Conductive and Tough Hydrogels Based on Biopolymer Molecular

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Functional Nanostructured Materials (including low-D carbon)

Conductive and Tough Hydrogel Based on Biopolymer Molecular Templates for Controlling in situ Formation of Polypyrrole Nanorods Donglin Gan, Lu Han, Menghao Wang, Wensi Xing, Tong Xu, Hongping Zhang, Kefeng Wang, Liming Fang, and Xiong Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10280 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018

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ACS Applied Materials & Interfaces

Conductive and Tough Hydrogel Based on Biopolymer Molecular Templates for Controlling in situ Formation of Polypyrrole Nanorods Donglin Gan1, Lu Han1, Menghao Wang1, Wensi Xing1, Tong Xu1, Hongping Zhang2, Kefeng Wang3, Liming Fang4, Xiong Lu1* Corresponding author: Xiong LU1 *, [email protected] 1

Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan, 610031, China 2

Engineering Research Center of Biomass Materials, Ministry of Education, School of Materials Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China

3

National Engineering Research Center for Biomaterials, Genome Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan, 610064, China 4

Department of Polymer Science and Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou, China;

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ABSTRACT Conductive hydrogels (CHs) have gained significant attention for their wide application in biomedical engineering owing to their structural similarity to soft tissue. However, designing CHs that combine biocompatibility with good mechanical and electrical properties is still challenging. Herein, we report a new strategy for the fabrication of tough CHs with excellent conductivity, superior mechanical properties and good biocompatibility by using chitosan framework as molecular templates for controlling conducting polypyrrole (PPy) nanorods in situ formation inside the hydrogel networks. First, polyacrylamide/chitosan (PAM/CS) interpenetrating

polymer

network

(IPN)

hydrogel

was

synthesized

by

UV

photopolymerization; Second, hydrophobic and conductive Py monomers were absorbed and fixed on CS molecular templates, then polymerized with FeCl3 in situ inner hydrophilic hydrogel network. This strategy ensured that the hydrophobic PPy nanorods were uniformly distributed and integrated with the hydrophilic polymer phase to form highly interconnected conductive path in the hydrogel, endowing the hydrogel with high conductivity (0.3 S/m). The CHs exhibited remarkable mechanical properties after the chelation of CS by Fe3+ and the formation of composites with the PPy nanorods (fracture energy 12000 J m−2; compression modulus 136.3 MPa). The use of a biopolymer molecular template to induce the formation of PPy nanostructures is an efficient strategy to achieve conductive multifunctional hydrogels.

Keywords: Conductive hydrogel, Tough hydrogel, Chitosan, Polypyrrole, Molecular Template


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1. INTRODUCTION Conductive hydrogels (CHs) are polymeric composites that consist of a conductive component containing highly hydrated polymer networks. CHs are promising candidate materials in the field of biomedical engineering because they show similar characteristics as soft tissue, such as flexibility, stretchability, and high water content. CHs can be potentially used in an extensive range of applications such as in artificial muscles,

1-2

cartilage,

3-4

electronic skin,5-6 and biosensors.7-8 Recently, CHs have also been used in drug delivery systems as they provide safe and efficient delivery of drugs by enabling controllable drug delivery through programmable electrical stimulation9-14. CHs are also used to provide electrical, electrochemical, and electromechanical stimulation to cells, such as mesenchymal stem cells,15 endothelial cells and nerve cells,16-18 to regulate various cell activities.

CHs are typically prepared by incorporating conductive nanomaterials or inherently conductive polymers into a hydrogel matrix. However, conductive nanomaterials such as graphene, carbon nanotubes (CNT), or Ag nanowires have the tendency to aggregate during hydrogel formation. This aggregation prevents the formation of conductive percolation paths; therefore, composite hydrogels based on conductive nanomaterials display poor electrical properties. Conductive polymers such as polythiophene (PT), polyaniline (PANI), poly (3,4ethylenedioxythiophene) (PEDOT), polypyrrole (PPy), and poly (paraphenylene-vinylenes) 1920

are also used to prepare CHs. For instance, a pure PPy hydrogel with a high electrical

conductivity of 0.5 S/m has been synthesized. However, the pure PPy hydrogel was brittle and had poor mechanical properties.21 To improve the mechanical properties of conductivepolymer-based hydrogels, conductive polymers have been copolymerized with synthetic polymers

such

as

polyacrylamide

(PAM),

polyacrylic

acid

(PAA),

poly(N-

isopropylacrylamide) (PNIPAM), and poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS).22-23 However, the conductivity of the CH networks deteriorated with the inclusion 3 ACS Paragon Plus Environment


of an insulating polymer block. In addition, the biocompatibility of commonly reported conductive-polymer-based CHs is not adequate because conductive polymers have very few active groups such as -NH2, -OH, that interact with tissue or cells. In summary, the preparation of CHs that combine biocompatibility with good mechanical and electrical properties is still challenging.

Most conductive polymers are hydrophobic, whereas typical hydrogel polymers are hydrophilic. Therefore, conductive polymers cannot be easily incorporated into hydrophilic hydrogels as homogeneous conductive reinforcement. Furthermore, it is also challenging to improve the compatibility of hydrogels and uniformly distribute the hydrophobic conductive polymer in the hydrophilic hydrogel network to construct a well-connected electrical path. To address this problem, natural polymers such as chitosan (CS), gelatin, cellulose, and hyaluronic acid have been used to prepare CHs. 24 This is because the molecular structures of natural polymers have a good affinity toward conductive polymers, which can lead to the integration of conductive polymers with hydrogel networks. For example, Banerjee et al. 25-27 reported that the hydroxyl groups of CS can be active sites that enable the homogenous blending of the conductive polymer with a hydrogel matrix. Qiu et al.28 reported the integration of PPy nanoparticles with gelatin-methacrylate and poly(ethylene glycol) diacrylate to form a cryogel. Furthermore, natural polymers also form physical crosslinks through hydrogen bonding or chain entanglement, which enhances the mechanical properties of hydrogels. In addition, these natural polymers can enhance cell adhesion and proliferation, which improves the biocompatibility of the CHs.

29-30 31-32

These previous studies inspired us

to employ natural polymers to prepare flexible and conductive hydrogels that overcome some of the CH-related challenges described earlier.


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Herein, we developed a tough and conductive polypyrrole and polyacrylamide/chitosan (PPyPAM/CS) CH with excellent conductivity, superior mechanical properties, and good biocompatibility. The hydrogel was obtained by employing an interpenetrating CS framework as the molecular template for controlling the in situ polymerization of the conductive-polymer in the inner hydrogel networks. The as-prepared CH displayed a compressive strength of 6.5 MPa, stretchability of > 250%, and a fracture energy of 12000 J m−2. The CH also had a compressive modulus of up to 136 MPa. The conductivity of the CH was as high as 0.3 S/m, which was comparable with that of a hydrogel entirely composed of a conductive polymer. The biocompatibility and electroactivity of the CHs improved the cell activity and skin tissue regeneration, as demonstrated by an in vitro cell culture and in vivo implantation for wound repair.

2. EXPERIMENTAL SECTION 2.1 Synthesis of Hydrogels Synthesis of PAM/CS Interpenetrating Polymer Network (IPN) hydrogel: The IPN hydrogel was synthesized by UV photopolymerization. First, different quantities of CS (0.25 g, 0.5 g, 0.75 g) were dissolved in 10 mL of deionized water at room temperature (RT). Then, fixed amounts of AM (2.6 g), N, N’-methylene bisacrylamide (1 wt.%), ammonium persulfate (APS), (1.5 wt.%) were added to this CS solution under continuous magnetic stirring. The obtained mixture was injected into a mold and exposed to UV light (365 nm, 2.8 mW/cm2) under the protection of nitrogen gas. The IPN hydrogel was ready after 5 min.

Synthesis of PPy-PAM/CS: The as-prepared IPN hydrogel was soaked in the pure Py monomer for 24 h under ambient conditions. The Py content was 10 v/v% and 20 v/v% in the PPy-PAM/CS hydrogel. The hydrogel was then stored in a sealed container at 4 °C. After 3 h, the Py monomers had permeated into the IPN hydrogel. Then, the IPN hydrogel was soaked 5 ACS Paragon Plus Environment


in a FeCl3 aqueous solution (0.924 mol/L) and stored at 3–4 °C for 12 h to allow Py to polymerize into PPy. The Py/FeCl3 mass ratio of was 1:3. The ratios of the different components used for preparation of the hydrogels are listed in Table S1.

2.2 Characterization Nanostructure: The fresh hydrogel sample was lyophilized at −80 °C. Then, the freeze-dried specimen was carefully slit and the cross-sectional morphology was observed by scanning electron microscopy (SEM, JSM 6390, JEOL, Japan).

Mechanical Properties: The hydrogels were molded into cylindrical specimens for compression testing and rectangular specimens for tensile testing. The mechanical property measurements of the hydrogels were conducted using a universal testing machine (5567, Instron, America) with a 2 kN load cell. The fracture energy was measured using a classical single edge notch test described in a previous study33. The tensile and compression test data were reported based on the average of four measurements. Details of the mechanical testing experiments are described in the SI.

Conductivity Measurement and Application as Sensors: The conductivities of the CHs were measured using a 2-point probe. The details of the conductivity tests are described in the SI. To demonstrate the sensing capability of the hydrogels, they were attached to a human wrist to sense the releasing and clenching of the hand. The current variations during the release/clenching cycles were recorded by an electrochemical system using the Amperometric i–t curve technique.


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Swelling Test: The swelling behavior was tested by soaking the hydrogel in a large amount of PBS solution at room temperature until swelling equilibrium was reached. The swelling ratio was calculated using the following equation: SR =

ௐೞ ିௐ೏ ୛೏

× 100%, (Equation 1)

in which Ws and Wd are the weights of swollen hydrogel and the nonswollen hydrogel, respectively. The average of four measurements was taken for each sample.

Drug Loading and Electrostimulated Release: Dexamethasone sodium phosphate was added in to the prepolymer solution when the hydrogel was synthesized. The content of the dexamethasone sodium phosphate in the CHs sample was 2 mg/g. The drug-loaded CHs were connected to a circuit, where they were used as the working electrode. The CHs were then soaked into PBS when the electrostimulated drug release was conducted, as shown in Figure 4. The voltages were set as −1 V and −3 V and a Pt plate was used as the reference electrode. The amount of dexamethasone sodium phosphate released was determined by measuring the absorbance of the collected solution at 242 nm using a UV-Vis spectrophotometer (Lambda 35, PerkinElmer, Hopkinton, MA, USA).

2.3 Electrostimulated Cell Culture Electrostimulated cell culture was conducted using the method described in our previous report34. Briefly, C2C12 (Stem Cell Bank, Chinese Academy of Sciences, SCSP-515) was cultured on PAM/CS, 10%PPy-PAM/CS, and 20%PPy-PAM/CS hydrogels with different electrostimulating potentials (0, 300, 600 and 900 mV) using a self-made high-throughput device. The electrostimulation was applied for 30 min each day. After 7 d, the C2C12 cultures on the sample were evaluated using fluorescence staining and a MTT assay. Further details are described in the SI. 7 ACS Paragon Plus Environment


2.4 Animal Experiments The wound healing experiment was conducted according to our recent report35. The experiments were performed in accordance with protocols approved by the local ethical committee and the laboratory animal administration rules of China. Full skin defects were created in five SD rats (about 200 g) and treated with PAM/CS, PPy-PAM/CS, and EGFloaded PPy-PAM/CS hydrogels. The defects without an applied hydrogel were treated as blank groups. After 0, 7, 14, and 21 d post-surgery, the percentage of wound closure was calculated from photographs of the wound. The surrounding skin was harvested and stained with hematoxylin & eosin (H&E) to evaluate the skin tissue regeneration. Further details are described in the SI.

3. RESULTS AND DISCUSSION 3.1 Design Strategy and Preparation The tough and conductive PPy-PAM/CS hydrogel contained a chemically and physically crosslinked hybrid network, which was produced by a three-step procedure (Scheme 1). First, a PAM/CS interpenetrating polymer network (IPN) hydrogel was synthesized by UV photopolymerization of acrylamide (AM) monomers in the presence of CS. The photoinitiated polymerization was used because this process is mild, which can well preserve the microstructure of the hydrogel. In particular, the entanglement zone of the CS chain can be maintained well in the hydrogel, which plays an important role for the in situ formation of PPy nanorods in the hydrogel network. In addition, use of the toxic additive is avoided during photo-initiated polymerization, and therefore the hydrogel has good biocompatibility. Second, the PAM/CS IPN hydrogel was swollen to adsorb pyrrole (Py) monomers. The Py monomers were immobilized in the PAM/CS IPN hydrogel through noncovalent interactions between the active functional groups of the CS chains and the hydrogen atoms of the Py monomers, as 8 ACS Paragon Plus Environment

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confirmed by density functional theory calculations and thermogravimetric (TG) analysis (details displayed in Supporting Information). Finally, the Py-absorbed PAM/CS IPN hydrogel was immersed into an FeCl3 solution and the Py monomers were oxidized into PPy on CS molecular templates. The PPy nanorods formed in the area where chitosan chain entanglement was present, where a large amount of Py monomers aggregated. At the same time, the CS chains were physically crosslinked through chelating bonds between the CS chains and the Fe3+/Fe2+ ions. The interaction between PPy and the CS chains also contributed to the physical crosslinking. After this step, the IPN in the hydrogel turned into a chemically and physically crosslinked hybrid network, which endowed the hydrogel with superior mechanical properties.

Introducing CS as an interpenetrated biopolymer framework in the hydrogel for absorbing Py and controlling the in situ formation of PPy nanorods was critical for fabricating such a conductive and tough hydrogel. First, CS has an exceptional affinity for the Py monomers. The CS chains interact with PPy chains through the hydrogen bonding of the active amino and hydroxyl groups on the chitosan with the hydrogen atoms of PPy 36. After CS was introduced in the PAM, the Py monomers were readily immobilized and the absorption ratio was as high as 20 wt.%. Thus, with the assistance of the CS framework, the Py monomers could permeate throughout the PAM/CS IPN hydrogel, as demonstrated in Figure S2a. Second, the interpenetrating CS chains in the hydrogel had three-dimensional nanostructures; therefore, the CS chains acted as templates that control the polymerization of Py to PPy for building a well-connected conductive path. During this progress, PPy nanorods also formed in the CS chain entanglement zones, which were also uniformly distributed in the hydrogel, endowing high conductivity to the hydrogel. Third, CS improved the mechanical properties of the hydrogel. The CS frameworks in the IPN network bridged the conductive polymer and PAM networks. The CS could interact with PAM through electrostatic interactions between the 9 ACS Paragon Plus Environment


active groups of CS and the amide groups of PAM. Furthermore, CS was physically crosslinked by Fe ions (Fe3+, Fe2+), which further enhanced the mechanical properties of the CHs. Without CS, the bare PAM hydrogel could not even maintain its shape after immersion into an FeCl3 solution (Figure S2b), because Fe ions break the internal noncovalent bonds in the PAM network. Finally, CS also improved the biocompatibility of the hydrogel so that the hydrogel was able to make intimate contact with cells, thereby enhancing the cell activity and tissue regeneration.

3.2 Nanostructures in the Hydrogel The SEM micrographs in Figure 1 prove that CS facilitated the absorption of Py and acted as a template to control the formation of PPy nanorods. The pure PAM hydrogel showed microporous structures with large flake-like substructures (Figure 1a). The PAM/CS IPN hydrogel had a seaweed-like architecture (Figure 1b), indicating that the CS and PAM chains were entangled with each other and formed dense structures. When the Fe ions entered the IPN hydrogel, the seaweed-like structure was destroyed and a dense structure appeared (Figure S3a). This structure was attributed to the chelation between Fe ions and CS. After Py was absorbed into the PAM/CS IPN hydrogel and polymerized in the presence of the FeCl3 solution, the hydrogel became more porous (Figure 1c), which suggested that the PPy broke the dense crosslinking between the CS and Fe ions. The porosity of PAM/CS, PAM/CS/PPy20%, PAM/CS/Fe3+ hydrogel was 57%, 34%, 15%, respectively (Table S2). The high-magnification micrograph showed that nanorod-like PPy appeared on the wall of the hydrogel micropores (Figure 1d), which were completely different to the nanospherical PPy formed in the aqueous solution (Figure S3b).

The formation of PPy nanorods was ascribed to the templating effect of CS. During the in situ formation process, the nanorods form in entanglement zones of CS in the PPy-PAM/CS 10 ACS Paragon Plus Environment

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hydrogel. XRD analysis further proved the role of the CS entanglement zones in the PPy formation. As shown in Figure S4, XRD spectra demonstrate the change in the CS entanglement zones during the PPy formation process. In the hydrogel without PPy, there is a strong peak in the CS spectrum at 21.8°, indicating the entanglement zones of CS37. In the hydrogel with PPy, the peak became weak, which shows that the entanglement zones were partially disrupted by PPy. During the PPy polymerization process, the PPy chain grow along the CS chain templates because of the noncovalent interactions between the active functional groups of the CS chains and the hydrogen atoms of the Py monomers. In particular, a large number of Py monomers aggregated in the entanglement zones of CS, and therefore weakened the hydrogen bond between CS chains. Consequently, the entanglement zones of CS were reduced. In previous reports, the polymerization of PPy in the aqueous solution generally utilizes high-speed homogenization or ultrasonication to disperse the Py monomers homogeneously in the solvent38-39. The oil-in-water emulsions form under this condition. Thus, the Py monomers were polymerized and grown in the emulsions to form the PPy with nanospherical structure. The current study provides alternative way to control the formation of PPy, and demonstrated that the nanostructures of PPy can be manipulated by biopolymer molecular templates.

3.3 Mechanical Properties The PPy-PAM/CS hydrogel showed outstanding mechanical properties. As shown in Figure 2a, the hydrogel was highly stretchable and flexible. The hydrogel was strong enough to sustain a weight of 500 g. It could also be tied and stretched to 2 times its initial length. Figure 2b shows the typical compressive stress–strain curve of the hydrogel. The compressive strength of the hydrogel was improved after the CS was interspersed into the PAM hydrogel (Figure S5). After PPy was in situ polymerized in the presence of FeCl3, the PPy-PAM/CS hydrogel reached a high compressive strength of 5 MPa, which was 10 times higher than that 11 ACS Paragon Plus Environment


of the pure PAM hydrogel. The compressive strength increased with increasing CS concentration and reached the highest value of 6.5 MPa when the CS content was 0.075 wt. % (Figure 2c). Furthermore, the compression modulus of the PPy-PAM/CS hydrogel was also affected by the concentration of CS and reached a maximum of 136.3 MPa, which was 20 times higher than that of the pure PAM hydrogel (Figure 2d). The tensile strength and fracture strain of the PPy-PAM/CS-0.075 hydrogel were also high—0.8 MPa and 260%, respectively (Figure 2e, f). The tensile modulus of the PPy-PAM/CS hydrogel increased from 7.5 MPa to 50.1 MPa with increasing CS content (Figure 2g). The fracture energy increased 10-fold from 1.2 kJ m−2 to 12000 J m−2 when the CS content increased from 0 to 0.075 wt.%, respectively (Figure 2h). In short, both CS interpenetration and PPy nanorods reinforcement contributed to the excellent mechanical properties of the hydrogels. First, the mechanical strength and fracture energy of the CS-PAM hydrogel increased with increasing CS content. Second, after the formation of the PPy nanorods, the fracture strain was significantly reduced, whereas the mechanical strength and fracture energy was substantially enhanced. With the synergistic effects of CS interpenetration and PPy nanorods reinforcement, the current hydrogel exhibited good mechanical properties.

The superior mechanical properties of the hydrogel were attributed to the chemically and physically crosslinked hybrid network. The covalently crosslinked PAM network bore loads up to large deformations, and the CS interpenetration further strengthened the network. The various noncovalent physical bonds in the hydrogel network played an important role in dissipating energy during this deformation, which mainly comprised two aspects. First, the CS framework formed physical crosslinks through hydrogen bonding and chain entanglement to enhance the mechanical properties of hydrogel. Second, the Fe crosslinking further enhanced the mechanical properties of the hydrogel as the active groups of CS formed coordinative bonds with Fe ions. In addition to the crosslinked hybrid network, the in situ 12 ACS Paragon Plus Environment

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formed rigid PPy nanorods also enhanced the mechanical properties of the hydrogel, acting as nano reinforcements with excellent interaction with the matrix polymer chains.

Rheological tests were conducted to characterize the viscoelastic properties of the hydrogel (Figure S6). When the CS content increased, the storage moduli of the PPy-PAM/CS hydrogels increased and reached 100 kPa, which indicated that the crosslinking density of the hydrogel increased with the CS content because the extent of the noncovalent bonding between CS and PAM increased. We further investigated the effect of PPy on the crosslinking density of the hydrogel by testing the swelling behavior of the conductive hydrogels (Figure S7). The swelling of the PAM/CS hydrogel in PBS increased with time until a swelling equilibrium was reached after 100 h. Compared with the pure PAM hydrogel, the equilibriumswelling ratio of the hydrogels decreased after the PPy was incorporated. The low swelling ratio of the PPy-PAM/CS hydrogel was attributed to the improved crosslinking density in the hydrogel owing to the more rigid underlying network and chain entanglement through chelation between the Fe ion and the active groups on the CS and PPy chains. The low swelling ratio allowed the hydrogels to maintain their shape and prevent them from shedding when implanted for tissue regeneration.

3.4 Conductivity and Use as Sensors The hydrogels had good conductivity. As shown in Figure 3a, the hydrogel could be incorporated into a circuit that lights up an LED. Figure 3b shows that the conductivity reached a maximum of 0.3 S/m with a PPy concentration of 20 v/v%. The hydrogel can be used as a sensor, which is vital for the human body monitoring applications (Figure 3c, d). The sensitivity of the sensor was evaluated using the ∆I/Ioff value, in which ∆I and Ioff are the electric current change with and without movement or strain, respectively. The sensor can monitor the human movements, such as the release and clenching of a hand. The hydrogel 13 ACS Paragon Plus Environment


could sense the current variation in the circuit during the clenching of each hand. Furthermore, the hydrogel was also sensitive to small loads. After a weight of 20 g was loaded on the CHs, a distinctive peak of ∆I/Ioff = 50% was observed. These results suggest that the CH could be used as a smart bracelet to monitor human body movement.

3.5 Electrostimulated Drug Delivery The hydrogel can also be used for programmable drug release by accurately controlled electrostimulation, as demonstrated by the electrically stimulated release of a model drug from the hydrogel. To prepare a drug-loaded hydrogel, dexamethasone was chosen as the model drug and mixed with the pre-polymer solution. After polymerization, the drug was trapped in the hydrogel network and doped into the PPy chains by electrostatic interaction (Figure 4a). As shown in Figure 4b, the PAM/CS IPN hydrogel had an unstimulated burst release of the model drug after 30 min. In contrast, the PPy-PAM/CS hydrogel could not release dexamethasone without electrical stimulation. After the application of electrical stimulation, the PPy-PAM/CS hydrogel displayed a sustained, controllable release of the drug (Figure 4c and 4d). The cumulative release was improved by increasing the PPy content of the hydrogel, and the release rate was controlled by the stimulation voltage. The release of the drug from the hydrogel was attributed to the redox reaction of PPy. During the oxidative polymerization process, the free anionic drug could be incorporated in the PPy backbone as a dopant molecule. When the PPy was electrochemically reduced under negative potentials, the anionic drug was released because the positive charge state of the polymer backbone was changed 40-41.


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3.6 Electrostimulated Cell Culture The hydrogel can be used to regulate cell behavior. We evaluated the morphology, adhesion, and spreading of C2C12 (Muscle myoblast, mouse) on hydrogels with different conductivities and electrostimulation voltages (Figure 5a). The electrostimulation of C2C12 was performed in the potential range of 0 to 900 mV (Figure 5b). Fluorescence images indicated that the adhesion of C2C12 on the PPy-PAM/CS hydrogel was better than on the PAM/CS hydrogel. The electrostimulation affected the proliferation activity of the cells. When the electrostimulating potentials were less than 600 mV, the proliferation activity of C2C12 increased with increasing electrostimulation potentials in both 10v/v% PPy-PAM/CS and 20v/v% PPy-PAM/CS. When the potential was larger than 600 mV, the proliferation activity of C2C12 was decreased. The composition of the hydrogel also affected the cell proliferation. Considering both the external electric voltage and the PPy content, the highest proliferation activity appeared when the potential was 300 mV and the PPy content was 20 v/v% (Figure 5c). The aspect ratio of the cells, which is one of the indicators of the earliest stage of myotubes formation, indicated the multidirectional growth of C2C12.42-43 The aspect ratio of C2C12 was also affected by the electrostimulation. The C2C12 on PPy-PAM/CS hydrogels were more elongated than those on the PAM/CS hydrogel (Figure 5d) after electrostimulation. The aspect ratios of C2C12 on the 10 v/v% PPy-PAM/CS sample under electrostimulation voltages of 0, 300, 600, 900 mV were 1.1, 2.6, 4.1, 2.7, respectively. This result indicated that an optimal electrostimulation was needed to promote the elongation of C2C12 on the conductive hydrogel. Furthermore, the conductivity of the hydrogel also had a strong influence on the elongation of C2C12. The highest aspect ratio was also obtained when the potential was 300 mV and the PPy content was 20 v/v%. Interestingly, cell elongation was observed on the 20%PPy-PAM/CS hydrogel even without electrostimulation, which was 15 ACS Paragon Plus Environment


because the conductive hydrogel can itself deliver the electrosignals to the cells to improve the activity of cells.44 In short, cell activity could be promoted under electrostimulation, and most importantly, the conductive hydrogel itself improved the activity of the C2C12.

3.7 Hydrogel Used for Wound Repair The in vivo implantation studies indicated that the PPy-PAM/CS hydrogel could promote skin wound repair (Figures 6a). After 21 days, the wound treated with a PPy-PAM/CS hydrogel was smaller than those treated with the PAM/CS hydrogel and the blank group (Figure 6b). The wound treated with the PPy-PAM/CS hydrogel was covered with hair, whereas the wounds of the blank group were not closed and were covered with bloody scabs. The skin wound closure ratio was used to quantify the wound healing ability of the hydrogel (Figure 6c). The wounds treated with PPy-PAM/CS hydrogel had a healing ratio of 95.4%, which was higher than that of the blank groups (85%) and the PAM/CS hydrogel (90%). After loading the PPy-PAM/CS hydrogels with epidermal growth factor (EGF), the repairing ability of PPyPAM/CS was further improved. Histomorphological evaluation of wound regeneration also indicated that the PPy-PAM/CS hydrogel could accelerate tissue regeneration (Figure 6d). The wounds treated with PPyPAM/CS hydrogel were covered by regenerated epithelial tissue. The new epithelial tissue was as thick as that of healthy skin. The new tissue only left 1900 µm and a few blood vessels appeared, which was better than that of blank groups (4900 µm) and PAM/CS groups (2700 µm). In sharp contrast, the wounds treated with EGF-loaded PPy-PAM/CS hydrogel showed the best repair among all the wounds. These wounds also displayed the highest reappearance of blood vessels and hair follicles in the regenerated-tissue area. These results indicated that the PPy-PAM/CS hydrogel was favorable for wound healing. 16 ACS Paragon Plus Environment

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These in vitro cell culture and in vivo wound-repair results suggest that the PPy-PAM/CS hydrogel promoted cellular activity such as cell adhesion, proliferation, elongation of electrically excitable cells, and regeneration of skin tissue. The good cell affinity and woundrepairing ability of the PPy-PAM/CS hydrogel was attributed to the CS-template-induced PPy nanorods in the hydrogel. PPy is an electroactive material that promotes the propagation of electrical signals between cells and improves cell excitability by forming tight contacts with the cell membrane45-49. The electrical signals are important factors in controlling the physiological conditions in tissue and cells50-52. A recent report has demonstrated that PPy itself could support the growth, adhesion, and differentiation of fibroblasts, mesenchymal stem cells, and neural cells53-54. However, PPy does not have good biocompatibility and cannot support cell activity for a long term. In the present study, PPy was closely bound with CS templates, which improved the biocompatibility of PPy. The CS possesses many advantages for wound-repair dressings such as hemostatic activity and antibacterial activity. Furthermore, it has many active hydroxyl and amino groups for promoting cell adhesion. In short, the PPy integrating with CS templates results in a hydrogel with good bioactivity and electroactivity for tissue engineering.

4. CONCLUSIONS In summary, although CS and PPy are commonly used to prepare conductive hydrogels, herein we designed a novel biopolymer molecular template-based method to prepare tough, conductive, biocompatible hydrogels that are mainly composed of CS and PPy. A CS molecular framework acted as a template supporting the formation of hydrophobic PPy nanorods in a hydrophilic hydrogel matrix. The PPy nanorods were uniformly distributed in the PAM/CS IPN hydrogel, thereby forming electric pathways to endow high conductivity to the hydrogel. This new strategy of in situ synthesis of PPy nanorods controlled by biopolymer 17 ACS Paragon Plus Environment


molecular templates overcomes the challenge of homogenously incorporating hydrophobic conductive polymers into hydrophilic hydrogels to form a percolating conductive network. The success of the demonstrated strategy is a breakthrough for developing conductivepolymer-based hydrogels.

The as-prepared hydrogel incorporated with biopolymer-molecular-template-induced PPy nanorods possesses excellent properties, which are described in detail as follows. 1. Good conductivity: Compared with the previously reported CS and PPy systems55-57, the conductivity of conductivity hydrogel (CH) is as high as 0.3 S/m, which is comparable to that of the hydrogel produced by a pure conductive polymer21, and higher than most values reported in composite conductive hydrogels (Figure 7). This is because the current hydrogel is prepared by a biopolymer template method, and Py is absorbed and fixed on the CS chain in the hydrogel. Then, PPy is synthesized along the chain of CS templates and forms nanorods, which are uniformly distributed in the hydrogel to endow the gel with good conductivity. 2. Good mechanical properties: The hydrogel is resilient, stretchable, and tough; attaining these properties was a challenge in previously reported conductive hydrogels46, 58. It can be compressed to 80% of its initial height with a compressive strength of 6.5 MPa, and the toughness is as high as 12 kJ/m2, which is comparable to that of the so-called “tough hydrogels.” The hydrogel was tougher and more robust than previously reported conductive hydrogels (Figure 7), which was ascribed to the chemically and physically crosslinked hybrid network in the hydrogel. 3. Electro-activity and biocompatibility: The PPy nanorods give the hydrogel good electroactivity and biocompatibility. This hydrogel favors propagation of electrical signals to cell/tissue, and consequently improves cell activity and accelerates wound regeneration. CS also endows the hydrogel with cell affinity and tissue adhesion.


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These properties are strongly required for versatile applications of conductive hydrogel, such as wearable electronic devices, electronic skin, and drug delivery systems. Thus, this biopolymer-molecular-template-induced PPy-nanorod composite multifunctional hydrogel holds promise in a variety of applications such as wearable electronic devices, wound dressings, sensors, and electrostimulated drug release systems.

SUPPORTING INFORMATION Supporting Information is available from the ACS Publications website or from the author. Detailed procedure for materials, fracture energy test, conductivity measurements, cell culture, animal experiments, porosity of the hydrogels, density functional theory study, and thermogravimetric analysis and additional figures and tables.

ACKNOWLEDGEMENTS This work was financially supported by the National key research and development program of China (2016YFB0700800), NSFC (81671824), Fundamental Research Funds for the Central Universities (2682016CX075,2682018QY02). The authors wish to acknowledge the assistance on materials characterization received from Analytical & Testing Center of the Southwest Jiaotong University.

CONFLICT OF INTEREST The authors declare no conflicts of interest.



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Scheme 1. Schematics of polypyrrole (PPy) composited conductive and tough hydrogels. (a) Synthesis of polyacrylamide/chitosan interpenetrating (PAM/CS IPN) hydrogels. (b) Pyrrole (Py) was absorbed into the IPN hydrogel, fixed on CS chains, and accumulated in the zone of CS entanglement, (c) PPy was in situ polymerized in the hydrogel under the controlling of CS molecular templates. PPy conductive pathway (purple line) intertwist along CS chains (purple line), and PPy nanorods aggregated on the chain entanglement zone of CS.


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Figure 1 Morphology of the various hydrogels. (a) PAM hydrogel. (b) PAM/CS IPN hydrogel. (c) PPy-PAM /CS conductive hydrogel. (d) Magnified image of circle area in Figure 2c, showing that PPy nanorods uniformly embedded in the hydrogel.



Figure 2 Mechanical properties of the hydrogels. (a) Photographs of PPy PAM/CS hydrogel (PPy 20v/v%, CS 5wt.%) under loading or twisting; (b). Typical compression stress-strain curves of the various hydrogels; (c). Compression strength of the hydrogel with different contents of CS; (d). Compression modulus; (e). Typical tensile curves; (f). Tensile strength of the hydrogel with different contents of CS; (g) Tensile modulus; (h) Fracture energy of PPy (20 v/v%)-PAM/CS hydrogel with various contents of CS;


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Figure 3 The conductive properties of the hydrogel. (a) PPy-PAM/CS hydrogel was connected to a circuit and illuminated an LED; (b) The conductivity of the PPy-PAM/CS hydrogel with different contents of PPy; (c) the hydrogel was attached to author’s wrist to real-time monitor human body motion by detecting electric current change; (d) The hydrogel as a stress sensor to detect load.



Figure 4 (a) Schematic representation of dexamethasone (DEX) load and release from the hydrogel. The drug was released by the redox process when negative potential was applied. b) Without electrostimulation, DEX was burst released from PAM/CS hydrogel, whereas no DEX was released from the PPy-PAM/CS hydrogel. (c) Under electrostimulation, DEX was released from hydrogels with different PPy contents. (d) Under electrostimulation, DEX was released from the 20%PPy-PAM/CS hydrogel at different potentials.


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Figure 5 Electrostimulation of C2C12. (a) high-throughput stimulation of C2C12 on the hydrogel with various contents of PPy and electric potentials. (b) Fluorescence micrographs of C2C12 cultured on PPy-PAM/CS and PAM/CS hydrogels. (c) Proliferation of C2C12 obtained by MTT analysis, (d) Cell aspect ratio of the C2C12




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Figure 6 Hydrogels for repairing full thickness defects on rats. (a) Schematics of hydrogel implantation. (b) Representative photos of gross appearance of defects treated with PAM/CS, PPy-PAM/CS, and EGF-loaded PPy-PAM/CS after implantation for different period. (c) Percent of wound closure. (d) hematoxylin & eosin staining of wound sections after 21 days S, sample. BV, blood vessel.



Figure 7 Conductivity and mechanical strength of recently reported composite conductive hydrogels from references.31, 58-65


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Table of Contents Graphic


Conductive and Tough Hydrogels Based on Biopolymer Molecular

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