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A facile and green approach towards electrically conductive hemicellulose hydrogels with tunable conductivity and swelling behavior Weifeng Zhao, Lidija Glavas, Karin Odelius, Ulrica M Edlund, and Ann-Christine Albertsson Chem. Mater., Just Accepted Manuscript • Publication Date (Web): 29 Jun 2014 Downloaded from http://pubs.acs.org on June 30, 2014
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Chemistry of Materials
A facile and green approach towards electrically conductive hemicellulose hydrogels with tunable conductivity and swelling behavior Weifeng Zhao, †, ‡ Lidija Glavas, † Karin Odelius, † Ulrica Edlund, † Ann-Christine Albertsson*, † †
Fiber and Polymer Technology, School of Chemical Science and Engineering, Royal Institute of Technology (KTH), Teknikringen 56-58 SE-100 44, Stockholm, Sweden ‡
College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, Sichuan, China KEYWORDS: Electrically conductive; Hemicellulose; Hydrogels; One-pot reaction; Ambient conditions. ABSTRACT: A one-pot reaction to synthesize electrically conductive hemicellulose hydrogels (ECHHs) is developed via a facile and green approach in water and at ambient temperature. ECHHs were achieved by cross-linking O-acetylgalactoglucomannan (AcGGM) with epichlorohydrin in the presence of conductive aniline pentamer (AP) and were confirmed by infrared spectroscopy (IR) and elemental analysis. All hydrogels had macro-porous structures, and the thermal stability of ECHHs was improved by the addition of AP. Hydrogel equilibrium swelling ratios (ESRs) varied from 13.7 to 11.4 and were regulated by cross-linker concentration. The ESRs can also be tuned from 9.6 to 6.0 by changing the AP content level from 10 to 40 % (w/w) while simultaneously altering conductivity from 9.05×10-9 to 1.58×10-6 S/cm. ECHHs with controllable conductivity, tunable swelling behavior, and acceptable mechanical properties have great potential for biomedical applications, such as biosensors, electronic devices and tissue engineering.
INTRODUCTION Hydrogels are three-dimensional cross-linked hydrophilic networks that can absorb up to thousands of times their dry weight in water.1 In general, these networks are either permanent (covalently cross-linked) or reversible (physically cross-linked).2 Due to their rubbery nature, which is similar to soft tissues, hydrogels have been extensively explored during the past two decades and are widely used in diverse biomedical applications, such as biomimetic mineralization,3 cancer therapy,4 contact lenses,5 scaffolds for tissue engineering,6, 7 vehicles for drug delivery,7, 8 coatings for medical devices,9 and extracellular matrices for biosensors.10 For certain applications, much attention has been given to the functionalities of hydrogels with a particular set of desired properties, such as self-healing ability,11, 12 DNA-inspired responsiveness,13 magnetism,14 degradability and renewability,15, 16 conductivity,17, 18, pH sensitivity,18 and thermo-sensitivity.19, 20 Research on conductive polymers (CPs), such as polypyrrole (PPy),21 polyaniline (PANi),22 and polythiophene (PTh),23 has expanded significantly in the biomedical field, as these polymers exhibit good compatibility with many biological molecules. Zha et al. prepared PPy nanoparticles as a potential organic photoacoustic contrast agent for deep tissue imaging. The preliminary results showed good biocompatibility with several vital organs (heart, liver, spleen, lungs and kidneys) in mice following a single imaging dose of PPy nanoparticles.21 PANi/poly[(L-lactide)-co-(ε-caprolactone)] fibers were
electrospun for the development of an electrically conductive, engineered nerve scaffold. The component fibers exhibited a conductivity of 6.41×10-3 S/cm and promising cytocompatibility with PC12 cells, and the electrical conductivity of the fibers affected the differentiation of PC12 cells.22 Three different CPs based on PTh were synthesized to investigate the growth and differentiation of primary myoblasts. The results indicated that these polymers may potentially be used to promote a specific cellular response without using certain biological agents.23 Due to the electrical conductivity and biocompatibility of CPs, the last decades have witnessed remarkable growth in the development of electrically conductive hydrogels (ECHs), which combine the unique advantages of conductive polymers and hydrogels.17, 24, 25 A conducting hydrogel based on poly(3,4-ethylenedioxythiophene) was developed with comparable electro-activity to conventional CPs, which has potential applications not only in medical electrodes but also in nerve guides, wound healing products, drug delivery and gene therapies.24 Recently, PANibased ECHs with a conductivity of 7.6×10-8 to 1.1×10-3 S/cm were prepared using the in-situ polymerization of aniline and could improve the biological response of PC12 and hMSC cells.25 However, their non-degradability still poses a limitation, resulting in much attention to recently developed degradable and electrically conductive hydrogels.26 Chen et al. developed new biodegradable electroactive hydrogels with aniline pentamer (AP) both grafted onto
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Scheme 1. Schematic synthesis of ECHHs using epichlorohydrin as a cross-linker in basic media.
gelatin and cross-linked with chitosan.27, 28 The preliminary study demonstrated that these hydrogels have the potential to be utilized in drug delivery and tissue engineering. In our previous study,17 degradable and electrically conductive hydrogels were obtained using poly(D,Llactide)-poly(ethylene glycol)-poly(D,L-lactide), glycidyl methacrylate, ethylene glycol dimethacrylate and aniline tetramer (AT), and a conductivity of 5.58×10-7 to 1.43×10-5 S/cm was achieved. Degradable and electrically conductive hydrogels from natural polymers were also investigated.29 These hydrogels, prepared by a one-pot reaction between chitosan and AT, possessed good film-forming properties, electrical conductivity (3.1×10-8-2.9×10-5 S/cm), and pH-sensitive swelling behavior.29 AcGGM-based hydrogels are being exploited due to their non-toxicity, biocompatibility, biodegradability and renewability.30-32 In our recent study, electrically conductive hemicellulose hydrogels (ECHHs) with high and controllable swelling were obtained by a combination of the conductivity from AT and the biodegradability and nontoxicity from hemicellulose (AcGGM) in a robust pathway.31 The swelling ratio can be tuned from 548 to 228 % by changing the AT content level from 10 to 40 % (w/w) while simultaneously altering conductivity by two orders of magnitude. The demonstrated pathway, however, involved an organic solvent (DMSO), elevated temperatures, and multi-step reactions. The design of ECHHs under greener conditions is desirable. We anticipate that a new synthetic route for ECHHs could be conducted in water at room temperature, which would reduce energy consumption and offer a more environmentally benign production method. Our aim is to design a greener one-pot pathway to ECHHs by cross-linking AcGGM in the presence of AP in basic water under ambient conditions. Our hypothesis is that the concentration of the chosen cross-linker, epichlorohydrin, and conductive AP will alter the swelling ratio and conductivity and enable control over the hydrogel properties.
EXPERIMENTAL SECTION Materials. N-phenyl-1,4-phenylenediamine, pphenylenediamine, succinic anhydride (SA), ammonium persulfate ((NH4)2S2O8), phenylhydrazine, ammonium hydroxide (NH4OH), hydrochloric acid (HCl), sodium hydroxide (NaOH) and epichlorohydrin (ECH) were purchased from Sigma-Aldrich and used as received unless otherwise stated. O-acetyl-galactoglucomannan (AcGGM) originating from spruce (Picea abies) was extracted from thermo-mechanical pulping (TMP) processed water, purified and concentrated by ultrafiltration (membrane cutoff 1 kDa), and lyophilized using a Lyolab 300 lyophilizer. The carbohydrate composition of the AcGGM isolate was 17 mol % glucose, 65 mol % mannose, and 15 mol % galactose. AcGGM had a number average molecular weight (Mn) of approximately 7,500 g mol-1 (DP ~ 40), a dispersity (Đ) of 1.3 and a degree of acetylation (DSAc) of 30 %, as determined by size exclusion chromatography (SEC) calibrated with MALDI-TOF.33 Synthesis of Aniline Pentamer (AP). AP was synthesized according to a previously described procedure.34 In brief, 4-oxo-4-(4-(phenylamino) phenylamino) butanoic acid was synthesized by a reaction of N-phenyl-1,4phenylenediamine and succinic anhydride. Subsequently, oxidative coupling of 4-oxo-4-(4-(phenylamino) phenylamino) butanoic acid and p-phenylenediamine with two equivalent amounts of (NH4)2S2O8 as an oxidant yielded AP in the emeraldine state (EMAP). The leucoemeraldine state of aniline pentamer (LMAP) was obtained by the reduction of EMAP by phenylhydrazine. 1H NMR of LMAP (400 MHz, DMSO-d6, δ): 12.10 (s, 2H, -COOH), 9.70 (s, 2H, -NH-CO-), 7.64 (d, 2H, -NH-), 7.53 (s, 2H, -NH-), 7.38 (d, 4H, Ar-H), 6.81-6.96 (m, 16H, Ar-H). 13C NMR(100 MHz, DMSO-d6, δ) 174.12 (-COOH), 169.46 (-NH-CO-), 140.77 (Ar-C), 138.29 (Ar-C), 137.11 (Ar-C), 135.66 (Ar-C), 130.84 (Ar-C), 120.42 (Ar-C), 119.51 (Ar-C), 118.35 (Ar-C), 117.51 (Ar-C), 115.40 (Ar-C), 31.26 (-CH2-), 29.54 (-CH2-). These results agree with previous reports,34, 35 and the
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Chemistry of Materials
spectra are shown in the Supporting Information (Figure S1).
Each spectrum was recorded as the average of 16 scans at a resolution of 4 cm-1 between 4,000 and 600 cm-1.
Preparation of Electrically Conductive Hemicellulose Hydrogels (ECHHs). ECHHs were prepared by a one-pot reaction with varying feed compositions, as specified in Scheme 1 and Table 1. Briefly, 100 mg of AcGGM was dissolved in 1 mL of a 2 M NaOH aqueous solution followed by the addition of varied amounts of AP (Table 1). The solution was vortexed until homogenous; 90 mg of epichlorohydrin was then added as a cross-linker. The cross-linking reaction was conducted at room temperature for 24 h, and the formed gels were subsequently neutralized with 2 mL of 1 M HCl, followed by washing in de-ionized water for 48 h; the deionized water was changed frequently. The ECHHs were kept in deionized water until further characterized. The ECHHs with 10, 20, 30 and 40 % (w/w) AP were annotated according to their equivalents of AcGGM, % (w/w) of cross-linker, and % (w/w) of AP. As an example, AcGGM/90C/10%AP contained 100 mg of AcGGM, 90 mg of epichlorohydrin and 10 % (w/w) AP. Hydrogels without AP were synthesized as reference samples and denoted in the same way, for example, AcGGM/90C. Hydrogels prepared from varied amounts of epichlorohydrin were used to investigate the effect of the degree of cross-linking on the swelling ratio (SR) of the hemicellulose hydrogels. Table 1. Composition of hydrogels.
Elemental analyses of dried hydrogels were performed on an elemental analyzer (Eurofins Mikro Kemi AB, report No. 1400551-1400555) for carbon (C), hydrogen (H) and nitrogen (N) with a carrier gas (He, at a flow rate of 100 mL min-1) at a combustion temperature of 1000 °C.
Sample name
AcGGM (mg)
Epichlorohydrin (mg)
AP (mg)
AcGGM/60C
100
60
AcGGM/90C
100
90
AcGGM/120C
100
120
AcGGM/60C/
100
60
10
100
90
10
100
120
10
100
90
20
10%AP AcGGM/90C/ 10%AP AcGGM/120C/ 10%AP AcGGM/90C/ 20%AP AcGGM/90C/
100
90
30
100
90
40
30%AP AcGGM/90C/ 40%AP
CHARACTERIZATION FTIR spectra were recorded using a Perkin Elmer Spectrum 2000 spectrometer (Perkin-Elmer Instrument, Inc.) equipped with a single reflection attenuated total reflectance (ATR) accessory (golden gate) from Graseby Specac (Kent, UK). FTIR was used to verify the molecular structure of the pristine AcGGM and hydrogels in a dry state.
After treatment of AcGGM in de-ionized water and a 2 M NaOH solution for 24 h, pH measurements were carried out using a pH-meter equipped with an Ag/AgCl electrode. 1
H NMR spectra of AcGGM after its treatment in deionized water and in a 2 M NaOH solution for 24 h were obtained using a Bruker Avance 400 MHz NMR instrument at room temperature with D2O as the solvent and residual H2O as the internal standard (δ = 4.79). For AP, d6-DMSO was used as the solvent and residual DMSO as the internal standard (δ = 2.50). Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) was used to verify structural differences between AcGGM before and after treatment with basic water for 24 h. MALDI-TOF analyses were performed on a Bruker Ultraflex MALDI-TOF mass spectrometer with a SCOUT-MTP Ion Source (Bruker Daltonics, Bremen) equipped with an N2 laser (337 nm), grid-less ion source, and reflector design. The positive ion spectra depicted are representations of the sums of 10 000 laser shots. The instrument operated at an acceleration voltage of 25 kV and a reflector voltage of 26.3 kV. DHB (10 mg/mL aqueous solution) and analyst (1 mg/mL aqueous solution) were mixed in a 5:2 ratio, and 0.3-0.5 µL was dropped onto a steel coordinate plate. Data were collected and analyzed with FlexAnalysis software (Bruker Daltonics). For the thermal stability of the hydrogels, thermogravimetric analysis (TGA) of the samples was performed using a Mettler-Toledo TGA/SDTA 851e. Approximately 15 mg of each sample was put into a 70-μL ceramic cup without a lid. TGA tests were conducted under an N2 atmosphere (flow rate of 50 mL/min) with a heating rate of 10 ˚C/min from 30 to 800 ˚C. The data were collected and analyzed with Mettler STARe software. Dynamic mechanical thermal analysis (DMTA) was used to determine the mechanical properties of the different hydrogels in the swollen state. The analysis was performed using a Q800 DMA analyzer (TA Instruments, USA) in compression mode. Hydrogel discs with an approximately 15-mm diameter and 5-mm thickness were punched; the discs were equilibrated at room temperature (25 °C) for 1 min and then heated to 40 °C with a temperature ramp of 5 °C/min and frequency of 1 Hz. The reported value is taken at 25 °C. The swelling ratio (SR) of the hydrogels was determined by immersing dry hydrogels in buffer solutions (prepared from Na2HPO4 and NaH2PO4, pH=7.2-7.4) at room temperature. The weights of the samples in the swollen state (ms,t) at different time points were measured after gently removing excess water with filter paper. The
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SR was calculated using Equation (1), where md denotes the weight of the samples in the dry state: , % 100 1 Their equilibrium swelling ratio (q) was calculated as following: , 2 where ms,e is the constant equilibrium weight in the swollen state. The cross-section morphology of the hydrogels was observed by Ultra-High Resolution FE-SEM (Hitachi S4800). The samples were lyophilized overnight in small vials, cross-sectioned, attached to the sample supports using carbon tape and coated with a 6.5-nm gold layer. Three-dimensional morphologies of the hydrogels were obtained using a Skyscan 1172 X-ray computed tomography (micro-CT) desk scanner supported by microtomographic reconstruction software. After serial reconstruction, the 3D objects were achieved using CTAn and CTVol software with an accurate adjustment of brightness and manipulation of the grayscale thresholds. The hydrogels were cut into 5-mm × 5-mm specimens and lyophilized overnight in small vials. The electrical and Hall effect measurements of 1 mol/L doped hydrogels were taken on specially designed Hallbar MOSFET structures with a single long channel and 4 additional voltage pads, which allow measurements of the potential inside of the channel. The Hall effect measurements were performed with a magnetic field of 0.5 T at 22 °C. After doping, the hydrogels were thoroughly dried in a vacuum oven for 48 h. Therefore, the conductivity of the hydrogel was not affected by water molecules. Hydrogel films, with an area of 5-mm × 5-mm and a thickness of 0.2 mm, were made by casting the solution onto a glass substrate with an area of 5 mm × 5 mm. Four tiny indium blocks were put in the corners of hydrogel films for better contact between the hydrogel and four probes.
RESULTS AND DISCUSSION The electrically conductive hemicellulose hydrogels (ECHHs) prepared herein combine the conductivity of aniline tetramer/pentamer and the biodegradability and renewability of AcGGM. A family of ECHHs was prepared by developing a facile synthesis at ambient conditions (water and room temperature), offering a greener pathway than those previously presented. The ECHHs were prepared by combining AcGGM and aniline pentamer (AP). The properties of the hydrogels, such as swelling behavior and conductivity, were varied by altering the cross-linking density and the amount of added AP.
Preparation of ECHHs. Polyaniline (PANi) has been proven to be a useful polymer with biomedical applications; however, the material has poor solubility in commonly used solvents.36 Aniline oligomers have better solubility than PANi and have been considered for use in
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the biomedical field. The residual oligomers after degradation could be readily consumed by macrophages during the normal wound healing process, reducing the risk of adverse responses.37 The preparation of conductive materials using aniline derivatives in aqueous media is strongly limited by their poor solubility in water. To prepare electrically conductive hydrogels, however, we design aniline oligomers with carboxyl end-groups and conduct the reaction in a green solvent (basic water). AcGGM is a water-soluble hemicellulose.38, 39 Hemicellulose-based hydrogels were prepared in a one-pot reaction via the cross-linking of AcGGM with epichlorohydrin in the presence of AP and NaOH. Apart from solubilizing AP, NaOH works as a catalyst and a proton scavenger for the crosslinking reaction.40 The as-prepared hemicellulose hydrogels and ECHHs were free-standing (Figures 1a and 1b). Their mechanical properties were investigated using dynamic mechanical thermal analysis and are discussed in detail below. Hydrogels without AP were transparent (Figure 1c), while the ECHHs were opaque (Figure 1d) due to the dark color of AP.
Figure 1. Freshly prepared AcGGM/90C hydrogel (a, c) and AcGGM/90C/40%AP hydrogel (b, d).
ATR-FTIR was used to confirm the structural composition of the final ECHHs. The ATR-FTIR spectra of AcGGM, AcGGM/90C hydrogel and AcGGM/90C/40%AP hydrogel are shown in Figure 2. Pristine AcGGM displays the characteristic C=O stretching at 1727 cm-1 corresponding to the acetylated pendant groups, some C-O-C vibrations at approximately 1022 cm-1 from the sugar units, and a hydroxyl band at 3000-3600 cm-1 (Figure 2a). The disappearance of the peak at 1727 cm-1 suggests de-acetylation of AcGGM in AcGGM/90C and AcGGM/90C/40%AP (Figure 2b). The ATR-FTIR spectrum of AcGGM/90C/40%AP in Figure 2c shows not only amide group absorption at 1656 cm-1, the characteristic peaks of the benzenoid ring at 1595 and 1508 cm-1, and the stretching vibrational bands of the quinoid ring at 1568 cm-1 from AP41 but also the signals from the AcGGM/90C hydrogel, indicating that AP was incorporated into the hemicellulose-based hydrogels.
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Chemistry of Materials Elemental analysis provided information of the proportions of nitrogen (N), carbon (C) and hydrogen (H) in the hydrogels;42 the data are given as average concentrations in Table 2. The weight ratios of nitrogen in the samples were < 0.3, 1.1, 1.7, 1.9 and 2.3 % (w/w) for the AcGGM/90C, AcGGM/90C/10%AP, AcGGM/90C/20%AP, AcGGM/90C/30%AP and AcGGM/90C/40%AP hydrogels, respectively. The amount of nitrogen increases with increased AP concentration, which demonstrates that AP is retained inside the formed hydrogels. However, the amounts of nitrogen in the hydrogel matrix are higher than those in the original solutions for all hydrogels (theoretical values, Table 2). Possible reasons for this behavior include unreacted AcGGM and epichlorohydrin eluting during the purification process, deacetylation occurring and/or small molecules (such as H2O) forming during the reaction that could be removed during purification. These reasons could lead to an overall increase in the nitrogen content of the samples. Table 2. The theoretical and experimental values (% w/w) of C, H and N proportions in the hydrogels. Sample name
Figure 2. ATR-FTIR spectra of AcGGM (a), AcGGM/90C hydrogel (b), and AcGGM/90C/40%AP hydrogel (c).
Measurements of pH and 1H NMR and MALDI-TOF analyses verified that deacetylation of AcGGM occurred in the alkaline solution. The AcGGM aqueous solution without added NaOH had a yellowish color with a pH of 4.10 (Figure 3a). However, the solution containing 2 M NaOH was brown, and the pH significantly increased to 12.20. After 24 h, there were no significant changes in either pH or color of the two different solutions. The AcGGM aqueous solutions with and without NaOH after 24 h were analyzed by 1H NMR in D2O, as shown in Figure 3b. The AcGGM solution without added NaOH showed a characteristic signal at 2.08 ppm that was attributed to the methylene protons of the acetyl groups.30 In contrast, this peak shifted to the high field (1.85 ppm) due to the deacetylation of AcGGM in basic solution. In addition, the signal of the acetyl groups in AcGGM has multiple peaks, as the acetyl groups in different sugar units have slightly different chemical shifts. However, AcGGM in basic water exhibited a single peak at 1.85 ppm that corresponded to the methyl groups after deacetylation. With the aid of MALDI-TOF-MS, an entire series of AcGGM fragments with satisfactory intensity were observed up to a size of 22 units (DP = 22). In Figure 3c, AcGGM without NaOH treatment shows mass fragments with molar mass differences of 204 and 162 that correspond to the molar masses of sugar units with and without an acetyl group, respectively. For hemicellulose in alkaline solution, a single molar mass difference of 162 was observed, confirming the deacetylation of AcGGM. Therefore, the cross-linking reaction using epichlorohydrin (in basic media) simultaneously deacetylated the hemicellulose (Scheme 1).
N(% w/w)
C(% w/w)
H(% w/w)
theoretical values
0
42.3
3.9
experimental values
