Wet-Spinning of Recombinant Silk-Elastin-Like Protein Polymer Fibers

Feb 2, 2009 - Recombinant Silk-Elastinlike Protein Polymer Displays Elasticity Comparable to Elastin. Weibing Teng , Joseph Cappello and Xiaoyi Wu...
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Biomacromolecules 2009, 10, 602–608

Wet-Spinning of Recombinant Silk-Elastin-Like Protein Polymer Fibers with High Tensile Strength and High Deformability Weiguo Qiu,† Weibing Teng,† Joseph Cappello,‡ and Xiaoyi Wu*,†,§ Department of Aerospace and Mechanical Engineering, Biomedical Engineering IDP and Bio5 Institute, University of Arizona, Tucson, Arizona 85721, and Protein Polymer Technologies, Inc., San Diego, California 92121 Received November 10, 2008; Revised Manuscript Received December 31, 2008

A recombinant silk-elastin-like protein copolymer SELP-47K containing tandemly repeated amino acid sequence blocks from silk, GAGAGS, and elastin, GVGVP, was fabricated into microdiameter fibers using a wet-spinning technique. Raman spectral analysis revealed the formation of antiparallel β-sheet crystals of the silk-like blocks. Dry SELP-47K fibers display the dependence of mechanical properties such as Young’s modulus on fiber diameter, suggesting more oriented and crystallized molecular chains in small-diameter fibers. Additionally, a brittle fracture mode was identified for dry fibers by SEM analysis of fracture surfaces. Hydration dramatically influenced the mechanical behavior of SELP-47K fibers. In contrast to the high tensile strength and limited strains to failure of dry fibers, fully hydrated SELP-47K fibers possessed strains to failure as high as 700%. Furthermore, upon chemical cross-linking, a tensile mechanical strength up to 20 MPa was achieved in hydrated fibers without compromising their high deformability. By combing the silk- and elastin-derived sequences into a single SELP-47K protein polymer, we demonstrated that protein fibers with high tensile strength and high deformability can be fabricated.

Introduction Protein fibers, which are fundamental building blocks of extraand intracellular matrices, play an important role in structure support, scaffolding, stabilization, and the protection of cells, tissues, and organisms.1 Fabricating performance protein fibers has been extensively pursued for many biomedical applications, such as tissue engineering and drug delivery. Among natural proteins, spider dragline silk is the strongest fiber. However, Shao and Vollrath2 demonstrated that under appropriate conditions silkworm silk can also form fibers with mechanical properties comparable to those of spider dragline silk. Interestingly, the crystallizable domains of spider dragline silk are comprised of poly(alanine) and poly(glycine-alanine) repeats,3 and those of silkworm silk consist of glycine- and alanine-rich GAGAGS (one-letter amino acid abbreviation is used) repeating motifs.4 Despite its excellent mechanical strength, the resilience of silk, which characterizes its capacity for shape and energy recovery under mechanical loading, is usually very poor.5 In contrast, fully hydrated elastin is highly resilient, especially at low frequencies.5 By combining polypeptide sequences derived from silkworm silk and mammalian elastin, silk-elastin-like proteins (SELPs), biosynthesized using recombinant DNA techniques,6 are designed to combine diverse physical, mechanical, and biological features. The incorporation of elastin-like blocks imparts stimuli sensitivity into SELPs, resulting in the formation of temperature and pH responsive hydrogels.7 Our recent tensile analysis8 also revealed excellent resilience (e.g., 90%) of preconditioned SELP films. Furthermore, previous studies demonstrated that SELPs with different monomer structures can be fabricated into microdiameter fibers.9,10 However, the secondary structures and mechanical properties * To whom correspondence should be addressed. Tel.: 1-00-520-6265854. Fax: 1-00-520-621-8198. E-mail: [email protected]. † Department of Aerospace and Mechanical Engineering. ‡ Protein Polymer Technologies. § Biomedical Engineering IDP and Bio5 Institute.

of SELP fibers have not been fully characterized. Subsequently, the structure-property relationship and formation mechanisms of SELP fibers remain poorly understood. In this study, a SELP-47K protein polymer with a monomer structure of (S)4(E)4(EK)(E)3, in which S is the silk-like sequence GAGAGS, E is the elastin-like sequence GVGVP, and EK is the penta-peptide sequence GVGKP, was fabricated into microdiameter fibers by using a wet-spinning technique. Like native silk in spider and insect glands, silk-based materials in solution often exist in a metastable silk I form, the structure of which remains poorly understood. Both β-turn11 and R-helix conformations12 have been proposed for silk I structures. Nevertheless, it has been well established that with mechanical shearing and elongation in the silk spinning process, silk molecules are aligned, so intermolecular hydrogen bonds can be formed and a silk I structure is converted into a silk II form characterized by an antiparallel β-sheet conformation.13 During this process, it is thought that water acts as a plasticizer inhibiting the β-sheet conversion.12 Thus, nonsolvents like methanol are often used as a coagulant in artificial silk fiber spinning to remove water and cause the formation of silk fibers.9 On the other hand, enhanced mechanical properties have resulted from an underwater spinning of silk fibers.14 This effect was attributed to enhanced molecular orientations, because the plasticization effect of water possibly provided silk molecules with more time to become aligned. Likely, both the molecular flexibility for peptide backbone alignment and the capability to “freeze” converted β-sheets are critical to the formation of strong silk fibers. In SELP-47K, the incorporation of elastin-like blocks enhances water solubility and likely provides more flexibility for SELP molecular reorientation and alignment. Raman spectroscopy was used to identify the formation of β-sheet crystals of SELP-47K fibers. To examine the hydration effects on material responses, mechanical properties of both dry and hydrated SELP-47K fibers were characterized. It is well-known that dry elastin usually

10.1021/bm801296r CCC: $40.75  2009 American Chemical Society Published on Web 02/02/2009

Wet-Spinning of Recombinant Protein Polymer Fibers

Biomacromolecules, Vol. 10, No. 3, 2009

displays low deformability but a high Young’s modulus, while fully hydrated elastin is highly deformable, with a strain to failure as high as 100% and a Young’s modulus as low as 0.2 MPa.15 Hydrophobic hydration of the elastin peptide backbone likely causes hydration-induced swelling, providing elasticity to elastin and elastin-like materials.16,17 In contrast, silk-based materials may contract significantly upon hydration.18,19 This is attributed to the disruption of intermolecular hydrogen bonds, the disorientation of amorphous or less crystallized molecular chains, and the conversion of extended β-sheet crystals back into less ordered structures, for example, random coil and β-turn. Therefore, “freezing” the molecular chains of the less crystalline region of silk-like fibers is key to retaining their high mechanical strength. In SELPs, it is anticipated that the silk- and elastinlike blocks will respond differently to hydration. Additionally, the chemical cross-linking of the elastin-like blocks may prevent the large disorientation of molecular chains and possibly “freeze” the less crystalline conformations, leading to enhanced material stability of SELP fibers.

Materials and Methods Sample Preparation. Frozen SELP-47K aqueous solutions at a concentration of 13% w/v were generously provided by Protein Polymer Technologies, Inc. (San Diego, CA). The SELP-47K solutions were lyophilized and redissolved in 98% formic acids (VWR) at a concentration of 25% w/v for fiber spinning. Wet-Spinning. A custom-made wet-spinning device was used for the fabrication of microdiameter SELP-47K fibers. Briefly, a wetspinning procedure consists of extrusion of protein solutions, formation of a protein thread, coagulation of the protein thread into a fiber, washing extra coagulant remaining in the fiber, air-drying, and collecting the fiber. In wet-spinning, an apparatus housing a ceramic capillary (Small Precision Tools, CA) of 28 or 38 µm in diameter is wired to a syringe pump (model R99-FM, Braintree Scientific, MA). SELP-47K fibers were spun into a coagulant bath of methanol/water, air-dried, and then collected by custom-made rotating Teflon mandrels driven by a DC-powered motor (Sterling Instrument, NY). Scanning Electron Microscopy. The microstructures (e.g., fiber diameter, surface morphology) of air-dried SELP-47K fibers were examined using a Hitachi-S3400 scanning electron microscope (SEM). To examine the fiber stability, some wet-spun fibers were extensively rinsed and then stored in 1× PBS at room temperature. After 1 year, fibers were taken out and air-dried for a SEM analysis. Likewise, the surface morphology of fractured dry fibers was also examined using a SEM. Raman Spectroscopy. Raman spectra of the wet-spun SELP-47K fibers were recorded on a Thermo Nicolet Almega microRaman system (Thermo Scientific). A solid-state laser with the wavelength of 532 nm was used as the exciting source. The non-negative least-squares (NNLS) analysis of Raman amide I and III spectra that was first proposed by Williams and Dunker20 and subsequently refined by Williams21,22 was pursued to estimate the secondary structural contents, including helix, β-strand, β-turn, and undefined conformation, as follows

Ax = b

(1)

f ) Fx

(2)

where A is a p × n matrix containing the normalized Raman amide I or III spectra of n reference proteins at p wavenumbers, column vector b contains the normalized Raman amide I or III spectrum of wet-spun SELP-47K fibers at the same p wavenumbers, column vector x is used to calculate percentage contents of secondary structures for SELP-47K fibers as a weighted sum of the secondary structures of the n reference proteins, F is an m × n matrix representing m classes of secondary structures of the n reference proteins, and column vector f then provides percentage contents of secondary structure for SELP-47K fibers. As

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detailed elsewhere,22 16 and 13 reference proteins were used in Raman amide I and III spectral analysis, respectively. Moreover, the A and F matrices in eqs 1 and 2 were directly taken from Williams’ work.22 Still, following the Williams’ procedure, the amide I band intensities of SELP-47K fibers from 1615 to 1710 cm-1 at an interval of 5 cm-1, and the amide III band intensities from 1225 to 1245 cm-1, from 1275 to 1310 cm-1 at an interval of 5 cm-1, and again at 1235 and 1285 cm-1 were tabulated into column vector b. Unlike proteins analyzed by Williams, SELP-47K contains less than 0.4% Tyr, Phe, and Trp, and thus subtraction of side chain bands due to these residues is unnecessary, evident by the minimal intensity of the Trp band at 1555 cm-1. Additionally, subtraction of the spectrum of buffer is not relevant, as the Raman spectra were collected on air-dried SELP-47K fibers. Nevertheless, base correction was performed using Grams 8.0 over the spectral region of 1800 to 1200 cm-1. Likewise, the NNLS analysis of the Raman amide I and III spectra of SELP-47K fibers was implemented using MATLAB. Cross-Linking. SELP-47K fibers were cross-linked overnight by glutaraldehyde (GTA; Mallinckrodt Baker) vapor in a vacuumed desiccator. The bottom of the desiccator was filled with 25% GTA in water while SELP-47K fibers collected on a glass spool were placed on a ceramic plate with holes, which separated the bottom chamber with GTA from the top chamber. Mechanical Analysis. The mechanical properties of both dry and hydrated (in 1× PBS at 37 °C) SELP-47K fibers were characterized using a high-resolution dynamic mechanical analyzer (DMA, PerkinElmer). Due to the difficulty in handling single fibers, only fibers with a diameter greater than 20 µm were mechanically analyzed. Dry fibers and hydrated fibers were elongated to break at a constant cross-head speed of 20 and 250 µm/min, respectively. The gauge length of dry fiber samples was 20 mm. Short samples with a gauge length of 5 mm were also analyzed for comparison. Due to the high extensibility of hydrated SELP-47K fiber samples and the limitation of the maximum travel distance of the drive shaft of DMA, short samples of 3 or 5 mm in length were used in mechanical analysis of hydrated samples. Data were successfully collected on 28 dry fiber samples with diameters ranging from ∼20 to 60 µm. Despite more than 10 trials, stress-strain analysis was only obtained on two fully hydrated fiber samples because of the limitation of the force measurement (∼10 µN) of the current facility. GTA cross-linking greatly enhanced the mechanical stability of fully hydrated SELP-47K fibers. As a result, nine fiber samples were successfully analyzed when fully hydrated in 1× PBS at 37 °C. The Young’s modulus, ultimate tensile strength, and strain at failure of SELP fibers were obtained from stress-strain analysis.

Results and Discussion Wet-Spinning of SELP-47K Microdiameter Fibers. Protein solutions of SELP-47K copolymers at a concentration of 25% w/v in formic acid were spun into fibers of meters in length (Figure 1A). The diameters of SELP-47K fibers can be regulated by the opening size of the spinneret and the postspinning draw of fibers during fiber collection. Fibers with diameters ranging from less than 10 µm to over 60 µm were fabricated. A SEM study demonstrated the formation of uniform, clean microdiameter fibers (Figure 1B,C). Interestingly, before they are airdried, two fibers can merge together and form interfiber bonding at the point of contact (Figure 1D,E). Additionally, the formation of hollow fibers was observed (Figure 1F). The wet-spinning of SELP-47K with a monomer structure of (S)4(E)4(EK)(E)3 results in the formation of fibers with smooth surfaces (Figures 1 and 2). In contrast, wet-spun SELP5 fibers with a monomer structure of (S)8(E)16 displayed horizontal lines on the surface along the fiber axis.9 Likely, the fewer silk-like blocks per repeat and the incorporation of lysine in SELP-47K are responsible for the dramatic difference in the fiber surface morphology. Our prior biodegradation studies revealed that a

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Figure 1. Wet-spun SELP-47K fibers. Microdiameter SELP-47K fibers of meters in length were collected on a glass spool (A); SEM micrographs of SELP-47K fibers with different diameters and surface morphologies (B, C); at the point of contact, two fibers can merge and form interfiber bonding before being air-dried (D, E); and some fibers are partially hollow (F).

Figure 3. Surface morphology of wet-spun SELP-47K fibers that had been hydrated in 1× PBS for 1 year.

Figure 2. Morphology of the end surface of a wet-spun fiber.

SELP5 film implant in rats retained nearly 100% of its original mass over the course of 7 weeks while a SELP8 film with a monomer structure of (S)4(E)8 lost over 80% of its original mass over the same time period.23 Given the similar molecular weights and the same ratio of the silk- to elastin-like blocks in SELP5 and SELP8, the doubled number of silk-like blocks per repeat in SELP5 thus dramatically enhances the stability of protein polymer structures, likely due to greater crystallization. SELP-47K has a monomer structure very similar to SELP8, except that the second valine of the pentapeptide GVGVP is replaced by a lysine in one of every eight elastin-like blocks. Compared to SELP5, the reduced number of silk-like blocks per repeat in SELP-47K leads to less crystallization of wetspun fibers. Additionally, the incorporation of lysine residues, which are positively charged in formic acid, further inhibits the formation of large, continuous fibrils of β-sheet crystals in the wet-spun SELP-47K fibers. The small texture features on the end surface of the SELP-47K fibers (Figure 2) are due to coagulation effects. When hydrated in 1× PBS at room temperature, no significant change of the SELP-47K fiber morphology occurred over a time period of 30 days (data not shown), suggesting excellent material stability of the wet-spun fibers. Significantly, the wet-spun fibers remained stable in 1× PBS at room temperature over a time period of 1 year (Figure 3). Surprisingly, all samples that were examined using a SEM displayed smooth horizontal lines on the surface along the fiber axis. In contrast, no horizontal lines were observed in fresh fibers (Figure 1). The mechanism of the dramatic morphological changes of wet-spun SELP-47K fibers hydrated in 1× PBS for 1 year was not well understood. Although SELP-47K is highly water soluble, the spinning of SELP-47K aqueous solutions into microdiameter fibers, as

Figure 4. Raman spectra of wet-spun microdiameter SELP fibers.

reported for a system of recombinant spider silks,24 was not achieved. Likely, the high surface tension of SELP-47K aqueous solutions prevented the break-up of solution droplets and thus the formation of a protein thread. Since, a low surface tension of protein solutions is critical to the formation of a protein thread at the orifice of the spinneret in wet-spinning,25 we lowered the surface tension of SELP-47K solutions by using 98% formic acid. Furthermore, sufficient entanglement of polypeptide chains is also necessary for the formation of protein fibers. As we observed (data not shown), no fibers were formed from SELP47K solutions at a concentration of 13% (w/v) while the wetspinning of 20% (w/v) SELP-47K solutions produced short fiber segments. Finally, clean, robust microdiameter fibers were obtained from the wet-spinning of 25% (w/v) SELP-47K solutions in formic acids. The wet-spinning of more concentrated SELP-47K solutions was not pursued because the clogging of spinnerets presented a consistent challenge. Raman Spectroscopic Analysis of SELP-47K Fibers. The Raman spectra of dry wet-spun SELP-47K fibers are given in Figure 4, and the band assignment is detailed in Table 1. In the 4000-2600 cm-1 spectral region, three major bands are ascribed to the methyl CH stretching doublet at 2972 and 2875 cm-1 and to the antisymmetric stretching of CH2 groups at 2935 cm-1.

Wet-Spinning of Recombinant Protein Polymer Fibers Table 1. Band Assignment of Raman Spectra wave numbersa 3400 br, sh 3287 m, br 2972 s 2935 s 2875 s 2771 vw 2729 w 1665 s 1557 vw 1450 s 1410 w 1334 m 1320 m 1228 s 1162 m 1119 w 1085 m 1024 m 1010 m 973 s 955 s 936 s 879 m 854 m 839 m 753 w, br 513 w, br 417 m b

approximate assignment of vibrational modeb ν(OH) stretching, H-bonded, and free ν(NH) stretching ν(NH) stretching, H-bonded ν(CH3) ν(CH2) νs(CH3) ν(C(CH3)2)26 ν(CH-CH3) aliphatic26 ν(CO) amide I ν(CC) δ(CH2) scissoring CH2 wagging31 δ(CH3) δ(CH2) ν(CN) amide III β-sheet,33 δ(CH2)13 ν(CC), δ(COH)33 ν(CC) skeletal stretching,30 F(C(CH3)2)31 ν(CC) skeletal random coil,33 β-sheet12 ν(CC) skeletal33 ν(CC) skeletal,33 ν(CN)31 F(CH3)33 F(CH3)33 ν(CC)31 F(CH2)33 ν(CC) of Pro ring34 Pro ring35 F(CH2) in-phase33 δ(CCC) of Val36 δ(CCC), δ(CCN)33

a br: broad; sh: shoulder; s: strong; m: medium; w: weak; vw: very weak. ν: stretching; δ: bending; F: rocking.

The CH2 symmetric stretching band is missing, likely due to an overlap with that of the CH3 symmetric stretching. Two weak bands at 2771 and 2729 cm-1 are assigned to the stretching vibration of C-(CH3)2 groups of valine and to that of CH-CH3 groups of alanine, respectively. Typically, Raman bands between 2800-2630 cm-1 are neglected in spectroscopic analysis owing to their significantly weak intensities relative to those bands above 2800 cm-1. However, Lawson et al.26 demonstrated that these bands are particularly useful in analyzing the relative locations of the methyl groups and the presence of CH2C and CH3C structural features. A broadband at 3287 cm-1 is assigned to the stretching of hydrogen-bonded NH groups. Furthermore, a broad shoulder observed at 3400 cm-1 suggests that the OH groups of serine (S) are largely hydrogen-bonded, because the characteristic band of free OH groups should be at ∼3500 cm-1. A similar red shift of ν(OH) stretching bands induced by hydrogen bonding also has been reported in L-serine crystals.27 Nevertheless, the stretching of free NH groups also contributes to the broad shoulder at 3400 cm-1. The NH and OH stretching bands, which are extremely sensitive to the changes of the hydrogen bonding structures, have been recently used to study the pH-induced conformational transitions in the poly-L-lysine model system.28 Analysis of the 1800-800 cm-1 spectrum of the SELP-47K fibers reveals the marker bands of silk II, including amide I at 1665 cm-1, amide III at 1228 cm-1, CC skeletal β-sheet stretching at 1085 cm-1, CH3 rocking at 973 cm-1, and CH2 rocking at 879 cm-1. These characteristic bands of silk II have been identified in silk processed from wild and cultivated cocoons,13 as well as in model peptides comprising the repeating GAGAGS hexamers.29 The observed marker bands of silk II in the SELP-47K wet-spun fibers suggests that the silk-like blocks, (GAGAGS)4, although segregated by the elastin-like blocks, form antiparallel β-sheets. Additionally, a band at 1162

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cm-1 ascribed to νCC and δC-OH has been observed at 1158 cm-1 in silk II-Cp but at 1173 cm-1 in silk I-Cp. This further confirms the existence of silk II in the SELP-47K wet-spun fibers. However, the relatively weak bands at 1410 cm-1 (δCRH2), 1320 cm-1 (amide III of the β(II)-turn conformation),29 1119 cm-1 (νCC skeletal stretching,30 and FC(CH3)231), and 956 cm-1 (FCH3) indicate the presence of silk I in the SELP-47K fibers, too. Noteworthy is the nonsymmetric amide III band at 1228 cm-1, which may overlap other characteristic amide III bands of silk I at high wave-numbers such as 1245 and 1270 cm-1. Interesting, these marker bands of silk I have also appeared in elastin-like proteins (ELP), including cyclo(VPGVG)3 and poly(VPGVG).32 Likely, the β(II)-turn conformation adopted by both silk I11 and ELP32 results in the same marker bands. Thus, the elastin-like blocks of the SELP-47K fibers certainly result in the weak marker bands of silk I. The silk-like blocks of silk I may also contribute to the appearance of these bands. But it remains challenging to differentiate the contributions from the elastin-like blocks and the silk-like blocks in the silk I form. The percentage contents of an R-helix, β-strain, β-turn and undefined structure may be estimated using the NNLS analysis of Raman amide I and III spectra, which was first proposed by Williams and Dunker20 and subsequently refined by Williams.21,22 The Williams model was applied to the SELP-47K fibers and the amide I and III spectral analyses led to consistent estimations of the β-turn content and of the undefined conformation content (Table 2). However, an error of around 10% was observed in estimations of the helix contents from the amide I and III analyses. In the amide III analysis, ordered helix that is assumed to be R-helix was not distinguished from disordered helix, which is a combination of 310, RII, and non-hydrogen-bonded helical residues. Likewise, estimations of the β-strand content from the Raman amide I and III analyses led to around 12% error. Despite the large discrepancy in the estimations of the helix and β-strand contents, both the Raman amide I and III spectral analyses suggest that β-strands, including both hydrogen-bonded β-sheets and non-hydrogen-bonded β-sheetlike structures, are the dominated conformers of wet-spun SELP-47K fibers. An attempt was also made to quantify the amount of silk I in the SELP-47K fibers by the I1410/I1450 intensity ratio. Monti et al.33 proposed to use the I1415/I1455 intensity ratio as a semiquantitative measurement of the amount of silk I, which is largely comprised of the β(II)-turn conformation. If compared to silk I chymotryptic precipitate (Cp), liquid silk, and film, the methylene bending and wagging bands were red-shifted from 1455 cm-1 to 1450 cm-1 and from 1415 cm-1 to 1410 cm-1, respectively, in the SELP-47K fibers. Thus, the I1410/I1450 intensity ratio was calculated for the SELP-47K fibers after the baseline correction. The intensity ratio is 0.21, which is much lower than that of 1.12 in silk I Cp. Noting that the I1415/I1455 intensity ratio is not equivalent to the percentage content of β-turns. In fact, an infrared spectral analysis from the same group revealed 55% β-turn conformation in silk I Cp.37 Compared to an estimation of 21.5% β-turn from the NNLS analysis of the amide I and III spectra (Table 2), the intensity ratio seems to underestimate the amount of β-turn conformation in the SELP-47K fibers. This may be due to the fact that ELP also displays a strong band at 1450 cm-1, although it largely adopts β(II)-turn conformation.32 Therefore, the intensity ratio of the CH2 wagging and scissoring modes seems inappropriate for semiquantifying the amount of β(II)-turn conformation in the SELP-47K fibers.

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Table 2. Secondary Structural Contents (%) Estimated by Raman Amide I and III Spectral Analysis undefined

β-turn amide I 21.5 a

ordered helix

amide III

amide I

amide III

amide I

amide III

21.5

11.8

10.0

8.3

7.8

disordered helix a

β-strand

amide I

amide III

amide I

amide III

9.8

-

48.6

61.0

Ordered and disordered helices were not distinguished in Raman amide III analysis.

Figure 5. Tensile stress-strain analysis of dry SELP-47K fibers (A). The Young’s modulus of dry fibers is a function of fiber diameter (B).

Mechanical Analysis of SELP-47K Fibers. The tensile analysis of dry SELP-47K fibers revealed a breaking elongation of less than 2% and an ultimate tensile strength of 20-80 MPa (Figure 5A and Table S1 provided in Supporting Information). The Young’s modulus of dry SELP-47K fibers is in the range of 1 to 5 GPa (Figure 5B), comparable to those of tendon collagen and dragline silk.5 However, the extensibility (i.e., the breaking elongation) of dry SELP-47K fibers is only a small fraction of that of tendon collagen and dragline silk, and its tensile strength is accordingly much lower than those of collagen and dragline silk. If compared to typical commercial silkworm silk (e.g., extensibility of 15% and tensile strength of 500 MPa),2 the wet-spun fibers are more brittle and much weaker. The significant reduction in the tensile strength and extensibility may be due to the incorporation of elastin-like blocks, because dry elastin is very brittle but displays good elasticity when hydrated.38 The wet-spun fibers display a strong dependence of mechanical properties (e.g., Young’s modulus) on fiber diameter (Figure 5B). Typically, fibers of smaller diameter possessed higher Young’s modulus and thus higher tensile strength. Liivak et al.39 also reported an increase in the maximum stress of wetspun silk fibers when the fiber diameters decreased. Such trends in fibers have been established as partially the result of an increased fiber orientation in smaller fibers, which may be obtained using smaller spinnerets or through larger postfabrication stretching. In smaller spinnerets, a protein solution thread experiences greater shear. Together with the elongational stress induced by the postfabrication stretching, the enhanced shear stress acting on the SELP-47K solution thread causes the silk-

Figure 6. Fracture surface of dry SELP-47K fibers.

like blocks to better crystallize, resulting in the formation of protein fibers with more orientated filaments. The fracture surface of dry SELP-47K fibers was analyzed by SEM (Figure 6). Consistent with the low deformability revealed by the tensile analysis, the smooth fracture surfaces suggest brittle facture as a failure mode for the dry SELP-47K fibers. Additionally, small kinks (marked by letters and arrows in the upper and lower figures) on the fracture surfaces indicate nonhomogenous microstructures of the dry fibers. We further speculate that the deformation of a less perfect round fiber may be localized and become unsymmetric under tensile strain. A crack may be first initiated in region A (Figure 6, upper), and propagate as catastrophic brittle failure. However, as the crack propagates, the accumulated strain energy will be gradually released. When the weakened crack front encounters small crystallized phases, the crack will propagate along the soft interface of the amorphous and crystal phases in the fiber-axial direction. Because no large, continuous crystal fibrils were formed in the wet-spinning of SELP-47K fibers, it is anticipated that the crack propagation will be back to its original direction, leaving small kinks (markers B, C, D, and E in Figure 6, upper and lower) on the fracture surfaces. This is dramatically different from the split longitudinal or fibrillation fracture of collagen fibers, which are composed of continuous fibrils.40 In contrast to dry SELP-47K fibers of high tensile strength but limited strain to failure, hydrated fibers display greater strain to failure but reduced tensile strength (Figure 7). These

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Table 3. Mechanical Properties of Hydrated, Cross-Linked SELP-47K Fibersa

Figure 7. Representative tensile stress-strain analysis of SELP-47K fibers fully hydrated in 1× PBS at 37 °C.

sample ID

φ (µm)

E (MPa)

σf (MPa)

εf (mm/mm)

1 2 3 4 5 6 7 8 9

21.6 22.5 24.9 26.0 26.4 27.4 28.8 41.0 49.8

5.7 3.4 1.9 5.6 4.8 3.2 4.3 0.9 3.0

5.4 4.8 11.4 4.4 6.3 13.3 20.8 5.7 0.8

1.9 2.5 3.5 2.4 2.4 5.3 7.4 3.1 0.6

a Modulus are calculated in the region of 10% strain. φ: fiber diameter; E: Young’s modulus; σf: tensile strength; εf: strain at failure.

demonstrated in Figure 8B, the tensile behavior of SELP-47K fibers with diameters in the range of 22 to 50 µm may be well described by the same master curve. Highly nonlinear elasticity is another characteristic of the cross-linked SELP-47K fibers in the true-stress and true-strain analysis. Impressively, the real tensile strength of hydrated, cross-linked SELP-47K fibers could be as high as 150 MPa. Compared to the noncross-linked SELP47K fibers, the significantly enhanced mechanical properties of cross-linked fibers suggest that cross-linking effectively modifies the mobility of the protein chains and the subsequent deformation of fibers at large strains.

Conclusion

Figure 8. Tensile stress-strain analysis of glutaraldehyde-crosslinked SELP-47K fibers fully hydrated in 1× PBS at 37 °C: engineering stress vs stretch ratio (A) and true stress vs true strain (B).

properties suggest that hydration, likely due to the plasticizing effect of water, decreases the protein chain crystallinity in the fibers. When the SELP-47K fibers were hydrated in 1X PBS at 37 °C, a reduction in Young’s modulus of more than 1,000fold was observed. The deformability of hydrated fibers, however, increased several hundred- fold, compared to that of their dry counterparts (Figure 5A). Accordingly, the ultimate tensile strength of SELP fibers decreased from several tens of MPa to about 1 MPa. Because the hydrated SELP-47K fibers were very soft (e.g., Young’s modulus of around 0.2 MPa), low signal-to-noise ratio was a big issue and a reproducible tensile stress-strain analysis was difficult to obtain. When cross-linked by glutaraldehyde, the hydrated SELP47K fibers were strengthened dramatically, possessing an ultimate tensile strength as high as 20 MPa (Figure 8A and Table 3). Although moderate reduction in their deformability was observed, the cross-linked SELP-47K fibers were stretched to over 200% strain (i.e., 300% elongation) without breaking. Strain to failure of some fibers even reached 700% strain. Impressively, the ultimate tensile strength of SELP fibers closely matched that of native collagen fibrous networks in bone tissue,41 although their Young’s modulus is still much lower. If considering the shrinkage of the cross sections of SELP-47K fibers under tension, true stress would be a better measurement of the real tensile stress acting on the cross section. As

Genetically engineered silk-elastin-like protein copolymer SELP-47K was spun into robust, clean microdiameter fibers using a wet-spinning technique. Raman spectroscopic analysis revealed the coexistence of structures consisting of silk I of a β-turn conformation and silk II in the form of antiparallel β-sheet. An estimation of 50-60% β-strand structures, together with the morphology analysis of wet-spun fibers and fiber fracture surfaces, indicate that no large, continuous fibrils were formed during the wet-spinning. Hydrated SELP-47K fibers were mechanically weak (e.g., Young’s modulus of 0.2 MPa and tensile strength of 1.2 MPa) but displayed large strains to failure (e.g., 700%). In contrast, dry SELP-47K fibers possessed high tensile strength (e.g., 40-60 MPa) but limited strains to failure (i.e., less than 2%). When chemically cross-linked using glutaraldehyde, the mechanical strength of hydrated SELP-47K fibers was enhanced dramatically. High tensile strength up to 20 MPa and deformability of 200-700% make the chemically cross-linked SELP-47K fibers very appealing for potential applications in tissue engineering. Acknowledgment. We thank Professor Robert Downs for assistance in collecting the Raman spectra of SELP-47K fibers. This work was partially funded by an NSF grant (CMMI0700323). Supporting Information Available. Raman spectrum of glutaraldehyde cross-linked SELP-47K fibers. A table including mechanical properties of dry SELP-47K fibers. This material is available free of charge via the Internet at http:// pubs.acs.org.

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