Intrinsically Fluorescent Silks from Silkworms Fed with Rare-earth

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Characterization, Synthesis, and Modifications

Intrinsically Fluorescent Silks from Silkworms Fed with Rare-earth Upconverting Phosphors Xiaoting Zheng, Menglu Zhao, Huihui Zhang, Suna Fan, Huili Shao, Xuechao Hu, and Yaopeng Zhang ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 8, 2018

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ACS Biomaterials Science & Engineering

Intrinsically Fluorescent Silks from Silkworms Fed with Rare-earth Upconverting Phosphors Xiaoting Zhenga,†, Menglu Zhaoa,†, Huihui Zhanga,b, Suna Fana,*, Huili Shaoa, Xuechao Hua and Yaopeng Zhanga,*

a

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

College of Materials Science and Engineering, Donghua University, Shanghai 201620, P.R. China. b

The Key Laboratory of High-Performance Fibers & Product, Ministry of Education,

College of Materials Science and Engineering, Donghua University, Shanghai 201620, P.R. China. CORRESPONDING AUTHOR Yaopeng Zhang E-mail: [email protected] Tel: +86-21-67792954. Fax: +86-21-67792855.

Suna Fan E-mail: [email protected] Tel: +86-21-67792954. Fax: +86-21-67792855.

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ABSTRACT: Fluorescent silk fibroin (SF) fibers have great potential in biomedical application and special functions for marking and tracking. How to fabricate fluorescent SF fibers with good fluorescence stability by a simple and environmentally-friendly method has yet to be explored. Here we successfully produced fluorescent SF fibers by using silkworms as bioreactors to introduce rare-earth upconverting phosphors (UCPs) into silk fibroin. The modified silk exhibited bright green colors under 980 nm laser. This directly feeding method to produce fluorescent SF fibers is green and environmentally friendly, and easy to use for mass production. Moreover, it provides an idea that SF fibers can be cooperated with more fluorescent materials which could exhibit different colors with certain wavelength of light for broad application. KEYWORDS: Fluorescent silk fibroin; Rare-earth upconverting phosphors; Feeding; Bioreactor; Mass production

1. INTRODUCTION Silk fibroin (SF) fiber has been widely used in textile industry, biological medicine and tissue engineering because of its lustrous appearance, extraordinary mechanical properties, biocompatibility, and controllable biodegradability 1-3. To meet a wide range of applications, SF has been integrated with various materials or chemically modified. Post-functionalization 4, amino acid modifications 5 and grafting reactions 6-8 were used for the SF chemical modification. Metallic compounds 9, oxidized graphene CNT

11

10

, and

were incorporated into SF fiber through dry spinning or electrospinning.

Moreover, a green way has been found to directly obtain enhanced SF fiber by feeding


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silkworms with inorganic nanoparticles modified artificial diet or mulberry leaves lately 12-15. Fluorescent SF fiber has immense potential in biomedical applications, including wounding dress, tissue engineering and drug delivery. Three methods have been reported to prepare fluorescent silk. The first one is to obtain silks with green, red and orange fluorescence through genetic engineering due to its great progress in recent years

21, 22

16-20

, which provides great potential

. However, efforts are still needed before

mass production to reduce cost and simplify process

23

. The second one is to post-

process silks with organic dyes 24 or inorganic nanoparticles 25-28. The method may be used in mass production, but many challenges, such as environmental pollution, fluorescent stability, the integration of additives within silk fibers, have to be overcome. The third one is to feed silkworms with dye additive to obtain intrinsically colored silk, which is more green and sustainable. Tansil et al. has reported pink colored silks by feeding silkworms with Rhodamine B modified diet way to produce pink colored silks

31

29, 30

. Trivedy et al. used similar

. However, the colored SF produced by feeding

silkworms is currently limited to textile manufacturing. Upconverting phosphors (UCPs), as important biological fluorescent labels, have been proved that can be applied in bio-imaging, and bio-generator. As 980 nm laser can penetrate biological tissue deeper than other lasers, silk materials with the function of upconverting fluorescence is more easily to be detected, even in vivo detection. In this work, we fed silkworms with UCPs modified diets and directly obtained the expected intrinsically fluorescent SF fibers. SEM and photoluminescence (PL) spectra as well as



images of modified SF fibers under 980 nm laser device were performed to confirm the existence of UCPs in the obtained SF fibers. These intrinsically fluorescent SF fibers may have great potential for application in the tissue engineering scaffolds with monitoring or bio-tracking features. 2. MATERIALS AND METHODS 2.1 Materials The eggs of Bombyx mori were provided by the Sericultural Research Institute of Guangxi, China. The artificial silkworm diet (powder) was purchased from the Sericultural Research Institute of Shandong, China. UCPs was purchased from Ningjing Zhiyuan Technology Co., Ltd (Shenzhen, China), which was prepared by hydrothermal method with lanthanide (Yb and Er) doping and oleic acid assisting, similar preparation process referring to the work of Wu et al 32. 2.2 Feeding Experiments Silkworm larvae were raised in a climatic chamber (Bilon HWS-350, Shanghai, China), as the growth of silkworms are easily affected by temperature, humidity, illumination and atmosphere. One hundred and twenty silkworm larva were divided into six groups. One group is control group fed with regular diet, the other five groups are experimental groups fed with UCPs modified diet from their second day of the fifth instar stage to spinning. The regular diet (Shangdong Sericultural Research Institute, China) was made of various plant powders, including mulberry leaf (38.4 wt%), defatted soybean (36.9 wt%), corn (9.0 wt%), agar (5.0 wt%), petioles (5.0 wt%), and other trace substances, including vitamin complex, choline chloride and citric acid.


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UCPs modified diet were prepared by mixing dry diet powder and UCPs solution (after ultrasonic treatment for 15-20 min) evenly and then microwaving for 5 min, pressing to be wafer finally. The mass fraction ratio of UCPs/dry diet powder was 0.03%, 0.06%, 0.09%, 0.12% and 0.15%. The temperature and humidity set in different growth periods of silkworms were the same to the work of Cai et al 14. 2.3 Silk Degumming Silkworm cocoons were harvest 6 days after spinning. They were dried for 1 h at 110 °C and 3 h at 80 °C in a vacuum drying oven, and then rolled out for 3-5 days in the air to dry thoroughly. Subsequently, the obtained cocoons were immersed in boiled 0.5 wt% aqueous Na2CO3 solution for 30 min and washed with deionized water to extract sericin thoroughly, then the SF fibers were obtained after drying overnight. The SF fibers obtained by feeding modified diet with former mass fraction ratio of UCPs/dry diet powder were assigned as UCPs-0.03%, UCPs-0.06%, UCPs-0.09%, UCPs-0.12% and UCPs-0.15%, respectively. The silk obtained by feeding regular diet was denoted by control. 2.4 Morphology Characterization The surface morphology of obtained SF fibers sputter-coated with platinum were observed through a Scanning Electron Microscope with energy dispersive spectra (EDS) analysis (Hitachi S-3000N) at 10 kV. The size of UCPs was observed with a Transmission Electron Microscope (TEM) (JEM-2100) at 200 kV and the UCPs were dispersed in ethanol.



2.5 PL Spectra and PL Images The fluorescence performance of SF fibers and UCPs were measured using a PL (JASCO FP-6600) instrument. They were excited with xenon lamp at 980 nm scanned in the range of 500-580 nm. The slit width of excitation and emission were both 0.8 mm. PL images of SF fibers were taken under a 980 nm laser device (Beijing Hi-Tech Optoelectronic Co., LTD, China) for 3 min. The excitation source is an external 980 nm semiconductor laser device connected to an optic fiber accessory with a diameter of 800 mm instead of the xenon source in the spectrometer. The power of the excitation source is adjustable from 0 to 1 W. The SF fibers were constructed as grid-like scaffold on a glass slide. First, paste a double-sided tape on a glass slide to form a 2 cm×2 cm square. Second, manually twist about 100 degummed silk filaments into a yarn, then crossly paste each yarn on the double-sided tape, each direction has six yarns. 2.6 Mechanical Properties The average diameter of each sample was obtained from ten points distributed along the fiber axis using optical microscope (BX-51, Olympus, Japan). For each sample, 15±5 single SF fibers were measured. Subsequently, these fibers were used to test mechanical properties using an Instron 5565 material testing machine at (20 ± 1) °C and (40 ± 5) % relative humidity. The experiment was carried out at an extension rate of 2 mm/min with a gauge length of 10 mm. 2.7 Fourier Transform Infrared Spectroscopy


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Infrared spectra were recorded by a Nicolet 6700 (Thermo Fisher, USA) with a diamond attenuated total reflectance (ATR) accessory at the resolution of 4 cm−1. A quantitative analysis of the secondary structure was carried out by operating spectra deconvolution 33, 34. 2.8 Synchrotron Radiation Wide-Angle X-ray Diffraction Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) was obtained at BL15U1 beamline at Shanghai Synchrotron Radiation Facility. The wavelength (λ) and the spot size of the X-ray were 0.07746 nm and 3 μm×2 μm, respectively. FIT2D (V12.077) and Peakﬁt (V4.12) software were applied to process the obtained data. Detailed process method can be found in our previous work 9. 2.9 Statistical Analysis All data results were expressed as mean ± standard deviations. One-way ANOVA test were performed to statistical analysis. P

Intrinsically Fluorescent Silks from Silkworms Fed with Rare-earth

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