distilled over CaH2 and used as prepared. PEG4K and PEG20k were purchased from Aldrich and purified by by repeated dissolution in DCM and precipitation in cold diethyl ether for three times and collected by filtering followed by drying under vacuum overnight. All the other chemicals were purchased from Aldrich and used as received. Synthesis of Polymer P1. A 100 mL round-bottomed flask with septum was charged with PEG4K (2 g, 0.5 mmol), 4-formylbenzoic acid (0.225 g, 1.5 mmol), 4-(dimethylamino)pyridine (0.015 g), and freshly distilled THF (20 mL). The whole system was evacuated and purged with argon three times. To the flask was then added a solution of N,N′-dicyclohexylcarbodiimide (0.412 g, 20 mmol) and freshly distilled THF (20 mL) via a syringe. Then, the whole mixture was allowed to stir at room temperature for 24 h. Finally, the reaction mixture was diluted with DCM, filtered through Celite to remove the undissolved solid, and the filtrate was concentrated by evaporation. The polymer P1 was obtained as a white solid by repeated dissolution in DCM and precipitation in diethyl ether three times followed by drying under vacuum at 50 °C overnight. Synthesis of Polymer P2. Because of relatively low reactivity of hydroxyl groups at both ends of PEG20K, 4-formylbenzoyl chloride 11079

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ACS Nano cylindrical glass disks using a UV light (361 nm) cured glue (NOA81, Norland Productes, Inc., USA). The thickness of coated silver layer is ∼50 nm. Then the two surfaces were mounted into the SFA chamber in a cross-cylinder configuration. The SFA chamber was saturated with water vapor. Approximately 100 μL of PEI aqueous solution (0.75 wt %) was directly injected between the two mica surfaces, and force measurement was conducted after the system was equilibrated for 15 min. After that, ∼70 μL of PEG or modified PEG aqueous solution (0.75 wt %) was injected between the two mica surfaces to investigate the changes of interaction forces. The film thickness and absolute separation distance were obtained in situ by employing an optical technique named multiple beam interferometry using the fringes of equal chromatic order (FECO). The measured force, F, between two curved surfaces was correlated to the interaction energy per unit area (W(D)) of two planar surfaces using the Derjaguin approximation.

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F(D) = 2πRW (D)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b05109. Movie S1 showing the self-healing behavior of the synthesized hydrogel (AVI) Polymer synthesis routes and more characterization data on the self-healing and strain-stiffening properties of the hydrogels (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: 780-492-1044. Fax: 780-492-2881. ORCID

Bin Yan: 0000-0001-5426-6179 Jacob N. Israelachvili: 0000-0001-8915-8741 Hongbo Zeng: 0000-0002-1432-5979 Author Contributions ⊥

B.Y., J.H., and L.H. contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Research Chairs program (H.Z.) and the Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, under Award DE-FG0287ER-45331 (J.N.I., SFA measurements, analysis, and interpretations of results). REFERENCES (1) Vito, R. P.; Dixon, S. A. Blood Vessel Constitutive Models 1995−2002. Annu. Rev. Biomed. Eng. 2003, 5, 413−439. (2) Shadwick, R. E. Mechanical Design in Arteries. J. Exp. Biol. 1999, 202, 3305−3313. (3) Fung, Y. C. Biomechanics: Mechanical Properties of Living Tissues; Springer Science & Business Media, 2013. (4) Freed, A. D.; Einstein, D. R. An Implicit Elastic Theory for Lung Parenchyma. Int. J. Eng. Sci. 2013, 62, 31−47. (5) Hjortdal, J. O. Extensibility Of the Normo-Hydrated Human Cornea. Acta Ophthalmol. Scand. 1995, 73, 12−17. (6) Kampmeier, J.; Radt, B.; Birngruber, R.; Brinkmann, R. Thermal and Biomechanical Parameters of Porcine Cornea. Cornea 2000, 19, 355−363. 11080

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