SilkâSilk Interactions between Silkworm Fibroin and Recombinant

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Silk-silk interactions between silkworm fibroin and recombinant spider silk fusion proteins enable construction of bioactive materials Linnea Nilebäck, Dimple Chouhan, Ronnie Jansson, Mona Widhe, Biman Mandal, and My Hedhammar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10874 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 29, 2017

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ACS Applied Materials & Interfaces

Silk-silk interactions between silkworm fibroin and recombinant spider silk fusion proteins enable construction of bioactive materials Linnea Nilebäck†, Dimple Chouhan‡, Ronnie Jansson†, Mona Widhe†, Biman B. Mandal‡,*, My Hedhammar†,* †

KTH Royal Institute of Technology, School of Biotechnology, AlbaNova University Center,

106 91 Stockholm, Sweden ‡

Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and

Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India

ACS Paragon Plus Environment

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ABSTRACT

Natural silk is easily accessible from silkworms and can be processed into different formats suitable as biomaterials and cell culture matrices. Recombinant DNA technology enables chemical-free functionalization of partial silk proteins through fusion with peptide motifs and protein domains, but this constitutes a less cost-effective production process. Herein, we show that natural silk fibroin (SF) can be used as a bulk material that can be top-coated with a thin layer of the recombinant spider silk protein 4RepCT in fusion with various bioactive motifs and domains. The coating process is based on silk assembly to achieve stable interactions between the silk types under mild buffer conditions. The assembly process was studied in real-time by Quartz Crystal Microbalance with Dissipation. Coatings, electrospun mats, and microporous scaffolds were constructed from Antheraea assama fibroin (AaSF) and Bombyx mori fibroin (BmSF). The morphology of the fibroin materials before and after coating with recombinant silk proteins was analyzed by Scanning Electron Microscopy and Atomic Force Microscopy. SF materials coated with various bioactive 4RepCT fusion proteins resulted in directed antibody capture, enzymatic activity, and improved cell attachment and spreading, respectively, compared to pristine SF materials. The herein described procedure allows a fast and easy route for construction of bioactive materials.

KEYWORDS Biomaterial, Silk fibroin, Recombinant spider silk, Functionalization, Self-Assembly


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INTRODUCTION Silk is a protein-based natural material with unique mechanical properties. It can be processed into many different formats, offering a versatility that makes it interesting for various medical applications, including cell culture, tissue engineering and drug delivery.1–6 Several strategies have been used to tune silk properties and to add specific functions to silk, which further expands the application areas.7–9 The current investigation aims at constructing cost-effective silk materials possessing bioactive properties by combining different silk types. Bioactivity can be obtained by producing recombinant silk proteins in fusion with different bioactive motifs and domains such as cell-binding motifs10,11 and antibody fragments12,13. Bioactive recombinant silk shows great potential, but the production is less cost-effective than natural silk. Yields are relatively low, especially compared to extraction of silk from silkworm cocoons, since silkworm larvae can be sustained at high density.9,14 Silk from the domesticated mulberry silkworm Bombyx mori, belonging to the family Bombycidae, is widely used for textile production and has also been extensively studied for other purposes, especially as a biomaterial due to its degradability.5,14 To a low cost, silk fibers can be harvested from B. mori cocoons or silk proteins (fibroins) can be extracted by dissolving cocoons in denaturing solvents and thereafter be reprocessed into new formats such as films, sponges, hydrogels and microparticles.2 Nonmulberry silks have recently started to be explored for use as biopolymers, showing slightly different characteristics than B. mori silk. Antheraea assama, a nonmulberry silkworm under the Saturniidae family, is mainly found in the Assam region in India. The A. assama silk is soughtafter due to its gold-shimmering silk.15,16 In contrast to B. mori silk fibroins (BmSF), A. assama silk fibroins (AaSF) contain Arginine-Glycine-Aspartic acid (RGD) motifs,15 which promotes cell adhesion. AaSF-based materials in various formats similar to BmSF derived materials can be


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generated from silk proteins extracted directly from the glands of A. assama or from solutions of the naturally spun fibers after dissolution in denaturing solvents. However, isolation of nonmulberry SF from spun fibers using conventional denaturing solvents yields very low amount of protein.17 Therefore, isolation from glands is a more viable option. Previous in vivo studies of such silk matrices have revealed non-immunogenicity of the material.18 Vascularization and tissue remodeling were observed under in vivo conditions making it a suitable substrate for tissue engineering applications.19 A recent study also demonstrated immuno-modulatory properties of 3D silk biomaterials where macrophage polarity was controlled in order to tailor the biological response.20 For wound healing applications and skin patches, electrospinning of SF can be employed for construction of materials with micrometer to millimeter thickness consisting of networks of fibers with diameters in the nanometer range.18 It is hypothesized that a fiber network may be an advantage when used as a biomaterial, since it resembles the fibrous structure of tissue extracellular matrix.21 For three dimensional cell culturing, microporous structures are considered promising as scaffolds. Freeze-drying can be employed to transform SF into suitable scaffold dimensions,19 although these typically lack bioactivity, which would be desired for enhanced interactions with cells and biomolecules. SF materials can be rendered bioactive by physical adsorption, or in the case of electrospinning, by co-spinning with functional biomolecules.6,18 The loaded substances will then diffuse out of the material over time. To achieve a prolonged effect, immobilization of functional motifs onto the material is preferred. This can be obtained by chemical coupling of active substances onto the amino acid residues exposed in SF.9,22 However, such a process leads to a risk for denaturation of folded protein domains (e.g. enzymes) or entrapment in a conformation


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that impair the activity. Construction of fusion proteins, where the DNA encoding a functional protein domain is fused to a silk protein sequence at the genetic level, allows production under native conditions upon translation into fusion proteins in host cells. The functional domain is then attached at a defined site and is retained in its active form during assembly into bioactive silk materials.23,24 It was recently shown that the recombinant silk protein 4RepCT25,26 assembles into thin coatings on various surfaces under mild buffer conditions and that the peptide motifs fused to them are functionally presented in the obtained coatings.27 We hypothesized that 4RepCT proteins are able to assemble also onto SF materials via silk-silk interactions. In that way the advantages of both silk types can be combined; the cost-efficient extraction of silk from silkworms for construction of large scaffolds in a wide variety of formats and the possibility to add functional groups to the material via a thin top-coating of 4RepCT fusion proteins leading to bioactive materials. In a previous study, Morgan et al. combined BmSF with a recombinant spider silk fused to RGD by mixing the silk solutions prior to spin coating. The influence of the mixing ratios on the coating properties was analyzed, but no obvious enhancement in cell binding was confirmed.28 Attempts have also been made to extract recombinant spider silk from cocoons from transgenic silkworms,29,30 but since rather harsh conditions are required to degum the threads and reformat them into alternative scaffolds, incorporated bioactive domains might loose it’s function during the processing. In this work, we coated SF materials in different formats with the recombinant partial spider silk protein 4RepCT fused to three different bioactive moieties; the fibronectin-derived cell adhesion motif FN11, the Protein A-derived Z domain with high affinity for the Fc region of antibodies31, and the enzyme xylanase, which degrades the polysaccharide xylan32. We present


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studies of the interactions between fibroin and recombinant spider silk, and show that this results in bioactive materials.

EXPERIMENTAL SECTION Preparation of silk fibroin solutions from B. mori silk cocoons and A. assama silk glands. Bombyx mori silk cocoons were used to isolate BmSF solution following a standard protocol.33 Briefly, cocoons were cut into small pieces for degumming to remove sericin. Pieces of cocoons were boiled with 0.02 M sodium carbonate for 2x15 min. The obtained degummed silk fibers were dried, detangled and dissolved in 9.3 M lithium bromide (LiBr) solution at 60 °C. Dissolved SF-LiBr solution was incubated at 60 °C for 4 h to become highly viscous and was subsequently dialyzed against Milli-Q water for three days using a cellulose dialysis membrane (MWCO 12 kDa, Sigma Aldrich) to obtain aqueous BmSF. AaSF was isolated from silk glands of Antheraea assama silkworms as previously described.17,18 Briefly, silkworms in the fifth instar larvae stage were sacrificed. Silk glands were collected and SF protein was extruded by squeezing the glands using forceps. The protein was gently washed with water three times and then dissolved in 1% (w/v) aqueous sodium dodecyl sulfate (SDS) solution. Dissolved SF solution was centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant SF solution was further dialyzed against Milli-Q water using a 12 kDa cellulose dialysis membrane for 4 h at 4 °C to obtain an aqueous AaSF solution. The SF concentration was measured using gravimetric method and was further diluted to the desired concentration. Fabrication of silk fibroin mats, scaffolds and coatings. Nanofibrous mats were prepared using electrospinning. Poly(vinyl alcohol) (PVA) (LobaChemiePvt. Ltd.; 1,700–1,800 degree of polymerization and 98-99 mol% hydrolysis) was mixed with SF to obtain optimum viscosity for


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electrospinning.18 Highly viscous PVA solution 13% (w/v) was prepared by dissolving PVA granules in lukewarm Milli-Q. Aqueous PVA solution was blended separately with the two different SF solutions (AaSF and BmSF) in a ratio of 4:1 (PVA: SF) (w/w). The electrospinning unit Super ES1 (E-spin nanotech) was used at the following settings: voltage = 25 ± 3 kV, flow rate = 0.800 ± 0.100 mL/h, tip to collector distance (d) = 15 cm, and rotating speed of drum collector = 500 rpm. Two types of nanofibrous mats were prepared: one hybrid mat of PVA + A. assama SF (PVA-AaSF, also called AaSF mat) and one hybrid mat from a blend of PVA + B. mori SF (PVA-BmSF, also called BmSF mat). Hybrid electrospun mats were incubated in 70% ethanol for 2 h to render the mats water insoluble by induction of β-sheet formation and to sterilize the materials. Subsequently, they were washed with sterile water three to four times to remove residual ethanol. Wet mats were treated under an UV lamp for 20-30 min in two cycles to sterilize both sides of the electrospun mats further. The mats were dried overnight at 37 °C and stored at -20 °C until use. Microporous scaffolds of BmSF and AaSF were fabricated using a freeze-drying process. Briefly, 300 µL of 2.5% (w/v) BmSF and AaSF solutions, respectively, were transferred to a 24well plate and frozen at -20 °C overnight followed by lyophilizing for 24 h. The freeze-dried scaffolds were treated with 100% ethanol for 1 h followed by 70% ethanol treatment overnight to induce β-sheets. The scaffolds were washed with Milli-Q three to five times for complete removal of ethanol. Water-soaked scaffolds were autoclaved for sterilization prior to use and stored in 70% ethanol at 4 °C. Aqeuous SF solution was freeze-dried using the drying manifold Alpha 1-4 LD plus (Martin Christ Gefriertrocknungsanlagen) for storage until preparation of coatings. Lyophilized SF was dissolved into 1 mg/mL solutions in Milli-Q for 1 h and centrifuged for 7 min at 10000 rpm to


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remove

nondissolved

particles.

The

supernatants

were

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diluted

in

20

mM

tris(hydroxymethyl)aminomethane (Tris), pH 8.0 to obtain 0.1 mg/mL solutions. The solutions were used for preparation of coatings according to the procedures described in each analysis section. Recombinant spider silk proteins. Recombinant spider silk proteins were provided from Spiber Technologies AB and stored frozen in 20 mM Tris buffer, pH 8.0. 4RepCT (23 kDa) was either used as is or as fusion proteins with bioactive motifs and domains; FN-4RepCT (4RepCT fused with a cell-binding peptide motif from fibronectin,11 24 kDa), Z-4RepCT (4RepCT fused with the antibody binding Z domain,23 33 kDa), and Xyl-4RepCT (4RepCT fused with the xylan degrading enzyme xylanase,24 43 kDa). Proteins were thawed, centrifuged briefly to remove aggregates, and diluted in 20 mM Tris, pH 8.0 to obtain 0.1 mg/mL solutions. Quartz Crystal Microbalance with Dissipation. Using AT-cut quartz crystals with metal film electrodes as sensors and monitoring their oscillating frequencies in response to an applied pulsating voltage, the Quartz Crystal Microbalance with Dissipation (QCM-D) technique allows observation of small changes at the sensor surfaces, such as adsorption of biomolecules. Adsorption of mass onto the sensor leads to a decrease in its oscillation frequencies and an increase in the dissipation of oscillations. The viscoelasticity of the adsorbed layer also influences the dissipation.34 Gold-coated QCM-D sensors (Q-Sense QSX 301, Biolin Scientific) were cleaned extensively and rendered hydrophobic by incubation in 2 mM 1-undecanethiol (Sigma Aldrich) in 99.5% ethanol (Solveco), as previously described.27 Excess alkylthiols were removed by sonication in pure 99.5% ethanol five times, five min each. Sensors were stored in 99.5% ethanol until use. QCM-D was performed using an E4 instrument (Q-sense AB) at 20.0 °C. Buffer and proteins


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were flowed over the sensors at 20 µL/min. A baseline was obtained with 20 mM Tris. 0.1 mg/mL protein solutions were freshly prepared before each measurement and kept on ice until entering the modules. First, AaSF or BmSF solutions were flowed over the sensors. Following a buffer flow to regain the baseline, 4RepCT protein variants were flowed through the system. Finally, the baseline was regained with Tris buffer. Except for AaSF followed by 4RepCT, for which one measurement was done, all measurements were performed in duplicates. If not otherwise stated, overtone 3 was chosen for data representation. Atomic Force Microscopy. AaSF, BmSF, and 4RepCT coatings were prepared on siliconized hydrophobic cover slips (Paul Marienfeld) by incubation in 0.1 mg/mL protein solutions at room temperature for 60 min, followed by three times wash with 20 mM Tris. Samples with topcoatings were prepared by a total adsorption time of 60 min (i.e. 30 min in AaSF or BmSF followed by 30 min in 4RepCT) with 20 mM Tris wash after incubation in each protein solution. Atomic Force Microscopy (AFM) imaging was done in a Bruker Dimension FastScan instrument, using ScanAsyst Fluid+ tips and PeakForce Tapping™. All images were obtained in a droplet of 20 mM Tris. Photographs. SF coatings were prepared as described in the AFM section, and mats and scaffolds were prepared as described in the fabrication section. Top-coatings were made by incubation in 0.1 mg/mL 4RepCT at room temperature for 60 min. Coatings were washed at least two times in Tris and dried before being photographed. The macroscopic appearances of AaSF and BmSF coatings, mats, and scaffolds top-coated with 4RepCT were documented by a Canon EOS 5D Mark II camera. Scanning Electron Microscopy. The morphology of silk coatings, mats, and scaffolds was analyzed using Field Emission Scanning Electron Microscopy (FESEM; Sigma 300, Zeiss). SF


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coatings and 4RepCT top-coatings were prepared as described in the previous section. For FESEM imaging, dried samples were placed on a stub covered with carbon tape, coated with gold by 130 s of sputtering, and characterized at an accelerating voltage of 2-4 kV. ImageJ software (Wayne Rasband, National Institute of Health) was used to determine the diameter of nanofibers in mats by measuring 50 randomly selected nanofibers from the magnified images. The pore size of scaffolds was also investigated using ImageJ software. Detection of hexa-histidine in combined silk coatings. SF coatings were prepared from 0.1 mg/mL AaSF or BmSF by incubation for 30 min at room temperature. Top-coatings were made by incubation of SF coatings in Xyl-4RepCT for another 30 min. Coatings were washed at least two times with 20 mM Tris after each incubation. Coatings were immersed in 2 µg/mL of fluorophore-labeled anti-His antibody (Penta-His Mouse IgG-Alexa Fluor 488, Product: 35310, Qiagen) in phosphate buffered saline (PBS) with 0.05% Tween 20 for 60 min at room temperature. After three washes with PBS/0.05% Tween 20, fluorescence microscopy was performed using an inverted Nikon Eclipse Ti fluorescence microscope. As negative controls, coatings of BmSF with 4RepCT and AaSF with 4RepCT were treated in the same way. Coating silk fibroin materials with recombinant spider silk for cell culture studies. SF coatings were prepared in hydrophobic 96-well plates (TC Plate 96 Well, Suspension, F, Sarstedt) by incubation of 0.1 mg/mL AaSF or BmSF solution in wells for 60 min at 37 °C. The coatings were washed two times with 20 mM Tris. SF coatings and mats (pre-wetted in sterile water) were top-coated by incubation in 0.1 mg/mL FN-4RepCT for 60 min at 37 °C. Excess silk solution was removed and coatings and mats were washed two times with 20 mM Tris. Cell culture. Human dermal fibroblasts of neonatal origin (HDF, ECAAC, Salisbury, UK) and the human keratinocyte cell line HaCaT (Cell line service, Germany) were cultured in DMEM


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nutrient mixture F12 ham (Sigma) supplemented with 5% fetal bovine serum (Sigma), penicillin (100 U/mL), and streptomycin (100 µg/mL). HDF were used at passage 9. Medium was changed every second to third day. Cellular stainings. Cells were harvested and seeded onto either coatings in 96 well plates (TC Plate 96 Well, Suspension, F, Sarstedt) or electrospun mats in 24 well plates at a seeding density of 5000/cm2. After 48 h (coatings) or 72 h (mats) of culture, cells were washed in PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X 100, and stained with PhalloidinAlexa Flour 488 or 594 diluted 1:500 for F-actin and Dapi 1:1000 for nuclei. Samples were viewed under a Nikon Eclipse Ti inverted fluorescence microscope and micrographs were taken using NIS elements BR (Nikon). Binding of IgG to Z-silk coated materials. SF coatings were prepared as described in the photograph section, and coatings, mats, and scaffolds of AaSF and BmSF were top-coated with Z-4RepCT for 60 min at room temperature followed by at least two times wash with 20 mM Tris. All materials were immersed in 0.05 mg/mL IgG-fluorophore (Rabbit Anti-Mouse IgG (H+L)-Alexa Fluor 488, Product: A-11059, Sigma-Aldrich) in PBS for 30 min. After three times of washing with PBS/0.05% Tween 20, PBS was added to all materials, which were then analyzed by fluorescence microscopy using an inverted Nikon Eclipse Ti fluorescence microscope. As control materials, BmSF and AaSF coated with 4RepCT were treated in the same way. Enzymatic activity assay. Coatings, mats (pre-wetted in 20 mM Tris) and scaffolds of AaSF and BmSF top-coated with Xyl-4RepCT were prepared in the same way as for the hexa-histidine detection. The enzymatic activity of xylanase in the silk materials was determined using a colorimetric reducing-sugar assay (Megazyme, Bray, Ireland), based on a method described


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elsewhere.35,36 In brief, enzyme-functionalized silk materials were immersed in a 1% (w/v) substrate solution (Wheat Arabinoxylan, Product: P-WAXYM, Megazyme) at pH 6.3. 20 mM Tris buffer was used as a negative control. The enzymatic reaction was allowed to proceed for 60 min at room temperature, after which the reaction was terminated by addition of copper reagent. Enzymatic product was visualized in a color-development step by addition of sodium arsenate, followed by detection of the product by measuring the absorbance at 520 nm using a CLARIOstar microplate reader (BMG LABTECH). Image analysis of fluorophore-labeled samples. Fluorescence intensity of Xyl-4RepCT topcoated SF coatings after staining with anti-His antibody was measured using the Intensity profile tool in NIS elements BR software. The intensity profile across the image was measured at 10 different positions and a pixel mean intensity was calculated for each image. For visual representation, the brightness and contrast of micrographs were enhanced by 100 and 80, respectively, using Adobe Photoshop CC 2015.0.1. However, this was not applied when extracting intensity data for quantification and subsequent statistical analyses. Fluorescence intensity of bound IgG-fluorophore to Z-silk coated SF materials was extracted from fluorescence images as Mean Gray Value (MGV) using ImageJ software. After removal of bright signal artifacts using the Remove outliers tool (radius: 10 pixels, threshold: 30), mean intensity MGV values were calculated by ImageJ using four different areas per material. Similar to the images of samples stained with anti-His antibody, the brightness and contrast of IgG stained micrographs for visual representation were enhanced by 100 and 80, respectively, for coatings and mats using Adobe Photoshop CC 2015.0.1. For scaffolds, the contrast was enhanced by 80 and the brightness was left unmodified. The modifications were not applied when extracting intensity data for quantification and subsequent statistical analyses.


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Cell coverage area (F-actin) on coatings was measured using NIS elements BR software. For each condition, 4-7 different areas from duplicate samples were analyzed. Statistics. Student’s t-test (or Mann Whitney U test) was used for statistical analysis of fluorescence intensity, spectrophotometric absorbance from enzymatic activity, and cell coverage area. P-values

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