Metal Ion Mediated Cellulose Nanofibrils Transient Network in

Feb 13, 2017 - The cations promote the formation of porous networks of nanofibrils by screening the repulsive negative charges on CNF surface and domi...
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Metal Ion Mediated Cellulose Nanofibrils Transient Network in Covalently Cross-linked Hydrogels: Mechanistic Insight into Morphology and Dynamics Jun Yang,* Feng Xu, and Chun-Rui Han Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, 100083, China S Supporting Information *

ABSTRACT: Utilization of reversible interactions as sacrificial bonds in biopolymers is critical for the integral synthesis of mechanically superior biological materials. In this work, cellulose nanofibrils (CNFs) reinforced covalent polyacrylamide (PAAm) composite hydrogels are immersed into multivalent cation (Ca2+, Zn2+, Al3+, and Ce3+) aqueous solution to form ionic association among CNFs, leading to the ionic−covalent cross-linked hydrogels. The cations promote the formation of porous networks of nanofibrils by screening the repulsive negative charges on CNF surface and dominate the mechanical properties and self-recovery efficiency of the hydrogels, resulting in mechanically reinforced ionic hydrogels with stiff (Young’s modulus 257 kPa) and tough properties (fracture toughness 386 kJ/m3). The in situ Raman spectroscopy during stretching corroborates the stress transfer medium of CNF, and the microscopic morphologies of stable crack propagation validates that the multiple toughening mechanisms occur in a balanced energy dissipation manner, enabling synergistic combination of stiffness and toughness. Moreover, the depth-sensing instrumentation by indentation test also demonstrates that the CNF ionic coordination contributes simultaneous improvement in hardness and elasticity by as much as 600% compared to those pristine gels. This work demonstrates a facile way to transfer nanoscale building blocks to bulk elastomers with tunable dynamic properties and may provide a new prospect for the rational design of CNF reinforced hydrogels for applications where high-bearing capability is needed.

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INTRODUCTION Hydrogels with the soft-and-wet property are acknowledged to be important functional materials due to their indispensable application in numerous academic and industrial fields.1,2 While a number of promising properties, such as biocompatibility, responsiveness to external stimuli, and biodegradability have been integrated into hydrogels, the relatively poor mechanical properties of most synthetic hydrogels remains a challenge, impeding their field applications that generally require high mechanical integrity.3,4 Recently, many efforts have been witnessed to improve the mechanical properties of hydrogels by optimizing their network structures, and typical toughening strategies include double-network structure,5 topological connection through slide-ring,6 and dynamic cross-linking systems.7,8 Among them sacrificial bonding is proposed as a dominate factor for the enhanced properties of biological materials such as bone, silk, and byssus.9,10 When subject to a stress, these sacrificial bonds (e.g., hydrogen bonds, metal coordination bonds) could sustain the stress at small deformation and preferentially rupture with original configurations survived, resulting in efficient energy dissipation mechanism and improved mechanical performance.11,12 The strategy to design high performance of polymeric hydrogels, including high strength and toughness, excellent fatigue resistance, is to introduce uniform cross-linked © XXXX American Chemical Society

structures and reversible sacrificial bonds (weak bonds) in the networks along with the strong bonds.3 In this regard, the strong bonds, either covalent or noncovalent, build the primary structure and maintain the integrity of the materials. The sacrificial bonds, on the other hand, damage upon stretching to dissipate energy and impart toughness to the materials via resisting crack propagation. Based on the sacrificial bond theory, Gong et al. designed a series of double-network (DN) gels that composed of two asymmetric network structures.5 Upon stress, the first rigid and brittle network preferentially ruptures to prevent crack propagation in the second network, leading to efficient energy dissipation.12 Despite their excellent mechanical properties, the multistep synthesis of DN gels is hampered by the limited components and lengthy preparation procedure. On the other hand, composite hydrogels possess the advantage of abundant raw material choices. Biopolymers, a class of renewable and abundant natural resources, can enrich composite hydrogels with various advanced functions for applications in tissue engineering, drug delivery, and catalysis.13−15 However, utilizing the sacrificial interactions of biopolymers remains to be explored for an integral synthesis of Received: December 26, 2016 Revised: February 3, 2017 Published: February 13, 2017 A

DOI: 10.1021/acs.biomac.6b01915 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules mechanically superior biological composites.13 Thus, it is important to develop tough, flexible, and processable composite hydrogels based on natural renewable biopolymers. Cellulose nanofibrils (CNFs) with native crystalline structure attract increasing interest due to their plant-based biocompatibility, excellent mechanical properties, and modifiability.16,17 These nanoparticles are composed of cellulose backbone chains intrinsically assembled into a parallel arrangement with the hierarchical structures that are held together by intra- and interhydrogen bonds. In this respect, one way to fulfill mechanical reinforcement would be to align CNFs along axial direction with closely packed individual fibrils through various interactions. According to previous work,18,19 there are two main issues that should be considered in preparing CNF reinforced composites, namely, (1) to achieve a uniform dispersion of the CNF and ensure homogeneous properties throughout the composite, and (2) to increase the interaction between CNFs and achieve an appropriate level of stress transfer. In this context, the dynamic interactions (e.g., electrostatic interaction and hydrogen bonds) within parallel aligned of CNF motifs would act as a crack propagation lock in extensional mode, rendering the improved mechanical properties. While numerous dynamic CNF hydrogels have been developed by reducing electrostatic repulsion between the surface carboxylate groups20−22 or adding gelling components (e.g., methycellulose,23 hydroxyethyl cellulose,24 and glycerol25), the role of dynamic CNF motif on the large strain (in nonlinear deformation regime) mechanical response, or more specifically, the rationale behind the effective load transfer mechanism is still unclear.26 To the best of our knowledge, no systematic effort has been afforded to utilize sacrificial bonding strategy to impart reversible CNF motifs such as cationmediated transient network into other elastomers by careful control of the dynamics, even though CNF can offer reinforcement with a high propensity for alignment under shear that generally driven by hydrogen bonding, electrostatic forces, and van der Waals interactions.16 In this work, inspired by interfacial interactions of natural hierarchical materials,9 we design tough and self-recoverable gels by constructing synergistic interfacial dynamic ionic bonds between 2,2,6,6tetramethylpiperidine-1-oxyl radical (TEMPO) oxidized CNF and cations in a second covalent polymer network. To this end, our work particularly involves the engineering of ionic coordination bonds acting as reversible sacrificial bonds via fibrils slipping, which is notably sought after in the bioinspired materials design strategy.27 It is envisioned that the cation induced ionic cross-linking method provides an unlimited platform for design of tough hydrogels, affording us an alternative way to synthesize a library of CNF based materials with tunable dynamic properties.

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Hydrogel Preparation. A two-step method was applied to synthesize ionic hydrogels. Initially, 10 mL of acrylamide (AAm, 10 wt %) solution with UV initiator Irgacure 2929 (6.25 mg, 0.2 mol % of AAm) and N,N-methylenebis(acrylamide) (MBA, 2.15 mg, 0.1 mol % of AAm) was prepared in deionized water. Then 4 mL of CNF aqueous suspension was added and sonicated (300 W) in an ice−water bath for 10 min to form a uniform mixture. After degassing by bubbling nitrogen for 5 min, the mixture was poured into rectangular tubes and subjected to UV irradiation (20 W, 365 nm wavenumber) for 60 min to achieve the covalently cross-linked composite gels (designated as PAAm-CNF-x, where x refers to the volume fraction of CNF in water). Afterward, the collected composite gels were immersed in aqueous solutions of the cations (Ca2+, Zn2+, Al3+, and Ce3+, 0.1 mol/L) at room temperature for 2 h to form ionic crosslinked gels. Finally, the ionic gels were soaked in deionized water for 24 h to remove superfluous cations. Characterization. The rheological tests were performed using a rheometer (Physica MCR 301) that operated in 25 mm parallel-plate configuration and 1 mm gap distance. The frequency sweep was conducted between 0.01 to 100 rad/s at a strain rate of 0.1%. The dynamic strain sweep was measured at a frequency of 6.28 rad/s. During all rheological measurements, a thin layer of low-viscosity silicone oil was applied to minimize water evaporation. The uniaxial tensile test was performed on the rectangular-shape specimens (10 mm in width, 6 mm in depth, and 35 mm in length) using a universal mechanical tester (Zwell 005) equipped with a 100 N load cell at room temperature. The initial distance (L0) between two clamps was 20 mm, and the stretching rate was set at 40 mm/min, which corresponded to the strain rate of 0.033 s−1. The fracture stress was calculated from the load divided by the cross-section area before deformation, and fracture strain was estimated from the clamp displacement divided by L0. The Young’s modulus was calculated by fitting the initial slope of the stress−strain curves within a strain range of 10−50%, and toughness was estimated by the area under the stress−strain curves until fracture point (the equation used in calculation toughness (T) was as below T = ∫ λλf0σ(λ)dλ, where λ0 and λf corresponded to the initial stretch and fracture stretch, respectively). For cyclic loading−unloading tests, the specimen was initially stretched to a predetermined strain and then unloaded at the same velocity (0.033 s−1). After each cycle, the samples were relaxed at room temperature for a certain waiting time (10−60 min) before next loading process, during which the specimens were coated with a silicon oil surface to minimize water evaporation. The energy dissipation was calculated by the area between loading−unloading curves. The residual strain ratio was indexed by a ratio of length change along the direction of stress after removal of stress with respect to the original length of the specimen. For notch-sensitivity test, a notch with 2 mm was imparted along the width direction of the specimens. To ensure repeatability, six specimens were measured for each sample and the average values were present. For metal content examination, the swollen ionic gels were thoroughly rinsed with deionized water for several times and freezedried, then analyzed using inductively coupled plasma mass spectrometry (ICP-MS, Elan DRCII). The in situ Raman spectroscopy was performed on the stretched hydrogels to examine stress transfer using a confocal laser Raman microscope (HORIBA Jobin Yvon) with a 785 nm near-infrared laser. The laser was focused at a fixed position with ∼2 μm spot, and the hydrogel strain rate was set at 0.017 mm/ min. UV−vis absorption spectra were recorded using a Cary 5000 spectrophotometer. The fractured hydrogel samples were freeze-dried and coated with gold for scanning electron microscopy (SEM, Hitachi S-3500) that operated at an accelerating voltage of 6 kV. For transmission electron microscopy (TEM), the samples were cryomicrotomed (Leica EM UC6) to collect the thin section (∼100 nm). The sections were stained with uranyl acetate solution (1.5 wt %) for 10 s, and applied for observation at an accelerating voltage of 100 kV (Hitachi H7600). Nanoindentation was conducted using a Hysitron Triboscope nanomechanical testing system. Indents were made by a three-side

EXPERIMENTAL SECTION

Materials. CaCl2 (dihydrate), ZnCl2 (anhydrous), AlCl3 (heptahydrate), and CeCl3 (heptahydrate) were purchased from Acros and used as received. 2-Hydroxy-4-(2-hydroxyethoxy)-2-methypropiophenone (Irgacure 2959) was received from Sigma. The cellulose nanofibrils were prepared from bleached softwood Kraft pulp using the TEMPO oxidation and sodium hypochlorite as the terminal oxidant. The concentration and surface carboxylate content of CNF in this work was 1.2 wt % and 1.47 mmol/g, respectively. The CNFs were 20−50 nm in diameter with aspect ratio of 60−100 based on TEM images (Figure S1). All other chemicals purchased were analytical grade unless otherwise noted and were used as received. B

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Figure 1. (a) Schematic illustration of ionic hydrogel synthetic process that includes the in situ polymerization to form composite hydrogels and then immersion in cation solution to produce ionic coordination. (b) Ionic coordination leads to CNF aggregation in ionic gels with increased opacity by visual observation and (c) corresponding absorbance in UV/vis spectra. pyramidal diamond indenter (Berkovich tip) with nominal radius of curvature of 100 nm, and the force resolution and displacement resolution were 1 mN and 0.1 nm, respectively. The hardness and the elastic modulus were obtained from the force−displacement curves. A typical indentation process contains four segments: approaching the sample surface at a constant strain rate (0.02 s−1), loading until the peak load, holding the peak for 10 s to minimize the influence of polymer creep on the unloading curves that used to calculate the elastic modulus, and finally unloading the indenter completely. A minimum of 20 indentations (100 μm between indentations to avoid interference) were performed for each specimen with a loading rate of 0.2 mN/s.

and high fatigue resistance that are superior to the counterpart composite gels without the ions. Since there is no structural change to CNF chains themselves by incorporating metal ions, the greater mechanical properties of the ionic gels should ascribe to the formation of more substantial interfibril attractive interactions.28 After immersing the CNF-PAAm composite gels into multivalent cations solution for 2 h, the ionic coordination occurred via supramolecular complexation between metal and carboxylate, where the translucent composite gels turned into opaque ionic gels along with the formation of ionically mediated CNF network (Figure 1b). As depicted in Figure 1a, the immersion of composite gels in cation solution leads to the screening of interfibril repulsive forces, which contribute to the formation of intermolecular domains and serve as junctions for chain association among CNF networks. For example, UV spectroscopy shows that the absorbance of PAAm-CNF-0.5 is around 0.12 at 500 nm (Figure 1c), while the PAAm-CNF-0.5Al3+ displays the higher absorbance of 1.1, implying the coalescence of CNFs. Notably, the CNF associating domains dictated by interfibril cation-carboxylate interaction play a vital role in network configuring of the ionic gels (polarized optical microscopy in Figure S2). Compared with the composite gels, where CNFs exhibit a random orientation of nanofibrils on a length scale of hundreds of nanomers (Figure 2a,b), the metal-mediated interconnected nanofibrils in ionic gels form highly porous networks with thicker junctions of overlapped strands (Figure 2b,c), implying the formation of more substantial interfibril cohesive adhesion that would contribute to viscous energy dissipation. Indeed, these well-ordered CNF arrays in TEM images (inset in Figure 2d) corroborate the existence of dominate intermolecular aggregation and chain association in parallel arrangement. The driving force of the fibril adhesion is primarily ascribed to the screening of interfibril “double-layer” repulsive forces that generated by carboxylate charges on the TEMPO-oxidized CNF surfaces,21,22 and enable CNFs to associate together through dominated cohesive interactions and van der Waals forces. Besides, the hydrogen bonding from the large number of hydroxyl and carboxylate groups in knitting bundles of cellulose chains within the fibrils is also expected to prevail, particularly at relatively low pH (weak acid of cation

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RESULTS As shown in Figure 1a, all reactants of acrylamide (AM, monomer), Irgacure 2959 (UV initiator), N,N′-methylenebis(acrylamide) (MBA, chemical cross-links) and CNF were added into a single water pot for the preparation of composite gels. The above mixture was sonicated in an ice−water bath and stirred homogeneously, then subjected to UV radiation for 60 min to initiate free-radical polymerization. Subsequently, the obtained composite gels were immersed into 0.1 mol/L aqueous solution of metal chloride (using CaCl2, ZnCl2, AlCl3, and CeCl3 as typical examples) to form ionically crosslinked CNF domains that utilized the binding affinity between metal cations and carboxylate groups on CNF surface. Finally, the obtained ionic gels were thoroughly rinsed with water to remove unbounded metal ions and denoted as PAAm-CNF-xMn+, where M and n referred to the cation and its valence, respectively. This metal-induced cross-linking procedure by strong metal-carboxylate bonding is straightforward, simply requiring immersion in metal ion solution at room temperature, leading to the formation of interwoven fibril networks. The distinctive benefits of this strategy include: (1) a simple one-pot process to produce the ionic network; (2) facile adjustment of the viscoelastic properties of the network by controlling cation ion valence and concentration; (3) reversible association of ions that contributes to efficient energy dissipation and high toughness of the ionic gels; and (4) dynamic rearrangement of CNF ionic associated network enables fast self-recovery and significant fatigue resistance property. These ionic gels display excellent fracture strength (1.1 MPa) and toughness (386 kJ/ m3), fast self-recovery (∼97% recovery ratio within 60 min), C

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with the increase in frequency and become significantly higher than the composite gels, which is ascribed to the relaxation of the coordination bonds and their contribution to the materials stiffness. In particular, both G′ and G″ increase with an increasing cation charge number in the order of Zn2+ < Ca2+ < Al3+ < Ce3+ (Figure S3), which may be justified that the increase in cation charge number leads to the stronger screening of electrostatic repulsion between CNFs and favors nanofibrils association thereby. Besides, the moduli are also affected by the cation radii, where the increase in ionic radii of divalent cations leads to an increase in moduli (Zn2+ (74 pm) < Ca2+ (99 pm)). This result can be rationalized by the Pearson’s hard and soft acids and bases (HSAB) theory.29 Furthermore, there appears a notable hysteresis for ionic gels in shear rheology by continuous dynamic frequency sweep experiment (Figure 3b), suggesting the CNFs can not recover their initial conformation after shear stress. This result reveals that some internal stress is produced during the formation of the percolated CNF ionic network and the complex dynamics of the network may arise from some unbinding of the transient ionic cross-links between distinct associating bundles (resting time-dependent frequency sweep in Figure S4). Indeed, this shift in moduli recorded for CNF ionic gels indicates that for the gels experienced longer resting time, more stored stress is relaxed. For strain sweep (Figure 3(c)), both G′ and G″ of the composite gels exhibit a slight decrease with increasing strain, which may relate to the disruption of hydrogen bonds at CNF interface under the high shear strain. Whereas for the ionic gels, the moduli display a more significant reduction with increased strain, indicating the disassociation of ionic cross-links. Next, we turn to the mechanical properties. Our intention is to modulate macroscopic mechanical properties of hydrogels by the reversible mechanism in ionic coordination associated CNF motifs. As shown in Figure 4a, the pristine PAAm shows comparably low stiffness and a rather ductile behavior with fracture strain around 350%. The composite gels take an intermediate position and exhibit higher mechanical properties than that of pristine PAAm gels. This improvement in properties is ascribed to the hydrogen bonds between PAAm and CNFs.18 While for the ionic gels, the reversible CNF ionic networks combined with PAAm permanent covalent bonds enable the ionic gels with superior mechanical performance. For example, PAAm-CNF-0.5-Ce3+ exhibited 4.1× and 8.5× higher in fracture strength and fracture energy than that of composite gels (the corresponding physical properties are summarized in Table S1) and displayed a pronounced yielding with an even longer inelastic plateau. This result favorably contrasts classical covalent cross-linked elastomers (i.e., hydrogels), for which an increase in stiffness always results in decreased stretchability and toughness.30,31 In the case of ionic gels, the inelastic deformation is coupled to the frictional sliding of the CNF against each other (Cryo-TEM below). Hence, the high stress level of the inelastic deformation region upon incorporation of CNF is strongly indicative of a dynamic ionic cross-linking scenario with stress-induced reversible supramolecular bonds. In fact, the contribution of ionic network can

Figure 2. TEM images of composite gels (a, b for PAAm-CNF-0.5) and ionic gels (c, d for PAAm-CNF-0.5-Al3+).

complex in water), where the carboxylate groups can be protonated to allow interfibril hydrogen bonding association. Before examining the effect of valence of cation on dynamic mechanical properties, the content of cation in the ionic gels was detected using ICP-MS. The initial sodium content of CNF owning to TEMPO oxidation was 1.42 mmol/g, which is consistent with a carboxylate content of 1.47 mmol/g. After immersing in metal solution and rinsing with water, almost no sodium was detected in the ionic gels, suggesting the thorough ion exchange of sodium with multivalent cations. According to ICP measurement, the multivalent cations basically meet the stoichiometric ratio of metal to carboxylate required by charge neutrality, where the molar amount of divalent cations (Ca2+, Zn2+) and trivalent cations (Al3+, Ce3+) is almost half and onethird to that of initial sodium content, respectively (Table 1). The divalent and trivalent cations possess higher binding energy and displaced solvation volume than monovalent cations do, and thus facilitate the cross-linking of intrafibril between multiple fibrils and improvement of mechanical properties.22 Consequently, one can expect that the cation-mediated crosslinks would lead to strong interactions with multiple carboxylate groups and enable dominating attractive forces in bridged CNFs by screening of electrostatic repulsion between neighboring nanofibrils. The small amplitude oscillatory shear rheology tests were performed by frequency sweep and strain sweep to gain insights into the structural arrangement (as the covalent cross-linking density in PAAm network is low, which may allow the CNFs to rearrange under shear). In Figure 3a, the storage modulus (G′) and loss modulus (G″) of the composite gels exhibit almost frequent independency over the whole frequency range, implying typical elastic character dominates the covalent network. In contrast, both G′ and G″ of the ionic gels increase Table 1. Metal Content in Ionic Hydrogels cation content (mmol/g) a

Na+a 1.42 ± 0.06

Ca2+ 0.65 ± 0.04

Zn2+ 0.68 ± 0.03

Al3+ 0.43 ± 0.02

Ce3+ 0.41 ± 0.02

Na+ content in composite gels before ion exchange D

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Figure 3. Dynamic rheological behaviors of hydrogels. (a) Dynamic frequency sweep (0.0158−100 rad/s, 1% strain), (b) hysteresis in repeated frequency sweep (color symbols and gray symbols correspond to first and the second consecutive dynamic frequency sweeps, respectively), and (c) strain sweep (0.01−100% strain, 1 rad/s; G′ solid, G″ open, red for PAAm-CNF-0.5, blue for PAAm-CNF-0.5-Zn2+, and green for PAAm-CNF-0.5Al3+).

Figure 4. Mechanical properties of hydrogels at room temperature: (a) tensile curves, (b) loading−unloading tests of hydrogels, (c) cyclic tensile loading−unloading curves with different testing times between two successive measurements, and (d) time-dependent recovery ratio (green columns) and residual strain (pink columns) of PAAm-CNF-0.5%-Al3+.

also be verified by addition of a stronger ligand (ethylenediaminetetraacetate, EDTA) to form chelating ligand with metal than the carboxylate groups, and cause the dissociation of CNF ionic network (Figure S5). The result indicates that the fracture strength of the ionic gels after EDTA treatment is almost the same as the corresponding composite gels, verifying the dissociation of interfibril attractive interactions.32 The cyclic loading−unloading tests were conducted to evaluate the energy dissipation of the ionic gels (Figure 4b). Compared with the covalent PAAm with elastic dominated properties, the composite gels dissipate a large amount of energy in cyclic test and the area of loop (energy dissipation per unit volume) increases with strain (Figure S6), implying the contribution of interfacial hydrogen to the energy dissipation. On the other hand, the ionic gels displayed even more significant energy dissipation as high as 386 kJ/m3 (PAAmCNF-0.5-Ce3+), which was 168× and 8.5× higher than the pristine PAAm gels and composite gels, respectively. This notable hysteresis loop stems from CNF dissociation induced rearrangement, involving intrachain and interchain reversible association−dissociation of ionic-bridged CNFs motifs upon stretching. Therefore, this mechanism enables an efficient stress transfer and energy dissipation process, contributing more significant viscous dissipation and shock absorbing capacity

than the dissociation of interfacial hydrogen bonds in the composite gels. In addition to the improvement in mechanical strength and toughness, another advantageous feature of ionic interaction is its ability to undergo repeated association to endow excellent self-recovery ability. For example, the recovery behavior of the ionic gels at a strain of 800% with different interval time between successive loading−unloading tests is shown in Figure 4c. Here we use recovery ratio (Wt/W0, W0 and Wt refers to the work in the initial cycle and after t time rest, respectively) to calculate recovery efficiency (Figure 4d), and the result indicates that the recovery ratio of PAAm-CNF-0.5-Al3+ exceeds 82% and 97% after 30 and 60 min, respectively. Besides, the residual strain gradually decreases with extension of resting time, and it is less than 6% after 60 min resting time. While it should be noted that the recovery ratio does not improve and leaves a small residual strain with waiting time after 4 h, implying the existing of some unrecoverable associations among CNFs. The nanoindentation technique was also employed to examine the resistance against penetration and creep displacement of the ionic gels that associated with different valence of metal ions (see Figure S7 for nanoindentation protocol). The averaged elastic modulus (E) and hardness (H, resistance to E

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Figure 5. Nanoindentation measurement of hydrogels: (a) elastic modulus, (b) hardness, and (d) plasticity index curves with respect to displacement into sample surfaces; (c) force−displacement curves of indentations made at a peak indentation load of 120 mN (black for pristine PAAm, red for PAAm-CNF-0.5, blue for PAAm-CNF-0.5-Zn2+, pink for PAAm-CNF-0.5-Ca2+, green for PAAm-CNF-0.5-Al3+, and wine for PAAmCNF-0.5-Ce3+).

Figure 6. Fatigue resistance of PAAm-CNF-0.5%-Al3+ gels by eight successive loading−unloading cycles of the (a) as-prepared samples and (b) recovered samples at room temperature. (c) The notch insensitivity of the ionic gels (solid and dash curves correspond to the normal and notched gels, respectively).

penetration) of the gels (Figure 5a,b) determined by the Oliver-Pharr model33 from the load-penetration curves between 300 and 500 nm are summarized in Table S2. The results show that at small penetration depths up to ∼200 nm, both E and H show notable deviations (indentation size effect), which may be ascribed to the specimen surface roughness and the interaction between the tip and sample. After indentation depth approaching 250 nm and onward, the curves exhibit stable trend for all the specimens, implying the CNFs are well distributed in the matrix along the indentation direction. Another important trend noted from the indentation measurement is that both E and H of the ionic gels display the constant increase with cation radii compared with composite gels, which is in agreement with the macromechanical test. Thus, this result further verifies that more energy is required to reduce entropy and raise enthalpy when the high valence coordinated gel network is deformed. The indenter tip force−displacement profiles with three typical regions are exhibited in Figure 5c. For all the specimens, no discontinuities are noted in the curves, suggesting no crack occurs during indentation test. During the initial loading segment, the force is increased at a constant velocity (0.5 mN/ S) and the curves steadily shift upward, where the ionic gels

coordinated with the higher cation valence exhibit a shallower depth of penetration into the gels due to an increase in hardness. By holding at a maximum load in the force− displacement curves for 25 s (time-dependent creep deformation in Figure S8), the gel viscoelasticity induced creep behavior appears, and the creep depth and displacement at maximum holding segments are inversion to the valence of cation. For example, the PAAm-CNF-0.5-Zn2+ gels showed a maximum displacement of ∼940 nm with permanent deformation at 420 nm, whereas PAAm-CNF-0.5-Al3+ with higher valence metal exhibited a lower permanent deformation (∼236 nm), suggesting the dynamic ionic cross-linking of CNFs motifs possesses a critical effect on the viscoelastic properties of the networks and dominates the creep resistance of the gels. From the nanoindentation testing, the plasticity index (χ), a parameter to define the relative plastic/elastic property of an elastomer at subject to external stress, was also calculated (Figure 5d). The result indicates that the ionic gels shows more significant viscoelastic properties with a higher plasticity index than the composite gels, that is, more energy is dissipated through plastic deformation (ductility) compared to the total energy dissipated. F

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Figure 7. Cryo-TEM images showing the morphological evolution of ionic gels (PAAm-CNF-0.5%-Al3+) under different strain ratios (see Supporting Information for sample preparation protocol). The initial ionic coordination associated CNF motifs are stretched into smaller sized domains (a, b), then disrupted into isolated fibrils with further increasing strain (c). Finally, the curled CNFs rearrange along the direction of the stress (represent by green arrows) to relax stress via ionic coordination that act as sacrificial bonds (d, e), yielding efficient energy dissipation.

Figure 8. SEM images of fracture morphology of PAAm-CNF-0.5-Al3+. The crazes with porous structures develop perpendicularly to the stretching direction (noted by arrows), and some pullout fibrils are noted with curled feature.

transferred to neighboring process zone (Figure 7). On the other side, the PAAm covalent cross-links bridge the network and stabilize crack propagation, leading to simultaneous toughening and stiffening mechanisms with increased crack resistance. Driven by this notch-insensitivity behavior, we examine the ionic gels toughening mechanisms by fracture morphology. There exists a broad range of failure crack in the process zone of ionic gels (Figure 8a), and some pullout CNFs can be found between neighboring polymer matrix which contribute to inelastic fracture energy dissipation due to the relatively large scale movement in the materials (Figure 8b). Besides, a large number of fibrils with slight curled-edge morphology are observed on the fracture surface of CNF-PAAm-0.5-Al3+ gels, reminiscent of many loading bearing biological composites with efficient energy dissipation mechanism such as bone and nacre.34 One can also note that these thick fibril bundles range from 50 to 100 nm, which is larger than the diameter of fibrils in individual ones, implying the bridged nanofibrils are strongly interacting by ionic bonds. Moreover, the crack path displays some crack branching and crack twisting phenomena (Figure 8c), where constrained microcrack occurs near to the major crack path. Together, this deflected crack propagation mechanism enables sufficient ability to delocalize stress concentration from crack tip due to the dynamic ionic association, yielding an increased resistance against stress slide and high shear modulus.

Furthermore, fatigue resistance of the ionic gels was examined by eight continuous loading−unloading tests without interval between each cycle. As illustrated in Figure 6a, except for the initial three cycles showing large hysteresis, the following cycles exhibit softening phenomenon respect to the first loading curve with almost closed loops and the stress− strain profiles almost overlapped, implying the fracture of CNF bridge cannot recover without resting. Another set of eight successive cycling test was conducted on the recovered gels after 24 h resting time at room temperature, and the result demonstrated that the fracture strength was even slightly higher than the initial value rather than getting worse (Figure 6b). This similar “self-stiffening phenomenon”34 in response to external mechanical stimuli may relate to the increase in physical cross-linking density that originates from the realignment of CNF chains after cyclic loading. Intriguingly, it is found that the ionic gels are remarkable in terms of fracture process with crack nucleation at the edge of the specimen and stable crack propagation during uniaxial deformation (Figure S9). Compared with the elongation of notched PAAm-CNF-0.5 gels decreases from 795% to 412% (Figure 6c), the PAAm-CNF-0.5-Zn2+ displays notch-insensitivity and the strain of notched samples still reaches 904%, slightly lower than its original elongation (983%). We correlate this result to the synergetic interactions between CNF ionic association and PAAm covalent network. Under stretching process, the dissociation of the ionic cross-links can effectively dissipate energy around the bridged fibrils, where stress is G

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Figure 9. (a) Raman shift toward a lower wavenumber that initially located at ∼1095 cm−1 upon axial tension for PAAm-CNF-0.5-Zn2+. (b) Shifts in the peak position of the Raman band at ∼1095 cm−1 with respect to strain for PAAm-CNF-0.5 (red), PAAm-CNF-0.5-Zn2+ (blue), and PAAmCNF-0.5-Al3+ (green).

increases from 0.05 to 0.2 M at the second immersion step (Figure 10a). Since the bundles of fibrils are parallel aligned

To better understanding the role of cation mediated CNF in fundamental stress transfer process, in situ Raman spectroscopy measurement during tensile loading of the ionic gels was performed. It has been suggested that the C−O stretching band shift in Raman spectrum represents direct loading deformation along the cellulose backbone.35 In other words, the down shift of glycosidic peak position at 1095 cm−1 under tensile strain model indicates the efficient load transfer. The evident downshift and peak broadening of the cellulose band with increased strain corroborate the molecular deformation of cellulose backbones and stress transfer from the matrix to the reinforcing phase (Figure 9a). Moreover, the shift gradient for trivalent cation (−1.45 cm−1/%, Figure 9b) coordinated gels is found to be higher than those divalent gels (−0.87 cm−1/%), which is consistent with the higher valence of cations lead to the greater stress transfer efficiency. For current CNF ionic cross-linking involved hybrid hydrogels, the improvement in mechanical properties is primarily attributed to the formation of cation mediated CNF ionic association with increased cross-linking density as compared with the composite gels. On the one hand, the crystalline CNFs in the ionically cross-linked domains act as load-bearings that effectively transfer stress and sustain deformation. On the other hand, the deassociation of the coordinates between the bridged cellulose chains act as sacrificial bonds to dissipate energy across the percolated fibril network. In this regard, the introduction of ionic cross-links makes the ionic gels stiffer than the composite gels, and the bridged fibrils with denser packed nanofibrillar networks promote CNF slide efficiently during tensile loading. Since the dynamic CNF network is able to dissociate and reassociate under stress to dissipate energy, the denser cross-linking structures alleviate stress concentration and resist crack propagation, resulting in dissociation and reassociation of dynamic CNF network under deformation. In fact, from the observation that the amount of CNF pull-out of the fractured gels increases with valence of cations (Figure S10), we can conjecture that more CNFs are involved in energy dissipation and the higher driving force is required for crack deflection. Taken together, the above microscopic features constrain the crack propagation and are favorable for efficient stress transfer with extremely high toughness.36−38 Except the preparation parameters of the cation valence, the mechanical properties of ionic gels can also be facilely adjusted by tailoring the cation concentration. Taking the Al3+ mediated ionic gel as an example (PAAm-CNF-0.5-Al3+), the fracture strength and Young’s modulus increase from 0.32 to 1.39 MPa and 52.1 to 435.5 kPa, respectively, with Al3+ concentration

Figure 10. (a) Tensile stress−strain curves of ionic gels prepared at different Al3+ concentrations from 0.05 to 0.2 mol/L. (b) TEM images shows the denser network structures with increasing of cation concentration.

with helical twists and interconnect via primary hydrogen bonding in CNF, the metal-carboxylate interactions are expected to form on surface of the same fibrils or adjacent fibrils, providing a dominate cohesive force in fibrous network. For the higher concentration of cations, as the interfibril distance is reduced during gelation, they possess a higher tendency to bridge fibrils and form a complex to enable fibrils to come together, considering the stronger electrostatic attraction.21 In this respect, it is reasonable to find that the higher concentration of cations tend to produce ionic gels with the higher mechanical stability as well as the denser gel structure (Figure 10b) due to the fact that they have a higher tendency than diluted cations to cross-link between adjacent fibrils. Thus, the increase of cation concentration allows increased ionic cross-linking degree with improved resistance against plastic deformation and yield stress, providing a facile way to tailor mechanical properties along with significant viscoelastic properties for potential tissue engineering applications.

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DISCUSSION In this work, we demonstrate the concept by engineering reversible cation cross-linked CNF motifs into a covalently H

DOI: 10.1021/acs.biomac.6b01915 Biomacromolecules XXXX, XXX, XXX−XXX

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CONCLUSIONS We have reported a facile and generalizable methodology for preparation of CNF reinforced hydrogels by tandem covalent cross-linking of PAAm and secondarily formed physical crosslinking ionic CNF domains in metal ion aqueous solution with varying charge numbers and ionic radii. The cation mediated ionic cross-linking reduces electrostatic repulsion between carboxylate groups and favors the CNF association into an interconnected network. The rheological measurements indicate that the transient bonds contribute to the improved material stiffness at small strain and dissociate at high strain. The attained ionic gels exhibit superior mechanical properties with excellent tensile strength and toughness in the order of Zn2+ < Ca2+ < Al3+ < Ce3+. Stable crack propagation and pullout toughening mechanisms occur in a balanced and dynamic manner, enabling synergistic combination of stiffness and toughness. The microscopic deformation behavior of the gels by performing Raman spectroscopy verifies the stress transfer medium role of CNF motifs. Besides, the creep behavior from nanoindentation measurements suggest the viscoelastic behavior of the ionic gels with a significant increase in hardness, elastic modulus, and plastic index. It is expected that the postformation of CNF network is applicable to other covalent polymer networks (such as poly(dimethylacrylamide), poly(acrylic acid), and poly(ethylene glycol)). Looking ahead, we expect that combining the biocompatibility and biodegradability of CNFs, this straightforward ionic cross-linking strategy for CNF based hydrogels may enrich exploration in biomedical field after loading with other functional molecules (e.g., drugs, catalysts, and fluorescent labels).

cross-linked polyacrylamide network. The metal−ligand bonds between metal cations and pendent carboxylate groups on CNF surface can cause reversible association and dissociation, i.e., act as sacrificial bonds under external stress to dissipate energy and facilitate chain orientation. Several multivalent cation metals (Ca2+, Zn2+, Al3+, and Ce3+) were chosen to serve as typical metal ion to form metal−carboxylate complexes and discuss the gels’ mechanics and crack propagation mechanism at the microscale combined with morphology characterization. Consistent with previous reports,39 the incorporation of multivalent cation leads to the reduction of Debye length of CNF and suppression of electrostatic repulsion between them, yielding dominant attractive interactions and gelation thereby (Figure S11). The results show that for a particular CNF concentration, the hydrogel strength increases with increasing charge number of the added cations and a decrease in network mesh size. Stiffening versus Toughening. It is extremely important, but generally challenging, to simultaneously improve the stiffness and toughness of polymeric materials.14 While this inverse relation does not hold true for the resulting ionic gels, and this feature is attributed to the distinct toughening mechanism. For these gels, the elastic architecture is maintained by both covalent cross-links and cation−carboxylate coordination bonds. The covalent network allows elasticity and maintains network stability. The ionic coordination bonds serve as dynamic cross-links in sacrificial way by providing a reversible structure for nanofibrils transition between association and dissociation. As exhibited in Figure 4b, the ionic gels show large hysteresis loops in loading−unloading cycles and the area of the loop increases as strain, implying an efficient energy dissipation process. In light of this result, the incorporation of sacrificial bonds into polymer matrix provides a broad opportunity to the attainment trade-off among the strength, modulus, and stretchability. Micromorphological Mechanisms. It has been proposed that the most effective ways of improving CNF-reinforced hydrogels include dispersing the bundled fibrils in the matrix and cross-linking these fragments via transient or covalent treatment to enhance rigidity.19 The images in Figure 2 demonstrate homogeneously dispersed nanopores with size of approximately 30 nm that are composed of closely packed nanofibrils, featuring the cation mediated self-assembly of CNFs. The curving and stretching morphologies in Figure 8 also emphasize the significance of flexibility of the fibrils and their ability to damp the applied stress. Collectively, this arrangement might be of importance in improving the toughness of the gels because the CNFs could bridge along the uniaxial direction without failing, yielding increased toughness by pull-out and crack bridging. The fibril “pull-out” from associated bundles involves continuous consumption of energy in overcoming the van der Waals attraction and hydrogen bonds between adjacent fibrils.18 In fact, this modular elongation process is similar to the events that occur in natural composites such as nacre where the adhesive polymer chains elongate continuously, resulting in energy absorption and increased fracture toughness.40 Since the microstructure of the CNFs is easily bridged and twisted that can be modulated in a continuous manner, this modulation would allow the release stress in the gels without breaking and provide toughness to the ionic gels accordingly.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b01915. Mechanical properties summary, CNC morphology, rheology, cyclic loading−unloading, digital picture, and SEM observation of hydrogels, and illustration of nanoindentation measurement (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 86-10-62337223. ORCID

Jun Yang: 0000-0002-1633-4465 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by Fundamental Research Funds for the Central Universities (2015ZCQ-CL-03), and National Natural Science Foundation of China (21404011, 21674013).

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