Decay behavior and stability of free radicals of silk fibroin with alkali/urea pretreatment induced by electron beam irradiation

doi:10.1016/j.polymdegradstab.2020.109344

Polymer Degradation and Stability

Volume 181, November 2020, 109344

https://doi.org/10.1016/j.polymdegradstab.2020.109344 Get rights and content

Highlights

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A simple pretreatment method to improve the yield and stability of the free radicals was reported.
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Crystalline transformation, compacted spatial structure are responsible for high concentration and stability.
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Radical concentration increased 10 times at 25 kGy. The half-life time of free radicals is 10 times.

Abstract

Silk fibroin determines the perseverance of silk-based materials. The samples of silk fibroin were pretreated combined alkali/urea system with rapid freezing in liquid nitrogen. The structural changes of unpretreated and pretreated silk fibroins were investigated by XRD, SAXS and FTIR. The results indicate that part of the amorphous region was transformed into silk-II crystalline structure, and the radius of gyration decreased due to the folding of molecular chains by hydrogen bonds. The free radicals of unpretreated and pretreated silk fibroin induced by electron beam irradiation were studied by electron spin resonance (ESR). The results indicate that the radical concentration increased by 10 times at 25 kGy and attained 8.04*10¹⁹ spins·g⁻¹ at 50 kGy after pretreatment, which is comparable to the saturated radical concentration of unpretreated silk fibroin above 200 kGy. And the half-life time of free radicals is 10 times longer than that of the unpretreated samples. The higher stability of the free radicals is considered to be related to the protein structure changes induced by pretreatment and this can provide convenience for silk irradiation grafting. The new pretreatment method may provide inspiration for designing protein structures to regulate free radical formation.

Introduction

Silk fibroin, derived from Bombyx mori cocoons, has been widely studied and used for material applications as a protein polymer. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, and controllable biodegradability [1]. And, it can be modified by various methods to meet different environmental needs, including solution blending, chemical-modification, microwave treatment, and radiation modification [2], [3], [4], [5], [6]. Among them, the radiation-induced grafting technique is considered to be one of the meaningful ways for silk fibroin modification. There has been a lot of research on silk fibroin irradiation [7], [8], [9]. The properties of the free radicals (such as yield and stability) induced by irradiation will directly affect the grafting efficiency. It is an important method to improve the performance of silk fibroin. Generally, the yield of free radicals of silk fibroin could restrict the grafting rate. To improve the grafting rate, the yield of free radicals induced by irradiation increased with absorbed dose. However, with the absorbed dose development, the surface of the silk fibroin turned yellow, the cracks occurred, and the molecular chain was broken [10], [11]. That means the mechanical properties of silk-based materials decreased [12]. Besides, the grafting degree of pre-irradiated silk fibroin may decrease with the storage time after irradiation, whose grafting degree would decrease by 50% in 30.0 min, owing to the rapid quenching of free radicals by oxygen and moisture when exposed to air [8]. Thus, increasing the yield and stability of free radicals may be more conducive to irradiation-induced grafting process. Furthermore, there is also controversy existed about the attribution of free radicals induced by irradiation [13], [14]. Therefore, it is necessary to further study the properties of free radicals and provide certain experimental basis for their assignation.

The free radicals are related to space and crystal structure of silk fibroin [15]. To transfer the crystalline structure and improve the yield and stability of free radicals, the silk fibroin was pretreated by the combination of the alkali/urea system and the rapid freezing in liquid nitrogen. The pretreated silk fibroin was dried in a vacuum and then irradiated at different doses at room temperature. The crystal and chemical structure changes were studied via FT-IR and XRD, respectively. Synchrotron radiation small angle X-ray scattering (SR-SAXS) was used to characterize the spatial structure changes. The free radicals formation, decay, and stability were characterized by electron spin resonance (ESR). (The higher free radicals concentration of 8.04*10¹⁹ spins/g was obtained at low dose of 25 kGy. The stability of the free radicals also enhanced and the half-life time was almost 10 times longer. This work can ameliorate the conditions and efficiency of the silk irradiation grafting process, and it may inspire designing structures to regulate free radicals formation.

Section snippets

Materials

Cocoons from B. Mori silkworms were purchased from the Gui Sheng cocoon silk industry and trade co. LTD. The Na₂CO₃, NaOH and urea were supplied by Sinopharm chemical reagent Co. Ltd.

Silk fibroin pretreatment and irradiation

Silk fibroin was prepared according to the earlier literature [17]. Cocoons were boiled for 25.0 min in an aqueous solution containing 0.02 M Na₂CO₃ and then rinsed thoroughly with deionized water to remove sericin and residual Na₂CO₃. The mixed solutions based on NaOH (7.0 g)/urea (9.0 g)/H₂O (84.0 g) and silk

Structural transformation

The structural changes of unpretreated (S₀) and pretreated (Sp₀) silk fibroin were characterized by FT-IR spectra in Fig. 1 (a). The silk fibroin has important characteristic information at amide-I band (1705 cm⁻¹–1595 cm⁻¹) and amide-II (1595 cm⁻¹–1465 cm⁻¹) [17], which are a typical polypeptides and proteins conformational characterization [18], [19]. The peak located at 1700 cm⁻¹of unpretreated (S₀) and pretreated (Sp₀) silk fibroin assigned to stretching vibration of C double bond O bond, and the peak

Discussion

The results indicate that the yield and stability of free radicals can be significantly improved after the pretreatment. Usually, the stability of free radicals is closely related to the crystalline structure of the polymers including the crystalline degree and the crystallites size. Their persistence mainly depends on the steric protection of the unpaired electron [30]. Moreover, free radicals migration would also decrease radical stability [30]. The crystal structure could also promote the

Conclusion

After pretreatment, the crystalline structure transformed and the spatial structure shrank promoted the yield increase of free radicals induced by silk fibroin irradiation. And the stability of the free radicals formed by EB irradiation enhanced greatly. The transformation mechanism is the molecular chain folding caused by hydrogen bonds formation. Furthermore, the debate about the attribution of free radicals irradiated by silk fibroin provides certain experimental data. This work can

CRediT authorship contribution statement

Hao Zhang: Data curation, Writing - original draft. Feng Tian: Software, Data curation, Writing - original draft. Haitao Lin: Supervision, Resources. Rongfang Shen: Software. Weihua Liu: Writing - review & editing, Formal analysis. Yuying Huang: Conceptualization, Validation, Project administration. Zhongfeng Tang: Conceptualization, Validation, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by National Key R&D Program of China (No. 2017YFA0403000), National Natural Science Foundation of China (51772308, 51763001, 51963002), and Qinghai Major Science and Technology Projects (No. 2017-GX-A3).

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Decay behavior and stability of free radicals of silk fibroin with alkali/urea pretreatment induced by electron beam irradiation

Highlights

Abstract

Introduction

Section snippets

Materials

Silk fibroin pretreatment and irradiation

Structural transformation

Discussion

Conclusion

CRediT authorship contribution statement

Declaration of Competing Interest

Acknowledgments

J. Ind. Eng. Chem.

Nucl. Sci. Tech.

Biophys. J.

Appl. Surf. Sci.

Carbohydr. Polym.

Int. J. Biol. Macromol.

Cell

Cell

Polym. Degrad. Stab.

Biomacromolecules

Polym. Degrad. Stab.

Materials fabrication from bombyx mori silk fibroin danielle

Nat. Protoc.

Grafting versus crosslinking of silk fibroin-g-PNIPAM via tyrosine-NIPAM bridges

Molecules

Electron beam-induced crosslinking of silk fibers using triallyl isocyanurate for enhanced properties

J. Appl. Polym. Sci.

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J. Micromec. Microeng.