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Aug 10, 2012 - Significantly Reinforced Composite Fibers Electrospun from Silk. Fibroin/Carbon Nanotube Aqueous Solutions. Hui Pan,. †. Yaopeng Zhan...
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Significantly Reinforced Composite Fibers Electrospun from Silk Fibroin/Carbon Nanotube Aqueous Solutions Hui Pan,† Yaopeng Zhang,*,† Yichun Hang,† Huili Shao,† Xuechao Hu,† Yuemin Xu,*,‡ and Chao Feng‡ †

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, P. R. China ‡ Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, P. R. China S Supporting Information *

ABSTRACT: Microcomposite fibers of regenerated silk fibroin (RSF) and multiwalled carbon nanotubes (MWNTs) were successfully prepared by an electrospinning process from aqueous solutions. A quiescent blended solution and a three-dimensional Raman image of the composite fibers showed that functionalized MWNTs (F-MWNTs) were well dispersed in the solutions and the RSF fibers, respectively. Raman spectra and wide-angle X-ray diffraction (WAXD) patterns of RSF/F-MWNT electrospun fibers indicated that the composite fibers had higher β-sheet content and crystallinity than the pure RSF electrospun fibers, respectively. The mechanical properties of the RSF electrospun fibers were improved drastically by incorporating F-MWNTs. Compared with the pure RSF electrospun fibers, the composite fibers with 1.0 wt % F-MWNTs exhibited a 2.8-fold increase in breaking strength, a 4.4-fold increase in Young’s modulus, and a 2.1-fold increase in breaking energy. Cytotoxicity test preliminarily demonstrated that the electrospun fiber mats have good biocompatibility for tissue engineering scaffolds.

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the spinning dope to prepare composite fibers. Carbon nanotubes (CNTs), as typical reinforcing agents with outstanding mechanical properties,28,29 have successfully enhanced numbers of polymers, such as polypropylene,30 polyamide 6,31 polyacrylonitrile,32,33 and so on.34−37 Due to their exceptional Young’s modulus and strength, CNTs might also be used to reinforce electrospun RSF fibers. Ko et al.18 utilized CNTs and tried to reinforce the electrospun RSF nano/microfibers from formic acid solution, and the mechanical properties of as-spun composite mats were not improved as expected. Only posttreatment with alcohol can make a significant modification on their mechanical properties.19,38 CNTs are not only a reinforcing agent but also a crucial functionalizing agent because of their high electrical conductivity.39−42 In this study, MWNTs were employed as reinforcing agents to modify the electrospun RSF fibers from aqueous solution. The dispersion of MWNTs in RSF solutions and electrospun fibers was investigated. In addition, the morphology, structure, and mechanical properties of the electrospun fibers and the biocompatibility of electrospun composite mats were examined.

INTRODUCTION Bombyx mori silk produced by silkworms with exceptional mechanical properties and luster has been used in textiles for thousands of years in human history. As well-known, silk fibroin offers a series of properties including mechanical superiority and biocompatibility. It also shows great potential applications in blood vessel engineering,1 drug delivery,2 and porous silk scaffolds.3,4 Furthermore, electrospinning possesses a unique superiority that enables the development of protein-based biomaterials. The diameter of electrospun fibers is in nano- or microscale.5−8 As a result, the materials produced by the electrospinning process exhibit greatly outstanding oxygen and water vapor permeability. Accompanying their biocompatibility, degradability, and processability, electrospun regenerated silk fibroin (RSF) fibers have been investigated widely for their potential applications in tissue engineering.9−14 To date, most nano/micro silk fiber mats are prepared from hash or toxic solvents, such as formic acid,15−19 hexafluoroisopropanol (HFIP),20,21 and hexafluoroacetone (HFA).7 The residual solvents in the fibers are very likely harmful to the growth of cells. Therefore, our group and other researchers replaced the organic solvent with water and prepared electrospun RSF fibers from aqueous solutions.5,6,22−27 However, the mechanical properties of the mats were still very poor for application. Normally, there are two ways to reinforce the RSF mats. One is to treat the electrospun RSF mats by methanol or ethanol, in which the RSF fibers experience some structure transition and crystalline transformation.5,6,19 The other way is to add a reinforcing agent into © 2012 American Chemical Society

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EXPERIMENTAL METHODS 2.1. Preparation of RSF/F-MWNT Electrospinning Dope. Cocoons of B. mori (produced in Tongxiang, China) were treated twice with 0.5 wt % Na2CO3 aqueous solution at Received: June 7, 2012 Revised: August 1, 2012 Published: August 10, 2012 2859

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100 °C for 30 min and rinsed with deionized water to remove sericin. The degummed silk was dissolved in 9.0 M LiBr aqueous solution at 40 °C for 2 h, and then dialyzed against deionized water for 3 days with a cellulose semipermeable membrane (molecular weight cutoff 14 000). The RSF aqueous solution was concentrated in forced airflow at 10 °C.43,44 The MWNTs (Shanghai Applied Nanotechnologies Co., China) synthesized via the thermal chemical vapor deposition (CVD) method, with a purity of 95%, were treated by refluxing in 2.6 M HNO3 at 90 °C for 8 h. After being washed with deionized water and dried, the acid-treated MWNTs were added into a sodium dodecyl benzene sulfonate (SDBS) aqueous solution, and mechanically stirred for 4 h. The redundant SDBS was removed afterward. The functionalized MWNTs (F-MWNTs) dispersed in water endured sonication for 2 h.45,46 The electrospinning dope was prepared by mixing the RSF aqueous solution (33 wt %) and F-MWNT aqueous solution (2 mg/mL) and further concentrating as described previously. The concentration of the solution was determined by the weight method. 23 An RS150 rheometer (Thermo-Haake Co., Germany) was employed to study the rheological behaviors of the 33% solutions at (22 ± 1 °C) using a titanium alloy parallel plate (Φ 20 mm). RSF/F-MWNT aqueous solution (33 wt %) was transferred to a 2.5 mL syringe capped with a 6-G needle (ID = 0.6 mm) as a spinneret. The electrospinning was performed by utilizing a voltage of 25 kV, a flow rate of 0.3 mL/h, and a grounded aluminum foil placed at a distance of 9 cm to collect random fibers. 2.2. Characterization. Electrospun mats were sputtered with platinum. Scanning electron microscopy (SEM) images were obtained on a JSM-5600LV (JEOL, Japan). Fifty fibers were randomly used to determine the average dimensions. A Raman spectrometer (Renishaw, 633 nm diode laser) with a resolution of 2 cm−1 was used to determine the secondary structure of the electrospun fibers. The Raman spectra of FMWNT powder were also obtained on the facility with a 532 nm diode laser. Quantitative analysis of the amide I region of the fibers was conducted under a deconvolution method reported by Trabbic47 and Hang.48 Two-dimensional (2-D) and three-dimensional (3-D) Raman microscopic imaging were used to characterize the dispersion of CNTs in the mats. A 20 μm square mat was first scanned to obtain the one-dimensional (1-D) spectra, which was then used to generate the 2-D and 3D Raman microscopic imaging by choosing the characteristic peak of tangential mode (G M) of MWNTs ranging from 1500 to 1650 cm−1. Wide angle X-ray diffraction (WAXD) patterns were obtained at the hard X-ray microfocus beamline (BL15U1) in the Shanghai Synchrotron Radiation Facility (SSRF), which has an energy ring of 3.5 GeV and a beam current attenuated from 210 to 140 mA. The wavelength (λ) of X-ray and the spot size at sample are 0.07746 nm and 10 μm × 10 μm, respectively. The diameter of the detector (Rayonix SX165) is 159 mm. Lanthanum hexaboride (LaB6), which has diffraction peaks at 0.152 and 0.214 nm, was used as a standard sample to fix the center of the diffraction circle and the sampleto-detector distance. Each mat was exposed for 20 s three times at different places, and a background for each sample was also gained simultaneously. FIT2D software was applied to deal with all the patterns. PeakFit v4.12 software was used to deconvolve the 1-D patterns by Gaussian functions, which were integrated to evaluate the crystallinity by

Crystallinity =

∑ Ic ∑ Ic + ∑ Ia

(1)

where Ic is the integrated intensity of crystal regions, Ia is the integrated intensity of amorphous regions. The mechanical properties of the electrospun nonwoven mats (6 mm × 25 mm) were measured using an Instron 5565 material testing instrument at (22 ± 2) °C and (55 ± 8) % of relative humidity (RH). The experiment was performed at an extension rate of 2 mm/min with a gauge length of 20 mm. The thickness of the sample was measured by a CH-1-S thickness measuremeter (Shanghai Liuling Instruments Co., Shanghai, China) with a resolution of 1 μm. Each sample was tested 10 times. 2.3. Biocompatibility Evaluation. 2.3.1. Leach Liquor Experiment on Electrospun RSF/F-MWNT Mats. The mats were sterilized in 75% ethanol for 30 min, then rinsed by phosphate buffer solution (PBS), immersed in Dulbecco modified Eagle medium (DMEM) for 5 h to obtain leach liquor. Logarithmic phase 3T3 cells suspension was added into a 96-well flat-bottomed culture plate at a density of 1 × 104 cells per well, and marginal wells were filled with PBS. The cells were then incubated at 37 °C in a humidified atmosphere containing 5% CO2. When the bottoms were over spread with cells, we discarded the substrate and washed the cells twice with PBS. A volume of 100 μL of 100% leach liquor was infused into experimental group and control group, respectively. The cells were incubated 12 h at 37 °C in a humidified atmosphere with 5% CO2. Then an inverted phase contrast microscope was used to observe the growth state of the cells. 2.3.2. Growth Testing of Lingua Mucosa Cells on Electrospun RSF/F-MWNT Mats. Separated Beagl’s lingua mucosa cells were grown on the electrospun mats, and incubated for 7 days in DMEM and Keratinocyte serum free medium (KSFM) mixed medium (volume ratio 1:1). The mats covered with cells were fixed by glutaraldehyde. After gradient ethanol dehydration and critical point drying, the mats with sputtered gold were observed with a Philips XL30 ESEM scanning electron microscope.

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RESULTS AND DISCUSSION 3.1. Dispersion of F-MWNTs in RSF/F-MWNT Aqueous Solutions and Electrospun RSF/F-MWNT Mats. 3.1.1. Dispersion of F-MWNTs in RSF/F-MWNT Aqueous Solutions. When MWNTs were oxidized by nitric acid, defects were created. Some CC double bonds at defect sites were broken and then bonded with hydrophilic groups of hydroxyl and carboxyl, which generated the ability to solubilize and disperse these nanotubes into matrixes.49−51 Furthermore, sonication was also helpful to break up MWNTs agglomerates. These procedures improved the dispersion of F-MWNTs in RSF aqueous solutions. Figure 1a shows the suspension of raw MWNTs in RSF solution, which was a control group. The agglomerates of MWNTs can be seen obviously. Figure 1b shows the newly prepared RSF solution blended with FMWNTs, which was then quiescently laid for 6 months and shown in Figure 1c. It can be seen that no obvious agglomerates were observed in solutions after MWNTs were functionalized to F-MWNTs. The pictures may indicate that FMWNTs dispersed well in RSF aqueous solutions after functionalization. 3.1.2. Dispersion of F-MWNTs in RSF/F-MWNT Electrospun Fibers. The mechanical properties of RSF/MWNT composite fibers depend greatly on the dispersion of MWNTs in the 2860

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Figure 1. Pictures of (a) suspension of raw MWNTs in RSF aqueous solution, (b) newly prepared RSF/F-MWNT aqueous solution, and (c) RSF/F-MWNT aqueous solution laid for 6 months.

Figure 3. Rheological behavior of 33 wt % RSF aqueous solutions with F-MWNT fractions: (a) 0, (b) 0.25 wt %, (c) 0.5 wt %, (d) 1.0 wt %, and (e) 1.5 wt %.

fibers. Well-distributed F-MWNTs in fibers can improve the mechanical properties, which drastically deteriorated when MWNT aggregation was up to a certain amount and size in fibers.18 3-D Raman microscopic imaging was used to evaluate the distribution of F-MWNTs in the electrospun RSF fibers. In the center of a composite mat, a square with an area of 20 μm2 was divided into thousands of small squares, which were scanned in series by a laser. Since the tangential mode (G M) peak of MWNTs (see Supporting Information Figure S1) was used to trace the 2-D and 3-D Raman microscope images, the red dots in Figure 2a and the red peaks in Figure 2b represent the F-MWNTs. From the random distributed pattern of red dots and peaks, it is known that F-MWNTs were distributed well in the fiber mats. 3.2. Rheological Behavior of RSF/F-MWNT Aqueous Solution. The rheological behavior of the RSF/F-MWNT aqueous solutions was studied to understand the influence of the F-MWNT fraction on the morphology of the electrospun RSF/F-MWNT fibers. Figure 3 shows the rheological behavior of 33 wt % RSF aqueous solutions with different F-MWNT fractions. It is known that all solutions exhibited obvious shear thinning behavior due to reduced density of macromolecular entanglement. In the case of the shear rates higher than 10 s−1, the viscosities of the pure RSF solution and the RSF solution with 0.25 wt % F-MWNTs remained approximately constant,

while the rheological curves of the solutions containing FMWNTs over 0.5 wt % kept declining slightly. Moreover, the viscosities of solutions rose with the increase of the content of F-MWNTs, therefore F-MWNTs may induce the entanglements of RSF molecules. In the pure RSF solution, the intermolecular and intramolecular entanglements of RSF molecules existed. In the blended RSF/F-MWNT solutions, however, the entanglements might be attributed to the intermolecular and intramolecular entanglements of RSF and F-MWNTs. It can be seen that the viscosities of the pure RSF solution and the RSF solution with 0.25 wt % F-MWNTs decreased dramatically, while the shear rate was from 0 to 1 s−1. The viscosities then increased gradually at shear rates ranging from 1 to 3 s−1. This result is different from the normal shearthinning behaviors of other blended RSF solutions. However, similar behavior was observed in the flow curve of native silk gland of B. mori, which was subjected to shearing for 30 s at each shear rate from 0.1 to 10 s−1.52 The critical shear rate where the viscosity abruptly increases in the flow curve is 4 s−1 instead. The first shear-thinning behavior may be caused by flow-induced molecular alignment, while the subsequent shearthickening behavior may result from nucleus formation before crystallization. When the weight fraction of F-MWNTs in the solution ranges from 0.5% to 1.5%, the strong interaction

Figure 2. (a) 2-D, and (b) 3-D Raman microscope images of electrospun RSF/F-MWNT mats with 0.5 wt % F-MWNTs. 2861

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Figure 4. SEM images of the electrospun RSF fibers reinforced by F-MWNTs with weight fractions of (a) 0, (b) 0.25 wt %, (c) 0.5 wt %, (d) 1.0 wt %, and (e) 1.5 wt %.

between RSF and F-MWNTs probably prevents the RSF molecular alignment and/or nucleus formation. Consequently, a monotonic decrease in viscosity with increasing shear rate is observed. 3.3. Morphology of Electrospun RSF/F-MWNT Mats. From the SEM images of electrospun fibers shown in Figure 4, it can be found that the pure RSF fibers are cylinder-like, while the composite fibers are ribbon-like. Clear amplified SEM and FESEM images of the electrospun fibers can be found in the Supporting Information (Figure S2). According to the fiber dimensions measured from SEM images, the pure RSF fibers had diameters around 2−4 μm, while the composite fibers showed widths of ca. 3 μm, and thicknesses ranging from 200 to 700 nm. Wang et al.27 and Chen et al.53 also observed some ribbon-like fibers in the electrospun mats from all-aqueous silk fibroin solution. They considered that the high viscosity of the highly concentrated RSF solution made the solution instable and inhomogeneous. Consequently, ribbon-like fibers were obtained. Another reason they pointed out was that the viscosity of the solution was too high for water to evaporate easily from the fibers surface, so that the fibers had to solidify on the aluminum foil and became flat. However, for the RSF/FMWNT system in our study, the formation of the ribbon-like fibers is possibly attributed to the electrical conductivity of the spinning dope. It is known that in the electrospinning process, the moving path of charged liquid jet was spiral, and an angle θ existed between the fiber axis and electric field E. That means ⎯→ → ⎯ the electrostatic force FE had a component F1 (F1 = FE cos θ) → ⎯ paralleled with the fiber axis and a vertical component F2 (F2 = → ⎯ → ⎯ FE sin θ). F1 pulled the fiber along the axis direction while F2 drew the fiber to be flat. Force analysis of liquid jet in electric → ⎯ field is shown in Figure 5. Due to the very weak F2 on the jet of the pure RSF aqueous solution with poor electrical conductivity, most pure RSF fibers exhibit circular cross sections. For the jet of RSF/F-MWNT aqueous solution with → ⎯ improved electrical conductivity, however, F2 becomes large and draws the liquid jet to be ribbon-like.

Figure 5. Force analysis of liquid jet in the electrospinning process.

3.4. Secondary Structures of Electrospun RSF/FMWNT Mats. In order to understand the mechanical properties of the composite materials, the secondary structures of the electrospun fibers were investigated by using Raman spectroscopy. Silk fibroin mainly has four types of secondary structures: random coil, α-helix, β-sheet, and β-turn conformation. The contents of these structures in fibers play important roles in their mechanical properties. The Raman spectra of pure RSF fibers, F-MWNTs, and F-MWNTreinforced RSF fibers are shown in Figure 6. F-MWNTs have two characteristic peaks at 1330 and 1580 cm−1 derived from disorder induced mode (D M) and tangential mode (G M),54,55 respectively. The two bands shifted a little to 1337 cm−1 and 1590 cm−1 in the electrospun RSF fibers with different FMWNT fractions ranging from 0.25 wt % to 1.5 wt %, respectively. This suggested that the F-MWNTs were incorporated with silk fibroin.18 It was able to find several characteristic bands of silk fibroin in samples a−e. The bands at 1667 cm−1 (amide I), 1259 cm−1 (amide III) and 1103 cm−1 correspond to β-sheet, random coil, and α-helix, respectively .56−58 The quantitative conformation contents of each sample were obtained by deconvolving the bands in amide I region of 1655 2862

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molecules. This may result from the increased conductivity of F-MWNT-incorporated RSF. As shown in Figure 5, the force ⎯→ → ⎯ along the fiber axis (F1 ) increased with the rise of FE relating to the conductivity of the spinning dope. Thus the drawing along the fiber axis induced the formation of β-sheet of RSF. From another perspective, plenty of hydrophilic groups such as hydroxyl and carboxyl on the surface of F-MWNTs could form some secondary bonds with hydrophilic domains of RSF molecules. This may also promote the conformation transition of RSF from intermediate conformation to β-sheet. 3.5. Crystalline Structure of Electrospun RSF/F-MWNT Mats. So far as we know, B. mori silk has two kinds of crystal structures, which are so-called silk I and silk II. Lotz et al.,59 Fossey et al.,60 and Asakura et al.61,62 have definitely stated the model of silk I. This paper will follow Asakura’s assignments, and it was confirmed that the conformation of one silk I chain is a repeated β-turn structure that is capable of forming intramolecular hydrogen bonds. Silk I exists in silk glands and undergoes conformation transition to silk II (extended β-sheet conformation) during the spinning process.61−63 According to Takahashi’s model,64 The reflections of silk II β-sheet structure were indexed using an orthorhombic unit cell with parameters a = 0.944 nm for the interchain direction, b = 0.895 nm for the intersheet distance, and c = 0.700 nm along the fiber axis (see Supporting Information Table S1).65 The composite fibers with 0 to 1.5 wt % F-MWNTs were abbreviated as S0, S0.25, S0.5, S1.0, and S1.5. The WAXD patterns of electrospun composite fibers are shown in Figure 7. In all samples, apparent diffraction peaks appeared at Dspacings of 0.209/0.226 nm, and 0.72 nm, corresponding to (420)/(040) reflection of silk II β-sheet structure and (110) reflection of silk I crystal,63 respectively. The main crystalline peaks at around 0.35, 0.37, 0.43, and 0.45 nm correspond to the reflections of (002), (021), (200), and (020) lattice planes of silk II, respectively. The broad peaks centered at 0.47 and 0.56 nm correspond to α-helix structure63 or short-range order.66,67 2-D WAXD patterns are shown in Supporting Information Figure S4. Figure 8 depicts the crystallinity of the composite fibers plotted against the weight fraction of F-MWNTs in each sample. The crystallinities of S0.25, S0.5, and S1.0 were higher than those of S0 and S1.5, and S1.0 exhibited the highest value. This may be because the F-MWNTs induced crystallization of silk,18 which is consistent with the results of Raman spectroscopy. In other words, the crystalline structures of S0.25, S0.5 and S1.0 were mainly β-sheet structure instead of silk I or α-helix. 3.6. Mechanical Properties of Electrospun RSF/FMWNT Mats. The mechanical properties of the electrospun nonwoven mats were tested, since people use mats instead of individual fibers in tissue engineering. The stress−strain curves are shown in Figure 9. The strength, Young’s modulus, and breaking energy of the pure RSF mats and the composite RSF mats are shown in Table S2 of Supporting Information. It can be seen that even a small addition of F-MWNTs in RSF leads to an obvious improvement of the mechanical properties. Compared to the pure RSF electrospun mats, the composite fiber mats with 1.0 wt % F-MWNTs exhibits a 2.8-fold increase in breaking strength, a 4.4-fold increase in Young’s modulus, and a 2.1-fold increase in breaking energy. The improvement of the mechanical properties may be mainly attributed to the FMWNTs with high strength and modulus performed as reinforcing agents. Meanwhile, well dispersed F-MWNTs of

Figure 6. Raman spectra of electrospun RSF fibers with F-MWNTs fractions of (a) 0, (b) 0.25 wt %, (c) 0.5 wt %, (d) 1.0 wt %, (e) 1.5 wt %, and (f) F-MWNTs.

± 5 cm−1 (random coil/α-helix), 1670 ± 5 cm−1 (β-sheet) and 1680 ± 5 cm−1 (intermediate conformation between random coil and β-sheet).58 In addition, the percentage of peak area of 1615 cm−1 band assigned to the phenyl group of tyrosine residues was used as an invariant internal standard to check the validity of each analysis. In our work, this value was controlled to be about 5% of the total peak area for the above four bands. The quantitative results and deconvolution details are given in Table 1 and Figure S3 (Supporting Information), respectively. Table 1. Quantitative Analysis of Raman Spectra in Amide I Region of Electrospun RSF/F-MWNT Fibers with Different F-MWNT Fractions content/%

a b c d e

weight fraction of F-MWNTs/%

phenyl group

α-helix/ random coil

βsheet

intermediate conformation

0 0.25 0.5 1.0 1.5

5.6 5.4 5.3 5.5 5.0

32.2 37.8 42.6 39.1 65.9

35.8 44.0 45.7 49.4 25.7

26.4 12.8 6.4 6.0 3.4

When the content of F-MWNTs increased from 0 to 1.0 wt %, the β-sheet and the random coil/α-helix contents increased while the content of intermediate conformation decreased from 26.4% to 6.0% obviously. However, it was noticed that the βsheet content drastically rose from 35.8% to 49.4%, followed by a small increase of random coil/α-helix. This indicates that even a small addition of F-MWNTs into RSF aqueous solution results in an obvious conformation transition of RSF from intermediate conformation to β-sheet. When the content of FMWNTs was up to 1.5 wt %, the β-sheet content of the composite fibers was as low as 25.7%, which was unexpected and much lower than those of other composite fibers and even the pure RSF fiber. It was noticed that the jet of 1.5 wt % solution is easily broken in the spinning process. The discontinuousness may be attributed to the F-MWNT aggregation. Thus weaker drawing was applied on the jet than other jets with F-MWNT contents below 1.5 wt %. Since drawing contributes to the conformation transition from random coil/α-helix to β-sheet, the β-sheet content of the fibers with 1.5 wt % F-MWNTs was very low. On the contrary, the addition of F-MWNTs up to 1.0 wt % into RSF composite fibers is effective to construct β-sheet conformation of RSF 2863

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Figure 7. WAXD patterns of electrospun RSF fibers with F-MWNTs fractions of (a) 0, (b) 0.25 wt %, (c) 0.5 wt %, (d) 1.0 wt %, and (e) 1.5 wt %.

Figure 8. Crystallinity of RSF/F-MWNT composite fibers versus weight fraction of F-MWNTs.

Figure 9. Stress−strain curves of electrospun RSF mats with FMWNT fractions of (a) 0, (b) 0.25 wt %, (c) 0.5 wt %, (d) 1.0 wt % and (e) 1.5 wt %.

1.0 wt % content in the electrospun fibers induced the maximal crystallinity corresponding to the highest β-sheet content, which may also contribute to the performance improvement of the mats. One of the possible reasons is that increased number of crystallites act as numbers of knots of molecules in the amorphous region68 and prevent the mats of 1.0 wt % from breaking. When the F-MWNT content was up to 1.5 wt %, the

mechanical properties of the mats deteriorated drastically. This may result from the critical agglomerates of F-MWNTs or the very low β-sheet content of 25.7% (Table 1). In our experiments, critical agglomerates of F-MWNTs in the spinning solutions were observed in the case of the contents of FMWNTs over 1.0 wt %, including 1.25 wt % and 1.5 wt %. 2864

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Figure 10. Inverted phase contrast microscope images of mouse 3T3 cells in leach liquors of electrospun mats of (a) RSF and (b) RSF/F-MWNT.

Figure 11. SEM of Beagl’s lingua mucosa cells on electrospun mats of (a) RSF and (b) RSF/F-MWNT.

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CONCLUSION F-MWNTs were well distributed in RSF aqueous solutions and dispersed in fibers by electrospinning process from the Raman images and quiescent blended solution. RSF/F-MWNT composite fibers electrospun from aqueous solutions were ribbon-like and had much smaller cross section than the pure electrospun RSF fibers. The composite fibers with 0.25 wt % ∼ 1 wt % F-MWNTs had higher β-sheet and α-helix/random coil contents than the pure RSF fibers. Even a small addition of FMWNTs (0.25 wt %) into RSF aqueous solution results in an obvious conformation transition of RSF from intermediate conformation to β-sheet. With increasing F-MWNTs fractions from 0 to 1.0 wt %, the crystallinity of the electrospun mats grew and the crystalline structure was mainly β-sheet (silk II) instead of silk I or α-helix. The mechanical properties of the reinforced mats were improved greatly by incorporating 0.25 wt % ∼1.0 wt % MWNTs. The tensile strength of the mats with an optimal content of F-MWNTs (1.0 wt %) was up to 3.24 MPa. The improvement of the mechanical properties may be attributed to the high strength and high modulus of FMWNTs, as well as the F-MWNT-induced crystallization of RSF. However, when the content of MWNTs was up to 1.5 wt %, the critical aggregation of MWNTs drastically deteriorated the structures and mechanical properties of this material. Cells cytotoxicity and growth experiments showed that the materials are nontoxic and have great potential for tissue engineering application.

Consequently, the mechanical properties of the 1.25 wt % sample might also be poorer than the 1.0 wt % sample. From the small elongation at breaking of all mats ranging from 2.14% to 4.06%, it is known that the mats are relatively brittle. 3.7. Biocompatibility of Electrospun RSF/MWNT Mats. Figure 10 shows the inverted phase contrast microscope images of mouse 3T3 cells in leach liquors of electrospun mats. Cultured cells in uniform size displayed good morphology and typical growth status like cobblestones. The fine growth state suggests that the cells grew and proliferated healthily. MTT result (see Figure S5 in Supporting Information) shows that the optical density (OD) values of RSF, RSF/MWNT, and the control group were on the similar level of 0.8 (p < 0.05), which indicated that the total numbers of cells on RSF mats and RSF/ F-MWNT mats were almost the same as the control group. Figure 11 shows the growth state of lingua mucosa cells of Beagl dog on electrospun RSF mats and RSF/F-MWNT mats. It can be seen that the lingua mucosa cells grew well, and the cells stretched pseudopodia into fiber mats for adhesion. The electrospun microfiber mats with high porosity can provide necessary space for cell growth and metabolism.69 The experiment demonstrated at least that lingua mucosa cells of Beagl dog can grow regular on the RSF mats and RSF/FMWNT mats. Meanwhile, due to limited pore sizes of mats, few cells infiltrated into mats, which corresponds with previous works.70,71 The preliminary results may indicate that electrospun RSF and RSF/F-MWNT mats had no obvious cytotoxicity for attachment, growth, and proliferation of 3T3 cells and lingua mucosa cells. Further studies are still needed to verify its biocompatibility for specific applications.

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ASSOCIATED CONTENT

S Supporting Information *

Amplified SEM and FESEM images, figures showing quantitative analysis of the secondary structures, 2-D WAXD patterns, MTT assay, and detailed mechanical properties of 2865

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RSF/F-MWNT mats. This information is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(Y.Z.) Phone: +86-21-67792954; Fax: +86-21-67792855; Email: [email protected]; Mailing address: College of Materials Science and Engineering, Donghua University, 2999 North Ren-min Road, Songjiang District, Shanghai 201620, P.R. China. (Y.X.) Phone: +86-21-64369181-58698; E-mail: [email protected]; Mailing address: Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, P. R. China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (50803011, 81170641), the Innovation Program of Shanghai Municipal Education Commission (12ZZ065), the Shanghai Rising-Star Program (12QA1400100), and the Fundamental Research Funds for the Central Universities.

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