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Mechanically Robust 3D Nanostructure ChitosanBased Hydrogels with Autonomic Self-Healing Properties Ali Reza Karimi, and Azam Khodadadi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10375 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016
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Mechanically Robust 3D Nanostructure Chitosan-Based Hydrogels with Autonomic Self-Healing Properties Ali Reza Karimi,*† Azam Khodadadi † †
Department of Chemistry, Faculty of Science, Arak University, Arak 38156-8-8349, Iran
ABSTRACT: Fabrication of hydrogels based on chitosan (CS) with superb self-healing behavior and high mechanical and electrical properties has become a challenging and fascinating topic. Most of the conventional hydrogels lack these properties at the same time. Our objectives in this research were to synthesize, characterize and evaluate the general properties of chitosan covalently cross-linked with zinc phthalocyanine tetra-aldehyde (ZnPcTa) framework. Our hope was to access an unprecedented self-healable three-dimensional (3D) nanostructure that would harvest the superior mechanical and electrical properties associated with chitosan. The properties of cross-linker such as the structure, steric effect, and rigidity of molecule played important roles in determining microstructure and properties of the resulted hydrogels. The tetra-functionalized phthalocyanines favor a dynamic Schiff-base linkage with chitosan to form a 3D porous nanostructure. Based on this strategy, the self-healing ability, as demonstrated by rheological recovery and macroscopic and microscopic observations, is introduced through dynamic covalent Schiff-base linkage between NH2 groups in CS and benzaldehyde groups at cross-linker ends. The hydrogel was characterized using FT-IR, NMR, UV/vis and rheological measurements. In addition, cryo-scanning electron microscopy (Cryo-SEM) was employed as a technique to visualize the internal morphology of the hydrogels. Study of the surface morphology of the hydrogel showed a 3D porous nanostructure with uniform morphology. Furthermore, incorporating the conductive nanofillers such as, carbon nanotubes (CNTs) into the structure can modulate the mechanical and electrical properties of the obtained hydrogels. Interestingly, these hydrogel nanocomposites proved to have a very good film-forming properties, high modulus and strength, acceptable electrical conductivity, and excellent self-healing properties at neutral pH. Such properties can be finely tuned through variation of the cross-linker and CNT concentration and as a result these structures are promising candidates for potential applications in various fields of research.
INTRODUCTION Self-healing hydrogels based on chitosan (CS), as one of the most practical soft matters with privilege of adopting three dimensional (3D) network, have attracted tremendous attention towards their applications in various fields of research ranging from biochemical to biomedical sciences due to their excellent biocompatibility, flexibility in fabrication, biodegradability and automatic self-repair capability after damage.1-3 Moreover, hydrogels based on chitosan are also electrically responsive biomaterials. However, the low sensitivities, conductivities and the soft nature of CS-based self-healing hydrogels make these compounds to be electrically non-conductive and mechanically weak after several on-off switching induced stimuli.4 In order to overcome these weakness, additives such as physical or chemical cross-linkers have been incorporated into CS-based hydrogels to improve their mechanical properties, chemical stability and conductivity capacities.5 In addition, molecular and geometric structures of cross-linkers also have notable effects in determining microstructures, as well as mechanical and electrochemical properties of hydrogels.6 Nowadays, the majority of endeavors to synthetic methods which adopt molecules as cross-linker have been developed to prepare 3D porous nanostructures.7 These materials exhibit large surface area, structural tunability and hierarchical porosity for mass transport. Therefore, 3D nanostructure hydrogels based on chitosan may find several promising applications for a wide range of technologies, such as drug releases, biosensors, medical devices and energy storage applications.8-11 To this end, the design of 3D nanostructured network and their corresponding morphologies has attracted intense attention because of their effectiveness in tuning the properties of hydrogels.12 In particular, chemical crosslinking is a significant method to create and modify 3D nanostructure with mechanical and chemical stabilities.13 Nevertheless, CS-based hydrogels have been extensively studied, and most of such hydrogels were prepared through covalent linkages using various cross-linkers,14,15 3D porous nanostructure CS-based hydrogels with self-healing behavior and high rheological properties, has not been reported. However, combining high mechanical properties of hydrogels with dynamic crosslinks have demonstrated to be very challenging.16-20
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Figure 1. Synthesis scheme of the dynamic covalently cross-linked hydrogel. Photograph and Chemical structure of (a) CS and (b) ZnPcTa before gelation. (c) Photograph and schematic of a proposed structure of the CS/ZnPcTa hydrogel through Schiffbased reaction. (d) Pore size of 3D porous nanostructure CS/ZnPcTa hydrogel.
In this context, we have recently developed a facile approach to prepare 3D porous nanostructure network based on chitosan with high rheological properties using zinc phthalocyanine tetra-aldehyde (ZnPcTa) as a cross-linker. ZnPcTa is particularly desirable planar component for evaluating this novel concept due to its size and rigidity.21 On the other hand, ZnPcTa can be implemented in the design of nanostructure to access self-healing hydrogel through dynamic covalent Schiff-base linkage between benzaldehyde groups at ZnPcTa β-ends and NH2 groups on chitosan (Figure 1). As it is evidenced form their name, the self-healing hydrogels can repair themselves automatically without any external stimuli and in the case of Schiff base hydrogels this can happen through a dynamic imine formation. Because of its dynamic equilibrium, the Schiff-base linkage could be considered as a quasi-covalent linkage, and the cleavage and regeneration of the imine bond keep occurring in the hydrogel network, which invoke the self-healing concept.22, 23 In addition, zinc phthalocyanines (ZnPcs) are good organic semiconductors and they have considerable importance owing to their potential applications in electronic devices and sensors because of their two-dimensional 18-π electron conjugated system.24 The presence of highly conjugated π-electron systems in ZnPc macromolecules not only imparts unique electrical properties but also introduces exceptional chemical stability.25 Therefore, ZnPcTa can improve the electrical and mechanical properties of hydrogels due to its delocalized π-electron system. As a result, it is significant to develop the strategies to design and construct the robust hydrogels with both the outstanding mechanical behavior and self-healing property. We anticipate that the mechanical and self-healing properties associated with these Schiff base can be controlled via modification of the ZnPcTa concentration. Besides significant mechanical and electrical properties of ZnPcTa as both the dopant and gelator, incorporation of a considerable amount of conducting nanofillers with large specific surface areas offers a great approach to the design the high performance nanocomposite hydrogels based on CS with superior electrical properties.26-29 Among the promising nanofillers used in the polymer matrices, carbon nanotubes (CNTs) possess high tensile strengths with excellent thermal, chemical stability and electrical conductivity.30-32 However, CNTs are difficult to disperse well in water for their highly intractable carbon nanostructures. The functionalization of CNT is considered as one of the most effective ways to improve the solubility of CNTs in water.33 In this study, we have shown the incorporation of carboxylic functional groups (COOH) into multi-walled carbon nanotubes (MWCNTs) that can give access to a well dispersed nanocomposite hydrogel. In fact, the electrostatic interaction between MWCNT-COOH and the CS might be involved in the improvement of the dispersion of Carboxyl MWCNTs, however, the addition of high content of carbon nanotubes often suppresses the hydrogel’s unique stimuli-responsive behaviors.
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In addition to the above-mentioned advantages of ZnPcTa as a gelator and improvement in hydrogel properties, ZnPcs present intense fluorescence in the near infrared region.34 This property has caused them to be extensively investigated in materials science,35 photochemistry or biomedical sciences.36-39 More importantly, ZnPcs are promising candidates as photosensitizers (PSs) due to their high triplet state quantum yields, long triplet lifetimes and effectively singlet oxygen generation capability. In spite of the high electron ability and electrochemical application, ZnPcs are restricted in solution because of their lower solubility in common organic solvents and water. Although, many peripherally substituted ZnPcs have been synthesized and their increasing solubility has facilitated electrochemical and spectroscopic studies of them in solution.40 It is interesting that the covalent conjugation of ZnPcTa to CS, which is soluble in acidic aqueous media41 can dramatically increase the solubility of the target ZnPc in water. Although there are reports on self-healable chitosan hydrogels based on covalently cross-linked concept, preparation of CS-based hydrogels with excellent self-healing behavior and high mechanical and electrical properties has become a challenging in recent years.19 Most of the conventional hydrogels lack these properties at the same time while hydrogels with these properties are of great interest in many applications. In this letter, we report for the first time a novel yet general strategy to prepare 3D porous nanostructured CS-based hydrogels with superb self-healing behavior and high mechanical and electrical properties using new cross-linker with multiple functional groups. There are some advantages of using ZnPcTa to produce new CS/ZnPcTa hydrogels: (1) Compared with traditional cross-linking chitosan with small molecules contain aldehyde groups, multi-aldehyde based on phthalocyanine as both the dopant and cross-linker leads to 3D porous nanostructure network with high rheological and electrical properties and tunable morphology by changing the cross-linker concentration. (2) The mechanical/electrical properties and self-healing behavior of hydrogels could be tuned using different concentrations of ZnPcTa. (3) The other effective approach to the preparation of ZnPcTa coupled with the chitosan as hydrophilic moiety is water solubility of ZnPcTa that is important for many applications of ZnPcTa. (4) More importantly, these self-healable hydrogels conjugated ZnPcTa as a new soft matters based on chitosan can be used as an effective photosensitizer (PS) for selective cancer therapy. In fact, a combination of these two potentially promising units (chitosan as a biocompatible and safe carrier and ZnPcTa as an effective cross-linker and photosensitizer) may improve the biological properties of synthesized hydrogels.
EXPERIMENTAL SECTION Materials. All solvents reagents were purchased from Sigma-Aldrich and were used without further purification. 4-(4-formylphenoxy)phthalonitrile (FPPht) was prepared according to literature procedure.42 Measurement and Statistical Data Analysis. Fourier transform infrared (FT-IR) data of FPPht, ZnPcTa, CS/ZnPcTa hydrogels and hydrogel nanocomposites were recorded on a Unicom Galaxy Series FTIR 5000 Spectrophotometer. Each spectrum was recorded over the region 4000-400 cm-1. 1HNMR measurements of FPPht, ZnPcTa and CS/ZnPcTa hydrogels were determined on a Bruker Avance 300 MHz spectrometer. DMSO-d6 and D2O were used as the solvents and the solvent signal was used for internal calibration (DMSO-d6: δ (1H) = 2.5 ppm), (D2O: δ (1H) = 4.79 ppm). Electronic spectral measurements of ZnPcTa and CS/ZnPcTa hydrogels were carried out using Perkin-Elmer Lamda double beem spectrophotometer in the range 190-900 nm. The surface morphology of CS/ZnPcTa hydrogels and hydrogel nanocomposites were obtained using Field Emission Scanning Electron Microscope (Mira 3-XMU) after sputter coating with a thin layer of gold (Au) under vacuum. Electrochemical impedance spectroscopy measurements were performed using an IVIUMSTAT in a solution containing 50 mM [Fe(CN)6]3−/[Fe(CN)6]4− at room temperature. The rheological experiments performed on a rheometer (MCR 300). All the experiments were carried out at room temperature using 40 mm parallel plate with plate gap of 1.0 mm. The hydrogel was placed between the parallel plates. The electrical conductivity of xerogel nanocomposite films was tested by four-point probe resistivity measurement system (JG 293015 Jandel) at room temperature. Synthesis of Zinc Phthalocyanine Tetra-aldehyde (ZnPcTa). Zinc phthalocyanine tetra-aldehyde (ZnPcTa) as a cross-linker was synthesized in two steps (Figure S1, Supporting Information). The first involves the nucleophilic aromatic nitro displacement of 4-nitrophthalonitrile (0.173 g, 1 mmol) 2 with 4-hydroxybenzaldehyde (0.122 g, 1 mmol) 1 in the presence of anhydrous K2CO3 (0.138 g, 1 mmol) as the base in 2 ml DMF. The mixture was stirred at room temperature for 24 h. After completion of the reaction, 5 ml acetone and 4 ml water was added respectively to the reaction mixture to give 4-(4-formylphenoxy)phthalonitrile (FPPht) 3. The resulting precipitate was washed with 5 ml hot water. In the next step (ZnPcTa) 4 prepared by cyclization of compound 3 (0.10 g, 0.42 mmol) with
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anhydrous metal salt Zn(CH3COO)2 (0.03 g, 0.16 mmol) in the presence of a few drops of DBU in 2 ml (DMAE) under microwave irradiation at 300 W for 15 minutes. The reaction mixture was then cooled to room temperature. After that, 2 ml ethanol was added and the product was filtered under reduced pressure. The green solid was washed several times with hot ethanol. Preparation of Covalently Cross-Linked Hydrogel. A 3% (w/v) chitosan solution was prepared by dissolving chitosan (0.1 g, medium viscosity, 75–80% deacetylated) in (1% v/v) aqueous acetic acid. The mixture was stirred using a vortex mixer at room temperature for 15 minutes to ensure that the chitosan was completely dissolved. The solution of cross-linker was combined in variable weight proportions to give the desired mass ratios of CS: ZnPcTa. As a typical hydrogel preparation, a solution of cross-linker was prepared by dissolving 0.012 g ZnPcTa in 2 ml aqueous DMF. With various hydrogels, the amount of ZnPcTa was different from 0.012 to 0.006 g. The as-prepared ZnPcTa solution was added to chitosan solution and the mixture was shaken, and gelation happened within ∼30 s. Therefore, a series of hydrogels with the different ratio of CS: ZnPcTa (%w/w) were prepared. Then the hydrogels were immersed in distilled water for 1 days to remove DMF and unreacted chemicals. Then, hydrogels were centrifuged (4000 r/min for 10 min at 25 °C) and they were rinsed with excess distilled water (3 times with 10 ml distilled water) and once washed with 10 ml ethanol. Then the residue of DMF was removed under reduced pressure. Preparation of Hydrogel Nanocomposites and Xerogel Nanocomposite Films. Hydrogel nanocomposites with the same cross-linking concentration and different amounts of MWCNT-COOH synthesized by adding various amounts of MWCNT-COOH to the chitosan solution. In a typical synthesis, 3% (w/v) chitosan solution was prepared by dissolving chitosan in of (1% v/v) aqueous acetic acid. After that, 0.5, 1 and 2 wt % (w/w) of MWCNTCOOH ( > 95%, OD 10-20 nm, ID 5-10 nm, L 10-30 um) were added to chitosan solution. Then the mixture was sonicated in an ultrasonic bath (Unique USC 1400 A) for 60 min. Afterwards, 12 wt% ZnPcTa solution was added to the MWCNT-COOH loaded chitosan solution. The mixture was again shaken, and gelation happened within ∼120-150 s. Then, the hydrogel nanocomposites were immersed in distilled water for 1 days to remove DMF and unreacted chemicals. Then, hydrogel nanocomposites were centrifuged (4000 r/min for 10 min at 25 °C) and they were rinsed with excess distilled water (3 times with 10 ml distilled water) and once washed with 10 ml ethanol. The residue of DMF was removed under reduced pressure. Moreover, xerogel nanocomposite films were obtained from solution casting and dried under vacuum. The mixtures of the solution were cast in Petri dishes, and the solvent was evaporated under vacuum at 40 °C after 48 h. The films were cut into a 1×1 cm square with a thickness ∼0.050 cm for the conductivity tests. Hydrogel Morphology. The internal morphologies and pore size of the hydrogels were examined by Cryo-SEM. The hydrogels were frozen in liquid nitrogen, placed inside the sample holder and the top section of the frozen gel was broken by a scraper and imaged at 1.5 kV. This method showed better images than normal SEM procedures using freeze-drying for hydrogel preparation. Morphological examination of the internal structure and uniform dispersion of the MWCNT-COOH in the hydrogel nanocomposites were carried out using SEM. Cell Culture and Biocompatibility Test. To evaluate the cytotoxicity of ZnPcTa, hydrogels and hydrogel nanocomposites, A435 cells (8×103 cells/well) were seeded in 96-well cell culture plates and then incubated in culture medium. After 24 h incubation, the culture medium was removed and the cells were washed with phosphate buffered saline (PBS). Then, 50 ml of PBS containing different concentrations (0-0.2 mg/ml) of ZnPcTa or ZnPcTa conjugated CS was added to in corresponding wells and co-incubated for another 24 h. The medium was replaced with 50 ml of MTT solution and further cultured for 4 h. Absorbance of the extract (OD value) was measured at 570 nm using a microplate reader. Cultures without ZnPcTa were used as control. Cell viability was calculated as Cell viability=
×
100%
Rheology Test. To understand the viscoelastic properties of hydrogel, the rheological experiments performed on a rheometer (MCR 300). All the experiments were carried out at 37 °C using a 40 mm parallel plate with plate gap of 1.0 mm. The hydrogel was placed between the parallel plate and the platform with special care to avoid evaporation of water. The storage modulus (G’) and loss modulus (G’’) were measured as a function of frequency (ω).
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Self-Healing Experiment. The rheology analyse of hydrogel was carried out to monitor qualitatively the selfhealing process. Moreover, elastic response of the hydrogel was analysed through strain amplitude sweep. Based on the strain amplitude sweep results, the continuous step strain measurement was carried out to test the rheology recovery behavior of the hydrogel. Furthermore, we demonstrated the self-healing behavior of the CS/ZnPcTa hydrogel by microscopic and macroscopic observations. Electrochemical Impedance Spectroscopy. To evaluate the electrochemical characteristics of the hydrogels and hydrogel nanocomposites, we performed a measurement using electrochemical impedance spectroscopy (EIS). The Nyquist plots (Zimaginary vs Zreal) of CS/ZnPcTa and MWCNT-COOH-loaded CS/ZnPcTa modified electrodes with 12 wt% ZnPcTa and different MWCNT-COOH contents; the curves were measured with the electrodes immersed in a solution of 0.1 M phosphate buffer (PBS, pH 7), 0.1 M KCl, 10 mM K3Fe(CN)6 and 10 mM K4Fe(CN)6. Electrical Characteristics of Xerogel Nanocomposite Films. The conductivity of self-healing xerogel nanocomposite films was tested by using four-probe method. The films were cut into a 1 × 1 cm square with a thickness ∼ 0.050 cm. The conductivity of each film was determined three times at different current values, and the average value was taken as the conductivity of the nanocomposite films. Moreover, the conductivity of self-repaired hydrogel with 12 wt% ZnPcTa and hydrogel nanocomposites with 12 wt% ZnPcTa and different MWCNT-COOH contents were investigated.
RESULTS AND DISCUSSION Synthesis and Characterization of Covalently Cross-Linked Hydrogels and Hydrogel Nanocomposites. In a typical synthesis, ZnPcTa as a cross-linker was prepared from 4-(4-formylphenoxy)phthalonitrile (FPPht) under microwave irradiation. Then, the Self-healing hydrogels could form after homogeneously mixing CS and zinc phthalocyanine tetra-aldehyde through dynamic Schiff-base crosslinkages between amine groups of CS and aldehyde groups on ZnPcTa termini. Therefore, hydrogels with 6, 9 and 12 wt% of ZnPcTa were prepared at the end of mixing process (25 °C,
