Clay Nanocomposite Hydrogels with Macroporous ... - ACS Publications

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Novel Biocompatible Thermoresponsive Poly(N‑vinyl Caprolactam)/ Clay Nanocomposite Hydrogels with Macroporous Structure and Improved Mechanical Characteristics Kun Shi,† Zhuang Liu,*,† Chao Yang,† Xiao-Ying Li,† Yi-Min Sun,§ Yi Deng,† Wei Wang,†,‡ Xiao-Jie Ju,†,‡ Rui Xie,†,‡ and Liang-Yin Chu*,†,‡ †

School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, P. R. China State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, Sichuan 610041, P.R. China ‡ State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan 610065, P. R. China §

S Supporting Information *

ABSTRACT: Poly(N-vinyl caprolactam) (PVCL) hydrogels usually suffer from the imporous structure and poor mechanical characteristics as well as the toxicity of cross-linkers, although PVCL itself is biocompatible. In this paper, novel biocompatible thermoresponsive poly(N-vinyl caprolactam)/clay nanocomposite (PVCL-Clay) hydrogels with macroporous structure and improved mechanical characteristics are developed for the first time. The macroporosity in the hydrogel is introduced by using Pickering emulsions as templates, which contain N-vinyl caprolactam (VCL) monomer as dispersed phase and clay sheets as stabilizers at the interface. After polymerization, macropores are formed inside the hydrogels with the residual unreacted VCL droplets as templates. The three-dimensional PVCL polymer networks are cross-linked by the clay nanosheets. Due to the nanocomposite structure, the hydrogel exhibits better mechanical characteristics in comparison to the conventional PVCL hydrogels cross-linked by N,N′-methylene diacrylamide (BIS). The prepared PVCL-Clay hydrogel possesses remarkable temperature-responsive characteristics with a volume phase transition temperature (VPTT) around 35 °C, and provides a feasible platform for cell culture. With macroporous structure and good mechanical characteristics as well as flexible assembly performance, the proposed biocompatible thermoresponsive PVCL-Clay nanocomposite hydrogels are ideal material candidates for biomedical, analytical, and other applications such as entrapment of enzymes, cell culture, tissue engineering, and affinity and displacement chromatography. KEYWORDS: hydrogels, stimuli-responsive materials, composite materials, porous materials, mechanical characteristics

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INTRODUCTION

hydrogels are used for medical and biorelated applications such as entrapment of cell or enzymes, tissue engineering, and so on, the mechanical characteristics, biocompatibility, and macroporosity are important factors.21−27 For instance, the macrospore structure enables to provide spaces for cell culture,21−23 and the good mechanical characteristics would make the PVCL hydrogels strong enough for artificial cartilage and scaffolds of tissue engineering.24−27 Therefore, development of PVCL hydrogels with good mechanical characteristics, biocompatibility and macroporosity is of great scientific and technological significance. To date, conventional PVCL hydrogels are normally crosslinked by small chemical molecules such as methylene bis(acrylamide) (BIS), and the polymerization usually occurs in ethyl alcohol, alcohol/water mixed solution or other organic solvent due to the poor solubility of monomer N-vinyl caprolactam (VCL) in aqueous solution.16,28−32 For example,

Thermoresponsive hydrogels can exhibit significant variation on their volume or other properties responding to external temperature,1−3 and thus they show remarkable potentials for versatile applications, such as optical systems,4 “on/off” switches for chemical reactions,5 artificial muscles or smart manipulators,6,7 drug delivery vehicles,8,9 chemical-/bioseparation platforms, and scaffolds for tissue engineering.9−11 Such smart hydrogel is a tridimensional network consisting of cross-linked polymer chains with temperature-dependent hydrophilicity; for instance, poly(N-isopropylacrylamide) (PNIPAM),12 poly(Nvinyl caprolactam) (PVCL),13 and poly(2-oxazoline)s.14 Other than the popular thermoresponsive polymer PNIPAM, PVCL has also attracted much attention because of its low biotoxicity15,16 and a dramatic phase transition in water at the lower critical solution temperature (LCST) about 33 °C.13,17−20 It has been reported that PVCL is biocompatible, which is a fact that has not been thoroughly verified for PNIPAM.13 Thus, compared with PNIPAM, PVCL is more promising for biomedical and bioanalytical applications.13 When PVCL © 2017 American Chemical Society

Received: March 30, 2017 Accepted: June 12, 2017 Published: June 12, 2017 21979

DOI: 10.1021/acsami.7b04552 ACS Appl. Mater. Interfaces 2017, 9, 21979−21990

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ACS Applied Materials & Interfaces

which was bought from Rockwood, was dried for 4 h at 150 °C before being used in the following experiments. N,N′-Methylene diacrylamide (BIS), ethanol and 4% (w/v) paraformaldehyde were all bought from Chengdu Kelong. Pluronic F-127 (Sigma-Aldrich) was used as surfactant in water phase. Lumogen red 300 (LR300) was used to color the VCL liquid. Streptomycin and penicillin (HyClone), fetal bovine serum (Gibco), the cell counting kit-8 (CCK-8, Dojindo), Dulbecco’s modified Eagle’s medium (DMEM, HyClone), Triton X100 (Sigma-Aldrich) and FITC phalloidin (Solarbio) were used in the experiments for cell cultivation and characterization. Pure water used throughout the experiments was produced from a water purifier (MilliQ Plus, Millipore). Preparation of Hydrogels. PVCL-Clay nanocomposite hydrogels were prepared with VCL as monomer, clay nanosheets as cross-linking agents, and Irg.184 as initiator by UV polymerization. Typically, to prepare uniform aqueous solution of clay, clay nanosheets (0.15 g) were dispersed in pure water (4 mL) with ice-bath and violent stir for 4 h. Next, 2.5 g of VCL was melted and dispersed in the clay solution by stirring the mixture at 40 °C for 30 s. The mixed solution was treated by ultrasonic (100 W) at room temperature for 20 min to form Pickering emulsions. Subsequently, Irg.184 (2 mg) that dissolved in 100 μL of ethyl alcohol in advance was added into the mixture. The reactant solution was transferred into a mold made up of a PTFE gasket with thickness of 2 mm, a polytetrafluoroethylene (PTFE) sheet, and a transparent quartz sheet with side length of 50 mm. The polymerization reaction was conducted under the irradiation of UV light (365 nm, 250 W) in a cold ice-bath for 5 min. The PVCL-Clay nanocomposite hydrogels were coded as “PVCLa-Clayb”, in which “a” represented the mass of VCL (g) and “b” indicated the mass of clay (g). For example, PVCL2.5-Clay0.15 meant that the PVCL-Clay nanocomposite hydrogel was prepared with 2.5 g of VCL and 0.15 g of clay in 4 mL of pure water. By replacing the clay with BIS (molar ratio of BIS to VCL was 2%) and F127 (1% w/v), the control sample of PVCL-BIS-F hydrogels were prepared as “PVCL2.5-BIS2%-F”. Another control PVCL hydrogel was prepared according to a reported method.28 VCL (2.5 g), BIS (molar ratio of BIS to VCL was 2%), and Irg.184 (2 mg) was dissolved in 4 mL of ethanol−water solution (1:1 of volume ratio) at room temperature. The resulting reaction liquid was then transferred into the PTFE mold and polymerized under the UV light in a cold ice-bath for 5 min. The conventional PVCL-BIS hydrogels were coded as “PVCL2.5-BIS2%”. Characterization of VCL-in-Water Emulsions and Hydrogels. The optical morphology of the VCL-in-water emulsions with LR300dyed VCL was observed by a laser confocal scanning microscopy (CLSM, TCS SP5-II, Leica). The reaction solutions before and after polymerization were observed by using the channel of red fluorescence. The microstructures of PVCL-Clay nanocomposite hydrogels, which were previously freeze-dried, were observed by a scanning electron microscopy (SEM, JSM-7500F, JEOL). The hydrogels were rapidly frozen under liquid nitrogen and freeze-dried with a freeze-dryer (FD1C-50, Beijing BoYiKang) at −48 °C for 48 h. To investigate the mass conversions of the VCL monomers and clay nanosheets to the polymer−clay cross-linked networks, the PVCL2.5Clay0.15 hydrogels were cleaned by fully swelling and shrinking in pure water repeatedly. The weights of freeze-dried PVCL2.5-Clay0.15 hydrogels were measured. Then, TGA (TG209F1, Netzsch) tests of the clay nanosheets and dried PVCL2.5-Clay0.15 hydrogels were conducted with the heating rate of 10 °C min−1 from 40 to 800 °C in nitrogen environment. Each mass conversion of monomers and clay nanosheets was calculated according to the dried hydrogel weight, feeding reactant weight and the TGA results. Moreover, the volume changes of the VCL droplets before and after the polymerization reaction were characterized using the CLSM. Typically, the reaction liquid of VCL2.5-Clay0.15 was first transferred into an ultrathin glass holder with space height of about 120 μm for easy observation. The cross-sections of a selected VCL droplet were then observed via layer-by-layer scanning technology using the CLSM. The volume changes of the VCL droplets were estimated according to the optical photographs and the scanning spacing.

Elenu et al.28 have prepared PVCL hydrogel using BIS as chemical cross-linker in alcohol/water mixed solution, and the prepared PVCL hydrogel exhibits good thermosensitivity. However, the hydrogels that chemically cross-linked by BIS molecules usually suffer from poor mechanical characteristics arising from the heterogeneous polymeric networks that resulted from the difference in reactivity between the monomer and the cross-linker.33 An number of cross-linked clusters are formed and the chain lengths between cross-linkers are highly variable. The short polymer chains are liable to fracture originating from the stress concentration.34 To improve the mechanical characteristics, researchers have prepared PVCL hydrogels using other cross-linkers such as ethylene glycol dimethacrylate (EGDMA) and poly(ethylene glycol) diacrylate (PEGDA) to create relatively homogeneous networks.35,36 However, both the organic chemical molecules are verified with notorious cytotoxicity.36,37 Instead of using organic chemical cross-linkers, Loos et al.31 have prepared hybrid PVCL hydrogels by in situ generating silica particles in the PVCL polymer solution. The silica particles that formed by sol−gel process act as physical cross-linkers for the PVCL polymers via the hydrogen bonds between the PVCL carbonyl functions and the remaining noncondensed silanol groups of the silica particles.31 However, this strategy for preparing PVCL hybrid hydrogels cannot induce macroporosity in the hydrogels, which may limit the application of silica-cross-linked PVCL hybrid hydrogels for cell proliferation.21−23 Therefore, in short, it still remains a challenge to fabricate PVCL hydrogels with improved mechanical characteristics, biocompatibility and macroporosity. Here, we report on a novel strategy to prepare a biocompatible thermoresponsive PVCL-Clay nanocomposite hydrogels with macroporous structure and improved mechanical characteristics. The proposed PVCL-Clay hydrogels are cross-linked by inorganic Laponite clay nanosheets to address the problem of cytotoxicity of small organic molecules. The safety of Laponite clay nanosheets has been confirmed by cytotoxicity test.38 The macroporosity in the PVCL-Clay hydrogel is induced by the residual unreacted VCL droplets. The VCL monomer is a liquid phase at 40 °C, and it is nearly insoluble in the clay-containing aqueous solution. The solubility of VCL monomer in water is only 4.1 mg/mL. After ultrasonic treatment, VCL-in-water Pickering emulsions are generated and stabilized by the clay nanosheets at the VCL/water interfaces.39,40 During the polymerization initiated by UV light, a part of VCL monomers convert to form the hydrogel networks. After polymerization, macropores are generated inside the hydrogels with the residual unreacted VCL droplets as templates. The prepared PVCL-Clay nanocomposite hydrogel networks consist of polymer chains that are quite homogeneous in length between the clay nanosheets, thus resulting in a dissipative architecture to distribute external force. Thus, the mechanical characteristics of the proposed PVCL-Clay nanocomposite hydrogels are improved compared with those reported PVCL hydrogels cross-linked by small chemical molecules.24 Such biocompatible thermoresponsive PVCL-Clay nanocomposite hydrogels with macroporous structure and improved mechanical characteristics are highly promising for many applications in biomedical fields.

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EXPERIMENTAL SECTION

Materials. N-Vinyl caprolactam (VCL) used without further purification was purchased from Sigma-Aldrich. 1-Hydroxy-cyclohexylphenylketone (Irg.184, TCI) was used as UV initiator. The hectorite “Laponite XLG” (Clay, [Mg5.34Li0.66Si8O20(OH)4]Na0.66), 21980


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Figure 1. Schematic diagram of the fabrication procedure of PVCL-Clay nanocomposite hydrogels. (a, d) Pickering emulsions of VCL monomer droplets dispersed in the Laponite clay aqueous solution with clay nanosheets stabilizing the interface, in which the initiator molecules are adsorbed on the clay surfaces (d). (b, e) Polymerized by UV-light irradiation, the three-dimensional networks are formed by cross-linking PVCL chains with clay nanosheets. (c, f) After washing, macropores are generated in the hydrogel with the residual unreacted VCL droplets as templates. The mechanical characteristics were first characterized by shearing the hydrogel samples with strongly rotating flows. The PVCL2.5Clay0.15, PVCL2.5-BIS2%, and PVCL2.5-BIS2%-F hydrogels with size of 1 cm × 1 cm were immersed in 3 mL of water in glass vials, which were stirred for 30 s using a mini-shaker (Lab Dancer, IKA). A digital camera was used to record this test procedure. Further, the mechanical characteristics of as-prepared PVCL-Clay nanocomposite hydrogels were also characterized quantitatively by using a tension tester (EZ-LX, Shimadzu) and a rheometer (TA-DHR-2). In the tensile tests, the asprepared hydrogels were cut into dumbbell-like sheets with the width and thickness of 2 mm and gauge length of 15 mm. All the samples were stretched at the speed of 100 mm/min at room temperature. The stretching stress of the hydrogel was defined as the ratio of tensile force to the area of real-time cross-section. The Young’s modulus (E) was defined as the slope of tensile stress−strain diagram at the strain of 100%. In the rheological tests, the as-prepared hydrogels with the thickness of 1 mm were cut into thin circular plates with the diameter of 25 mm. The distance between the two clamp plates were controlled at a constant distance of 900 μm during the tests. The rheological tests were performed over the frequency scope of 0.01 to 100 rad/s at constant shear strain (γ = 0.001). Assembly Performance of PVCL-Clay Nanocomposite Hydrogels. To show the assembly performance of the PVCL-Clay nanocomposite hydrogels, we stacked two subunits of PVCL2.5Clay0.15 hydrogels together, and then dried in the atmospheric environment with relative humidity of 70% at 25 °C for 36 h. After reswelling in deionized water at 25 °C, two subunits firmly connected together. SEM observation and manual stretching characterization of the bonded hydrogels were carried out subsequently. The assembling process was schematically illustrated in Figure S1. Thermoresponsive Property of Hydrogels. The thermoresponsive equilibrium volume change behaviors from 15 to 50 °C of the hydrogel sheets (1 cm × 1 cm) were recorded by a digital camera. Before each measurement, the hydrogel sheets had been kept at each predetermined temperature for 8 h in thermostatic water bath to ensure reaching the equilibrium state. The dynamic thermoresponsive shrinking behaviors of hydrogels were performed as follows. The full-swollen hydrogel sample with size of 1 cm × 1 cm was transferred into a sealed glass holder. The dynamic thermoresponsive shrinking behaviors of the hydrogel sheets were recorded by a digital camera. The temperature of the hydrogel samples was controlled by a temperature stage system (TS62, Instec) from 25 to 45 °C. Three measurements were conducted to obtain statistical data for each sample. Cell Cultivation. To demonstrate the biocompatibility of the PVCLClay nanocomposite hydrogels, we used PVCL2.5-Clay0.15 hydrogels

for cell cultivation. The hydrogels immersed in phosphate buffered saline (PBS) were first treated for sterilization in an autoclave (126 °C, 142 kPa, Shuangha YX280A) for 20 min. After that, the hydrogels were cut into standard disks with diameter of 1 cm to fit the 48-well plates (Costar). Then, human osteoblast-like cells (MG-63, American Type Culture Col-lection) were inoculated at a density of 5 × 103 cells/well on the hydrogels, and were cultivated in DMEM medium containing 100 μg/mL streptomycin, 100 μg/mL penicillin, and 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2. The culture media was replaced every 2 days. Cell Viability and Morphology on Hydrogels. The viability of the MG-63 cells on the hydrogels was assessed using CCK-8. The DMEM medium acted as negative control groups in the tests. After incubating for 1, 3, and 5 days respectively, the hydrogels were transferred into new wells and 250 μL of medium containing 25 μL CCK-8 (10%, v/v) was added for 2 h incubation. Subsequently, 100 μL of the supernatant was moved to a new 48-well cell cultivation plate. The optical density (OD value) of the supernatant was assessed using a microplate reader (Bajiu). The morphology of MG-63 cells on PVCL2.5-Clay0.15 hydrogels was observed via SEM. Before SEM observation, the cells were cultivated on the hydrogel sample for 3 days, and then fixed in glutaraldehyde solution (2.5%) for 1 h. Next, after the dehydration treatment with ethanol solutions of gradient concentration, the cell samples were naturally dried in vacuum environment at ambient temperature before sprayed with gold by a sputter coater. The cell morphology was also observed using CLSM. First, the cells were cultivated for 1 day or 5 days, and then immobilized with paraformaldehyde (4%) at ambient temperature. Afterward, the samples were cleaned using PBS and then permeabilized using Triton X-100 (0.1%), followed by incubation in BSA/PBS (1%) for 30 min to block nonspecific binding. The MG-63 cells were then stained for 30 min using 5 μg/mL of FITC phalloidin for CLSM observation. To investigate the thermoresponsive detachment of cells from the PVCL-Clay nanocomposite hydrogels, the ambient temperature of the MG-63 cells cultivated for 5 days was first decreased from 37 to 4 °C and kept for 2 h, and then increased to 20 °C. The residual MG-63 cells on the hydrogel surface were observed using CLSM, after changing medium and staining treatment.

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RESULTS AND DISCUSSION Fabrication Strategy. The fabrication strategy of the PVCLClay nanocomposite hydrogels is schematically illustrated in Figure 1. First, the VCL dispersed in pre-prepared clay solution is heated at 40 °C to melt into liquid. Because of the poor solubility 21981


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Figure 2. Qualitative mechanical characteristics tests of (a−c) PVCL2.5-BIS2% hydrogel, (d−f) PVCL2.5-BIS2%-F hydrogel, and (g−i) PVCL2.5Clay0.15 hydrogel. (a, d, g) Network structure diagrams of (a) PVCL2.5-BIS2% hydrogel, (d) PVCL2.5-BIS2%-F hydrogel, and (g) PVCL2.5-Clay0.15 hydrogel. (b, e, h) Optical images of (b) PVCL2.5-BIS2% hydrogel, (e) PVCL2.5-BIS2%-F hydrogel, and (h) PVCL2.5-Clay0.15 hydrogel before shaking tests. (c, f, i) Optical images of corresponding hydrogels after strongly shaking with a mini-shaker. Scale bar is 1 cm.

in water, the VCL liquid enables to disperse in the claycontaining aqueous solution under ultrasonic treatment, thus forming Pickering emulsions with clay nanosheets stabilizing the VCL/water interfaces (Figure 1a).39,40 Without the stabilization provided by clay nanosheets at the emulsion interfaces, the VCLin-water emulsions are quite unstable (Figure S2). The polymerization is triggered by UV light using Irg.184 molecules as initiators, which partly adsorb on the surface of clay nanosheets (Figure 1d).41 During the polymerization, the VCL monomers in the dispersed phase are preferentially cross-linked with clay nanosheets at the interface to form a hydrogel layer; meanwhile, the VCL monomers are polymerized into oligomers because the initiator is soluble in VCL liquid. The polymerization reaction consumes VCL monomers that have been dissolved in the continuous water phase, which would facilitate the diffusion and dissolution of VCL monomers from the dispersed phase into the continuous water phase based on the equilibrium theory. The oligomers that formed in the dispersed VCL phase also tend to

move into the continuous water phase, because the VCL oligomers are soluble in water phase. Both the VCL monomers and oligomers with active radicals could continually diffuse into the water phase, and the oligomers are cross-linked by the clay nanosheets to form hydrogel networks (Figure 1b, e), because hydrogen bonds can be formed between the Si−O−Si as well as SiOH groups on the clay surfaces and the amide (CONH(R)) groups of the PVCL polymers.24 As a result, the volumes of dispersed droplets are reduced to some extent (Figure 1e). However, the formed hydrogel networks would impede the VCL oligomers further transferring to the water phase. The clay nanosheets cannot pass across the VCL/water interface and enter into the VCL phase to cross-link the oligomers inside the dispersed VCL phase base on energy-trapped theory at the emulsion interface.42 Therefore, the unreacted VCL monomers and un-cross-linked oligomers are left in the droplets to generate macropores in the hydrogels. After washing, the unreacted monomer in the droplets are removed, and the pores are left at 21982


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Figure 3. (a, b) Stress−strain curves, (c, d) Young’s modulus, and (e, f) toughness of the as-prepared PVCL-Clay nanocomposite hydrogels with (a, c, e) different clay contents or (b, d, f) different VCL contents.

into pieces (Figure 2b, c). By ultrasonically dispersing the VCL monomers in an aqueous solution containing BIS and F-127, the PVCL-BIS-F hydrogel is prepared with macroporous structure (Figure 2d). The PVCL-BIS-F hydrogel is also easily damaged under the shaking test (Figure 2e, f). Because of the small molecular size of BIS, a cross-linker molecule only connects two polymer chains. However, the inorganic clay cross-linkers are nanodiscs with 1 nm of thickness and 30 nm of diameter, which can connect multiple polymer chains (Figure 2g). The topologically cross-linked chains between the clays may exhibit uniform length, which avoids the stress focus points. Thus, the PVCL-Clay nanocomposite hydrogels have remarkably improved mechanical characteristics because of their unique organic−inorganic network structures,24 even though they are porous. The PVCL-Clay nanocomposite hydrogel remains intact perfectly after the strong shearing (Figure 2h, i, Movie S1). The quantitative characterization of mechanical characteristics of the PVCL-Clay nanocomposite hydrogels is tested by a tensile machine. Unfortunately, the as-prepared common PVCL hydrogels cross-linked by BIS are too weak to be fixed on the tensile machine, and they cannot even sustain the external force when they are carefully moved by the tweezers (Movie S2).

the residual droplet positions (Figure 1c, f). By replacement of the unreacted VCL with water in the hydrogel, the color of asprepared PVCL-Clay nanocomposite hydrogel changes from the initial milk white to transparent after washing and swelling in water (Figure S3). Mechanical Characterization of PVCL-Clay Nanocomposite Hydrogels. The qualitative characterization of mechanical characteristics of the PVCL-Clay nanocomposite hydrogel comparing with those of control hydrogel samples is shown in Figure 2. The PVCL-BIS hydrogel is a conventional PVCL hydrogel cross-linked by BIS, and the PVCL-BIS-F hydrogel is a PVCL hydrogel prepared by ultrasonically dispersing the VCL liquid monomers in an aqueous solution containing surfactant Pluronic F-127 and BIS. All the hydrogels are sheared by strongly rotating flow in a bottle for 30 s using a mini-shaker (Movie S1). The mechanical characteristics are quite different because of their different network structures. As the PVCL-BIS hydrogel is prepared in ethanol−water solution that enables to completely dissolve VCL monomers and is cross-linked by BIS, the network structure of PVCL-BIS hydrogel is heterogeneous. So, the PVCL-BIS hydrogel exhibits poor mechanical characteristics. During the shaking test, the common PVCL-BIS hydrogel breaks 21983


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Figure 4. (a−c) CLSM micrographs and (d) SEM image of the (a, b) VCL2.5-Clay0.15 reaction solution and (c, d) PVCL2.5-Clay0.15 hydrogel. (a, b) CLSM micrographs showing the (a) red channel image and (b) transmission channel image of VCL-in-water emulsions. (c, d) CLSM micrograph of (c) the red channel image and (d) SEM image of polymerized PVCL2.5-Clay0.15 hydrogel. Scale bars are 50 μm.

stabilized by the clay nanosheets. As shown in Figure 4a, b, the sizes of droplets of dispersed VCL monomer phase are ranged about 5−50 μm. The emulsions in Figure 4a are prepared with VCL2.5-Clay0.15 reaction solution, which is composed of 2.5 g of VCL with dye LR300 as the dispersed phase and 4 mL of aqueous solution containing 0.15 g of clay as the continuous phase. The more the content of VCL monomers in the solution with constant clay concentration, the more the emulsion droplets generated (Figure S6). After polymerization, the dye LR300 in the original VCL emulsion droplets still stand inside the residual unreacted emulsion droplets (Figure 4c). After polymerization, the volumes of the VCL droplets are reduced to some extent. The volume change of the VCL droplets before and after polymerization is shown in Figure S7. In Figure S7, the morphology of the droplet changes from a sphere to an ellipsoid because of the limited ultrathin space. By scanning the droplets at different heights layer-by-layer using CLSM (Figure S7b), the volumes of the droplets before and after polymerization are calculated. After polymerization, the average volume of the VCL droplet is reduced to 80.54% compared with original volume before polymerization (Figure S7a). That is, the volume reduction of the VCL droplets is about 19.46% after the UV polymerization. Further, the constituent content of the washed clean PVCL2.5Clay0.15 hydrogel is measured by using a thermal gravimetric analyzer (TGA) (Figure S8). The results show that the PVCL2.5-Clay0.15 hydrogel contains ca. 86.4 wt % PVCL polymers and ca. 13.6 wt % clay. According to the weights of feeding reactants and the resultant PVCL2.5-Clay0.15 hydrogel in dried state, the mass conversion of the monomer VCL is estimated as ca. 18.96%, which is quite consistent with the results of average volume change of the VCL droplets. During the UV-

While, the as-prepared PVCL-Clay nanocomposite hydrogels possess good mechanical tensile characteristics, and can be stretched up by the tensile machine (Figure S4). The tensile results of the mechanical tests are shown in Figure 3a and 3b. The breaking elongation of the macroporous PVCL1.5-Clay0.15 hydrogels is ca. 370%, which is larger than that of the PVCL hydrogels cross-linked by EGDMA (∼239%)35 and is similar to that of the hybrid PVCL hydrogels cross-linked by in situ formed silica particles (180−440%).31 The fracture stress of the PVCLClay hydrogels is lower than that of the above-mentioned two previously reported hydrogels,31,35 because of the macroporous structure. The effects of clay and VCL contents in the initial reactants on the mechanical characteristics of the as-prepared PVCL-Clay hydrogels are investigated. With constant VCL content, both the Young’s modulus and the toughness of PVCL-Clay hydrogels increase with increasing the clay content (Figure 3c, e). Moreover, the elastic modulus G′ of PVCL-Clay hydrogels in the mechanical rheological tests also increases gradually with increasing the clay content during the whole range of the frequency sweep (Figure S5), because more clay nanosheets in the hydrogel networks could provide more energy dissipation units during the deformation of the nanocomposite hydrogels.24 With increasing the VCL content in the reaction mixture, both the Young’s modulus and the toughness of the hydrogels decrease (Figure 3d, f), because more macropores are formed in the hydrogel networks. Morphological Analyses and Assembly Performance of PVCL-Clay Nanocomposite Hydrogels. The macropores in the networks of the PVCL-Clay nanocomposite hydrogels are introduced by the templates of emulsion droplets, which are 21984


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Figure 5. SEM images of (a) PVCL-BIS hydrogel and (b−f) PVCL-Clay nanocomposite hydrogels with different contents of clay or VCL. (a) PVCL2.5BIS2% hydrogel; (b) PVCL2.5-Clay0.09 hydrogel; (c) PVCL2.5-Clay0.15 hydrogel; (d) PVCL2.5-Clay0.21 hydrogel; (e) PVCL1.5-Clay0.15 hydrogel; (f) PVCL3.5-Clay0.15 hydrogel. Scale bars are 100 μm.

initiated polymerization, the VCL monomers are preferentially cross-linked by clay nanosheets at the interface to form a hydrogel layer, which is a barrier for the VCL oligomers to further transfer to the water phase. As a result, only a part of VCL oligomers can enter the water phase and are cross-linked by the clay nanosheets to form three-dimensional polymer networks, while the residual unreacted VCL droplets act as templates for the formation of macropores in the PVCL-Clay hydrogels (Figure 4d). When the PVCL hydrogels are prepared with homogeneous reaction solution, such as the conventional PVCL-BIS hydrogels, uniform pores that resulted from the ice crystals during the freeze-drying are found in the SEM image of freeze-dried hydrogels (Figure 5a). Like the PVCL2.5-Clay0.15 hydrogel (Figure 4d), all the PVCL-Clay nanocomposite hydrogels show macroporous structures (Figure 5b−f). With the same content of VCL dispersed phase, no matter how the clay concentration in

the water solution changes, the macroporous structures of PVCL-Clay nanocomposite hydrogels are almost the same (Figure 5b−d). The increase in the clay concentration only causes denser cross-linkage. While, with increasing the VCL content, the prepared PVCL-Clay nanocomposite hydrogels possess more macropores due to the increased emulsion droplets. When the VCL content is low, the VCL-Clay reaction solution forms a small quantity of VCL emulsion droplets (Figure S6a), which results in a few macropores in the PVCL1.5Clay0.15 nanocomposite hydrogel (Figure 5e). By contrast, when the VCL content is high, the amount of the macropores in PVCL3.5-Clay0.15 nanocomposite hydrogel becomes much more (Figure 5f). On the basis of the hydrogen bonding between the clay nanosheets and PVCL polymers, different PVCL-Clay nanocomposite hydrogel subunits can be assembled easily by drying and reswelling treatment. During such a process, the thickness of 21985


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hydrogels show significant thermoresponsive characteristics. The volume phase transition temperature (VPTT) of the conventional PVCL-BIS hydrogels is around 32.5 °C, while the PVCLClay nanocomposite hydrogels containing different contents of clay exhibit the VPTT around 35 °C (Figure 7). The slight

PVCL-Clay hydrogel obviously decreases (Figure 6a, b). The original PVCL2.5-Clay0.15 hydrogels are macroporous (Figure

Figure 7. Thermo-dependent equilibrium swelling ratios of PVCL-Clay nanocomposite hydrogels with different contents of clay.

increase in VPTT of hydrogel results from the hydrophilicity of the added clay nanosheets.45,46 Referring to the volumes of the PVCL-Clay nanocomposite hydrogels at 50 °C, the swelling degrees at low temperature increase with decreasing the crosslinkage with clay nanosheets. Taking the PVCL2.5-Clay0.09 nanocomposite hydrogel as an example, the swelling ratio of hydrogel volume from 15 to 50 °C is 6.37. With increasing the content of clay nanosheets, the cross-linkage of PVCL-Clay nanocomposite hydrogel increases. Thus, the swelling degree of the PVCL-Clay nanocomposite hydrogel at 15 °C decreases.47 Compared with that of the PVCL-Clay nanocomposite hydrogels, the swelling degree of the conventional PVCL-BIS hydrogel is smaller because of the remarkable chemical cross-linkage. Figure 8 shows the dynamic thermoresponsive shrinking behavior of the PVCL-Clay nanocomposite hydrogels with the different contents of clay or VCL. After completely swelling, the shrinking behavior of the PVCL-Clay nanocomposite hydrogels is induced by rapidly increasing the temperature from 25 to 45 °C within 1 min, and keeping it constant at 45 °C for 30 min. The shrinking rate of PVCL-Clay nanocomposite hydrogels is different from that of the PVCL-BIS hydrogel, and varies with the clay content. As shown in Figure 8a, both shrinking rate and shrinking degree of PVCL2.5-Clay0.09 hydrogels are more significant than that of PVCL-BIS 2% hydrogel. While, with increasing the content of clay nanosheets, the shrinking rate and shrinking degree of the PVCL-Clay nanocomposite hydrogels are decreased because of the growing cross-linkage (Figure 8c, Figure S11). When the clay content is fixed, the VCL content also significantly influences the thermoresponsive shrinking rate and shrinking degree (Figure 8b), because the monomer content decides both the macroporosity and the density of polymer chains. The large macroporosity will result in rapid responsive rate,48 whereas the increased chain density between the clay nanosheets that resulted from the increased monomer content can restrict the shrinking rate due to the low flexibility of polymer chains.46 With increasing the VCL monomer content, the macroporosity in the hydrogel is increased, thus resulting in the increased shrinking rate; meanwhile, the density of the polymer chains between the clay nanosheets is also increased, thus causing

Figure 6. (a, b) Optical photographs of the cross-section of PVCL2.5Clay0.15 hydrogel in (a) completely swelling state and (b) dryingreswelling state. (c) SEM image of the hydrogel shown in b. (d) SEM image and (e) optical image of the combined PVCL2.5-Clay0.15 hydrogel via drying-reswelling treatment. (f) Optical image of the combined PVCL2.5-Clay0.15 hydrogel that stretched by hands to demonstrate the excellent bonding performance. The scale bars are (a, b) 0.2 cm, (c, d) 100 μm, and (e, f) 1.5 cm.

5c, Figure S9a); while, after drying, the macropores in the network collapse due to the dehydration. Because of the rearranged hydrogen bonding between the clay nanosheets and PVCL polymers,43,44 the macropores are conglutinated. When the PVCL-Clay nanocomposite hydrogel is treated by drying and reswelling, the macropores in the hydrogels have difficulty recovering their shapes because of the new hydrogen bonds that are formed between the Si−O−Si as well as SiOH groups on the clay surfaces and the amide (CONH(R)) groups of the PVCL polymers. Thus, the reswelled nanocomposite hydrogel shows honeycomb-like structures rather than the original macroporous structures (Figure 6c, Figure S9b). Processed by a stackingdrying-reswelling procedure (Figure S1), the two PVCL2.5Clay0.15 hydrogel blocks connect very well (Figure 6e). At the interface of the hydrogel blocks, lots of bonding are newly generated across the interface because of the hydrogen bonding reinteractions of clay nanosheets and PVCL polymer chains at the interface (Figure 6d). In consequence, the two hydrogel blocks are tightly locked together by the reassociated noncovalent interactions, and the combined hydrogels are tight enough to tolerate the stretching test by hand (Figure 6e, f). During the elongation, the overlap regions of the bonded PVCLClay nanocomposite hydrogels do not show any slippage (Figure S10), and the position of the fracture gap is outside the joints (Movie S3). Thermoresponsive Properties of PVCL-Clay Nanocomposite Hydrogels. All the PVCL-Clay nanocomposite 21986


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Figure 8. (a, b) Dynamic thermoresponsive shrinking behaviors of the PVCL-Clay nanocomposite hydrogels with different contents of (a) clay or (b) VCL when immediately increasing the temperature to 45 °C. (c, d) Maximal corresponding shrinking rates of PVCL-Clay hydrogels with different contents of (c) clay and (d) VCL, in which the maximal shrinking rates are defined as the maximal slope values of curves in a and b.

the decreased shrinking rate. Thus, with increasing the VCL content, the thermoresponsive shrinking rates of PVCL-Clay nanocomposite hydrogels increase first and then decrease because of the competitive effect between the macroporosity and the chain density (Figure 8d). Cell Cultivation of PVCL-Clay Nanocomposite Hydrogels. To demonstrate the biocompatibility of the PVCL-Clay nanocomposite hydrogels, the PVCL2.5-Clay0.15 hydrogels are selected for cultivation of human osteoblast-like cells (MG-63) at 37 °C. As shown in Figure 9a, the cells are adhered very well on the hydrogel surface for 1-day cultivation. For 5-day cultivation, MG-63 cells are obviously proliferated very well, and almost cover the hydrogel surface (Figure 9b). Typically, as shown in the fluorescence image in Figure 9c, MG-63 cells display filamentous morphology, which means the F-actin is fully developed and the cells adhere quite well on the hydrogel surface. Almost all the MG-63 cells show an extended morphology (Figure 9d), which indicates MG-63 cells present favorable spreading with many pseudopodium on the hydrogel surface. By using cell counting kit-8 assay on the cells, the optical density (OD) value of cell viability obviously increases from 1 day to 5 days (Figure 9e), which shows a good cell proliferation on the macroporous PVCL-Clay nanocomposite hydrogel. The results show that the PVCL-Clay nanocomposite hydrogels are biocompatible and are beneficial to cell culture. On the one hand, both PVCL and clay possess no toxicity to the cells; moreover, the clay with electric charge can effectively adsorb adhesive proteins and nutriment to the benefit of cell cultivation.38 On the other hand, the macroporous structure of the hydrogel does a favor to the mass transfer of oxygen and nutrients, and also provides more spaces for cell proliferation, thus resulting in good cell adhesion and proliferation on the macroporous PVCL-Clay hydrogel.21−23

After the MG-63 cells are cultivated on the PVCL-Clay hydrogel for 5 days, when the temperature is decreased from 37 to 4 °C, the MG-63 cells show limited spreading and the cell shape becomes cycloidal, which means the F-actin is poorly developed. As a result, the cells are detached from the hydrogel surface and few cycloidal cells is residual on the substratum (Figure 9f). Such cell detachment behavior is attributed to the well-defined temperature sensitivity of the PVCL-Clay hydrogel, which is switched to be hydrophilic at low temperature.38 By temperatureregulated detachment that is quite convenient for operation, the PVCL-Clay hydrogels could be used for subculture of cells.

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CONCLUSION In summary, a novel strategy for fabricating biocompatible thermoresponsive PVCL-Clay nanocomposite hydrogel with macroporous structure and improved mechanical characteristics has been successfully developed. The VPTT of the PVCL-Clay nanocomposite hydrogels is around 35 °C. The macropores in the hydrogels result from the residual unreacted monomer-inwater Pickering emulsion droplets that stabilized by clay nanosheets. Cross-linked by the clay nanosheets, the proposed nanostructured hydrogels with unique network structures exhibit improved mechanical characteristics compared with the conventional PVCL hydrogels that cross-linked with BIS. The thermoresponsive shrinking rates of PVCL-Clay nanocomposite hydrogels are affected by both clay and VCL contents. Combining the biocompatibility of PVCL, hydrophilic clay nanosheets with negative charge, and macroporous structure, the PVCL-Clay hydrogels provide promising substrates for cell culture. And, the cell detachment can be easily achieved by decreasing the environmental temperature, which is attributed to the temperature-regulated hydrophilicity of PVCL polymer. 21987


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Figure 9. Morphology and viability of MG-63 cells cultivated on PVCL-Clay nanocomposite hydrogels. (a, b) CLSM images of the cells on the PVCL2.5-Clay0.15 hydrogel for (a) 1-day cultivation and (b) 5-day cultivation. (c) CLSM image of the 5-day cultivated cells on the PVCL2.5-Clay0.15 hydrogel with high magnification. (d) SEM micrograph of the cell cultivated on the PVCL2.5-Clay0.15 hydrogel for 3 days, in which the false color is added for better illustration. (e) Viability of the cells cultivated on the PVCL2.5-Clay0.15 hydrogel for 1, 3, and 5 days. (f) CLSM image of the residual 5 day-cultivated cells on the PVCL2.5-Clay0.15 hydrogel after the cell detachment by decreasing the temperature. The scale bars are (a, b, f) 50 μm, (c) 25 μm, and (d) 10 μm.

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With flexible assembly performance, the proposed biocompatible thermoresponsive PVCL-Clay nanocomposite hydrogels with macroporous structure and improved mechanical characteristics are ideal material candidates for medical and biorelated applications, such as entrapment of enzymes and cells, tissue engineering, affinity and displacement chromatography, sensors, and so on.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b04552. Schematic illustration of the bonding process of the PVCL-Clay nanocomposite hydrogel subunits; results about the stability of VCL monomer droplets, optical images of the PVCL-Clay hydrogel, TGA data of the clay 21988


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nanosheets and the dried PVCL2.5-Clay0.15 hydrogel, morphology characterization of the same VCL emulsion template before and after UV polymerization, optical images of the tensile test on the as-prepared PVCL2.5Clay0.18 hydrogel, rheological characteristics of the asprepared PVCL-Clay hydrogels, optical micrographs of emulsion droplets of VCL-in-(clay aqueous solution), optical images of the cross-section of PVCL2.5-Clay0.15 hydrogel, stress−strain curve of the combined PVCL2.5Clay0.15 hydrogel, and optical images of the dynamic thermoresponsive shrinking behaviors of hydrogels (PDF) Movie S1, mechanical shaking tests with the PVCL-Clay nanocomposite hydrogels (AVI) Movie S2, mechanical behavior of the conventional PVCL hydrogel cross-linked by BIS (AVI) Movie S3, stretching test with the bonded PVCL-Clay nanocomposite hydrogels (AVI)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.-Y.C.). *E-mail: [email protected] (Z.L.). ORCID

Yi Deng: 0000-0002-1765-5244 Xiao-Jie Ju: 0000-0003-1086-338X Liang-Yin Chu: 0000-0002-2676-6325 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge support from the National Natural Science Foundation of China (91434202, 81621062), the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R48), and State Key Laboratory of Polymer Materials Engineering (sklpme2014-1-01, sklpme20173-03).

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Clay Nanocomposite Hydrogels with Macroporous ... - ACS Publications

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