Design of self-healing and electrically conductive silk fibroin-based

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Design of self-healing and electrically conductive silk fibroin-based hydrogels Lichao Liu, Yueying Han, and Shanshan Lv ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04871 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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ACS Applied Materials & Interfaces

Design of self-healing and electrically conductive silk fibroin-based hydrogels Lichao Liu1‡, Yueying Han1‡, Shanshan Lv1*

1State

Key Laboratory of Organic-Inorganic Composite Materials, College of Chemical

Engineering, Beijing University of Chemical Technology, 15 BeisanhuanDong Road, Chaoyang District, Beijing, China 100029

ABSTRACT Self-healing and electrically conductive silk fibroin (SF)-based hydrogels were developed based on the dynamic assembly/disassembly nature of supramolecular complexes and the conductive nature of polypyrrole (PPy). The self-healing properties of the hydrogels were achieved through host-guest interactions between β-cyclodextrin (β-CD) and amino acid side chains (tyrosine, tryptophan, phenylalanine and histidine) on SF. PPy deposition was achieved via in situ polymerization of pyrrole using ammonium persulfate as an oxidant and laccase as a catalyst. The PPy-coated hydrogels behaved as an elastomer and displayed excellent electrical properties, with adjustable electrical conductivities ranging from 0.8±0.2 to (1.0±0.3)×10-3 S·cm-1. Furthermore, possibility of potential utilization of the hydrogels in electrochemistry applications as flexible yet self-healable electrode materials was explored. This study not only shows great potential in expanding the role of silk-based devices for various 1

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applications, but also provides a useful approach for designing multifunctional self-healing protein-based hydrogels.

KEYWORDS Self-healing; electrically conductive; β-cyclodextrin; polypyrrole; silk fibroin-based hydrogels.

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INTRODUCTION Self-healing and electronically conductive hydrogels have emerged as promising materials for a broad range of applications such as wearable electronics, artificial skin, soft robotics, energy storage materials in supercapacitors, bioelectrodes, implantable amperometric biosensors, electro-stimulated drug release devices, and neural prostheses. Such self-healing and electronically conductive properties are highly relevant in applications in which it is essential that material structures can be maintained/restored and consequently improving the reliability and durability to accomplish their functions, especially in sophisticated in vivo environment under high loads during biomedical applications, and other high risk situations such as deep-sea and space travel. 1-3 Hence, considerable efforts have been devoted to empower hydrogels with conductive properties and self-healing ability 4, 5. For instance, Sottos, White, Moore, Hersam and co-workers developed self-healing electrical circuits with microcapsules containing gallium-indium liquid metal, hexyl acetate and polymer-stabilized carbon nanotubes and/or graphene flakes as precursor healing agents, which was released to restore the conductive pathway upon damage 6-8. Park and co-workers developed a conductive and self-healing hydrogel by polymerization of pyrrole within agarose matrix 9. Li, Yu and co-workers developed a hybrid gel by incorporating an acetonitrile-based supramolecular gel with cubic architecture within

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polypyrrole aerogel matrix, which combined high conductivity and self-healing property 10.

Among many polymer materials for constructing hydrogels, silk fibroin (SF) exhibits a number of superior properties including impressive biocompatibility, suitable stability, unique mechanical properties and versatile processability. Thanks to its various amino acid compositions, SF displays a very good ability to be functionalized and crosslinked. In addition, SF could be naturally derived from renewable feedstock, Bombyx mori silk, which is cultivated more than 480000 ton per year all over the world. 11 For these reasons, in recent years, SF has become a precious starting material highly attractive for developing innovative applications. Physically crosslinked hydrogels could be obtained from regenerated SF solutions through transition of the protein conformation from amorphous to intermolecular β-sheet structures. 12 Covalently crosslinked hydrogels could be synthesized using various chemical and enzymatic crosslinkers 13. In our previous studies, SF-based hydrogels with tunable tensile strength and elasticity were fabricated through Ru(II) mediated photochemical crosslinking of tyrosine residues 14-16.

The SF-based hydrogels, at a starting point, offers advantageous features in

serving as a highly flowable platform promising for self-healing and electrically conductive hydrogels. However, we noticed that little attention has been dedicated to SF as substrate for design of self-healing and electrically conductive hydrogels. 4


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Therefore, in this study, we endeavor to develop a new strategy for design of SF-based hydrogels that synergize self-healing ability and electrical conductivity.

Electrical conductivity could be achieved by incorporating electrically conductive nanomaterials (such as graphene, carbon nanotubes and Ag nanowires) and polymers (such as polythiophene, polyaniline and polypyrrole (PPy)) into the SF-based hydrogels 17.

PPy is one of the most widely used inherently conductive polymer because of its high

conductivity, good biocompatibility and non-cytotoxicity 18. PPy can be easily synthesized from pyrrole monomers using in situ chemical (e.g., with ammonium persulfate, ferric chloride and copper(II) chloride), electrochemical and enzymatic polymerization (e.g., with laccases, peroxidases and hydrogen peroxide) 9, 19, 20. Additionally, it has been reported that conjugation with other biocompatible materials would not significantly affect the conductivity of PPy 21. For example, Freddi and co-workers coated degummed silk fabric with PPy by in situ oxidative polymerization using ferric chloride as catalyst 22. Otero 23 and Mo 19 also used the ferric chloride-mediated chemical polymerization method to coat PPy on electrospun SF and poly(L-lactic acid-co-e-caprolactone)/SF fibers, respectively. Yang, Wang and co-workers fabricated PPy-coated SF composite scaffolds by 3D bioprinting and electrospinning 15. Despite progress in conductive SF fibers, achieving synergistic

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features of high conductivity, decent mechanical properties and self-healing ability in SF-based hydrogels still remains a challenge.

Self-healing ability could be achieved by diverse strategies through either physical crosslinking or dynamic covalent crosslinking. 24-26 β-cyclodextrin (β-CD), a type of cyclic oligosaccharides comprised of α-1,4-linked glucopyranose units with a hydrophilic outer surface and a hydrophobic inner cavity, is suitable to form physical inclusion complex with a large variety of guest molecules including aromatic amino acid residues (tyrosine, Tyr, Y; tryptophan, Trp, W; phenylalanine, Phe, F) in peptides/proteins 27. β-CD can be easily grafted onto polymer chains, especially large biomacromolecules 28-30.

For example, Nouri and co-workers grafted SF fabrics with β-CD in the presence of

citric acid as crosslinking agent and sodium hypophosphite as catalyst via esterification reaction, although their purpose was not relevant to fabricating self-healing supramolecular hydrogels 31-33. In spite of advances in dynamic crosslinking strategies, self-healing SF-based hydrogels were rarely reported 34, 35, particularly those which can meet the conductivity requirement.

With these above problems in mind, we aimed to develop a new strategy for design of self-healing and electrically conductive SF-based hydrogels based on the self-healing 6


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properties of the classical β-CD supramolecular complexes and the electrical properties of the well-known conductive polymer, PPy (Figure 1). β-CD-CHO was synthesized using Dess-Martin periodinane (DMP) 36, and conjugated onto SF with lysine (Lys, K) amines via Schiff base formation. The β-CD conjugated SF was photo-chemically crosslinked to form dual-crosslinked hydrogels. The Tyr-targeting photo-chemical crosslinking reaction led to formation of dityrosine, providing covalent crosslinking sites in the dual-crosslinked hydrogels. 14-16 Considering that Tyr, Trp, Phe and His were suited for host-guest interactions with β-CD 27 with the apparent K values of 105, 85, 107 and 2.8 M-1, respectively 37, the grafted β-CD would bind with neighboring amino acid side chains on SF (Table S1), providing supramolecular crosslinking sites in the dual-crosslinked hydrogels. Based on the dynamic characteristics of host-guest interactions between β-CD and amino acids, it was hypothesized that the dual-crosslinked hydrogels would achieve self-healing ability. The as-prepared self-healing hydrogels were then employed as molecular templates for in situ polymerization of pyrrole monomers using laccase as a catalyst and ammonium persulfate (APS) as an oxidant under mild conditions. It was hypothesized that PPy would uniformly deposit and integrated with the SF to form sufficient interconnected conductive path in the hydrogel networks, and thereby achieve electrical conductivity. The morphological, mechanical, conductive and self-healing properties of the resultant PPy-coated SF-based dual-crosslinked hydrogels were investigated.

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Figure 1. Schematic representation of network structure of the designed silk fibroin (SF)-based hydrogels. Self-healing properties could be achieved through host-guest interactions between grafted β-CD and intrinsic amino acid residues (Tyr, Trp, Phe and His) on interactive surfaces of photo-chemical crosslinked hydrogel pieces. Electrical properties could be achieved through in situ synthesis of polypyrrole.

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RESULTS and DISCUSSION Successful conjugation of β-cyclodextrin (β-CD) on to silk fibroin (SF) was confirmed by a well-established phenolphthalein spectrophotometric method 38 (Figure S1). Photo-chemical crosslinking of the β-CD-conjugated SF led to formation of stretchable and compressible hydrogels. Visual observation confirmed self-healing properties of the SF-CD dual-crosslinked hydrogels (Figure 2A and Movie S1). In order to further investigate the self-healing behavior, we attempted to measure the interactions between two pieces of the SF-CD dual-crosslinked hydrogels by tracing the force changes upon detachment of adhered/healed hydrogel building blocks. Using a customized microbalance, a curve of in situ detected interaction force versus change in position was obtained. The maximum detected mass corresponded to dissociation force of the two pieces of hydrogels during detachment. 24 In the SF-CD dual-crosslinked hydrogel system, the protein concentration and β-CD content could be kept unchanged, while the chemical crosslinking density could be adjusted. Hydrogels with variable APS content were prepared. Instron tests showed a Young’s modulus range of 22.5±9.5 to 60.7±6.7 kPa (Figure 2B and S2). The dissociation force measured by the microbalance was found to decrease with increasing APS content (Figure 2C).

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Self-healing mechanism primarily lies in reversible and dynamic equilibrium of certain interactions and requires a “mobile phase” (dangling side chains containing functional groups) to initiate the self-healing process. The side chains must be long and flexible enough to make the functional groups accessible to each other across the interface between two separate hydrogel pieces. At the same time, the hydrogel network must be sufficiently deformable to minimize steric hindrance of the interacting functional groups to allow reforming of broken bonds/crosslinks. 24-26 On the one hand, considering that molecular pores in the hydrogels ranging from 2.3 to 17.3 μm with an average equivalent spherical diameter (ESD) of ~5 μm were larger than the side chains and thus would not interfere significantly with the mobility of the side chains (Figure 2D), the reduction in dissociation force could be attributed to decrease in the compliance of the hydrogel with increasing rigidity/Young’s modulus. On the other hand, considering that more Tyr residues were consumed in photo-chemical crosslinking reactions and thus less available for interacting with β-CD, the reduction in dissociation force may also be attributed to decrease in the number of binding events.

Moreover, a chemically crosslinked SF-based hydrogel bearing non β-CD was prepared in a similar manner. In control experiments, while pairs of SF-based chemically crosslinked hydrogels did not stick together, a piece of SF-CD dual-crosslinked hydrogel and a piece of SF-based chemically crosslinked hydrogel stuck to each other to form a 10


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combined gel (Movie S1). These different behaviors were also studied by microbalance tests. The dissociation force between two pieces of SF-based chemically crosslinked hydrogels was found to be (2.8±0.8)×10-5 N. Meanwhile, the dissociation force between one piece of SF-based chemically crosslinked hydrogel and one piece of SF-CD dual-crosslinked hydrogel under the same condition was (4.4±0.5)×10-3 N, and that between two pieces of SF-CD dual-crosslinked hydrogels was (1.1±0.1)×10-2 N. These results demonstrated that β-CD enhanced the apparent interaction forces between hydrogel building blocks from non-specific interaction to more than one order of magnitude. In this sense, through the combination of the SF-based hydrogel and the β-CD supramolecular complexes, this study provided a general strategy to obtain self-healing protein-based hydrogels.

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Figure 2. Formation of SF-CD dual-crosslinked hydrogels. A) Photographs of a healed SF-CD dual-crosslinked hydrogel sample after rejoining the sliced samples. Change of B) Young’s modulus and C) dissociation force as the APS concentration increased. The modulus and dissociation force were determined and reported as the mean±standard error (n≥3). D) SEM images of freeze dried scaffolds of the hydrogels showed interconnected porous structures.

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Furthermore, since the SF-CD dual-crosslinked hydrogels possessed interconnected porous structures, it would be sufficient to allow free diffusion of solutions in and out of the hydrogel structure. Thus, the highly porous entangled networks are expected to facilitate transfer of ions and electrolytes, suggesting possibility of application in electrochemical studies. To explore this possibility, we employed the hydrogels as dielectric materials. Briefly, pieces of hydrogel in conjunction with electrodes (copper anode and carbon cathode) as current collectors embedded in a solution of electrolyte (0.25 M Na2SO4) were wiring to a power source supplying controllable constant AC potential (Figure 3A). The distance between the electrodes was kept relatively unchanged, and current values were recorded. When there was no hydrogel between the electrodes, the current was 0.02 A. When there was a piece of hydrogel between the electrodes, the current was 0.056±0.005 A. When a piece of hydrogel was cut into halves and pulled apart, the current dropped back to 0.02 A. When the two halves were brought into contact, the current increased to 0.043±0.005 A, indicating restoration of the electrochemical function. Several pieces of hydrogels could be combined; as the number of pieces of hydrogels increased, the current decreased almost linearly with R2>0.98 (Figure 3BC). These results obeyed the Ohm's law in terms that the current (𝐼) through the hydrogel between two points was proportional to the voltage (𝑉) across the two points, and that the resistance (𝑅) was proportional to the distance between the two electrodes (𝐿), equal to the number of healed pieces of hydrogels (𝑛) multiplied by the

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thickness of a piece of hydrogel (𝑙). Additionally, the SF-CD dual-crosslinked hydrogels could be further tailored by introducing azobenzene (Azo) groups (Figure S3).

Figure 3. Electrical performance of the SF-CD dual-crosslinked hydrogels in 0.25 M Na2SO4. A) Illustration of electrochemical tests on pieces of hydrogel samples, sliced samples and self-healed samples. Change of current as B) applied potential and C) thickness of adhered hydrogels increased. The current was determined and reported as the mean±standard error (n≥3). It is of note that no appreciable current could be detected without the electrolyte. The detectable current was mainly attributed to ionic mobility in and out of the hydrogel network, and thereby the SF-CD dual-crosslinked hydrogels could be treated as dielectric materials.

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After confirmation of the self-healing properties, PPy was deposited on the hydrogels (Figure 4AB). It is of note that PPy could form H-bond and covalent interactions with SF due to the presence of Ser/Thr, Lys and Tyr residues 39 (Table S1 and Figure 4C), and thereby be fixed instead of physically adsorbed on the hydrogels, favorable to obtain good compatibility between SF and PPy. In the enzymatic polymerization method, laccases were employed as the catalysts. An advantage of the present study is that the photo-chemical crosslinking reaction has been used to immobilize and stabilize various enzymes in SF-based hydrogels 15. It was noticed that upon addition of laccase, a solution of pyrrole slowly turned to a black color. For comparison purposes, if the solution is purged with N2 to remove all dissolved O2 prior to the addition of laccase, almost no black color appeared. In the chemical polymerization method, APS, which has been often used as the oxidant, was employed. In this sense, a possible advantage of the present study is that APS was used as the initiator in the photo-chemical crosslinking reaction of SF-based hydrogels. Co-production of polymer aggregates was observed due to occurrence of unavoidable solution-phase polymerization 9. Success in polymerization of pyrrole in the hydrogels was proved by color change from yellow to grey/black (Figure 4D). FTIR and Raman spectra confirmed presence of PPy in the hydrogels (Figure S4). The presence of PPy in the hydrogels was also confirmed by SEM images. The hydrogels prepared by the enzymatic method were uniformly coated with thin layers of PPy, while those prepared by the chemical method were coated with

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globular, much rougher and more evident particles of PPy (Figure S5). The different surface morphology was probably due to the effect of the deposition methods. EDX mapping images of the hydrogels were also taken (Figure S6). It is of note that weight percentage of nitrogen in pyrrole is close to that in SF, which makes it difficult to differentiate SF and PPy based on nitrogen. Instead, increase in the weight percentage of carbon indicated formation of PPy on the hydrogels. (Table S2)

Figure 4. Chemical and enzymatic polymerization of pyrrole. Reaction schemes of A) the chemical method using ammonium persulfate (APS) and B) enzymatic method using dioxygen (O2) and Trametes versicolor laccase. C) Scheme of the interactions 16


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between PPy and SF through amino acid side chains (Ser, Thr, Lys and Tyr). DE) Photographs of products obtained from polymerization reactions. F) Photographs of healed PPy-coated SF-CD dual-crosslinked hydrogel samples after rejoining the sliced samples.

The mechanical and viscoelastic properties of the PPy-coated SF-CD dual-crosslinked hydrogels were measured by mechanical tests and Dynamic Mechanical Analysis (DMA) (Figure S7). Deposition of PPy via the chemical and enzymatic methods increased the compressive modulus at 15% strain to 206.0±12.5 kPa and 2.0±0.8 MPa, respectively. Hysteresis became larger after coating PPy. This could be attributed to PPy, which is relatively rigid and would deteriorate elasticity of the hydrogels. In contrary to the fact that two pieces of SF-based chemically crosslinked hydrogels would rupture below 20% strain (n ≥ 3) during compressive tests, two pieces of SF-CD dual-crosslinked hydrogels before and after coating PPy remained intact up to 70% and 60% strain, respectively, indicating a strongly healed interface (Figure 5). Interface between hydrogel pieces was further inspected by an optical microscope. The self-healing behavior was demonstrated by observing disappearance of the gap between two interactive surfaces 9 (Figure S8). Additionally, the dissociation force between two pieces of PPy-coated SF-CD dual-crosslinked hydrogels prepared by the chemical and enzymatic methods was measured to be (5.5±0.6)×10-3 and (3.8±0.6)×10-3 N, respectively. The difference in the dissociation force could be also 17



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attributed to decrease in the compliance of the hydrogel along with increase in rigidity, which was consistent with the results of the microbalance tests above (Figure 2BC). It is interesting to find that conduction during the healing process could accelerate healing. Upon applying a potential difference of 5 V for 90 s through wiring the hydrogels to a power source, the dissociation force between two pieces of PPy-coated SF-CD dual-crosslinked hydrogels prepared by the chemical method increased from (5.5±0.6)×10-3 N to (8.5±0.1)×10-3 N, which might be caused by electropolymerization of pyrrole moieties during the healing process. It is worth mentioning that the dissociation force between two pieces of SF-CD dual-crosslinked hydrogels before and after coating PPy could not be compared based on rigidity only, as change in roughness induced by deposition of PPy (Figure 2D and S5) would also affect the distance between the interactive molecules on the interactive surfaces 24.

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Figure 5. Representative stress-strain curves and photographs obtained from compression tests on the hydrogels with and without self-healing behavior. AE) SF-based chemically crosslinked hydrogel samples, BF) SF-CD dual-crosslinked hydrogel samples, PPy-coated SF-CD dual-crosslinked hydrogel samples prepared by the CG) chemical and DH) enzymatic methods, respectively.

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Retaining the flexibility and self-healing properties of the SF-CD dual-crosslinked hydrogels (Figure 4EF and 5, and Movie S2) makes the PPy-coated hydrogels suitable for soft electronic devices that can recover electrical performance. Electrical conducting ability was investigated via measurement of the conductivity of the resultant hydrogels by the cyclic voltammetry (CV) method. Through measuring the electric potential-current curve, change of materials’ electrochemical performance during the charge and discharge processes could be derived. The CV curve of the electrode materials for supercapacitors was expected to be a closed rectangular curve. The SF-CD dual-crosslinked hydrogels did not display appreciable conductivity, but it became conductive after PPy coating (Figure 6 and S9AB). The PPy-coated SF-CD dual-crosslinked hydrogels prepared via the chemical polymerization method achieved a high conductivity of 0.8±0.2 S·cm-1 (n≥5). The conductivity of the PPy-coated SF-CD dual-crosslinked hydrogels prepared via the enzymatic polymerization method was (1.0±0.3)×10-3 S·cm-1. As main components of the hydrogels showed good cytocompatibility (Figure S10), the PPy-coated SF-CD dual-crosslinked hydrogels represented a promising candidate for application as cardiac repair patch material, as the electrical conductivity of myocardial tissue has been reported to be 1.6×10-3 S·cm-1 longitudinally and 5×10-5 S·cm-1 transversally 40. We also cut the hydrogel samples into halves, and measured the conductivity before and after self-healing. Upon cutting, the conductivity dropped to much lower than that of the original intact hydrogel. After

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self-healing, the conductivity of the hydrogels prepared via the enzymatic polymerization method remained above (0.8±0.1)×10-3 S·cm-1, suggesting reformation of an integrated conductive network 10. The incomplete restoration of the conductivity might be most likely caused by physical defects such as poor alignment of the sliced hydrogels (Figure S9CD). In case that an LED bulb was involved and the power source was linked into the circuit by two copper wires, cutting and self-healing of the conductive network could also be observed from that an LED light went off and on (Figure 6EF).

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Figure 6. Electrochemical tests on PPy-coated SF-CD dual-crosslinked hydrogels prepared by the enzymatic and chemical polymerization methods. A-D) Representative 22


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cyclic voltammetry curves and Nyquist plots obtained from intact, sliced and self-healed hydrogel samples. EF) Illustration of the self-healing behavior by an LED bulb. It is of note that the experiments involving the LED bulb was performed in air (instead of a solution of electrolyte), and thereby the conductivity was mainly attributed to the electrical property of PPy. Yet ionic mobility of buffer solutions possibly trapped in the hydrogel network during the sample preparation and storage processes might also contribute.

CONCLUSIONS Collectively, these results demonstrated that by combining the unique desirable properties of conductive polymers and supramolecular hydrogel, the newly designed PPy-coated SF-CD dual-crosslinked hydrogels can be used to make reversibly self-healable and conductive materials. Specifically, a new kind of SF-based hydrogel with both the supramolecular physical crosslinking and photo-chemical crosslinking was developed. The dynamic manner of the host-guest interactions of β-CD and amino acids imparted self-healing properties to the hydrogels. Moreover, ammonium persulfate (APS) and laccase were successfully used to coat PPy on the self-healing SF-based hydrogels, gave rise to features of tunable electrical conductivity. Furthermore, implantation of the hydrogels in electrochemical studies showed that the hydrogels 23



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recovered their mechanical strength and retained their electrical conductivity after being cut and healed. It is of note that there might be a limitation of the present study. The relative low content of Lys (amine groups) on SF resulted in shortage of β-CD. Consequently, the binding events between β-CD and guest molecules were limited. To circumvent this issue, exploration of suitable covalent conjugation methods under physiological conditions deserves more study in the future. Nevertheless, the availability of self-healing and electrically conductive silk-based hydrogel materials is expected to widen the applications of silk in development of innovative silk-based smart devices. This study demonstrates a useful strategy in designing self-healing and electrically conductive protein-based hydrogels through taking unique advantages of supramolecular chemistry and polymer nanoscience.

EXPERIMENTAL SECTION Materials. Dess-Martin periodinane (DMP) was obtained from Energy Chemical. β-cyclodextrin (β-CD) (99.999%) was purchased from Adamas (Shanghai, China). Ammonium persulfate (APS) and tris(2,2’-bipyridyl) dichlororuthenium (II) hexahydrate ([Ru(bpy)3]2+Cl-2) were purchased from Sigma-Aldrich. Laccase from Trametes versicolor was purchased from Yuanye (Shanghai, China). Pyrrole (purity 99.5%) was purchased from Innochem. Analytical reagent grade calcium chloride (CaCl2), sodium 24


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carbonate dehydrous (Na2CO3), absolute ethanol (CH3CH2OH) and all other reagents were purchased from Sinopharm Chemical Reagent Beijing Co. Ltd., and used without further purification. Preparation of regenerated silk fibroin (SF). Regenerated SF were prepared as previously described 14. Briefly, raw silk (produced in Zhejiang, China) was boiled in 0.05 w.t. % Na2CO3 for 90 min at 90°C, and rinsed thoroughly with deionized water, then dried overnight at room temperature. The degummed silk was dissolved in a solution of CaCl2:CH3CH2OH:H2O (1:2:8) at 60°C for 2 h, and then centrifuged at 8000 r.p.m. for 12 min at 4°C to remove aggregates. The supernatant was continuously dialyzed against deionized water using a cellulose dialysis membrane (MWCO 12400 Da) for 3 days to remove salts, and then lyophilized to obtain regenerated SF. SF conjugated with β-CD (SF-CD) through Schiff base formation. In a typical experiment, 0.20 g of β-CD was dissolved in 5 mL of DMSO. 2 equiv. of DMP was added and the reaction mixture was stirred for 1 h at room temperature. Addition of 150 mL of acetone and cooling at -10°C allowed isolation of the crude product β-CD monoaldehyde (β-CD-CHO) by filtration. Complete removal of the periodinane by-product was accomplished by repeated dissolution of the β-CD-CHO in DMSO and precipitation with acetone. The complexed acetone and DMSO is removed by dissolving the β-CD-CHO in water, stirring for 1 h, and lyophilization. 36 The β-CD-CHO was dissolved in phosphate saline buffer (PBS, 100 mM, pH 7.4). A given volume of 25



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regenerated SF solutions was mixed and incubated with the β-CD-CHO solution at 25°C to yield SF-CD. SF conjugated with azobenzene groups (SF-Azo) through diazonium coupling reaction. An aqueous solution of sodium nitrite (170 mg) was added to a solution of 4-ethynylaniline (270 mg) in distilled water (20 mL) and 12 N HCl (0.80 mL) at 0°C. The reaction mixture was stirred for 75 min at 0°C. 41 Next, the regenerated SF solution in borate buffer (100 mM borate, 150 mM NaCl, pH 9) was added drop-by-drop to the mixture. After stirring for 45 min, the yellow color changed to orange. λmax = 340 nm (Azo). 42 Longer reaction times resulted in protein gelation. Preparation of SF-based hydrogels. Chemically crosslinked SF-based hydrogels were prepared as previously described 15. In a typical experiment, 40 mg of SF with/without modification were solubilized in 186 μL of PBS. 10 μL of APS (1M) solution and 4 μL of [Ru(bpy)3]2+Cl-2 (20mM) solution were then added and mixed with the above protein solution by vortexing. The final solution irradiated for 10 min using a 100 W fiber optical white light source placed 4 cm away from the mold. Afterwards, the hydrogels were removed from the mold and stored in PBS for 2 h and washed thoroughly to remove remaining reagents. The concentrations were adjusted to 100-200 mg·ml-1 SF/SF-CD, 130-400 μM Ru(II), 6-50 mM APS in PBS.

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Polypyrrole (PPy) coating via in situ polymerization. In the case of laccase-mediated polymerization of PPy, 200 mM pyrrole was prepared in sodium acetate buffer (200 mM, pH 4.5). SF-CD dual-crosslinked hydrogels containing 0.72 U·mL-1 laccase were incubated in the solution of pyrrole for 36 h at 50°C. The mixture was saturated with O2 by bubbling O2 stream before the reaction. In the case of chemical polymerization of PPy, 200 mM pyrrole was prepared in PBS (200 mM, pH 4.5). SF-CD dual-crosslinked hydrogels were incubated in a mixture of APS and pyrrole for 36 h at 4°C under stationary condition. After the reactions, the hydrogels were washed with the buffer thoroughly in order to eliminate unreacted pyrrole, as well as unfixed PPy. 20 Amino acid analysis. SF was dissolved in concentrated HNO3 and completely hydrolyzed. The resultant solution was used for amino acid analysis, which was carried out on Hitachi L-8900 Amino Acid Analyzer. 16 Determination of β-CD by phenolphthalein spectrophotometry. Samples were added into phenolphthalein dissolved in NaHCO3/Na2CO3 buffer solution (pH 10). After thorough mixing, absorbance of the phenolphthalein colorimetric solution was measured at 550 nm and ambient temperature.38 The absorbance of the phenolphthalein colorimetric solution with and without addition of pure β-CD was also measured under the same condition, and referred to as positive and negative control, respectively.

27



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UV/Vis spectroscopy. The absorbance was measured on a SHIMADZU (UV-2600) UV/VIS spectrophotometer. Fourier transform infrared spectroscopy (FTIR). The FTIR spectra were obtained using an FTIR spectrometer (FTIR, Bruker Optics VERTEX70, USA) in the transmission mode in the range of 400 to 4000 cm-1 with a resolution of 4 cm-1 at room temperature. Raman spectroscopy. The Raman spectra with a resolution of about 1 cm-1 were recorded at room temperature using a Renishaw inViaTM confocal Raman microscope. The samples were illuminated by a 514 nm Argon ion laser. Mechanical tests. Mechanical measurements were conducted at room temperature on Instron 3367 tensometer with a 100-N load cell and a custom-made force gauge at a cross-head speed of 25 mm·min-1 (as required by GB/T6669-2008 ISO1856-2000). The modulus was determined from the stress-strain curve as the slope at 15% of strain. For tensile tests, hydrogel samples were prepared in a custom-made mold (with a length of 10 mm, a width of 5 mm and a thickness of 1 mm). For compression tests, hydrogel samples were prepared in a custom-made cylinder mold (with a diameter of 5 mm). The moduli were determined and reported as the mean±standard error (n≥3). Dynamic mechanical analysis (DMA) was conducted on hydrogel samples at 1% strain in a frequency range of 0.1-10 Hz using Instron Q800 DMA.

28


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Microbalance tests. Interactions between hydrogel building blocks were evaluated on a customized microbalance setup as previously described 24. Briefly, a piece of hydrogel was hung from a microbalance detector with a soft thread. Another piece of hydrogel was immobilized at the bottom of a beaker with its position parallel to the top one. The beaker was placed in an apparatus that could be driven upward/downward at a constant speed. The apparatus moved upward, allowing the top piece of hydrogel settle onto the bottom one. When difference in the detected mass exceeded 7 mg, the apparatus moved upward a distance of 1 mm to ensure sufficient contact between the two pieces of hydrogels for 90 s. Then the apparatus moved downward until the two pieces of hydrogels detached. After completion of a programmed approach-detach cycle, the interacting forces (represented by the detected mass) were plotted versus change in position (represented by the distance moved by the apparatus). The driving forces (in newton, N) were obtained from the detected mass (in gram, g) by multiplying the standard acceleration due to gravity (g≈9.8 N·kg-1). Scanning Electron Microscopy (SEM). The hydrogel samples were shock-frozen in liquid nitrogen and lyophilized. The obtained samples were fractured and coated with gold for SEM observation using a scanning electron microscope (HITACHI S-4700, Japan). Electrochemical tests. Constant current tests on the hydrogel samples were conducted in 0.25 M Na2SO4 using a GENTEK 12305 power supply. The constant current (𝐼) 29



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applied to the surface created a voltage (𝑉) difference and then the conductivity was calculated as follows:

𝑉=𝐼×𝑅

(1)

L

(2)

𝑅 = 𝜌𝑆

𝐿

(3)

𝐿=𝑛×𝑙

(4)

σ = RS

where σ (S·cm-1) is the electrical conductivity, 𝐿 (cm) is the distance between the reference electrode and working electrode, 𝑆 (cm2) is the cross-sectional area of the sample, 𝑅 (Ω) is the ohmic resistance, ρ (Ω·cm) is the resistivity, 𝑙 is the thickness of a piece of hydrogel and 𝑛 is the number of healed pieces of hydrogels. The conductivities of the hydrogel samples were measured using the cyclic voltammetry method in a three-electrode system (CHI760E electrochemical workstation, Chenhua Ltd, China) at room temperature in a 1M NaCl solution with a scan rate of 50 mV·s-1. AC impedance tests, also known as electrochemical impedance spectroscopy (EIS) tests, were done in the frequency range of 0.01 to 1×105 Hz and the AC signal amplitude of 5 mV at the open circuit voltage of 0.22 V. The EIS data were analyzed by building an equivalent circuit to associate with Nyquist plots, where Z’ and Z” are the real part and imaginary part of impedance, respectively. 30


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Cell studies. L929 mouse fibroblasts purchased from Peking Union Medical College Hospital (PUMCH; Beijing, China) were cultured according to the recommended protocols. MTT assays were performed to assess cytotoxicity. Prior to each test, samples were treated with ultraviolet (UV) radiation for sterilization. Cells were seeded in a standard 96-well plate at a density of 100000 cells per well in triplicate. After 24h, various amounts of the samples were added into the wells. Cells were incubated for another 24h, and then assayed following the manufacturer’s protocol using a microtiter plate reader (Multiskan FC, Thermo Scientific, USA). Cell viability was calculated as an indicator of the cell proliferation rate.

Supporting information. Supplemental figures and additional experiments regarding grafting of azobenzene (Azo) groups onto SF (SF-Azo) and cytotoxicity evaluation.

Declarations of interest: none

Corresponding Author *To whom correspondence should be addressed. Email: [email protected], Phone: (86)10-64411656, Fax: (86)10-64434784. 31



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Author Contributions L. S. conceived the idea, designed the overall experiments, analyzed the data, and wrote the manuscript. L. L. and H. Y. designed, conducted and analyzed the data of individual experiments, and edited the manuscript. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

ACKNOWLEDGMENT The authors would like to thank Prof. Feng Shi for inspiring discussion, thank Mr. Peichen Xie for kind assistance on micro-balance tests, thank Miss Changyu Wang for kind assistance on MTT assays, thank Mr. Shouchuan Li and Mr. Haoquan Zhang for kind assistance on mechanical tests, thank Dr. Ning Xiao and Dr. Yan Huang for kind assistance on electrochemical tests, and thank Dr. Qiang Lyu (Soochow University) for kind assistance to buy raw silk. This work was supported by the National Key Research Program of China [grant number: 2016YFA0201700/2016YFA0201701], the National Natural Science Foundation of China [grant number: 31400813], and the Foundation of Beijing University of Chemical Technology [grant number: XK180301].

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Design of self-healing and electrically conductive silk fibroin-based

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