Article pubs.acs.org/Biomac
Sticky Situation: An Investigation of Robust Aqueous-Based Recombinant Spider Silk Protein Coatings and Adhesives Thomas I. Harris,†,‡ Danielle A. Gaztambide,†,‡ Breton A. Day,§,∥,‡ Cameron L. Brock,§ Ashley L. Ruben,† Justin A. Jones,*,§ and Randolph V. Lewis*,†,§ †
Departments of Biological Engineering, §Biology, and ∥Nutrition, Dietetics, and Food Sciences, Utah State University, Logan, Utah 84322, United States S Supporting Information *
ABSTRACT: The mechanical properties and biocompatibility of spider silks have made them one of the most sought after and studied natural biomaterials. A biomimetic process has been developed that uses water to solvate purified recombinant spider silk proteins (rSSps) prior to material formation. The absence of harsh organic solvents increases cost effectiveness, safety, and decreases the environmental impact of these materials. This development allows for the investigation of aqueous-based rSSps as coatings and adhesives and their potential applications. In these studies it was determined that fiber-based rSSps in nonfiber formations have the capability to coat and adhere numerous substrates, whether rough, smooth, hydrophobic, or hydrophilic. Further, these materials can be functionalized for a variety of processes. Drug-eluting coatings have been made with the capacity to release a variety of compounds in addition to their inherent ability to prevent blood clotting and biofouling. Additionally, spider silk protein adhesives are strong enough to outperform some conventional glues and still display favorable tissue implantation properties. The physical properties, corresponding capabilities, and potential applications of these nonfibrous materials were characterized in this study. Mechanical properties, ease of manufacturing, biodegradability, biocompatibility, and functionality are the hallmarks of these revolutionary spider silk protein materials.
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INTRODUCTION Spiders have long been recognized for their silk fibers and webs that have endured millions of years of evolutionary pressure.1−3 The lustrous and ornate properties of spider silks are only surpassed by the properties that these assembled protein structures possess. The most prominent features that sparked modern investigations into spider silks are the mechanical properties of these natural materials. Spider silks possess combinations of strength and elasticity that often match or surpass manmade materials. More recently discovered properties include biocompatibility, biodegradability, and adaptive capabilities that rise from the modular nature of the materials.2−8 All of these properties, or combinations of them, position spider silks as a significant biomaterial. Various types of spider silks exist and are necessary for the spider’s survival and lifecycle.9 These silks have different functions and forms that may better suit a specific silk type to an appropriate role. On the upper end of complexity are the orb weavers that can produce six types of silks and one glue.1,10−12 These silk proteins serve vital and different functions such as web spinning, prey capture, egg sac formation, anchoring, and adhesion. These last two functions are often overlooked because of their underlying nonfibrous structures and the large focus on investigating fiber properties. However, these © XXXX American Chemical Society
two functions are extremely important for maintaining web integrity as well as acquiring prey.1,13 Generally speaking in orb weavers, anchoring is carried out by piriform attachment disks and the aggregate protein acts as an adhesive coating on the capture spiral.1,14−18 Other spider species also possess similar systems with various levels of sophistication for protein adherence systems.13,15,19−21 Spiders are not the only organisms to benefit from adhesive proteins that interact with various surfaces or components.22−24 Many biological entities employ natural adhesives, which often have environmentally adaptive properties that exceed synthetic adhesive and coating structures. Some systems, like mussels and barnacles, are fairly complex and rely on multiple components and processes to function.24−28 Other bioadhesives are generally more similar to spider silks, which rely on only a few key proteins or morphologies. Other insects such as wasps, bees, silkworms, midges, and caddisflies use adhesive silks and fluids to construct or organize crucial formations.28−32 These organisms primarily use glues to construct basic structures like nests, lifeline attachment, or to assemble and maintain silk Received: August 23, 2016 Revised: October 1, 2016
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DOI: 10.1021/acs.biomac.6b01267 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 1. Production of rSSps coatings and adhesives. Generalized flow diagram starting with protein sequences used and corresponding secondary structures (purple, β-sheets; red, 310-helices; and green, β-spirals) with respective expression systems. Processing steps of purified rSSps are shown along final coating and adhesive products.
fibers. Unlike spider silks, these other adhesive silks are rarely used in web construction and prey capture, which require more robust properties. A few synthetic systems have been designed that rely on simple synthetic polymers that mimic the properties of natural adhesives.22,24,25,33−35 Most of these natural and synthetic glues appear to use similar methods of adherence and attachment. This method, for silks, begins with physical entanglements, interactions, and ultimately relies on secondary, noncovalent bonds.25,36 Many of these adhesive and coating systems have been actively pursued due to their impressive abilities to function under diverse conditions, rendering them superior to manmade glues.23,24,30,34 Additionally, the adherence mechanisms are attractive because of the lack of chemical processes such as cross-linkers or catalysts. The structures and proteins involved in these bioadhesives are biocompatible, biodegradable, and perform under a variety of environmental conditions.29,37 For example, silkworm adhesives and coatings have been developed and rely either on sericin (a simple and naturally forming adhesive) or fibroin proteins that are easily manipulated through various methods, such as pH or electrogelation.38 With regard to recombinant spider silk proteins (rSSps), the difficulty of solubilization, use of organic solvents, and a primary focus on reproduction of synthetic fibers have limited investigations to characterize and explore nonfibrous systems. Previous studies for recombinant spider silk coatings in vivo have demonstrated decreased inflammation, cell adhesion, and capsular fibrosis in animal models and future clinical studies are being developed for these coatings.5,39,40 No such studies exist for adhesives, to the authors’ knowledge, even though the natural spider silk adhesives have been characterized. Additionally, these few studies are usually for specific applications or settings and do not focus heavily on the characters or structures of the rSSps used.
Due to these unique mechanical and biological properties, numerous research projects have been initiated to produce rSSps and other silks in recombinant and transgenic hosts. However, large-scale production of spider silk proteins that make up these silks has proven unsuccessful in the past due to the inability to cultivate spiders, and isolate a specific and pure product.41,42 Additional limitations are the heterologous expression of rSSps comparable to the large size of the natural spider silk proteins (>250 kDa), efficient purification, and recovery of these proteins from production systems, and solubilizing these recombinant proteins in water.43−46 These limitations have been addressed in recent research and initial solutions are being investigated.47,48 Conventionally, chaotropic or organic solvents have been used to solubilize the recovered rSSps.49 The issues with these common solubilization methods, such as toxicity, difficult handling, and cost make any large-scale or industrial production approaches unlikely, if not impossible. It is the overall goal of this research to characterize the feasibility of potential applications using a rapid and scalable approach for solubilizing rSSps in a water-based solution. Following this solubilization method, it has been found that a diverse array of materials can be created including, but not limited to, hydrogels, sponges, lyogels, films, fibers, fibrous mats, coatings, and adhesives.50 This research focuses on robust coatings and adhesives formed with rSSps from recombinant versions of the major ampullate spidroins (MaSp1 and MaSp2): the most characterized of the spider silks (Figure 1). Although these silk proteins are naturally used as fibrous structures in the spider’s dragline, it has been found that they also have impressive adhesive and coating capabilities. A substantial amount of research and characterization on bioadhesives and coatings has been performed on other insect silk systems.29,32,38,51 With regards to rSSps, and other silks, the B
DOI: 10.1021/acs.biomac.6b01267 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
compressor was used for coating applications. A layer of silk was applied from 50 cm away for 15 s and then allowed to dry for 3 min before another layer was applied. Spraying and drying was repeated until desired thickness or coverage was achieved. Special precautions were taken when creating an initial base layer to avoid beading or running of the liquid rSSps dope on the substrate surfaces. Dip Coating Formation. Substrates were fully immersed in a prepared rSSps dope for 5 s, removed, and allowed to dry for 5 min, multiple layers were applied to achieve thicker and smoother coatings. Spray-Dip Coating Formation. An initial spray coat of solvated rSSps (standard procedure) was applied to the surface of a substrate before applying a dip coating. This led to the formation of a uniform coating and reduced beading or pooling of the solution during drying. Field Emission Scanning Electron Microscopy (FE-SEM). The prepared coatings of interest were imaged by field emission scanning electron microscopy using a FEI Quanta FEG 650 (FEI). The coated samples were cut to reveal the coating or placed in an upright fashion on conductive copper tape, which was then mounted on an aluminum stub. No deposition of a conductive coating was used since the samples were imaged using the low vacuum mode at 0.22 Torr (29.33 Pa), with a primary beam strength of 8 kV, a dwell time of 15 μs, and a large field detector (LFD). Finally, images and measurements were taken for each sample at 100×, 750×, and 5000× magnifications and saved for later retrieval and analysis. Coating Surface Testing. Various characterization tests were performed on coated substrates to determine the surface properties of the coatings. Tests to determine the coefficients of friction for noncoated and rSSps coated samples were performed on silicone and polyurethane (PU) substrates. All samples were tested in triplicate at a constant rate of 1 mm/min, with a normal force of 1.25 N, an initial gauge length of 20 mm, and a collection rate of 120 Hz. The samples were pulled from stand still to determine the necessary force to start movement and then maintain motion. The data were analyzed with Microsoft Excel and the static (eq 1) and kinetic (eq 2) coefficients were determined.
use of organic solvents and a primary focus on reproduction of synthetic fibers have caused limited investigations to characterize and explore nonfibrous systems.10,45,52 From naturally fiber forming proteins, presented here are two nonfiber materials, adhesives and coatings, created from synthetic analogs of MaSp1 and MaSp2 along with synthetic chimeric rSSps designed from two separate silks. These constructs were selected primarily based upon the availability of sufficient amounts from their respective expression systems for testing and characterization. Additionally, these proteins are better understood and characterized both in natural and synthetic systems.53,54 Finally, the novelty and potential observed for these fibrous proteins in nonfibrous forms was considered appropriate for investigation. Various parameters and capabilities of these coating and adhesive systems have been identified and characterized for the first time in these particular systems. To our knowledge, this is the first in-depth data on normally fiber forming rSSps as coatings and adhesives. The relationships between the material’s formation, structures, and properties were investigated and described. These initial results further demonstrate the unique properties of non-fiber-based materials formed from spider silks.
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MATERIALS AND METHODS
Goat Derived Protein Purification. As previously reported, shorter versions of the natural Nephila clavipes MaSp1 and MaSp2 (rMaSp1 and rMaSp2) proteins were expressed in the milk of transgenic goats and then purified through a process of tangential flow filtration, precipitation, washing, and lyophilization.46 Bacterially Derived Protein Purification. Synthetic chimeric proteins flagelliform-tyrosine-serine (FlYS3 and FlYS4) of N. clavipes flagelliform silks were expressed in E. coli and purified. These constructs contain repetitive GPGGX motifs from flagelliform and strength motifs polyalanine from major ampullate silks. The previously published method for molecular design, fermentation, and purification of chimeric construct FlYS expressed in E. coli was used.49 Due to large purification volumes and the tendency to self-assemble under shear conditions; ammonium sulfate precipitations and washing methods were used instead of dialysis to separate the rSSps from the elution fraction. Precipitation and extraction of the protein was performed with a 3 M solution of ammonium sulfate and a separation layer of isopropanol (IPA) that was 5% (v/v) of the final volume. This solution was allowed to stir for 12 h and then the precipitated rSSps were removed from the IPA phase by filtration. The protein was washed with diH2O (NanoPure Thermo Fischer) until the flow-through conductivity was
