Synthetic Engineering of Spider Silk Fiber as Implantable Optical

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Synthetic Engineering Spider Silk Fiber as Implantable Optical Waveguides for Low-Loss Light Guiding Xin Qiao, Zhigang Qian, Junjie Li, Hongji Sun, Yao Han, Xiaoxia Xia, Jin Zhou, Chunlan Wang, Yan Wang, and Changyong Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01752 • Publication Date (Web): 06 Apr 2017 Downloaded from http://pubs.acs.org on April 7, 2017

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ACS Applied Materials & Interfaces

Synthetic Engineering Spider Silk Fiber as Implantable Optical Waveguides for Low-Loss Light Guiding Xin Qiao†, Zhigang Qian‡, Junjie Li†, Hongji Sun†，Yao Han†, Xiaoxia Xia‡ Jin Zhou†, Chunlan Wang†, Yan Wang* † and Changyong Wang*† †Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences, 27 TaipingRoad, Beijing, 100850, P.R. China ‡State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China

KEYWORDS: Genetic engineering, Spider silk protein, Optical waveguides, Implantation, Biodegradation

ACS Paragon Plus Environment

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ABSTRACT: A variety of devices used for biomedical engineering have been fabricated using protein polymer because of their excellent properties, such as strength, toughness, biocompatibility and biodegradability. In this study, we fabricated optical waveguide using genetically engineered spider silk protein. This method has two significant advantages: (1) recombinant spider silk optical waveguide exhibits excellent optical and biological properties, and (2) biosynthesis of spider silk protein can overcome the limitation to the research on spider silk optical waveguide due to the low yield of natural spider silk. In details, two kinds of proteinbased optical waveguides made from recombinant spider silk protein and regenerative silkworm silk protein were successfully prepared. Results suggested that the recombinant spider silk optical waveguide showed smoother surface and higher refractive index when comparing with regenerative silkworm silk protein. The optical loss of recombinant spider silk optical waveguide was 0.8 ± 0.1dB/cm in the air and 1.9 ± 0.3dB/cm in mouse muscles, which were significantly lower than that of regenerative silkworm silk optical waveguide. Moreover, recombinant spider silk optical waveguide can meet the demand to guide and efficiently deliver light through biological tissue. In addition, recombinant spider silk optical waveguide showed low toxicity to cell in vitro and low level inflammatory reaction with surrounding tissue in vivo. Therefore, recombinant spider silk optical waveguide is a promising implantable device to guide and deliver light with a low loss.

1. INTRODUCTION Modern implantable medical devices have been developed for a variety of functions, such as tissue engineering, drug delivery systems,

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optical devices for diagnosis or treatment3-6 and


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electronic devices.

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Among the various implantable medical devices reported, optical

waveguides have many important applications. For instance, optical waveguide devices fabricated by poly(dimethylsiloxane) (PDMS), owing to its optical transparency, can be biologically inserted into living tissue for real-time observations and analyses in the cellular and sub-cellular scales.8-9 Also, they are frequently inserted into brain to deliver light to deep targets in the field of neuroscience and optogenetics. Currently, implantable optical waveguides are largely fabricated from glass, silicone and metals.10-11 Although these implantable optical waveguides are highly efficient, they are not well suited for biological applications due to the lack of biocompatibility and biodegradability. 12-13 To overcome these limitations, protein-based optical waveguides with excellent performance need to be developed. Fibers and proteins typically produced by Bombyx mori silkworms and spiders have been widely used in the biomedical fields, because of their distinctive physical and biological properties, such as excellent strength, toughness,

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biocompatibility and biodegradability.17

Recently, they have drawn people’s increasing attention for their potential as optical waveguides. Meanwhile, development of protein-based optical waveguides in biomedical applications is promoting the demands for more kinds of optical materials and interfaces. Except the biocompatibility with the biological system,18-19 optical waveguides must overcome the limitation that the light-guiding efficiency inside living tissues.20 Parker and Ghosh et al. have reported a new method of directly writing 3D optical waveguides from an aqueous solution of regenerated Bombyx mori silk fibroin.21-22 These optical waveguides have the ability to manipulate and transport light in a controlled and efficient manner. Therefore, the use of these biocompatible and biodegradable optical waveguides will provide a new way to transmit light for various applications, such as optical therapy and real-time sensing inside living tissues.

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Kujala et al. have shown the waveguide properties of natural degummed Bombyx mori silk.17 The reported waveguide properties make this optical waveguide have huge potential to deliver optical power efficiently in the photomedicine field. The Huby et al. have demonstrated the optical fiber behavior of native spider dragline silk from the spider Nephila clavipes. They proved light could propagate in native spider dragline silk in air and physiological liquid.

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However, the disadvantage of the optical fiber based native spider dragline silk is that the optical loss is higher than that of many synthetic polymer fibers. Silkworm silks, easily domesticated and farmed for large-scale production, have a variety of commercial applications, such as textiles and biomedical materials.25 The easy availability of silkworm silks is one of important reasons that researches on optical waveguide based on regenerated silk protein could be easily carried out and mainly focused on. Unlike silkworm silks, the large-scale harvesting of spider silks by farming spiders is not feasible due to (1) spiders’ cannibalistic nature26-27 and (2) the difficulty of breeding and low production rate in captivity. The difficulty of obtaining should be responsible for few research conducted on the optical waveguides from spider silk. To overcome the limited availability, production of spider silk proteins by genetic engineering technology is the most promising alternative pathway. Based on the studies about spider silk genes, genetic engineering spider silk proteins have been expressed in many kinds of hosts including bacteria, yeast,

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plant as well as in the milk of

transgenic mammal.32 The major ampullate spidroins (MaSp) of the dragline silks from the spider Nephila clavipes and Araneus diadematus are best characterized. All these major ampullate spidroins have multiple repetitive and consensus core sequences consisting of some distinct consensus motifs.14, 33 Production of spider silk proteins mimicking the native protein MaSp of Nephila clavipes in E. coli have been realized. 34-38 Purified proteins can be processed


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and assembled into different morphologies for a variety of applications. However, there is no report about researches on optical waveguide based on recombinant spider silk protein. It can be seen that optical waveguides fabricated with natural silkworm silk and regenerative silkworm silk have been well characterized previously. 17, 21-22 Conversely, few research focus on optical waveguides of spider silk due to the difficulty in production on a large scale, which makes these waveguides of spider silk particularly attractive to researchers. To overcome the limitation of low spider silk production for application as optical waveguides, a new method to fabricate optical waveguide using spider silk protein was developed via genetic engineering technology in this study. Genetic engineering spider silk protein has been expressed in E. coli with high cell-density culture and purified using Ni-NTA immobilized metal affinity chromatography. Two kinds of protein-based optical waveguides made from recombinant spider silk protein and regenerative silkworm silk protein were successfully fabricated. The optical loss influenced by surface microstructures of optical waveguides was assessed by optical microscopy and scanning electron microscope (SEM). In order to evaluate the efficiency of guiding light, parameters of refractive index and optical loss were measured. Also, the ability to guide and deliver light in biological tissue was assessed in muscle of Sprague-Dawley (SD) rat. In addition, the biocompatibility of optical waveguides was evaluated using cytotoxicity experiment of optical waveguides in vitro and in vivo. 2. EXPERIMENTAL SECTION 2.1 Construction of recombinant plasmids The monomer protein sequence was selected based on the 31 amino acids consensus repeat GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG mimicking the native core domain of the


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major ampullate dragline Spidroin protein 1(MaSpI) sequence from the spider Nephila clavipes. 39-41

The corresponding monomer DNA sequence was designed to suit for codon usage in E. coli and synthesized (Figure 1A). The synthesized DNA sequence, flanked by compatible restriction sites SpeI and XhoI at the 3′ end and NdeI and NheI at the 5′ end, was cloned into the NdeI-XhoI site of the expression vector pET-19b (Novagen, WI, USA) to create the plasmid called pET-19b -1mer MaSpI (Figure 1B). The expression vector pET19b also contained a unique restriction site (PvuI) in the ampicillin resistance gene (selection marker). Two fragments (PvuI-NheI fragment and SpeI-PvuI fragment) each containing a copy of a monomer DNA gene were obtained by simultaneously performing two double digestion reactions. The PvuI-NheI and SpeI-PvuI fragments were ligated together by using T4 ligase (New England Biolabs, Ipswich, MA, USA), thus effectively doubling the size of the monomer DNA gene insert and restoring the unique restriction site (PvuI) located in the ampicillin resistance gene of the plasmid (Figure 1C). After 4 rounds of cloning, this strategy increased the number of DNA sequence repeats insert and allowed the plasmid (pET-19b -16mer MaSpI) to express the recombinant silk protein having 16 repeats of 31 amino acids monomer. Finally, the expression plasmid of pET-19b -16mer MaSpI was identified by restriction digest analysis with NdeI and XhoI. Note: All molecular biology procedures abovementioned were performed following the manufactures’ and standard protocols.34 2.2. Expression and Purification of Recombinant Spider Silk Protein 2.2.1. The expression of protein


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The expression and purification of recombinant spider silk protein is illustrated in Figure 2A. Firstly, the recombinant plasmid pET-19b -16mer MaSpI was transformed to competent cells E. coli BL21 (DE3) (Tiangen Biotech Co., Beijing, China), a common efficient expression host suitable for pET expression system. After transformed competent cells were plated and cultivated on selective LB agar plate (containing 50 µg/mL ampicillin) overnight, a single colony was selected and inoculated into a 15 mL tube containing 4mL liquid LB medium with ampicillin (50 µg/mL). Bacteria were cultivated at 37 °C and 200 rpm in the incubator and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma, MO, USA) at 30 °C when the OD600 reached 0.4~0.6. Cells were harvested after 5 h of induction by centrifugation at 10,000 rpm for 10 min at 4 °C. Protein identity and expression were confirmed and analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 2.2.2. High cell-density culture of recombinant E. coli For large-scale expression, the high cell-density culture (HCDC) of recombinant E. coli was performed in a 5 L fermentor (Bailun Bio-Technology, Shanghai, China). The selected colony was inoculated into the 4 mL LB medium with ampicillin (50 µg/mL) in a 15 mL tube and cultured at 37 °C and 200 rpm for 10 h. 4 mL of LB medium containing recombinant E. coli was transferred into 400 mL R/2 medium (pH=6.80) supplemented with 10 g/L glucose and 0.7 g/L MgSO4·7H2O at 30 °C and 200 rpm. Once the OD600 reached 1.6-2.0, this seed culture was then inoculated into fermentor containing the same R/2 medium.41 The nutrient was kept by adding feeding solution containing 700 g/L glucose and 20 g/L MgSO4·7H2O according to the strategy called pH-stat feeding.41 Meanwhile, the dissolved oxygen concentration was maintained above 35% of air saturation by increasing the purity of oxygen and by accelerating the stirring speed up to 700 rpm. When the OD600 reached ~40, expression was induced by 1


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mM IPTG for 4-6 h. Cells were sampled at the time of 1, 2, 3, 4, 5 h, respectively after induction and were harvested for purification at by centrifugation 10,000 rpm for 10 min at 4 °C. Finally, protein expression at different time was analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 2.2.3. Purification of protein The expressed proteins with N-terminal His-tag were purified by Ni-NTA immobilized metal affinity chromatography (IMAC). The bacterial pellet was suspended in lysis buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 5 mM imidazole) and disrupted by sonicating with ultrasonic homogenizers for 10 min. After centrifugated at 8,000 rpm for 20 min, the supernatant was heat treated at 80 °C for 20 min to eliminate most of native E. coli. proteins. The resultant protein solution were further centrifugated at 9500 rpm for 20 min at 4 °C and then loaded onto a NiNTA resin (Qiagen, CA, USA) column that had been equilibrated with the lysis buffer abovementioned. Those unbound proteins were removed after several successive washing of the Ni-NTA resin with washing buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole). Then, recombinant proteins containing His-tag were eluted and recovered with elution buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 150 mM imidazole). To remove the salts and imidazole, the eluted recombinant protein solution was dialyzed in cellulose ester snake skin membranes with MWCO 12,000-14,000 Da (Spectra/Por Biotech, CA, USA) against 1 mM NH4HCO3 solution at 4 °C. Finally, the solution was filtered by 0.22 mm syringe filters (Millipore, MA, USA) and lyophilized. The purity of recombinant protein was analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 2. 3. Preparation of Regenerated Silkworm Silk Solution


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The preparation of regenerated silkworm silk solution is illustrated in Figure 2B. 5g of Bombyx mori silk cocoons were measured and cut into dime-sized pieces. Pieces of silk cocoons were both boiled in 0.02 M solution of Na2CO3 and Milli-Q water for 30 min. Fibers were washed with Milli-Q water for 3 times to remove sericin and dried thoroughly. Finally, degummed silk fibroin was dissolved in 9.3 M LiBr solution at 60 °C for 4 h to prepare a 20% (wt/vol) solution. The solution completely dissolved would be highly viscous and transparent in the color of golden. To remove the LiBr, the mixed solution was dialyzed in cellulose ester snake skin membranes with MWCO 1000 Da (Spectra/Por Biotech, CA, USA) against Milli-Q water at 4 °C for 24 h. The impurities were removed by centrifuging twice at 12,000 rpm at 4 °C for 20 mins. To improve the concentration of regenerated silkworm silk solution, aqueous solution was concentrated to 400 mg/mL by 10% (wt/vol) polyethylene glycol (Sigma, MO, USA) with molecular weight of 6000 for 12 h. 42 The regenerated silkworm silk solution should be stored at 4 °C。

2.4. Fabrication of optical waveguides The purified lyophilized recombinant spider silk proteins were dissolved with 1, 1, 1, 3, 3, 3hexafluoroisopropanol (HFIP; Sigma, MO, USA) to prepare a 400 mg/mL solution. The solution was shaken at 37 °C until all proteins were completely dissolved overnight. Meanwhile, the regenerated silkworm silk solution was directly used for the next experiment. Next, recombinant spider silk protein solution and regenerated silkworm silk solution were respectively pipetted into polytetrafluoroethylene tubes with inner diameters of 800 µm. Protein solutions in the molds were heated at 60 °C for at least 7 days allowing HIFP and water to evaporate completely. After being fabricated, optical waveguides could be easily removed from the molds.


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2.5. Waveguide Characterization 2.5.1. The microstructure observation The characteristic and surface microstructures (i.e., surface defects, breaks and diameter) of optical waveguides were observed and analyzed by an optical microscopy (Olympus, Japan) and a scanning electron microscope (Hitachi S-4800N scanning electron microscope.). Optical microscopy images of silk waveguides were observed under 5×5 magnification with common white light. Meanwhile, SEM images of silk waveguides were photographed with an applied voltage of 5 kV and at room temperature. Prior to SEM photographing, each sample was gold coated for 30s by using a sputter coater device (Leica, Vienna, Austria). 2.5.2. Buckling force experiment The buckling force of the flexible optical waveguides can be estimated with the equipment developed by ourselves. The standard curve (weights-force) must be draw by calibrating the equipment using weights of 1, 2, 3, 4, 5, 7, 10, and 15 grams. 2 cm long waveguides were fixed vertically to the equipment and were pushed forwards to the mechanical sensor by motor. Then, the data were recorded with the mechanical sensor and analyzed with the software of Matlab. The scatter diagram of force vs. time plots was graphed to get the maximal force point. 2.6. Optical Experiments In order to assess the possibility as implantable optical waveguides, the optical behavior and parameters (refractive index, waveguide loss) of those samples are essential to measured. 2.6.1. Measurement of refractive index


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The refractive index of the optical waveguides was measured using the equipment of spectroscopic ellipsometer (SENTACH SE850). Samples were prepared into the standard form of film covering the silicon substrate with spin coater. Then, these samples were measured at wavelengths ranging from 350 nm to 1700 nm at incident angles of 70 and 75 degree. The measured data were read and fitted with the existing standard models using the software of SpectraRay III to export the refractive index of the optical waveguides automatically. 2.6.2. Measurement of the optical loss The optical losses of those optical waveguides in air and muscle were measured with the cutback method according to previous studies.16, 43 In this work, the optical losses described by the attenuation coefficient α were also assumed to be constant, according to the attenuation law of Beer-Lambert. First, 635 nm laser light was coupled into those optical waveguides that light guided inside, and the input power was assumed P0. The output power P1 was measured with an optical power meter at the length of L1. Then, this waveguide was cut to a length L2 (L2＜L1) and P2 was measured at once. The lengths of optical waveguides were precisely measured by an optical microscope with software of ImageJ2X. Output power of every waveguide was recorded repeatedly at various lengths (n≥3). Finally, the optical loss is calculated according to the BeerLambert law with the output power at different lengths according to equ (1)

α=

× log

(1)

whereas the output powers Pj and Pi were measured respectively at lengths Lj and Li (Lj > Li.). In order to improve the accuracy, several optical waveguides were used here (n=3).


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2.6.3. Light delivery to deep tissue To assess whether the recombinant spider silk optical waveguide is an ideal implantable optical device to guide and deliver light in biological tissue, the optical waveguide was inserted into muscle of SD mice. First, the laser light at 635 nm was delivered to the muscle surface without a waveguide, and the light penetration length was measured soon afterwards. Then, the recombinant spider silk optical waveguide was inserted into muscle and the light penetration length was measured when laser light of same intensity launching. Meanwhile, the laser light was observed whether can be delivered into the deep tissue along the optical waveguide. 2.7. Biocompatibility of optical waveguides 2.7.1. Cytotoxicity of optical waveguides in vitro To evaluate cytotoxicity and biocompatibility, optical waveguides samples sterilized by the method of γ-irradiation were incubated in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin at 37 °C for 48 h to extract soluble toxic substances. The murine fibroblasts L929 cell line was commonly chosen for cytotoxicity and biocompatibility study in vitro due to highly stable and fast growing features. Cells were cultured in extracting culture medium from incubated optical waveguides samples and maintained in an incubator at 37 °C with 5% CO2 and 95% relative humidity. To evaluate the cytotoxicity of the optical waveguides, a standard colorimetric 2-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay was performed. About 5×103 cells were seeded into each well of 96-well plates with 200 µL normal


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D-MEM, D-MEM containing leach liquor of regenerative silkworm silk optical waveguides or D-MEM containing leach liquor of recombinant spider silk optical waveguides, respectively, and incubated at 37 °C in 5% CO2 for 1 day. After 1 day, 20 µL of MTT solution (5 mg/mL in PBS) was added in each well and incubated at 37 °C for 4 h. Next, the medium was removed and the purple formazan was solubilized by 200 µL of DMSO. After mixing, the absorbance was measured at λ = 472 nm by a Spectra Max M2 plate reader (Molecular Devices, CA, USA). The experiment above was performed repeatedly at the time of 2 days, 3 days and 4 days. The normal D-MEM medium without cells was used determine the background absorbance, and the experiment was performed in triplicate (n = 5). Meanwhile, cell viability can also be assessed by using LIVE/DEAD Kit (Invitrogen, CA, USA), which allowed identification of live cells (green color) from dead cells (red color). About 5×103 cells were seeded into 48-well plates with normal D-MEM supplemented with 10% (v/v) FBS, and incubated at 37 °C in 5% CO2 for 1 day. After 1, 2, 3 and 4 days, cells were incubated with calcein AM (4 µM) and ethidium homodimer-1 (2 µM) simultaneously for 30 mins at 37 °C to stain live cells (green) and dead cells (red). After staining, cells were washed twice with PBS and observed using fluorescence microscope (Leica, Wetzlar, Germany). 2.7.2. Subcutaneous Implant Test To assess whether the genetically engineered spider silk optical waveguide is an ideal implantable optical device, which can be left and absorbed within the body after surgery, the biodegradation and biocompatibility of optical waveguides were studied in mice. Immunocompetent male SD rats (weight 200-220g) were used for biocompatibility study in vivo. After mice received abdominal nesthesia by pentobarbital natrium, non-biodegradable surgical


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sutures, regenerated silkworm silk optical waveguides and genetically engineered spider silk optical waveguides of same length were implanted subcutaneously in 3 groups of mice. After implantation, the incisions were then closed using 6-0 surgical nylon sutures. Mice were housed for 3 weeks. After mice were executed by CO2 asphyxiation, the inflammatory response was assessed by histological staining. The samples of muscle around implant were cut off and immersed immediately in 4% formalin solution for over 24 h. Then, the samples were cut into 5µm sections and stained with hematoxylin & eosin (H&E). After staining, slides were observed and imaged using a bright-field microscope with a 10× objective lens. Thirty replicates of every type of sample were implanted into thirty different mice, which ensure ten mice can be studied at every time point. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Military Medical Science (Beijing, China), according to the Guide for the Care and Use of Laboratory Animals. 2.8. Statistics All statistic analyses of the data are performed with the software of GraphPad Prism 5.0 (San Diego, California, USA). Data are measured in triplicate at least. To test for significant differences between culture medium in MTT assay, two-way ANOVA with one repeat was used for comparison. p

Synthetic Engineering of Spider Silk Fiber as Implantable Optical

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