Influence of shear on the structures and properties of regenerated silk fibroin aqueous solutions

Abstract

In this paper, the structures and properties of regenerated silk fibroin (RSF) aqueous solutions under shear were investigated by using a polarizing microscope, an optical shearing instrument, a rotational rheometer, a Raman spectrometer and a synchrotron radiation small angle X-ray scattering facility. It was found that after being sheared at a constant shear rate for a suitable time, the RSF aqueous solution showed gradually decreased viscosity and enhanced birefringence before protein denaturation. Meantime, rod-like structures were formed and conformational transition happened in the solution. These results indicated that the RSF molecules in the solution might gradually form rod-like liquid crystal structures after applying sufficient shear. Moreover, it was also found that the increase of the shear rate could reduce the critical shear time for the solution to begin to form the liquid crystal structure.

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1. Introduction

Silkworms can produce fibers from a silk fibroin aqueous solution under atmospheric pressure and room temperature^1,2 and the spinning process in vivo attracts great attention.^3–5 It is well known that shear stress, which gradually changes along the silk gland, is very important to the formation of silk fiber.^6,7 Furthermore, silk fibroin dope undergoes continuous shear when it flows along the silk gland before forming a fiber, which means a suitable shear time affects the spinning process of the silkworm as well. Therefore, in the study of biomimetic spinning, many attempts had been focused on the effect of shear on the regenerated silk fibroin (RSF) aqueous solution.^8–10 Rossle et al.^11,12 investigated the structural evolution of RSF aqueous solution under shear and found the shear stress resulted in the elongation of RSF molecules. Zhou et al.¹³ suggested that the sol–gel transition of RSF aqueous solutions could be induced by shear. Liu et al.¹⁴ studied the conformational transition of RSF aqueous solutions under shear, which gradually changed from random coil/α-helix to β-sheet conformation. Meanwhile, reports have revealed that other factors such as pH value and metal ions (for example Ca²⁺) could also affect the formation process of silk fiber,^15,16 which had not been considered simultaneously with the factor of shear in the above literatures.

In this work, in order to biomimic the formation of the silk fibroin dope in silk gland, the pH value and the Ca²⁺ concentration of the RSF aqueous solution were adjusted. Then the structures and properties of the RSF solutions during shear were investigated using polarizing microscope, optical shearing instrument, rotational rheometer, Raman spectrometer and synchrotron radiation small angle X-ray scattering (SAXS) facility. The goal of current work is to extensively investigate the effect of shear on the formation of silk fiber and provide a useful guide for future study.

2. Experimental methods

2.1. Sample preparation

Bombyx mori cocoons from Zhejiang province, China, were degummed and a 20 wt% RSF aqueous solution was prepared as reported.¹⁷ To prepare a dry-spinnable RSF aqueous solution, the pH value and Ca²⁺ concentration of the solution were adjusted. The solution was firstly mixed with a buffer reagent of 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES)–trishydroxymethylaminomethane (Tris) to form a RSF solution with a pH value of 4.8. A 3 mol L⁻¹ CaCl₂ aqueous solution was then added into the RSF system to adjust the concentration of Ca²⁺ to 2.4 mmol g⁻¹.¹⁷ Then the mixture was condensed in forced airflow at 10 °C and the resultant RSF aqueous solution with a corrected RSF concentration of 41 wt% and Ca²⁺ concentration of 3.9 wt% was prepared for measurements.

2.2. Characterization

The optical properties of RSF aqueous solutions under shear were observed by a BX51 polarizing microscope (Olympus Co., Japan) with a CSS450 optical shearing system (Linkam Co., UK).

The rheological measurements of RSF aqueous solutions were performed on a RS150L rheometer (Thermo Haake, Germany) at 25 ± 0.1 °C equipped with a parallel plate configuration with a diameter of 20 mm.

Raman spectra of RSF aqueous solutions were obtained using a LabRam1B microscopy Raman spectrometer (Dilor Co., France). A He–Ne laser at 632.8 nm was used to generate an intensity of 6 mW on the samples and the data were recorded from 900 to 1800 cm⁻¹. The quantitative analysis of the amide I region was conducted using a deconvolution method reported by Zhou et al.¹⁸

Small-angle X-ray scattering (SAXS) experiments for RSF aqueous solutions were carried out at the BL16B1 beamline in Shanghai Synchrotron Radiation Facility with a spot size of 1 mm × 1 mm and a wavelength of 1.24 Å. During the SAXS experiments, the samples were continuously sheared by the CSS450 optical shearing system (Linkam Co., UK) using two parallel stainless steel plates coated with Kapton film and the exposure time for each SAXS pattern was 200 s. The detector-to-sample distance was calibrated by lanthanum hexaboride to 5020 mm. Patterns of solvent (deionized water) were recorded as background. All two-dimensional (2D) detector images were analyzed using FIT2D software (V12.077) to produce one-dimensional (1D) intensity profiles. Then the radius of gyration (R_g) for RSF aggregates in the solutions under different shear conditions was calculated from the SAXS results according to the Guinier extrapolation.¹¹ The shapes of RSF aggregates were determined by DAMMIN software¹² and the results were visualized by Pymol software.

3. Results and discussion

3.1. Optical properties of RSF aqueous solutions

Fig. 1 shows the polarizing microscope images of RSF aqueous solutions sheared at 0.5 s⁻¹ for different shear times. It was clear that at the beginning of shear, the RSF aqueous solution showed black under polarized light (Fig. 1a). As the shear time increased to about 900 s, part of the solution became birefringent (Fig. 1c), which implied that the RSF aqueous solution was gradually transformed from isotropy into anisotropy and an ordered structure was formed in the solution due to the continuous shear. This shear time was named as critical shear time in this paper. Meanwhile, the critical shear times when the solution began to show birefringence under different shear rate were also recorded in Fig. 2 and 3, which showed similar results of the RSF solutions sheared at 1 s⁻¹ and 2 s⁻¹, respectively. By combining Fig. 1–3, it was found that the increase of shear rate and shear time could both lead to the birefringence, implying the increase of shear time could lead to the birefringence, and the increase of the shear rate could reduce the critical shear time for the solution to became birefringent. These results indicated that the RSF aqueous solution could change into anisotropy after being sheared at a constant shear rate for an appropriate time.

Fig. 1 Polarizing microscope images of the RSF aqueous solutions under different shear times ([small gamma, Greek, dot above] = 0.5 s⁻¹).

Fig. 2 Polarizing microscope images of the RSF aqueous solutions under different shear times ([small gamma, Greek, dot above] = 1 s⁻¹).

Fig. 3 Polarizing microscope images of the RSF aqueous solutions under different shear times ([small gamma, Greek, dot above] = 2 s⁻¹).

3.2. Rheological behavior of RSF aqueous solutions

Fig. 4 shows the rheological results of the RSF aqueous solutions under different shear rates and different shear times. It was found that although the shear rates were different, the rheological behaviors of the solutions were similar. When the shear time was short, the viscosity of the solution was low. Then the viscosity gradually increased with the extension of shear time until it reached a peak value. Moreover, the shear time corresponding to the peak value of the viscosity was consistent with the time when the solution began to show birefringence (i.e. the critical shear time). This indicated that at this moment, some RSF molecules were orderly arranged and the liquid crystal structure might begin to form in the RSF solutions due to the shear. The formation of liquid crystal resulted in the decrease of the viscosity after the critical shear time due to the orientation of the RSF molecules and formed ordered structures in the solution. With further extension of the shear time, the viscosity of the solutions decreased continuously, which meant the orientation of the RSF molecules was promoted and more liquid crystal structures were formed in the solutions. At last, the viscosity of the solutions was unstable due to the denaturation of protein. In addition, by comparing the changes in the viscosity of RSF aqueous solutions under different shear rates and different shear times, it was also found that the increase of the shear rate could reduce the critical shear time for the solution to begin to form the liquid crystal structure.

Fig. 4 The rheological results of the RSF solutions under different shear rates and different shear times.

3.3. Secondary structure of RSF aqueous solutions

Fig. 5 shows the Raman spectra of RSF aqueous solutions sheared for different shear times. And Table 1 gives the quantitative analysis results of the amide I region in these Raman spectra. The main absorption bands of secondary structures were assigned as follows: 945 cm⁻¹, 1105 cm⁻¹ as α-helix conformation, 1252 cm⁻¹ as random coil (in the amide III region), 1665 cm⁻¹ as β-sheet (in the amide I region), 1680 cm⁻¹ as β-turn structure (intermediate conformation). The band at 1085 cm⁻¹ was also assigned to the β-sheet conformation. It was found that the initial RSF aqueous solution was mainly in random coil/α-helix and intermediate conformation. After being sheared for about 560 s, the content of the random coil/α-helix conformation was decreased, while that of the β-sheet conformation was increased. These implied that the conformation of silk fibroin in the solution gradually changed from random coil/α-helix to β-sheet during shear. Meanwhile, combined with the above optical and rheological results of RSF aqueous solution, it was clear that these conformations became gradually ordered and the liquid crystal structures with birefringence began to form. As the shear time further extended (660 s), the conformational transition to β-sheet continued, accompanied with the enhancement of birefringence (see Fig. 2-c) and the development of the liquid crystal structures in the solution as well as the decrease of the solution viscosity. When the shear time was too long (e.g. 1000 s), the denaturation of protein might occur since the transparent RSF solution became white and opaque gradually.^19,20

Fig. 5 Raman spectra of the RSF solutions sheared for different times ([small gamma, Greek, dot above] = 1 s⁻¹).(a) 0 s, (b) 560 s, (c) 660 s, (d) 1000 s.

Table 1 Quantitative analysis results of the amide I region in Raman spectra of RSF aqueous solutions sheared for different times ([small gamma, Greek, dot above] = 1 s⁻¹)

Shear time (s)	Random coil/α-helix (%)	β-Sheet (%)	Intermediate conformation (%)	Phenyl (%)
0	48	10	37	5
560	40	15	40	5
660	25	39	31	5
1000	20	40	35	5

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3.4. Aggregation behaviors of RSF aqueous solutions

The SAXS scattering results can be used to obtain the information on the shapes and structures of RSF aggregates in aqueous solutions. The R_g values of the RSF aggregates in aqueous solutions under different shear rates and times were obtained from the Guinier analysis.¹¹ As shown in Table 2, the R_g value of the initial RSF aggregate in aqueous solution was about 30 nm. When the shear time extended properly, this value was gradually increased, which meant the RSF molecules were aggregated under shear. As the shear time extended overly, the R_g value of the RSF aggregate began to decrease due to the denaturation.

Table 2 The R_g of RSF aggregate in aqueous solutions under different shear rates and different shear times

Shear time (s)	Rg (nm) [small gamma, Greek, dot above] = 0.5 s^−1	Shear time (s)	Rg (nm) [small gamma, Greek, dot above] = 1 s^−1	Shear time (s)	Rg (nm) [small gamma, Greek, dot above] = 2 s^−1
0	30.4	0	30.4	0	30.4
400	31.9	300	31.5	200	32.0
900	32.3	560	31.8	460	33.4
1000	35.5	660	38.1	520	37.3
1200	27.2	1000	26.1	800	27.7

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The shapes of RSF aggregates simulated by DAMMIN software are given in Fig. 6. It was clear that the initial RSF molecules were disordered in the aqueous solutions. After being sheared for a while (e.g. Fig. 6A-b), the RSF molecules were gradually ordered along the direction of shear and an elongated RSF aggregate with a ratio of the length to diameter (L/D)²¹ of 12 was appeared, which meant a rod-like structure was formed in the solution. As the shear time increased (e.g. Fig. 6A-c), the aggregation of the RSF molecules was further promoted and the L/D ratio of the rod-like structure was increased to 14. When the shear time extended enough (e.g. Fig. 6A-d), more and more RSF molecules were ordered to form a rod-like structure with a L/D ratio of 16. This was consistent with the L/D ratio of rod-like liquid crystal model for natural silk fibroin reported by Viney et al.²¹ Their results indicated that the liquid crystal structure domain in the natural silk fibroin aqueous solution was rod-like and its L/D ratio was approximately 15–30. Our results showed that, after being applied sufficient shear, the RSF molecules could also form a rod-like liquid crystal structure as the natural silk fibroin in vivo. With further increasing shear time, the RSF aggregates became smaller due to protein denaturation (e.g. Fig. 6A-e). Fig. 6B and C show similar results as Fig. 6A. Although the shear rates were different from 0.5 s⁻¹ to 2 s⁻¹, a rod-like liquid crystal structure could be formed in all the RSF aqueous solutions after appropriate shear time. These simulation results were also in agreement with the optical and rheological results above.

Fig. 6 Molecular shape of RSF simulated by DAMMIN software under different shear conditions (the L/D ratio of the rod-like structure in the aggregate is labelled on the top right of the figures) (A) shear rate = 0.5 s⁻¹, (B) shear rate = 1 s⁻¹, (C) shear rate = 2 s⁻¹.

4. Conclusion

In this work, the effect of shear on the structures and properties of RSF aqueous solutions was investigated. After being sheared at a constant shear rate for a suitable time, the RSF aqueous solution showed gradually enhanced birefringence and decreased viscosity. Meanwhile, its conformation was gradually transformed from random coil/α-helix to β-sheet and a rod-like structure in the elongated RSF aggregate was formed in the solution under the condition. These indicated that a liquid crystal structure was gradually aggregated in the solution after being applied sufficient shear. Furthermore, the increase of the shear rate could reduce the critical shear time for the solution to begin to form the liquid crystal structure. These results were helpful to understand the formation mechanism of silk fiber and provide a useful guide for in vitro study in future.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (21274018), the Specialized Research Fund for the Doctoral Program of Higher Education (200802550001), the Programme of Introducing Talents of Discipline to Universities (No. 111-2-04), DHU Distinguished Young Professor Program (A201302) and the Fundamental Research Funds for the Central Universities (2232013A3-11).

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