Robust Protein Hydrogels from Silkworm Silk - ACS Sustainable

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Robust Protein Hydrogels from Silkworm Silk Zhao Li, Zhaokun Zheng, Yuhong Yang, Guangqiang Fang, Jinrong Yao, Zhengzhong Shao, and Xin Chen ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.5b01463 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 8, 2016

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ACS Sustainable Chemistry & Engineering

Robust Protein Hydrogels from Silkworm Silk Zhao Li†, Zhaokun Zheng†, Yuhong Yang‡, Guangqiang Fang†, Jinrong Yao†, Zhengzhong Shao†, Xin Chen*,†

†State Key Laboratory of Molecular Engineering of Polymers, Collaborative Innovation Center of Polymers and Polymer Composite Materials, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai, 200433, People’s Republic of China

‡Research Centre for Analysis and Measurement, Fudan University, Shanghai 200433, People's Republic of China

* Corresponding author. E-mail address: [email protected]

KEYWORDS: Silk fibroin, Surfactant, Hydrogels, Mechanical properties, Multifunction, Biocompatibility, Catalysis.

ABSTRACT: Silk protein is a promising natural material applied in various fields, but the application of silk protein-based hydrogel is quite limited because of its long gelation time and poor mechanical properties. Here, we present a facile way to prepare strong silk protein hydrogels simply by adding surfactant into silk fibroin aqueous solution and incubating at 60 °C. The resulting silk protein hydrogels demonstrate fairly good mechanical properties, for example, the silk protein hydrogel made by adding sodium dodecyl sulfate (SDS) has the compressive and tensile modulus of 3.0 and 3.3 MPa respectively, which are close to some 1

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tissues in the body, such as cartilages, tendons and ligaments. The effect of different types of surfactant on the formation of strong silk protein hydrogel, and the possible reason for the improvement of the mechanical properties of the hydrogel are also discussed. In addition, we show that such a strong silk protein hydrogel maintains good biocompatibility when adding proper amount of surfactant. Finally, we use a Fe3O4-loaded silk protein hydrogel as an example to demonstrate its application on the catalytic field. All these results imply that such a natural, sustainable, strong, and biocompatible protein-based hydrogel holds a great promise as a multi-functional material in various application fields.

INTRODUCTION

Hydrogels that contain large amount of water in their three-dimension network structures, are mechanically-stable materials, which only swell but do not dissolve in aqueous solutions.1-4 It is known that the hydrogels have a wide application, but always are limited due to the poor mechanical performance, especially when high strength is needed.5,6 In recent years, many researchers have devoted themselves to developing hydrogels exhibiting excellent mechanical properties. Among all of the efforts, double-network hydrogels, topological hydrogels, and nanocomposite-hydrogels, reported by Gong, Okumura, and Haraguchi separately, have drawn much attention because of their substantially improved mechanical behavior.7-12 However, these hydrogels are not flawless as their biocompatibility is unsatisfactory while their degradation product is probably toxic, which severely limits their utilization in the biomedical fields.13-15 Silk fibroin is an abundant, cheap, and sustainable natural material extracted from Bombyx mori silkworm silk, and holds the distinctive biological properties including biocompatibility, 2


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biodegradability, and minimal inflammatory response in vivo. Silk fibroin hydrogels are easily prepared from aqueous silk fibroin solution by forming β-sheet structures.16,17 Generally speaking, the gelation process of silk fibroin can be regulated by heating, variation of pH,18 treatments with alcohol,19 addition of metal ions,16 and shearing (such as ultra-sonication20 or vortexing21). Silk fibroin hydrogels, to some extent, are able to meet the demand of biomedical application, such as drug delivery22,23 and cell adhesion,24 in terms of their excellent biocompatibility and biodegradability. Unfortunately, the clinical use of this ideal material is often limited due to its relatively long gelation time. Although increasing protein or Ca2+ ion concentration, raising temperature or decreasing pH are able to reduce the gelation time, the effect is far from satisfactory.16 Other attempts, such as blending other polymers may weaken the biocompatible and biodegradable nature of silk fibroin.25 In 2012, Lu and his coworkers reported that by adding surfactant sodium dodecyl sulfate (SDS) into silk fibroin solution, they are able to cut the gelation time down to about 15 min under mild conditions.26 Two years later, Park et al. continue to use other surfactants, like polyethylene glycol octylphenyl ether (Triton X-100), sodium dodecylbenzene sulfonate (SDBS), octyltrimethylammonium bromide (OTAB), dodecyltrimethyl ammonium bromide (DTAB), and hexadecyltrimethyl ammonium bromide (HTAB) to study their effect on the acceleration of the gelation time of silk fibroin solution.27 However, both studies mainly focused on the gelation time and possible reasons for the decrease in the gelation time of the hydrogel with the addition of the surfactant. Neither of them mentioned the mechanical properties of the formed silk fibroin hydrogels. We guess it probably because they both used

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low concentration silk fibroin solution (4.0 wt% by Lu26 and 2.5 by Park27), so the corresponding silk fibroin hydrogels are very weak. In our previous work,28 when we prepared the spinning dope to spin silk fibroin-carbon nanotube hybrid fiber, we used SDS to disperse carbon nanotubes uniformly in high concentration (>10 wt%) regenerated silk fibroin (RSF) solution. Once in a while, we found when the SDS concentration reached a certain extent, it easily induced the gelation of the high concentration RSF solution and formed a tough hydrogel. Therefore, in this work, we tried to use the surfactant to develop a facile way for the fabrication of strong RSF hydrogels, as normally the RSF hydrogels are very weak. We proposed the possible mechanism how the surfactant enhanced the mechanical properties of the RSF hydrogel. In addition, we also showed the multifunctional potential of such a natural protein hydrogel made from cheap, abundant, and sustainable silk fibroin.

EXPERIMENTAL SECTION Preparation of RSF/Surfactant Hydrogels. RSF aqueous solution was prepared from B. mori silkworm cocoons following a conventional procedure as described in our previous work.28 In brief, raw B. mori silkworm silks were degummed twice with 0.5 wt% NaHCO3 solution at 100 °C for 30 min and then washed with distilled water and allowed to air dry at room temperature. The degummed B. mori silk fibers were dissolved in 9.3 mol/L LiBr aqueous solution at 60 °C for 1 h. After dialysis against deionized water in a Visking dialysis tube (MWCO: 10−12 kDa) for 3 days at room temperature, the solution was filtered and resulting RSF solution was about 5 wt%. Then the solution was concentrated by reverse

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dialysis with PEG aqueous solution for about 24 h to reach a concentration about 17 wt%. The concentration of the RSF solution was measured by constant weight method. Different kinds of surfactant aqueous solution, i.e., SDS, SDBS, Triton X-100 (shortened as Triton in the text), OTAB, dodecyl dimethyl benzyl bromide (DDBAB), and cetylpyridinium chloride (CPC) solutions were added quantitatively into RSF solution to make the final silk fibroin concentration of 10, 12.5, and 15 wt% with the final surfactant concentration from 5 to 80 mmol/L. Then, the mixed solutions of RSF and surfactant were put in the oven at 60°C to form hydrogels. The samples were named as mRSF-X-n, in which m, n and X represent the concentration of silk fibroin, the concentration of surfactant, and the type of surfactant, respectively. Mechanical Tests of RSF/Surfactant Hydrogels. Mechanical properties of the RSF/surfactant hydrogels were tested on an Instron 5565 universal testing machine at 25±5 °C and 50±5% relative humidity. For uniaxial compression test, cylindrical samples with the diameter of 12.5 mm and the height of 6 mm were used, and the compression rate was 0.06 mm/s. As for tensile tests, the sample was 50 mm in length, 10 mm in width, and about 1 mm thick. During testing, the gauge length was 20 mm and the cross-head speed was 0.1 mm/s. 20 samples were used for both compression and tensile tests. The significance of differences in mechanical properties was determined by one-way ANOVA in the statistics package in OriginPro 8. Cell Viability. To evaluate the cytotoxicity of the RSF/surfactant hydrogel, the metabolic viability of cells cultured with medium supplemented with the hydrogel extract was measured. The use of hydrogel extract is a common way to briefly evaluate the 5



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biocompatibility of the hydrogel, especially when the component contained in the hydrogel is suspected to be toxic, which was applied by many research groups.29-31 Specifically, RSF/SDS hydrogel was cut into small cubes (0.5 × 0.5 × 0.5 cm) and immersed in deionized water for 3 days to remove the uncombined SDS.26 Afterwards, the RSF/SDS hydrogel was sterilized by UV radiation and then immersed in DMEM medium at 37 °C for 24 h to obtain the hydrogel extract . Mouse fibroblast cells (L929) were seeded into a 96-well tissue culture plate at a seeding density of 10,000 cell/well, which is according to the method used by many research groups.32-34 After 24 h culturing with pristine DMEM medium, the DMEM medium was replaced by the hydrogel extract. Then, after 24 h and 72 h of incubation, the cell metabolic viability was measured by using a CCK-8 assay, respectively. We used the ethanol induced RSF hydrogel as a negative control during the test. The significance of differences in cell viabilities was determined by one-way ANOVA in the statistics package in OriginPro 8. Fabrication of Fe3O4-loaded RSF/SDS Hydrogels. RSF/SDS hydrogel was immersed in an aqueous solution of iron ions with a molar ratio of [Fe2+]/[Fe3+] = 2/3 at 4 °C. After 24 h, the hydrogel was transferred into 28 wt% ammonia water for 6 h at room temperature to allow the in situ formation of Fe3O4 nanoparticles in the hydrogel matrix. Catalytic Effect of Fe3O4-loaded RSF/SDS Hydrogels. Fe3O4-loaded RSF/SDS hydrogel, used for H2O2 detection, was cut into a small cube (0.5 × 0.5 × 0.5 cm) and put into 2 mL of 0.2 mol/L acetate buffer (pH = 4.0) with 530 mmol/L H2O2 and 816 mmol/L 3,3’,5,5’-tetramethylbenzidine (TMB). After the reaction taken place at 40 °C for 20 min, the Fe3O4-loaded RSF/SDS hydrogel cube was removed and the solution was tested by a

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wavelength-scan mode from 450 nm to 800 nm on a Shimazu UV-2550 UV-Vis spectrometer. Other Characterizations. The morphology of lyophilized RSF hydrogel samples were observed with a Hitachi S-4800 high-resolution SEM (HRSEM) at 20 kV after sputtering with gold. X-ray diffraction (XRD) patterns were recorded on a Bruker D8 Advance X-ray diffractometer with CuKα radiation in the range of 20-80°. Raman spectra was collected with a Renishaw inVia Reflex spectrometer with a He-Ne laser providing 6 mW of energy at 785 nm and a typical acquisition time of 10 s.

RESULTS AND DISCUSSION The Preparation and Mechanical Properties of RSF/SDS Hydrogel. We first tried to use SDS to induce the high concentration RSF solution ([RSF] >10 wt%, same meaning throughout the text) to form hydrogel, as SDS is the most frequently used surfactant. Generally, after a period of heating in the oven at 60 °C, RSF/SDS mixed solution (left centrifuge tube in Figure S1A) gradually turned into white translucent hydrogel while the pure RSF solution (right centrifuge tube Figure S1A) still remained in liquid form. The final formed RSF/SDS hydrogel is shown in Figure S1B, which is elastic and maintain the shape of the mold. We shall point out that we are able to make different shape of the hydrogel only according to the shape of the mold. Afterwards, we investigated the influence of SDS concentration on the gelation time of RSF/SDS hydrogel. Since high concentration RSF solution is able to gelate spontaneously at room temperature in a few days, we presume that if the RSF/SDS solution does not form 7



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hydrogel after heating for 48 h, the SDS concentration is too low to promote the gelation process in order to exclude the interference of natural protein denaturation. The results are shown in Table S1. Generally, the lower the RSF concentration or the higher the SDS concentration was, the shorter the gelation time we found. When the SDS concentration was 5 mmol/L, all three RSF solutions with different concentration we chose did not gelate. However, with the increase in SDS concentration, the gelation time was significantly shortened, for instance, at the highest RSF concentration (15 wt%), when the SDS concentration reached 20 mmol/L, the gelation time was able to be cut down to about 30 min. In the meantime, the increase in RSF concentration led the relatively longer gelation time at the same SDS concentration. Of course, if the SDS concentration is too high, for instance, 15 mmol/L when RSF concentration is 10 wt%, or larger than 20 mmol/L when RSF concentration is 12.5 wt% and 15 wt%, the uncontrollable gelation occurred during the mixing, which resulted in an inhomogeneous hydrogel. Normally, the naturally formed RSF hydrogels are very weak, but the RSF/SDS hydrogels we made have an outstanding mechanical performance compared to those natural ones. Intuitively, as shown in Figure 1A, three small cylindrical RSF/SDS hydrogels (sample 15RSF-SDS-20, with a diameter of 12.5 mm and a height of 6 mm) hold up a weight as heavy as 2 kg. Another, as shown in Figure 1B, a 0.5 kg weight can be lifted up by a rod-like RSF/SDS hydrogel (also sample 15RSF-SDS-20, 18 mm × 5 mm × 3 mm) safely. Therefore, these images give us the first impression on the strength of the RSF/SDS hydrogel, which undergoes a qualitative leap compared to natural counterparts. Then, we conducted quantitative analysis of the mechanical properties of RSF/SDS hydrogels by both 8


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compression and tensile tests. Figure 1C shows the typical compression curves of RSF/SDS hydrogels with different RSF concentration. The corresponding Young’s modulus and compressive stress are listed in Table 1. It clearly indicates that the RSF/SDS hydrogel gets stronger with the increase in RSF concentration (i.e., the solid content in the hydrogel), which is very reasonable. From Table 1 we can find that when RSF concentration increases from 10 wt% to 15 wt%, the corresponding compressive modulus of RSF/SDS hydrogel goes up more than 4 fold, namely from 0.62 MPa to 2.98 MPa. The similar trend is found on compressive stress, i.e., from 0.28 MPa to 1.07 MPa at 50% compression. In the meantime, although the increase of SDS concentration shorten the gelation time, it seems not significantly affect the mechanical properties of the hydrogel. The compressive stress of the hydrogels made from 10 mmol/L SDS is a little bit lower than that of 20 mmol/L SDS, but the compressive modulus is almost the same (no significant difference according to the statistical analysis).

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Figure 1. (A) Three small cylindrical RSF/SDS hydrogels (sample 15RSF-SDS-20, with a diameter of 12.5 mm and a height of 6 mm) sustain a 2 kg weight, (B) a rod-like RSF/SDS hydrogel (sample 15RSF-SDS-20, 18 mm × 5 mm × 3 mm) hangs a 0.5 kg weight, (C) typical stress-compression curves of RSF/SDS hydrogels with different RSF concentration ([SDS] = 20 mmol/L), (D) typical stress-strain curve of RSF/SDS hydrogel ([RSF] = 15 wt%, [SDS] = 20 mmol/L). Table 1. Comparison of the mechanical properties of RSF/SDS hydrogels* Compression modulus

Compressive stress at

(MPa)

compression=50% (MPa)

10RSF-SDS-20

0.62±0.17a

0.28±0.04c

12.5RSF-SDS-20

1.39±0.15a

0.68±0.06c

15RSF-SDS-20

2.98±0.73a, b

1.07±0.18c, d

15RSF-SDS-10

2.92±0.20b

0.92±0.09d

Sample

*All values measured were expressed as mean ± standard deviations (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA). Different letters indicate different analytical groups. a: p = 3.1×10-20; b: p = 0.24; c: p = 1.7×10-18; d: p = 9.9×10-3. 10


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In most cases, even “strong” hydrogel only can subject to compression, seldom can be stretched. However, our RSF/SDS hydrogels are strong enough to prepare specimens for tensile tests. Figure 1D shows a representative stress-strain curve of the RSF/SDS hydrogel (sample 15RSF-SDS-20). The corresponding Young’s modulus is 3.28±0.57 MPa, the breaking stress is 0.74±0.12 MPa, and the breaking strain is 134±21%. In other words, when the solid content in RSF/SDS hydrogel is about 15 wt%, both the compressive and tensile modulus are around 3.0 MPa, which means the mechanical properties of such a RSF/SDS hydrogel are quite close to those of natural elastomers like skin, cartilage, tendon and ligament.

Investigation on Other RSF/Surfactant Hydrogels. Surfactant, according to the different types of electric charge, can be divided into three types: anionic surfactant, nonionic surfactant and cationic surfactant.27,35 In order to investigate if all types of surfactants are able to make strong RSF hydrogels, we tested other five surfactant, one more anionic surfactant SDBS, one nonionic surfactant Triton, and three cationic surfactant OTAB, DDBAB, CPC. The result shows that only SDBS and Triton have a similar gelation accelerating effect as SDS, in other word, we are only able to obtain RSF/SDBS and RSF/Triton hydrogel. However, the ability of both SDBS and Triton to accelerate the gelation of RSF solution is weaker than that of SDS. At the same surfactant concentration, the gelation time is longer for SDBS and Triton than for SDS (Table S2). For instance, at 20 mmol/L surfactant concentration, SDS needs about 35 min, SDBS needs 45 min, and Triton even needs 2 h. For nonionic surfactant Triton, only increasing its concentration to 80 mmol/L, it reaches a satisfactory gelation time of 40 min. For three cationic surfactant OTAB, DDBAB, CPC, they 11



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are not able to induce the formation of a stable RSF hydrogel, but result in silk fibroin aggregation or precipitation. These phenomenon, i.e., when we used different kind of surfactant to induce the gelation of high concentration (>10 wt%) RSF solution is similar to those reported by Park and his coworkers when they used low concentration (2.5 wt%) RFS solution.27 That is, both anionic and nonionic surfactant are able to shorten the gelation time of RSF solution, and the effect of anionic surfactant is much faster than the nonionic surfactant. In case of cationic surfactant, only silk fibroin aggregations are found because of the strong electrostatic interactions between silk fibroin chains and surfactant molecules. The main purpose of this work is to produce a tough protein hydrogel, so we are more care about the mechanical properties of the resulting RSF hydrogels. Table 2 compares three RSF hydrogels made from same protein concentration (15 wt%) and formed within 1 h. There seems to be no obvious difference among the mechanical properties of these RSF hydrogels, according to the statistical analysis. Therefore, it may imply that the mechanical performance of RSF hydrogels induced by different surfactant mainly depend on their solid content. Surfactant significantly contributes to promote the gelation of the silk fibroin molecules to form a three-dimensional network, but itself has little effect on the improvement of the mechanical properties. However, we believe the structure of these RSF/surfactant hydrogels should have some difference from naturally formed pure RSF hydrogels. Table 2. Comparison of the mechanical properties of different RSF/surfactant hydrogels* Young’s modulus

Compressive stress at

(MPa)

compression=50% (MPa)

15RSF-SDS-20

2.98±0.73a

1.07±0.18b

15RSF-SDBS-20

2.86±0.16a

1.00±0.12b

Sample

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15RSF-Triton-80

2.75±0.47a

1.14±0.11b

*All values measured were expressed as mean ± standard deviations (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA). Different letters indicate different analytical groups. a: p = 0.84; b: p = 0.14. Discussion on the Possible Reason for the Formation of a Strong RSF/Surfactant Hydrogel. It is no doubt that the driving force for the formation of RSF hydrogel is the appearance of physical crosslinking points, which are β-sheet structures from the self-assembly of the silk protein chains.26,27 The addition of the surfactant promotes hydrophobic interactions not only between silk fibroin molecules but also between silk fibroin molecules and long alkyl chains of surfactant.26,27 Therefore, it is easier for the formation of β-sheet structures, in other word, it requires shorter time to form β-sheet structures, thus accelerates the gelation process. However, far from this, the addition of surfactant also changes the structure of the resulting RSF hydrogel, which we think is the main reason for the enhancement of the mechanical properties. We know the β-sheet content in RSF hydrogel (in final equilibrium state) no matter prepared by which method normally is the same, as the formation of β-sheet structure is a thermodynamic process. We also used FTIR spectroscopy to estimate the β-sheet content in the hydrogels (spectra not shown), and the results confirmed that it was almost identical among three RSF hydrogel samples prepared by different methods, i.e., RSF/SDS hydrogel, naturally formed RSF hydrogel, and ethanol induced RSF hydrogel. However, although the β-sheet content is almost the same, the structure of these hydrogels has a large difference. Figure 2 shows the SEM images of lyophilized samples of the liquid nitrogen frozen RSF hydrogels. It clearly demonstrates that RSF/SDS hydrogel (Figure 2A) has the most homogenous structure than the naturally formed 13



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RSF hydrogel (Figure 2B) and ethanol induced RSF hydrogel (Figure 2C). Therefore, we may attribute the outstanding mechanical properties of RSF/SDS hydrogel to its relatively “tight” structure derived from the uniform and small pores. It is easy to understand that small pore size distributes stress more evenly in the hydrogel network when resisting stress concentration, and small pores as well as increased pore number can act as a barrier against crack propagation.16 Of course, such a unique structure in RSF/SDS hydrogel should arise from the effect of the surfactant during the gelation process. As we discussed above that the hydrophobic interactions between silk fibroin and surfactant chains speed up the self-assembly of silk protein to form β-sheet structure, however, in the same time, the interference of surfactant among the silk fibroin chains may prevent them from forming large β-sheet domains. Thus, the surfactant may induce the silk fibroin to quickly form numerous but small-sized β-sheet domains as the crosslinking points, which leads the homogenous structure in RSF/surfactant hydrogels.

Figure 2. SEM images of different freeze-dried RSF hydrogel. (A) RSF/SDS hydrogel, (B) naturally formed RSF hydrogel, (C) ethanol induced RSF hydrogel.

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Potential Use of RSF/SDS Hydrogel as a Multi-functional Material. Among all the RSF/surfactant hydrogels, because SDS is the most frequently used surfactant and it is able to induce the gelation of RSF hydrogel in the shortest time, we chose RSF/SDS hydrogel as an example to explore the potential of RSF/surfactant hydrogel as multi-functional materials. We know the most attractive character of protein-based hydrogel is its biocompatibility, so it is important to know whether such an advantage still remains in the RSF/SDS hydrogel. We first put the RSF/SDS hydrogels in deionized water for 3 days, as reported the previous work by Lu and his coworkers,26 most of the uncombined SDS molecules in the hydrogel released in first 2 days. Then, we prepared hydrogel extract and used it to test the in vitro cytotoxicity on L929 mouse fibroblasts cells.29-34 Figure 3 demonstrates that all the cell variability after both one-day and three-day incubation with the hydrogel extract are fine. Even for the sample with the high SDS concentration (15RSF-SDS-20), the cell survival rate is above 80%. There is almost no significant difference for cell variability between the hydrogels with different SDS concentration. Only the highest SDS concentration sample 15RSF-SDS-20 shows a significant difference from other samples at one day if we set p < 0.05, but still shows no significant difference if we set p < 0.01. All these results confirm that the RSF/SDS hydrogel does not exhibit obvious cytotoxicity,26 so it is still suitable to be used as a biocompatible material.

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Figure 3. In vitro cytotoxicity of L929 cells incubated in RSF/SDS hydrogel extracts for 1 and 3 day with different SDS concentration. Data were expressed as mean ± standard deviations (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA), * denotes p < 0.05. Moreover, we consider to add some other properties on the biocompatible RSF/SDS hydrogel in order to make it to be qualified as a multi-functional material. In this research, we tried to apply it as a Fe3O4 nanoparticle carrier to detect H2O2.36 The in situ fabrication of Fe3O4-loaded RSF/SDS hydrogel was convenient, just by immersing the RSF/SDS hydrogel into an iron ions aqueous solution till the diffusion equilibrium was reached, followed by soaking in ammonia water to incubate the Fe3O4 nanoparticles. Eventually, the white RSF/SDS hydrogel turned into black, indicating the successful loading of Fe3O4 nanoparticles. XRD and Raman spectroscopy were carried out to confirm the formation of Fe3O4 nanoparticles rather than γ-Fe2O3. Figure 4A is the XRD pattern, from which we can see the position and relative intensity of all the peaks are consistent with an inverse spinel Fe3O4 with a face-centered cubic structure (JCPDS No.79-0416). Moreover, the Raman spectrum exhibits a single peak at 666 cm-1 (Figure 4B), which also accords with the characterization 16


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of Fe3O4 reported in the literature.37

Figure 4. (a) XRD patterns and (b) Raman spectroscopy spectra of pristine RSF/SDS and Fe3O4-loaded RSF/SDS hydrogel, (c) the illustration of the oxidation reaction of TMB by H2O2 catalyzed with Fe3O4-loaded RSF/SDS hydrogel, (d) UV-Vis spectra of the H2O2/TMB mixed solution with pristine RSF/SDS and Fe3O4-loaded RSF/SDS hydrogel after 20 min at 40 °C. After the successful preparation of Fe3O4-loaded RSF/SDS hydrogel, we investigate its catalysis ability for the oxidation reaction by H2O2. The catalytic reaction of Fe3O4-loaded RSF/SDS hydrogel follows the condition as reported in the literature,36 i.e., H2O2 (530 mmol/L) was used as the substrate and TMB (816 mmol/L) as the chromogenic agent. Figure 4C visually demonstrates that if the Fe3O4-loaded RSF/SDS hydrogel is not put into the H2O2/TMB mixed solution, it remains colorless. However, after putting the hydrogel into the mixed solution, the solution turns blue, which indicates the Fe3O4 nanoparticles in RSF hydrogel successfully catalyze H2O2 to oxidize TMB. The extent of the change in color 17



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reflected the extent of the reaction process. This color change process is able to be monitored with UV-Vis spectrophotometer quantitatively. Figure 4D shows clearly that before the addition of the Fe3O4-loaded RSF/SDS hydrogel, the absorption between 450 and 800 nm can be neglected, but after the addition, there is an obvious characteristic peak centered at 650 nm emerged, which is able to calculate the catalytic efficiency if one wants to get a quantitative result. In addition, after the reaction, the hydrogel is able to remove from the solution completely (Figure 4C). Therefore, we demonstrate here that the in situ fabricated Fe3O4-loaded RSF/SDS hydrogel not only shows the peroxidase-like catalytic activity, but also can be easily taken out from the reaction solution, adding the merits of utilization convenience and repeatability.

CONCLUSION

In this work, we present a facile way to produce strong RSF hydrogels simply by adding surfactant into high concentration RSF solution (>10 wt%). For instance, the compressive and tensile modulus are as high as 3.0 MPa and 3.3 MPa when the solid content in RSF/SDS hydrogel is about 15 wt%. We find only anionic and nonionic surfactant are able to induce the formation of RSF hydrogel. The mechanical properties of the resulting RSF hydrogels mainly depend on the solid content in the hydrogel, while the type and concentration of the surfactant has little effect. Thus, we propose a possible reason for the significant improvement of RSF hydrogel by adding surfactant. That is, although the total β-sheet content in the hydrogel almost does not change, the surfactant speeds up the formation of β-sheet structures, and more importantly confines their size. Therefore, these numerous but small-sized β-sheet 18


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domains act as the crosslinking points, leading more homogenous structure in RSF/surfactant hydrogels than those in naturally formed and ethanol induced RSF hydrogel. As a result, such a homogenous structure in RSF/surfactant hydrogel is able to endure more stress on it, showing the outstanding mechanical properties. In addition, the in vitro cytotoxicity test with L929 cells indicates the RSF/surfactant hydrogels keep the good biocompatibility of the pristine RSF hydrogel. We also show that by in situ forming Fe3O4 nanoparticles in RSF hydrogel, the resulting Fe3O4-loaded RSF hydrogel is able to act as a peroxidase-like catalyst to catalyze H2O2-related oxidation reaction efficiently and conveniently. We believe that such a strong and biocompatible hydrogel derived from a natural, abundant, and sustainable protein material may have a great potential as a multi-functional material in difference application areas. ASSOCIATED CONTENT Supporting Information. Digital photos of RSF solution and RSF/SDS hydrogel as-prepared, the comparison of gelation time of RSF/SDS solutions, the comparison of gelation time of other RSF/surfactant solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author * E-mail address: [email protected]. Fax: +86 21 5163 0300. Tel: +86 21 6564 2866. Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Z.L. and Z.Z. contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (No. 21274028, 21574023 and 21574024). The authors sincerely thank Miss Suhang Wang, Mr. Kunyuan Luo, and Miss Han Cao for their valuable suggestions and discussions.

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For Table of Contents Use Only

Robust Protein Hydrogel from Silkworm Silk Zhao Li, Zhaokun Zheng, Yuhong Yang, Guangqiang Fang, Jinrong Yao, Zhengzhong Shao, Xin Chen*

Strong protein hydrogel was successfully made from cheap, abundant, and sustainable silk fibroin from Bombyx mori silkworm silk by adding surfactant.

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Robust Protein Hydrogels from Silkworm Silk - ACS Sustainable

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