Enzymatic Degradation of Films, Particles, and Nonwoven Meshes

Article pubs.acs.org/journal/abseba

Enzymatic Degradation of Films, Particles, and Nonwoven Meshes Made of a Recombinant Spider Silk Protein Susanne Müller-Herrmann and Thomas Scheibel* Lehrstuhl Biomaterialien, Universität Bayreuth, Universitätsstraße 30, D-95447 Bayreuth, Germany S Supporting Information *

ABSTRACT: The performance of biomaterials in vivo is largely influenced by their stability and the rate and extent to which they degrade. Materials for tissue engineering applications, for example, have to be mechanically stable to support cell adhesion and proliferation without collapsing. On the other hand they need to be replaced gradually by native extracellular matrix and have to be (slowly) biodegradable. Therefore, it is of critical importance to be able to tune the degradation behavior of a biomaterial. Recombinantly produced spider silk proteins have been shown to be versatile biopolymers for medical applications. They can be processed into a variety of morphologies, and by chemical or genetic modification the properties can be adjusted to specific demands. Furthermore, in vivo experiments confirmed the lack of immunological reactions toward certain spider silks. In this study the degradation behavior of the recombinant spider silk protein eADF4(C16) in solution as well as processed into particles, films and nonwoven meshes was analyzed, and the impact of crosslinking of the scaffolds was assessed thereon. In addition to two bacterial proteolytic model enzymes, protease type XIV from Streptomyces griseus (PXIV) and collagenase type IA from Clostridium histolyticum (CHC) used in all experiments, several recombinant human matrix metalloproteinases (MMPs) and one elastase were used in studying degradation of soluble eADF4(C16). For soluble eADF4(C16) all degradation kinetics were similar. In case of eADF4(C16) scaffolds significant differences were observable between PXIV and CHC. All scaffolds were more stable toward proteolytic degradation in the presence of CHC than in the presence of PXIV. Further, particles were degraded significantly faster than films, and nonwoven meshes showed the highest proteolytic stability. Chemical cross-linking of the scaffolds led to a decrease in both degradation rate and extent. KEYWORDS: recombinant silk proteins, cross-linking, biodegradation, MMPs, protease XIV, collagenase IA, scaffolds for biomedical applications

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INTRODUCTION Materials interacting with human tissue have to provide certain requirements. These biomaterials must be biocompatible, not toxic, not mutagenic, and should not induce a strong immune or inflammatory response nor cause an allergic reaction.1−3 Furthermore, the mechanical properties such as tensile strength or elastic modulus are very important and must be adapted to tissue-specific conditions.1,3,4 Another crucial factor is the degradation behavior of the material, which strictly depends on the application.3,5,6 In wound healing, for example, the degradation process involves a complex interaction of cellular and extracellular components,7 setting high demands on biomaterials used as a wound closure device. In all phases proteolytic enzymes participate in regulating mechanisms8 with the expression level of different proteases varying largely during the various phases of wound healing.9 The most important proteases are matrix metalloproteinases (MMPs) and serine proteases.10,11 An elevated concentration of these proteases is also associated with chronic wound healing.9,12,13 However, also certain diseases, such as cancer or cardiovascular diseases, lead to an elevated plasma level of MMPs, which needs to be © XXXX American Chemical Society

considered in the design of biomaterials, e.g., for applications as drug delivery devices.14,15 Importantly, proteases are also produced in healthy individuals, yet to a lesser extent, as they are involved in a range of normal physiological processes such as nerve growth, immune response, and bone remodeling.16 The interaction of these proteolytic enzymes with the biomaterial has a severe impact on the specific application. Because of its biocompatibility and mechanical properties, spider silk is considered an attractive material for biomedical applications. However, large scale production of spider silk is limited by the cannibalistic behavior of spiders. Therefore, a method for recombinant production of spider silk proteins in Escherichia coli was established by our group17,18 among others. Here, we used eADF4(C16), which is based on the sequence of one dragline silk protein of the European garden spider (Araneus diadematus). It consists of 16 repeats of a consensus sequence (C-module) (GSSAAAAAAAASGPGGYGReceived: December 3, 2014 Accepted: March 3, 2015

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DOI: 10.1021/ab500147u ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

(5 kV) using a needle-to-collector distance of 15 cm. The flow rate was adjusted to 5.25 μL/min with a syringe pump. Nonwoven meshes were post-treated with methanol vapor overnight. For in vitro experiments, round nonwoven samples with a diameter of 11 mm were prepared using a punch. Cross-Linking. Cross-linking (CL) of particles, films, and nonwoven meshes was performed by incubating the samples in a solution containing 10 mM Tris buffer pH 7.5, 5 mM ammonium persulfate (APS), and 50 μM Tris(2,2′-bipyridyl)dichlororuthenium(II) (Rubpy, Sigma-Aldrich, Germany) in the dark overnight. For initiation of the cross-linking reaction, samples were exposed to a tungsten lamp for 5 min. Afterward, samples were washed three times with TCNB buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij 35, pH 7.5). Particles were alternatively cross-linked by adding APS (10 mM) and Rubpy (100 μM) to the eADF4(C16) solution before initiation of particle formation (CL-b). After incubation for 1 h in the dark, particles were exposed to a tungsten lamp for 5 min. Afterward, samples were washed three times with TCNB buffer. This procedure leads to a different extent of cross-linking in comparison to the “normal” route of adding the cross-linking agents after particle formation.45 Enzymes and Buffers. Recombinant human matrix metalloproteinases (MMPs) 2, 8, 9, and 13 (R&D Systems, USA), human neutrophil elastase (polymorphonuclear (PMN) elastase, Merck, Germany), protease from Streptomyces griseus, type XIV (PXIV, Sigma-Aldrich, USA), and collagenase from Clostridium histolyticum, type IA (CHC, Sigma-Aldrich, USA) were employed as model proteases. MMP-2, MMP-8, MMP-9, and MMP-13 were activated by adding p-aminophenylmercuric acetate (APMA, Sigma-Aldrich, USA) according to the manufacturer’s protocol. Experiments were performed in TCNB buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij 35, pH 7.5) at 25 °C for direct comparability. In vitro Degradation of Soluble eADF4(C16). For analysis of in vitro degradation of soluble spider silk proteins, lyophilized eADF4(C16) was dissolved in 6 M GdmSCN and dialyzed against 10 mM Tris buffer, pH 7.5. After centrifugation (17,000xg, 30 min) the concentration was adjusted to 1 mg/mL in TCNB buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij 35, pH 7.5). For overnight experiments, soluble eADF4(C16) was incubated in the presence of protease mix “normal range” (protease mix N: MMP-2 200 ng/mL, MMP-8 10 ng/mL, MMP-9 100 ng/mL, MMP-13 1 ng/ mL, PMN elastase 40 ng/mL; total enzyme concentration: 351 ng/ mL), protease mix “wound range” (protease mix W: MMP-2 250 ng/ mL, MMP-8 100 ng/mL, MMP-9 400 ng/mL, MMP-13 50 ng/mL, PMN elastase 250 ng/mL; total enzyme concentration: 1050 ng/mL), CHC 1050 ng/mL, and a control without enzymes. Additionally, the effect of single MMPs was analyzed by incubating soluble eADF4(C16) in the presence of 200 ng/mL MMP-2, MMP-8, MMP-9, MMP-13, and PMN elastase, respectively. For time-dependent experiments, soluble eADF4(C16) was incubated in the presence of protease mix N (MMP-2 200 ng/mL, MMP-8 10 ng/mL, MMP-9 100 ng/mL, MMP-13 1 ng/mL, PMN elastase 40 ng/mL), protease mix W (MMP-2 250 ng/mL, MMP-8 100 ng/mL, MMP-9 400 ng/mL, MMP-13 50 ng/mL, PMN elastase 250 ng/mL), MMP-2/PMN elastase (250 ng/mL each), PXIV 351 ng/mL, and CHC 351 ng/mL. Samples were taken after 1, 5, 15, and 30 min and after 1, 2, 4, 6, and 9 h. In vitro Degradation of Particles, Films and Nonwoven Meshes. For in vitro experiments of eADF4(C16) scaffolds, particles, films, and nonwoven meshes (non-cross-linked and cross-linked) were incubated in the presence of PXIV and CHC (175 μg/mL in TCNB buffer), respectively, for 15 days. Control experiments without enzymes were conducted in TCNB buffer. One milliliter of solution was used per 2 mg of scaffold. Buffers or enzyme solutions were changed every 24 h. The experiments were performed in triplicates and the protein concentration in the supernatant was determined by UV spectroscopy on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). Scaffold samples were taken after 1, 3, 6, 10, and 15 days, washed with TCNB buffer twice and with distilled water three times. Particle samples were stored in distilled water at 4 °C or

PENQGPSGPGGYGPGGP). eADF4(C16) can be processed into various morphologies such as particles,19,20 films,21,22 nonwoven meshes,23 hydrogels, 24 or capsules.25,26 The possibility of using organic solvents (e.g., HFIP or formic acid) as well as aqueous buffers for processing in combination with the option of genetic or chemical modification of the protein20,23,27 allows adjustment of the materials’ properties to specific demands. The aim of this study was to analyze the in vitro degradation behavior of eADF4(C16) processed into particles, films, and nonwoven meshes. Films and nonwoven meshes made of recombinant spider silk proteins represent promising materials for coating of implants, wound healing devices or in tissue engineering applications,23,27,28 whereas particles are more likely to be suitable for drug delivery devices.29 Furthermore, the effect of cross-linking of the scaffolds was investigated. For degradation experiments, the bacterial model enzymes protease type XIV from Streptomyces griseus (PXIV) and collagenase type IA from Clostridium histolyticum (CHC) were applied. Along with α-chymotrypsin30−33 and trypsin,31,34 these enzymes are among the most widely used proteases for the assessment of the proteolytic stability of silk proteins (protease Type XIV30,32,33,35−41 or Type XXI,42 and collagenase Type IA32,35,43 or Type F42). To get a better idea of the relevance of these results regarding in vivo conditions, we started with a set of degradation experiments using soluble eADF4(C16). We compared the proteolytic stability of soluble eADF4(C16) in the presence of PXIV and CHC. Additionally, the recombinant human MMPs 2, 8, 9, and 13 as well as PMN elastase were included in this analysis.

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MATERIALS AND METHODS

Production of eADF4(C16). Production of the recombinant spider silk protein eADF4(C16) in E. coli and its purification were performed as described previously.17 Briefly, eADF4(C16) was purified after incubating cells in lysis buffer (0.2 mg/mL lysozyme in 50 mM Tris/100 mM NaCl, pH 7.5) at 4 °C for 30 min and cell disruption by ultrasonication. Cell fragments were separated by centrifugation, and soluble E. coli proteins were precipitated by heat denaturation (80 °C, 20 min). After centrifugation, soluble silk proteins in the supernatant were salted out using 20% ammonium sulfate at room temperature, the pellet was washed with urea and distilled water and lyophilized. Particle Formation. Lyophilized silk proteins were dissolved in 6 M guanidinium thiocyanate (GdmSCN) and dialyzed against 10 mM Tris buffer (Tris (hydroxymethyl)-aminomethane-HCl), pH 7.5. After centrifugation (17,000xg, 30 min) the concentration of the solution was adjusted to 0.5 mg/mL. For initiation of particle formation, an equal volume of 2 M potassium phosphate buffer, pH 7.5 was added to the silk protein solution, followed by incubation for 1 h. After centrifugation for 15 min at 17 000g, the particles were washed with Tris buffer three times and stored therein at 4 °C. Film Fabrication. Films were cast from a 5 mg/mL solution of eADF4(C16) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) into polystyrene well plates (Nunc, Langenselbold, Germany). For each film 0.5 mg of eADF4(C16) were used per cm2. Films were allowed to dry at a relative humidity of 30% and were post-treated with 100% methanol (level of purity pro analysi, p.a.) under identical conditions to induce β-sheet formation in order to render silk films water insoluble.44 Electrospinning to Produce Nonwoven Meshes. For fabrication of nonwoven meshes, eADF4(C16) was dissolved in HFIP at a concentration of 15% (w/v) and filled in a 1 mL syringe with a blunt 21 G needle which was connected to a high voltage power supply (−25 kV). Fiber mats were collected on aluminum foil placed on a collector plate connected to a second high voltage power supply B


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ACS Biomaterials Science & Engineering lyophilized and stored at −20 °C. Films and nonwoven meshes were air-dried and stored at room temperature. SDS-PAGE. Proteolysis of soluble silk proteins was stopped upon inactivation of the proteases at 95 °C in Lämmli buffer for 5 min, and samples were stored at −20 °C before applying them to a SDS-PAGE gel (12.5% Tris-HCl gel). Scaffolds were dissolved in 6 M GdmSCN after proteolysis, dialyzed against 10 mM Tris buffer, pH 7.5, and analyzed by SDS-PAGE (12.5% Tris-HCl gel). Subsequently, SDSPAGE gels were silver stained. Equal volumes of scaffold samples were applied, except for the 15 d sample of non-cross-linked particles incubated in PXIV solution. Because of the high degree of degradation, twice the volume was applied in that case. Mass Spectrometry. For analysis of degradation products, mass spectrometry (MS) was performed on silk proteins in solution or the supernatant of degradation experiments. Samples were prepared using matrix solution (α-cyano-4-hydroxycinnamic acid 10 mg/mL in acetonitrile, 0.1% trifluoroacetic acid in H2O). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was performed on a Bruker Reflex III (Bruker, Bremen, Germany) equipped with a 337 nm N2 laser in the linear mode and 20 kV acceleration voltage. Scanning Electron Microscopy (SEM). After incubation of soluble eADF4(C16) in PXIV or CHC solution for 30 min, 20 μL were dried on Thermanox plastic coverslips and washed with distilled water four times. After incubation in buffer or enzyme solutions for various times, eADF4(C16) particles, films, and nonwoven meshes were washed three times with distilled water and air-dried (particles were dried on Thermanox plastic coverslips). After sputtering with platinum, SEM images were taken with a 1450EsB Cross Beam (Zeiss, Germany) at an accelerated voltage of 3 kV. Fourier Transform Infrared (FTIR) Spectroscopy. FTIRanalysis of protein secondary structure of films, particles and nonwoven meshes before and after incubation in buffer or enzyme solutions was performed on a Bruker Tensor 27 spectrometer (Bruker, Bremen, Germany). FTIR-spectra were recorded by placing the samples onto a Ge crystal. Spectra were measured by attenuated total reflection (ATR) with a resolution of 4 cm−1, and 60 scans were averaged. Analysis of the amide I band (1590−1720 cm−1) by Fourier self-deconvolution (FSD) was performed to determine individual secondary structure elements. Band assignments were made according to Hu et al.46

Table 1. Average Plasma/Serum Levels of Proteolytic Enzymes in Healthy Individuals (normal range) and during Wound Healing (wound range) average plasma/serum level proteolytic enzyme

normal range (ng/mL)

wound range (ng/mL)

MMP-2a MMP-8b MMP-9c MMP-13d PMN elastasee

200 10 100 1 40

250 100 400 50 250

a 15,56,59,63−67 b 50,52,55,67−69 c 15,50,55,57−60,63,65,67−69 d 12,51,52,59,60,62,66

;

e12,53,54,61,70,71

;

;

;

the destruction of phagocytosed material.53,71 More detailed informations about these enzymes can be found in Table S1 in the Supporting Information. As detailed long-time degradation studies of silk protein scaffolds are not feasible using recombinant human MMPs because of their low availability, short-time activity, and high costs, we performed our experiments with collagenase IA from Clostridium histolyticum (CHC), which has been previously used for degradation studies of silk protein scaffolds.32,35,43 This crude collagenase is in fact a mixture of several enzymes, including type I and type II collagenase.73 Because of the functional relation of these two enzymes to MMPs,74 CHC is likely to act as model enzyme for wound proteases. We analyzed this feature with soluble eADF4(C16), which was incubated both with CHC and with MMP-2, -8, -9, and -13 and PMN elastase in order to evaluate the extent of conclusions to be drawn from using CHC as a model enzyme. Unlike in other studies carried out in PBS buffer at 37 °C, here all experiments were performed in TCNB buffer, as it is important to guarantee maximum activity of the employed MMPs, and because phosphate-containing buffers induce precipitation of soluble eADF4(C16). The temperature was set to 25 °C to ensure stability of eADF4(C16) in solution for the duration of the experiment, because at higher temperatures the silk protein self-assembles into fibrillar morphologies under these buffer conditions (data not shown), which would negatively influence the read-out. Degradation of eADF4(C16) in Solution. Analysis of the degradation of soluble eADF4(C16) is important, because information can be obtained about the presence of recognition sequences in the primary structure of the protein. These results can not directly be transferred to processed silk proteins, because recognition motifs might be buried due to the secondary structure of the proteins. Yet the absence of recognition sequences shows that silk protein scaffolds will be stable in the presence of the respective protease. Besides, during degradation of the scaffolds protein fragments are likely to be released into solution, and further degradation of these fragments (especially larger ones) is a crucial factor concerning the biocompatibility of the material. Soluble eADF4(C16) was incubated overnight in the presence of protease mix N, protease mix W, CHC, and no enzyme as control. Analysis by SDS-PAGE and MALDI-TOF MS showed that all enzymes led to a complete degradation of the silk proteins overnight (Figure 1a). Additionally, the experiment was performed in the presence of MMP-2, -8, -9, -13 or PMN elastase, each individually, in order to analyze the contribution of single MMPs to silk degradation. In the presence of MMP-8, -9, and -13, no degradation of soluble

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RESULTS AND DISCUSSION The proteolytic environment in vivo under normal conditions (healthy individuals) and during wound healing can be extrapolated from published plasma/serum levels of various proteases. We selected matrix metalloproteinases (MMPs) 2, 8, 9, and 13 and polymorphonuclear (PMN) elastase for our experiments, because they are among the most relevant proteases in wound healing.9,13,47−49 Because plasma/serum levels of these proteases vary largely not only during wound healing but also in healthy individuals, concentrations summarized in Table 1 represent average values published in a variety of studies.12,15,50−71 MMPs are proteases that are involved in the remodeling and degradation of the extracellular matrix. They comprise a family of zinc-dependent endopeptidases and are divided into six subclasses (collagenases, gelatinases, stromelysines, matrilysins, membrane-type MMPs, and other MMPs).10,47,72 For our analysis two gelatinases (MMP-2, MMP-9) and two collagenases (MMP-8, MMP-13) were selected. The unique property of mammal collagenases is their ability to cleave the triple helix of fibrillar collagen which in turn can subsequently be degraded by gelatinases.47 PMN elastase is stored in polymorphonuclear granulocytes and released upon activation. It plays an important part as mediator of inflammation and tissue injury, as well as in C


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Figure 1. (a) SDS-PAGE followed by silver staining of soluble eADF4(C16) after incubation in TCNB buffer overnight in the presence of 1 protease mix N (normal range: MMP-2 200 ng/mL, MMP-8 10 ng/mL, MMP-9 100 ng/mL, MMP-13 1 ng/mL, PMN elastase 40 ng/mL), 2 protease mix W (wound range: MMP-2 250 ng/mL, MMP-8 100 ng/mL, MMP-9 400 ng/mL, MMP-13 50 ng/mL, PMN elastase 250 ng/mL), 3 CHC 1050 ng/ mL, 4 control without enzymes (left panel). MALDI-TOF mass spectra of samples 1−3 are shown in the right panel. (b) SDS-PAGE followed by silver staining of soluble eADF4(C16) after incubation in TCNB buffer overnight in the presence of 1 MMP-2 200 ng/mL, 2 MMP-8 200 ng/mL, 3 MMP-9 200 ng/mL, 4 MMP-13 200 ng/mL, 5 PMN elastase 200 ng/mL (left panel). MALDI-TOF mass spectra of samples 1 and 5 are shown in the middle panel and of samples 2−4 in the right panel.

preferring Leu at P1’ which is not present in eADF4(C16). The other amino acids preferred in positions P1 to P3′, namely Gly, Ala, Pro, and Ser, are similar for all MMPs. Because these amino acids comprise almost 85% of the sequence of eADF4(C16), all MMPs should digest this spider silk protein to a similar extent. However, the predicted cleavage pattern of MMP-2 is the most unspecific one, partly explaining the gained result. The cleavage pattern of PMN elastase has Ala as one of the preferred amino acids at P1. In eADF4(C16) there is one polyalanine stretch in the sequence of each C-module. PMN elastase is known to have a broad substrate specificity, degrading nearly every component of the extracellular matrix as well as a variety of diverse proteins such as cytokines or clotting factors,77 explaining the detected effect of PMN elastase on soluble eADF4(C16). Our results show that eADF4(C16) can be degraded by specific human wound proteases, which is an important requirement for its application in biomedical devices. However, not all wound proteases possess the ability to cleave the protein. This might allow the adjustment of silk proteins, such as for controlled drug release dependent on the surrounding tissue. For example, MMP-9 is upregulated in tumor cells78 or in chronic wounds.79 Thus, particles for drug delivery or wound coverage devices could be tailored by the specific introduction of specific MMP-9 cleavage sites into the spider silk sequences. To get more insights into the degradation kinetics, we analyzed eADF4(C16) degradation in a time-dependent manner using protease mix N, protease mix W, CHC, and protease XIV from Streptomyces griseus (PXIV), respectively. PXIV has been previously used to analyze degradation of silk scaffolds.32,35,38,40,41 It is a mixture of at least ten proteases with

eADF4(C16) was observable (Figure 1b). MMP-2 and PMN elastase on the other hand were both capable of degrading the silk protein almost completely overnight (Figure 1b). Remaining amounts of full length protein detected after overnight incubation in the presence of MMP-2 were most likely due to a decrease in enzyme activity over time. An activity loss of about 50% after 2 h at 37 °C has been previously reported for MMP-275, which reflects one limitation for longtime degradation studies. Strikingly, no larger fragments/ intermediates of eADF4(C16) were detectable. The theoretical degradation pattern of the different MMPs76 (Table 2) did not predict this result. All MMPs have similar cleavage patterns, Table 2. Theoretical Cleavage Patterns of Proteases Used in This Studya proteinase

cleavage patternb

MMP-2 MMP-8 MMP-9 MMP-13 PMN elastase PXIV CHC

−/p/−/− † li/−/−/− g/pas/−/g † l/−/g/− g/pa/-/g † l/−/g/− g/P/−/g † l/−/ga/− −/−/−/viat † −/−/−/− nonspecificc −/P/X † G/P/−d

a

Information on MMP-2, -8, -9, -13 and PMN elastase was taken from the MEROPS website (http://merops.sanger.ac.uk/).76 bPreferred amino acids at the subsites P4/P3/P2/P1 † P1′/P2′/P3′/P4′ shown in the usual one-letter code, upper- and lower-case letters represent the strength of the preference of specificity; † is the cleavage site. cProduct information Sigma-Aldrich, USA. dProduct information Sigma-Aldrich, USA, X usually represents a neutral amino acid. D


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Figure 2. SDS-PAGE followed by silver staining. (a−e) Incubation of soluble eADF4(C16) in TCNB in the presence of (a) protease mix N (normal range: MMP-2 200 ng/mL, MMP-8 10 ng/mL, MMP-9 100 ng/mL, MMP-13 1 ng/mL, PMN elastase 40 ng/mL), (b) protease mix W (wound range: MMP-2 250 ng/mL, MMP-8 100 ng/mL, MMP-9 400 ng/mL, MMP-13 50 ng/mL, PMN elastase 250 ng/mL), (c) MMP-2 and PMN elastase 250 ng/mL each, (d) PXIV 351 ng/mL, (e) CHC 351 ng/mL. Samples d and e were additionally analyzed by (g) MALDI-TOF MS or airdried for (f, h) SEM analysis.

a broad substrate specificity similar to pancreatic serine enzymes such as chymotrypsin.80,81 Although PXIV is more likely a suitable mimic for digestive enzymes, it was here used as an additional model enzyme to allow comparison with previously published experiments. Additionally, the experiment was performed with a mixture of only MMP-2 and PMN elastase based on our first results. Degradation by protease mix N (Figure 2a) was slower than by protease mix W (Figure 2b), which was expected because of the higher enzyme concentration of the latter. After 9 h, degradation of eADF4(C16) was still incomplete in case of protease mix N, whereas eADF4(C16) was completely degraded by protease mix W. When comparing silk degradation kinetics in the presence of protease mix W with that of the MMP-2/PNM elastase mix (250 ng/mL each, like in protease mix W) (Figure 2b, c) no significant differences were observable. An identical proteolysis pattern as in case of the complete protease mixture was observable, indicating that MMP-8, -9, and -13 had apparently no impact on degradation of eADF4(C16) or its degradation products. The concentration of CHC and PXIV was adjusted to 351 ng/ mL, corresponding to the overall enzyme concentration in protease mix N. Degradation kinetics of both enzymes were

comparable (Figure 2d, e), and no significant differences to the proteolysis pattern of protease mix W (or MMP-2/PNM elastase alone) could be observed, despite the lower enzyme concentration indicating that these proteases act as decent in vitro models for the long-time analyses of the proteolytic stability of this particular spider silk protein. Interestingly, no protease-specific degradation patterns could be identified. Silk proteins were either not degraded or completely degraded with one intermediate fragment appearing with a molecular weight of 35 kDa. This intermediate was further examined by incubating eADF4(C16) solutions in the presence of PXIV or CHC for 30 min followed by analysis using MALDI-TOF MS (Figure 2g). Strikingly, no soluble degradation product could be detected in the corresponding molecular weight range. Therefore, such samples were air-dried and analyzed using SEM. For both enzymes small particles were visible (Figure 2f, h). Apparently this intermediate was stabilized against proteolysis due to a kinetically favored selfassembly into submicroparticles with higher protease-resistance than the soluble protein, leading to the temporary accumulation of the intermediate. Importantly, in contrast to salted-out particles, these intermediate particles were soluble in Lämmli E


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Figure 3. SEM pictures of eADF4(C16) particles (non-CL, CL-b, and CL) before incubation (t0) and morphological changes after incubation in TCNB buffer in the presence or absence of PXIV or CHC (175 μg/mL) for 1−15 days, respectively.

after (CL) or before (CL-b) particle formation as described previously.29 Non-cross-linked and cross-linked particles revealed no morphological differences in SEM analysis (Figure 3). The surface of the particles was rather smooth, and particle sizes were well below 1 μm with the size of the majority of particles in the range between 300 to 500 nm. Secondary structure analysis by FTIR spectroscopy and FSD revealed no significant differences between non-CL particles and CL particles (Figures S1 and S2).29 CL-b particles, however, exhibited slightly higher α-helical, β-sheet, and β-turn contents and a lower random coil content. Cross-linking of films did also not induce morphology changes: films showed flat surfaces with only few small holes (Figure 4). Yet, cross-linked films showed a slightly decreased β-sheet content when compared to non-CL films according to FSD analysis (Figure S2). Nonwoven meshes produced by electro-spinning from HFIP solutions were post-treated with methanol vapor.23 Liquid methanol cannot be used for nonwoven meshes as it leads to “melting” of the fibers creating film-like structures.23 Nonwoven meshes were composed of homogeneous fibers with diameters below 1 μm and a smooth surface (Figure 4). Crosslinking did not alter the mesh surface morphology, yet the secondary structure was influenced, with significantly increased β-sheet content and decreased random coil content and only minor changes in contents of α-helices and β-turns (Figure S2).

buffer and thus chemically less stable than particles made of full length protein upon salting out, which explains their degradation in the reaction mixtures over time (Figure 2b−e). Degradation of eADF4(C16) Scaffolds. All experiments of eADF4(C16) scaffolds were performed with the model proteases PXIV and CHC only, because of the high amount of protease needed, the low availability of recombinant human proteins (MMP-2 and PMN elastase), as well as the short-time in vitro stability thereof. Further, the concentration of the enzyme solution was increased by a factor of 500 from 351 ng/ mL (which reflects wound range) to 175 μg/mL due to the considerably slower degradation kinetics of the scaffolds compared to soluble silk proteins. Degradation experiments would not be feasible with lower enzyme concentrations, as it would take months to years to observe any effects. Further, it has to be noted that degradation products released into solution could not be detected due to their low concentration. Importantly, the used enzyme concentration was in the same range as in similar degradation studies of silk protein scaffolds published previously32,35,40,41 and indicates that degradation in vivo might be much slower, which should be kept in mind. Buffer conditions and temperature were not altered compared to experiments with soluble silk proteins. Scaffold Characterization Prior to Enzymatic Degradation. Particles were formed by an all aqueous process and in distinct cases cross-linked either by addition of APS and Rubpy F


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Figure 4. SEM pictures of eADF4(C16) (a) films (non-CL and CL) and (b) nonwoven meshes (non-CL and CL) before incubation (t0) and morphological changes after incubation in TCNB buffer in the presence or absence of PXIV or CHC (175 μg/mL) for 1−15 days, respectively.

Scaffold Characterization after Incubation in TCNB Buffer. All scaffolds, non-cross-linked or cross-linked, were stable in TCNB buffer, and incubation for 15 days did not lead to any hydrolysis or dissolution thereof (Figure 5). Further, no morphological changes were observed using SEM (Figures 3 and 4). Scaffold Characterization upon Proteolysis. Particles. Non-cross-linked particles were almost completely degraded after incubation in the presence of PXIV for 2 days (99 wt % degraded) (Figure 5a). Within 15 h of incubation, particles started to fuse and form aggregates, partly with elongated structures (Figure 3). In comparison, degradation by CHC was much slower (93 wt % degraded after 15 days). Particle formation in the presence of APS and Rubpy (CL-b) resulted in uniform cross-linking throughout the particles.45 Diffusion of cross-linking reagents into the particles (CL) preferentially cross-linked the particle surfaces.29 CL-b particles showed a diminished degradation behavior in the presence of both enzymes (Figure 5a), with both degradation rate and degree of degradation being reduced compared to non-cross-linked particles. Degradation by PXIV reached a plateau at day 7 (88 wt % degraded), and morphological changes were similar to those of non-cross-linked particles, but to a lesser extent (Figure 3). Degradation by CHC was not terminated after 15 days (68 wt % degraded) (Figure 5). Particles started to “melt” and collapsed, but individual particles were still recognizable. Cross-linking of particles after assembly (CL) led to a considerable reduction in eADF4(C16) degradation (Figure 5a). In the presence of PXIV, degradation of CL particles was observable for 8 days (24 wt % degraded) before it apparently stopped, and in the presence of CHC a plateau was already reached after 3 days (6 wt % degraded). Furthermore, no

significant changes in morphology were visible (Figure 3). These results showed that the cross-linking method significantly influenced the proteolytic stability of particles. Films. Sixty weight percent of non-CL films were degraded upon incubation with PXIV for 15 days but a plateau was not reached (Figure 5b). Before incubation with the proteases films had a smooth surface, but after 3 days in PXIV solutions the surface was very rough and had a beadlike structure (Figure 4). At day 15, the surface appeared smoother again and was partly covered with elongated rough structures with small holes. Degradation using CHC was much slower and also not finalized at day 15 (13 wt % degraded) (Figure 5b). After 15 days the surface was rougher compared to PXIV treated films with similar, but less, elongated structures on the surface (Figure 4a). eADF4(C16) in cross-linked films was degraded with slower kinetics than in non-cross-linked films in the presence of PXIV, but there was no significant difference between CL and non-CL films in the presence of CHC (Figure 5b). At day 15, degradation was not yet finalized, in the presence of neither PXIV (29 wt % degraded) nor CHC (12 wt % degraded). Similar structures as on non-cross-linked films could be observed on cross-linked films (Figure 4a). Even though only 12 wt % of the cross-linked films were degraded in the presence of CHC, the surface structure of the silk films was altered significantly, presumably because of the deposition of self-assembled degradation products. Nonwoven Meshes. Nonwoven silk meshes were slightly more stable in the presence of proteases than films (Figure 5c). For the first 6 days the degradation rate of non-cross-linked films and non-cross-linked nonwoven meshes was identical in the presence of PXIV. In contrast to films, degradation of nonwoven meshes stopped after day 6 (46 wt % degraded). G


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Figure 5. Percentage of degraded eADF4(C16) after incubation of the different scaffolds in TCNB buffer in the presence or absence of PXIV or CHC (175 μg/mL) for 1−15 days, respectively (orange triangles, PXIV; blue-green circles, CHC; black squares, TCNB). (a) Particles, (b) films, (c) nonwoven meshes. Cross-linking (CL) was obtained by incubation of the scaffolds in Tris buffer containing APS and Rubpy in the dark overnight and subsequent exposition to a tungsten lamp for initiation of the cross-linking reaction. Alternatively, particles were cross-linked by addition of the cross-linking agent before particle formation (CL-b).

Morphological analysis revealed that within the first 6 days fibers started to fuse and collapse (Figure 4b). During the following 9 days, further morphological reorganization was observable. With some remaining fibers with a beadlike surface structure, the majority of the samples looked like that of noncross-linked films after 15 days of incubation in the presence of PXIV. Incubation of non-CL nonwoven meshes in the presence of CHC yielded no significant degradation (about 5 wt % degraded) (Figure 5c). However, “melting” of the fibers was observable with areas of highly fused structures, which was caused by the coverage of the nonwoven meshes with similar structures like those observable on degraded films (Figure 4).

Cross-linked nonwoven meshes showed almost no degradation in the presence of either PXIV or CHC (2 and 0.5 wt % degraded, respectively) (Figure 5c). However, the morphology of the nonwoven mats changed significantly, especially in case of incubation with PXIV (Figure 4b). Even though the porous structure seemed to be largely intact in some areas, there were other areas where fibers were completely fused, again because of the coverage with structures with similar morphologies as found in similarly treated films. Additionally, the nonwoven scaffolds were covered with large particulate aggregates, and remaining fibers appeared more ribbon-like with increased diameters. CL nonwoven meshes incubated in the presence of H


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Figure 6. Analysis of non-cross-linked scaffolds before (t0) and after incubation with PXIV or CHC (175 μg/mL in TCNB buffer) for 1 to 15 days by SDS-PAGE followed by silver staining (*15 day sample: twice the volume was applied).

ever, certain secondary structure elements are more readily degraded by some proteases than others. Hu et al. reported a higher activity of CHC toward random coil regions compared to β-sheet regions in silk fibroin scaffolds,43 whereas Numata et al. described a high activity of PXIV toward β-sheet structures.36 Here, such correlation could not be confirmed indicating that proteolytic degradation is a more complex process influenced by a variety of factors, which cannot be easily predicted. Despite of a comparable secondary structure composition of non-CL particles and nonwoven meshes before proteolytic degradation (Figure S2), non-CL nonwoven meshes were much more stable in the presence of PXIV than non-CL particles presumably due to a different surface composition, structure and morphology. Degradation of nonwoven meshes was not only slower, they were also degraded to a lesser extent. This effect was even more pronounced in the presence of CHC. eADF4(C16) fibers in nonwoven meshes are very smooth, therefore possessing a small surface area; because of the spinning process, protein chains are highly aligned, leading to a closer packing of the silk proteins compared to particles or films.42 Characterization of the Remaining Scaffolds after Incubation in the Presence of Proteases. MALDI-TOF MS analysis detected no larger fragments in the supernatants after incubation of the scaffolds in the presence of proteases for 1 day (data not shown). In vivo, released degradation products can potentially induce an immune response. Yet the absence of immunogenicity of a eADF4(C16) scaffold (as a film) was previously shown in vivo where silicone implants coated with eADF4(C16) films were implanted into rats.28 For a period of 12 months, no significant immunological reaction was detectable. Furthermore, a remaining eADF4(C16) film could be identified on the explants, verifying the proteolytic stability of spider silk protein films also in vivo for this time period. After incubation in the presence of proteases for up to 15 days, the remaining non-cross-linked scaffolds were denatured using GdmSCN and analyzed by SDS-PAGE (Figure 6). In

CHC for 15 days were only slightly fused with an overall intact porous structure. Aggregates were also visible, yet to a lesser extent than on nonwoven meshes degraded by PXIV. This finding is in accordance with previous findings in which degradation of silk fibroin fibers with PXIV revealed particulate debris thereon within 7 days.38 Because of a decrease in fiber diameter degradation through a surface erosion process was suggested. Here, a similar process happened with CL nonwoven meshes in the presence of CHC (Figure 4b, arrow in the bottom right SEM image) and PXIV. The behavior of nonwoven silk scaffolds differed also from that of the others due to an apparent rearrangement of silk proteins in the remaining scaffolds. This process presumably led to a decreased accessibility of the remaining mesh structure toward proteolytic degradation and further prevented release of degradation fragments. Our hypothesis was supported by SEM analysis showing increased fusion of the fibers (Figure 4b) as well as by SDS-PAGE showing an increased fragmentation of the silk proteins in the remaining scaffold (Figure 6). The higher resistance of fibers toward enzymatic degradation in comparison to films was most striking for CL samples, in accordance with previous observations.42,82 Comparing all results revealed several factors influencing the degradation behavior of spider silk scaffolds. Even though degradation of eADF4(C16) in solution was comparable in the presence of PXIV and CHC, there were significant differences concerning the proteolytic stability of the employed scaffolds against these proteases (Figure 5). The accessibility of cleavage sites on a scaffold is highly important in combination with the kind of applied enzyme. Degradation in the presence of PXIV was always faster than in the presence of CHC confirming its high degrading potential for silks compared to other proteolytic enzymes.35,39,42 The initial secondary structure composition of silk protein scaffolds had been previously shown to impact the degradation behavior.30,36,37,83 Generally, a higher β-sheet content reduces degradation rates, whereas increased random coil contents accelerate enzymatic degradation.32,83−85 HowI


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For nonwoven meshes and CL-b particles treated with PXIV for 15 days, this peak was most pronounced. For both samples, a plateau was reached in the degradation profile after 6 to 7 days even though further morphological changes were observable. Additionally, analysis of the secondary structure composition revealed a significant decrease in ß-sheets and a significant increase in random coil content (Figure S2). These observations can be explained by proteolytic cleavage within ßsheet-rich regions without release of any degradation products, leading to an increase in terminal carboxyl groups. The predominantly random coil structure, which was not detected in any other sample, on the other hand most likely induced the detected “melting” of the samples. In the case of CL-b particles, we would not expect the release of further degradation products even during prolonged degradation times due to the crosslinking of the particles. In the case of non-cross-linked nonwoven meshes, however, it is surprising that less than 50% of the scaffold was dissolved. Considering a ß-sheet content of about 14%, an α-helix content of 15%, and a random coil content of 46%, it is remarkable that the sample was insoluble in the aqueous environment. Therefore, it cannot be excluded that further degradation will be observed during longer incubation times in the presence of PXIV. For the remaining three samples (CL films/PXIV, CL nonwoven meshes/PXIV, and CL films/CHC), the formation of a peak at 1740 cm−1 can be correlated with a decrease in ßsheets and an increase in random coil content, yet the effect was less significant. CL nonwoven meshes were highly stable both in the presence of PXIV and CHC, with only negligible amounts of released degradation products. However, morphological changes were observable in terms of a more ribbonlike fiber morphology and coverage with particulate aggregates. Again, secondary structure analysis revealed a decrease in ß-sheets and an increase in random coil content. Apparently, the degradation of crystalline regions on the surface of fibers led to the formation of particulate debris covering the nonwoven meshes. Cross-linking of the fibers seems to have prevented most of the degradation products to be released into solution and rather have caused collapsing yet not fusion of the fibers and the formation of particulate aggregates. The fact, that these aggregates were only detected in nonwoven mesh samples can be attributed to the aforementioned alignment of the protein chains during the spinning process. Evaluation of CHC and PXIV as Model Enzymes. Riley et al. demonstrated that pretreatment of a biosynthesized extracellular matrix by Clostridial collagenase promotes human keratinocyte proliferation and migration in vitro.88 Furthermore, an in vivo study confirmed promotion of wound healing by direct application of Clostridial collagenases. This is interesting, because during wound healing, keratinocytes respond to the extracellular matrix. For example, production of MMP-1, -2, and -9 by keratinocytes leads to a remodeling of the matrix and induces keratinocytes to migrate into the wound and close it.88 This further supports our proposed comparability of CHC and human wound proteases due to their functional relation. Compared to mammalian collagenases Clostridial collagenase possesses the ability to cleave collagen more unspecifically.88 Concerning eADF4(C16) in solution, we observed no differences in the degradation products in the presence of CHC and MMP-2/PMN elastase. Yet, in the case of eADF4(C16) processed into scaffolds, cleavage sites for human proteases might be less available compared to cleavage

case of particles incubated in the presence of PXIV, no full length eADF4(C16) was detectable after 1 h of incubation, and later four defined fragments appeared, presumably representing fragments with eight repeat sequences (C8) and smaller. After 15 days of incubation, particles of eADF4(C16) were almost completely degraded in the presence of PXIV. Particles incubated in the presence of CHC started to degrade within the first 24 h (Figure 6). After 3 days, no full length eADF4(C16) was detectable, and a number of apparently stable fragments could be detected between 6 and 15 days of incubation. Films degraded with PXIV showed a fragment with slightly lower molecular weight than the full length protein between day 3 and 10, and this fragment remained stable even after 15 days. Incubation of films in the presence of CHC led to only minor proteolysis of eADF4(C16) with full length protein remaining even after 15 days. After 10 days of incubation fragments appeared and got pronounced after 15 days, which is consistent with the degradation kinetics. Inconsistencies in the amount of full length protein might be due to dilution effects during dialysis and/or uneven staining of the gels. eADF4(C16) in nonwoven meshes incubated in the presence of PXIV started to degrade within the first day appearing as a smear. Full length eADF4(C16) was still detectable after 15 days with defined degradation fragments visible after 6 days, which was pronounced after 15 days. In case of nonwoven meshes incubated in the presence of CHC almost no eADF4(C16) degradation was observable within the first 10 days, and only weak degradation bands could be detected after 15 days. It should be noted, that unlike in case of soluble eADF4(C16), where only one additional degradation product could be detected, several degradation bands occurred in case of the scaffolds, most likely because soluble silk proteins are wellaccessible for proteases and therefore more readily degradable. As mentioned before, one intermediate accumulates because of its kinetically favored self-assembly into submicroparticles. However, these self-assembled particles are less stable than particles produced from full length protein by salting out. Structural Changes upon Proteolysis. The secondary structure of the scaffolds during proteolytic degradation was analyzed by FTIR spectroscopy and FSD analysis of the amide I band (Figures S1 and S2). All scaffolds retained their starting secondary structure after incubation in TCNB buffer for 15 days. Surprisingly, there was no correlation between the secondary structure composition before treatment with proteases and the degradation kinetic or degree of degradation. Likewise, changes in the secondary structure composition could not be correlated to either morphology or cross-linking of the scaffolds. For both PXIV and CHC, results of FTIR analysis were inconclusive. A remarkable feature was the occurrence of a peak at 1740 cm−1 after 6 to 15 days in spectra of CL-b particles, CL films, non-CL, and CL nonwoven meshes incubated with PXIV and CL films incubated with CHC (Figure S1). In the literature, this peak has been assigned to (deionized forms of) glutamic acid, aspartic acid and terminal −COOH.86,87 eADF4(C16) does not possess any aspartic acid and only one glutamic acid per C-module. Therefore, the appearance of the peak at 1740 cm−1 might be an indication for increased fragmentation of the remaining silk proteins, which are, however, not released into solution, and thus an increased number of free carboxyl-termini is presented. Yet the formation of a peak at 1740 cm−1 after proteolytic degradation of silk scaffolds has not been reported before. J



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sites for CHC. So far, we conducted one preliminary experiment by incubation of silk films in the presence of PXIV, CHC, and MMP-2/PMN elastase, respectively, for 2 h (Figure S3). After proteolytic degradation, small holes were visible in the surface of the films, with a similar morphology in case of CHC and MMP-2/PMN elastase. Prolonged degradation times might alter the outcome in similar experiments. However, we could show that also after film formation cleavage sites are available for human proteases. Therefore, we suggest that in vitro degradation experiments of silk-based biomaterials intended for wound healing purposes should be performed in the presence of CHC rather than PXIV. Because of its caseinolytic activities PXIV, on the other hand, is presumably better suited as model protease for digestive enzymes.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +49 (0) 921 55 7361. Fax: +49 (0) 921 55 7346. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Hendrik Bargel for assistance with SEM analyses and Martin Humenik for assistance with MALDI-TOF MS measurements. This work was supported by the Deutsche Forschungsgemeinschaft (SFB840 TP A8).

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CONCLUSIONS The assessment of the degradation behavior of a biomaterial is of great importance for its putative application due to diverse demands concerning proteolytic stability. Examination of the in vitro degradation of the recombinant spider silk protein eADF4(C16) in the presence of different proteases illustrates the importance of a thorough analysis of the respective proteolytic environment in vivo in the future. Unlike predicted by their cleavage patterns, only two out of five recombinant human proteases were able to digest soluble eADF4(C16), namely MMP-2 and PMN elastase vs MMP-8, -9, and -13. And even though proteolysis of soluble silk proteins was comparable for two bacterial model proteases, there were significant differences concerning degradation of eADF4(C16) scaffolds. All scaffolds were degraded more rapidly by PXIV, which rather reflects a model for digestive enzymes, than by CHC, reflecting more woundlike protease properties. However, this is in line with the fact that spiders can digest/eat their own silk. Additionally, the morphology of the scaffolds had a major impact on the proteolytic stability. In the presence of PXIV or CHC, particles were degraded faster than films, and nonwoven meshes exhibited the highest resistance toward proteolytic degradation. Surprisingly, we did not observe a correlation either between the initial secondary structure composition of the scaffolds and their degradation behavior or between the applied protease and an increase/decrease of certain secondary structure elements. Chemical cross-linking of the scaffolds increased the proteolytic stability, as expected, and for particles we showed that by altering the cross-linking process further adjustments concerning degradation kinetics and degree of degradation could be made. However, it must be taken into consideration that in the case of eADF4(C16) scaffolds, the protease concentration used in this study (and in similar studies published by other groups) was considerably higher compared to physiological levels. Thus, under in vivo conditions, scaffolds are most likely to persist for a significantly longer period of time than shown in our in vitro experiments.

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ABBREVIATIONS MMP, matrix metalloproteinases; PMN elastase, polymorphonuclear elastase; PXIV, protease type XIV from Streptomyces griseus; CHC, collagenase type IA from Clostridium histolyticum; Tris, Tris (hydroxymethyl)-aminomethane-HCl; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; p.a, pro analysi; APS, ammonium persulfate; Rubpy, Tris(2,2′-bipyridyl)dichlororuthenium(II); TCNB, Tris/CaCl2/NaCl/Brij 35; CL, cross-linked; APMA, p-aminophenylmercuric acetate; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; FTIR, Fourier transform infrared; ATR, attenuated total reflection; FSD, Fourier self-deconvolution; SEM, scanning electron microscopy

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S Supporting Information *

The following files are available free of charge on the ACS Publications website at DOI: 10.1021/ab500147u. Detailed information about the expression and targets of the employed wound proteases and secondary structure composition of the scaffolds, and additional SEM images of degraded silk protein films (PDF) K


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