Study on Cast Membranes and Electrospun Nanofibers Made from

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Study on Cast Membranes and Electrospun Nanofibers Made from Keratin/Fibroin Blends Marina Zoccola,*,† Annalisa Aluigi,† Claudia Vineis,† Claudio Tonin,† Franco Ferrero,‡ and Marco G. Piacentino† CNR-ISMAC, National Research Council, Institute for Macromolecular Studies, 13900 Biella (BI), Italy, and Department of Materials Science and Chemical Engineering, Polytechnic of Torino, 10129 Torino (TO), Italy Received May 23, 2008; Revised Manuscript Received August 14, 2008

Keratin regenerated from wool and fibroin regenerated from silk were mixed in different proportions using formic acid as the common solvent. Both solutions were cast to obtain films and electrospun to produce nanofibers. Scanning electron microscopy investigation showed that, for all electrospun blends (except for 100% keratin where bead defects are present), the fiber diameter of the mats ranged from 900 (pure fibroin) to 160 nm (pure keratin). FTIR and DSC analysis showed that the secondary structure of the proteins was influenced by the blend ratios and the process used (casting or electrospinning). Prevalence of β-sheet supramolecular structures was observed in the films, while proteins assembled in R-helix/random coil structures were observed in nanofibers. Higher solution viscosity, thinner filaments, and differences in the thermal and structural properties were observed for the 50/50 blend because of the enhanced interactions between the proteins.

Introduction Fibroin and keratin are biocompatible polymers that are attracting a lot of attention as biomaterials for biomedical applications, particularly in the emerging field of tissue engineering.1-3 Electrospinning is the simplest way of producing fibers with a nanoscale diameter. In this process, a polymer solution in a spinneret is subjected to an electric field generated by a high voltage power supply. A polymer jet is ejected when the electric field overcomes the surface tension, travels toward a grounded collector, and is stretched and deposited as a nonwoven mat on the collector. By adjusting solution properties and operating parameters, very small fiber diameter and high porosity nonwoven mats can be obtained. This makes electrospinning a promising technique for the fabrication of tissue-engineered scaffolds.4 An important improvement patent on electrospinning was issued in 1934,5 but only in recent years has electrospinning been widely studied and applied for many synthetic and natural polymer processing,4,6,7 including silk fibroin spinning. Fibroin is a high molecular weight polypeptide containing at least two fibroin proteins, the light and heavy chains, 25 and 325 kDa, respectively.8 The protein chains are aligned along the fiber axis, bonded together by a close network of interchain hydrogen bonds, with adjacent -(ala-gly)n- sequences forming the well-known β-sheet crystals.9 Silk fibroin has been electrospun as a pure polymer,10 blended with other polymers and used as composite with other materials in fibroin-based nanofibers. Sukigara et al. modeled and optimized the process parameters and studied the morphological, chemical, and mechanical properties of fibroin nanofibers obtained by electrospinning.11-13 Min et al.14 reported the production of silk fibroin nanofiber nonwovens for cell culture of normal human keratinocytes and * To whom correspondence should be addressed. Phone: +39-0158493043. Fax: +39-0158408387. E-mail: [email protected]. † CNR-ISMAC. ‡ Polytechnic of Torino.

fibroblasts. Formic acid was used as the spinning solvent and the nanofibers were insolubilized by a treatment with an aqueous solution of methanol. Fibroin has been electrospun with chitosan into a continuous fibrous structure.15 A variety of compositions of fibroin/ poly(ethylene oxide) aqueous blends have also been successfully electrospun.16 Moreover, electrospinning of silk fibroin/chitin blend solutions have produced nanofibers with a phase-separation structure, which were stabilized by a water vapor treatment and tested for cell attachment and spreading.17 Silk fibroin-based nanofiber scaffolds containing bone morphogenetic protein 2 and nanoparticles of hydroxyapatite have been prepared via electrospinning and used for in vitro bone formation from human bone marrow-derived mesenchymal stem cells.18 Solvents used to electrospin silk fibroin and its blends are formic acid, 1,1,1,3,3,3-hexafluoro-2-propanol and, more recently, water.10 In the present work, silk fibroin was blended with regenerated keratin from wool. Keratin is a fibrous protein characterized by a high stability due to intramolecular covalent (disulfide) bonds. It is abundantly present in nature as wools, hairs, furs, nails, horns, mammal claws and quills, bird feathers, and the epidermis of reptiles. Wool fibers are composed of spindle-shaped cortical cells made up of intermediate filaments, almost completely R-helical, surrounded by an amorphous protein matrix and a sheath of overlapping cuticle cells. Wool keratins have a molecular weight ranging from 45 to 60 kDa of the microfibrils from the cortical cells to 6-28 kDa of the protein from the matrix.19 Wool keratins show cell adhesion sequences, RGD and LDV, which have been found in several extracellular matrix proteins, such as fibronectin.20 Wool keratin has been regenerated in the form of sponges that were reported to be good scaffolds for cell cultivation.20 Sponges have been fabricated by lyophilization of an aqueous wool keratin solution after controlled freezing20 or by a compression-molding/particulate-leaching method consisting of

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extraction of keratins from wool, spray drying to give S-sulfo keratin powder, mixing of keratin with urea, compressionmolding together with sieved NaCl particulates above the melting temperature of urea, and finally, removal of the salts and urea in water to create interconnected pores in a continuous S-sulfo keratin matrix.21 Hybrids of keratin sponges have also been produced with calcium phosphate (hydroxyapatite) that gave keratin additional functions, allowing differentiation of the osteoblast patterns.22 Lee at al.23 blended keratin and fibroin and studied the secondary structure of silk fibroin/S-carboxymethyl kerateine films, observing a transition from random coil to β-sheet secondary structure of fibroin, probably determined by the presence of polar amino acids in keratin, which act as polar solvent. In addition, the authors found that films cast from silk fibroin and S-carboxymethyl kerateine (SCMK) showed decreased blood coagulation compared with silk fibroin or keratin alone.24 Little data are available about the electrospinning of keratin. Keratin has been electrospun in blends with poly(ethylene oxide) in water, producing regularly shaped nanofibers mainly at the 50/50 ratio and 7-10% polymer concentration.25 In the present work, silk fibroin and keratin extracted from wool were dissolved in formic acid and mixed in different proportions. The solutions were characterized for their rheological behavior and then they were both electrospun to produce nanofibers and cast in polyester plates to obtain blend films for structural comparative studies. The morphology of the nanofiber was investigated and correlated with the properties of the original solutions. Emphasis was given to the effect of the electrospinning process on the secondary structure of the proteins.

Experimental Section Preparation of Keratin/Fibroin Blend Solutions. A combed sliver of degummed silk (pure fibroin-free sericin) was extracted with petroleum ether in a Soxhlet apparatus, dissolved in a ternary solvent system of CaCl2/H2O/EtOH (1/8/2 mol ratio) for 2 h at 70 °C with vigorous shaking, and dialyzed in a cellulose tube (molecular weight cut off 12.000-14.000) against deionized water for 3 days at room temperature to remove salts. Then the aqueous solution of silk fibroin was filtered (5 µm pore size filters), cast onto polyester plates, and dried at room temperature. Literature data show that fibroin can be electrospun into continuous and regular nanofibers at concentrations ranging from 12 to 15% w/w, using formic acid as spinning solvent.14 Thus, the concentration, 15% w/w, was selected to electrospin fibroin blended with a low molecular weight polymer such as wool keratin. Fibroin solutions were prepared by dissolving in 15% w/w formic acid (concentration g95%, boiling point 100.8 °C, from Sigma-Aldrich), and the fibroin film was regenerated from water with stirring for 2 h at room temperature. Keratin was extracted by sulfitolysis from a combed sliver of Australian Merino wool, 19.5 µm fineness.19 A fiber sample was cleaned by Soxhlet extraction with petroleum ether to remove fatty matter, washed with distilled water and conditioned at 20 °C and 65% r.h. for 24 h. A total of 7.5 g of cleaned and conditioned wool fibers were cut into snippets some millimeters long, put in 150 mL of aqueous solution containing urea (8 M) and meta-bisulphite (0.5 M), adjusted to pH 6.5 with NaOH (5 N), and treated by shaking for 2 h at 65 °C. Successively, the keratin solution was treated as described above for fibroin, to obtain a 15 wt % solution of keratin in formic acid. The pure solutions of fibroin and keratin were mixed in different proportions (100/0, 90/10, 70/30, 50/50, 30/70, 10/90, and 0/100 w/w keratin/fibroin) and stirred for 2 h at room temperature to prepare blend

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Figure 1. Experimental and theoretical values of viscosity for keratin/ fibroin blend solution obtained at 42.8 s-1 shear rate.

Figure 2. Conductivity of keratin/fibroin blend solutions as a function of keratin content.

solutions at 15 wt % total protein concentration. Every solution was measured for viscosity and conductivity. Preparation of Regenerated Keratin/Fibroin Blend Films and Nanofibers. Solutions were both cast on polyester plates at 50 °C overnight to obtain regenerated keratin/fibroin blend films and electrospun to produce nonwoven mats of nanofibers. About 3 mL of each keratin/fibroin solution were loaded in a syringe linked to a capillary pipe with a metallic needle tip (inside diameter 0.20 mm). The anode of the power supply (HVA b2 Electronics) was connected with the metallic tip. The cathode (collecting screen) was a rotating stainless disk of 5.5 cm diameter covered with an aluminum sheet. A voltage of 30 kV was applied and a constant volume flow rate of 0.005 mL/min was supplied to the electric field through the needle. The collecting screen was placed at a distance of 10 cm from the syringe. Measurements and Characterizations. Viscosity was measured with an Anton Paar Physica MCR 301 rheometer equipped with a PTD 200 Peltier temperature control device at 25 °C ( 0.1 °C with coneplate geometry (75 mm diameter, 1° angle, and 45 µm truncation). The shear rate was logarithmically increased from 0.4 to 10000 s-1. Conductivity was measured with an Eutech Instruments multiparameter tester PC300 calibrated with a 1.413 mS/cm (at 25 °C) standard solution. The morphology of the nanofibers was examined by a Leica Electron Optics 135 VP scanning electron microscope. The average fiber diameter and its standard deviation were determined by measuring 100 fibers selected randomly from each mat produced. FT-IR spectra of the samples dried at 105 °C for 1 h were obtained with a Nexus Thermo Nicolet spectrometer by using a single bounce ZnSe micro ATR with a circular form 0.72 mm diameter. A total of 100 scans were taken from 4000 to 650 cm-1 with a resolution of 4 cm-1 and a gain of 8.0. A spectrum was collected in two different areas of each sample. The spectra were processed with the OMNIC software.

Keratin/Fibroin Nanofibers Differential scanning calorimetry (DSC) was performed with a Mettler Toledo DSC 821 calorimeter calibrated by an indium standard. The calorimeter cells were flushed with 100 mL/min nitrogen. The runs were performed on conditioned samples (20 °C, 65% r.h.) from 30 to 400 °C at a heating rate of 10 °C/min.

Results and Discussion Solution Properties. All the keratin/fibroin blend solutions showed a shear-thinning behavior and the magnitude of the viscosity decreased with increasing the keratin content (from 0.44 Pa s of pure fibroin to 0.15 Pa s of pure keratin at 100 s-1 shear rate), with the exception of the keratin/fibroin 50/50 blend solution, where the viscosity value at different shear rates was higher than in other fibroin-richer solutions (data not show). Figure 1 shows the viscosities of keratin/fibroin blend solutions versus the keratin content at 42.8 s-1 shear rate obtained by experimental measurements, compared with the theoretical viscosities calculated following the additivity rule for ideal mixtures26

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ln ηT )

∑ wi ln ηi

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(1)

i

where wi is the weight fraction of the ith component, ηi is the solution viscosity at 42.8 s-1 shear rate, and ηT is the theoretical viscosity of the blend. One measurement was carried out for each solution, except for the 50/50 w/w keratin/ fibroin blend where a second measurement confirms the first one (0.42 and 0.41 Pa s at 42.8 s-1 shear rate). For the blends with a keratin content greater than 30%, the measured viscosity had a positive deviation with respect to its theoretical value at all shear rates. This synergistic effect on viscosity is a consequence of keratin-fibroin interactions, which promote the formation of network structures.27 Positive deviation decreased with increasing shear rate, due to the shear-thinning behavior.28 Maximum synergy was observed for the keratinfibroin 50/50 w/w. On the other hand, the viscosity of the keratin/fibroin 10/90 and 30/70 blend solutions were slightly

Figure 3. SEM pictures and fiber diameter histograms of electrospun nanofibers with different keratin/fibroin mixing ratio. (A) Pure fibroin, (B) 10/90, (C) 30/70, (D) 50/50, (E) 70/30, (F) 90/10, (G) pure keratin, and (H) average diameter and standard deviation of nanofibers vs keratin content in the blends.

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Figure 4. IR spectra of keratin/fibroin blend films: (a) pure fibroin, (b) 10/90, (c) 30/70, (d) 50/50, (e) 70/30, (f) 90/10, and (g) pure keratin.

Figure 5. IR spectra of pure fibroin films and nanofibers.

lower than expected by the additive rule, indicating a negligible interaction between the keratin and the fibroin chains (Figure 1).29 Conductivity of the blend solutions increased with increasing the keratin amount (figure 2) until a plateau at keratin concentration higher than 70% was reached, because the polar amino acids content of keratin is higher than that of fibroin. Nanofiber Morphology. Figure 3 shows the microscopic features of the nanofiber mats obtained from the different solutions, the respective fiber diameter histograms, and the average fiber diameter versus the keratin amount in the blends. Mats were made of continuous nanosized filaments randomly collected on the rotating screen with round or flat cross sections. Flat nanofibers were present in large amounts in fibroin rich blends (10/90 and 30/70 w/w keratin/fibroin) and can be attributed to uneven evaporation of the solvent from the skin and the core of the jet during drawing that caused the filament to collapse.30 From 100% keratin solution, thin nanofibers with bead defects were produced due to instability of the jet by effect of the surface tension in the presence of electrical forces.31 As shown in Figure 3, the nanofiber diameter and its standard deviations were influenced by the keratin amount in the blends. Pure fibroin nanofibers were flat and showed the highest mean

diameter and standard deviation (945 and 483 nm, respectively). As the keratin amount increased, the nanofibers became thinner and more homogeneous. Very thin and very homogeneous nanofibers were produced with the 90/10 and 100/0 keratin/ fibroin blends (mean diameter of 233 and 169 nm and standard deviations of 57 and 49 nm, respectively), although a large number of beads were evidenced with pure keratin. Notwithstanding the highest viscosity of the solution, finest nanofibers were produced electrospinning the 50/50 keratin/fibroin blend (mean diameter 207 nm and standard deviation 66 nm). In general, when solutions show high viscosity, nanofibers with larger diameters are obtained. This occurs when the viscosity increase is determined by the polymer concentration increase. In this case, the total polymer concentration for each blend solution is the same (15% w/w), therefore, the highest viscosity of the 50/50 blend solution is due to the stronger interactions between the two proteins present in equal amounts in this blend. Probably, this synergistic effect on the protein interactions during the solvent evaporation in the electrospinning process promotes the formation of finest nanofiber. In regard to the histogram diameter distributions, as the keratin amount in blend increases, distributions are more similar to Gaussian curves also due to the lower standard deviation of diameters distributions. Moreover, at the electrospinning conditions tested, no bimodal distributions were found as a consequence of jet instability and its consequent branching. Structural Characterization of Keratin/Fibroin Films and Nanofibers. The structural characterization of keratin/ fibroin films and nanofibers was conducted analyzing the bandshape of amide I, whose absorption falls in the 1700-1600 cm-1 range. In fact, the amide I band, connected mainly with the CdO stretching vibration, is known to be sensitive to the secondary structure of the proteins.32 Before spectra acquisition, the samples were dried at 105 °C for 1 h to eliminate the water absorption band (1650 cm-1) that interferes with the amide I peak. The spectra of the dried samples were baseline corrected and smoothed with the Savitsky-Golay method (9 points).33 For each blend, two spectra without any significant differences were collected in different areas of the sample. Figure 4 shows the IR spectra of pure keratin, keratin/fibroin blends, and pure fibroin films prepared by casting from formic acid. The amide

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Figure 6. Amide I shape of pure keratin and keratin/fibroin blend film and nanofibers.

I of pure keratin film (Figure 4, spectrum g) is a doublet peak of CdO stretching vibration at 1650 and 1620 cm-1, with the maximum at 1620 cm-1. On the basis of literature data, the absorption peak at 1650 cm-1 suggests the presence of a R-helix/ coiled coil structures in the keratin chains, whereas the absorptions between 1610 and 1633 cm-1 are typically seen for β-sheet structures.32,34,35 Therefore, the maximum at 1620 cm-1 indicates the propensity of keratin cast from formic acid to form β-sheet structures.36 Likewise, with keratin, the peak at 1620 cm-1 that can be seen in the IR spectra of the pure fibroin film (Figure 4, spectrum a) confirms the β-sheet crystallization mode of fibroin regenerated from formic acid.37 With the exception of keratin/fibroin 50/50, all the blend films showed an absorption at 1620 cm-1, higher than that at 1650 cm-1, confirming the

prevalence of the β-sheet secondary structure. On the contrary, in the keratin/fibroin 50/50 blend film, the absorption at 1650 cm-1 is more intense than that at 1620 cm-1, suggesting a propensity of the protein chains to the R-helix-coiled structure assembling, probably because of the strongest keratin-fibroin interactions, which occur in this blend, as demonstrated by viscosity measurements. Structural changes induced by the electrospinning process were studied comparing the IR spectra of the nanofibers with films produced by casting from the same solutions. In Figure 5, the intense absorption bands of the pure silk film at 1620 cm-1 (amide I), 1520 cm-1 (amide II), and the shoulder at 1260 cm-1 (amide III) reveal that silk film prepared by casting

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Figure 7. DSC curves of pure fibroin and pure keratin film and nanofibers.

Figure 8. DSC curves of nanofibers: pure fibroin (a), keratin/fibroin 10/90 (b), keratin/fibroin 30/70 (c), keratin/fibroin 50/50 (d), keratin/fibroin 70/30 (e), keratin/fibroin 90/10 (f), and pure keratin (g).

from formic acid solution crystallized principally in the form of the β-sheet secondary structure (as described above). On the other hand, the silk fibroin nanofibers spectrum shows a shift to higher wavenumbers of the amide I (1650 cm-1) and amide II (1535 cm-1) bands and a decrease of the absorption band at 1260 cm-1. The nanofibers are collected randomly on the rotating disk due to the low rotating speed of the collector, as shown in SEM pictures (Figure 3). Moreover, using an ATR accessory for sampling, the depth of penetration of radiation is short (few micrometers) and little scattering occurs, so the shift to higher wavenumbers of the amide I and amide II suggests the prevalence of random coil or R-form (silk I) conformation of the silk protein in the electrospun fibers.38 Random coil and silk I structures cannot be differentiated by infrared spectroscopy, because of their overlapped absorption bands.39 However, the rapid evaporation of the solvent, that occurs in the electrospinning process, hinders the formation of β-sheet structures. Moreover, other authors10 have shown that fibroin electrospun mats are characterized by an amorphous structure and confirmed the FTIR data by wide-angle X-ray diffraction analysis. Other medium intensity bands were observed in the skeletal stretching region (1000-900 cm-1). In particular, the band at 1015 cm-1 arises from the -(gly gly)- sequence, while those at

1000 and 980 cm-1 are attributed to the -(gly ala)- periodic sequence.40 All these bands are modified in the nanofiber spectrum, suggesting changes in the secondary protein structure induced by the electrospinning process itself. Analogous behavior was observed for pure keratin and keratin/fibroin blend nanofibers. As shown in Figure 6, in the nanofibers made of pure keratin and keratin/fibroin, the amide I peak at 1620 cm-1, related to the β-sheet structure, disappears. One can postulate that the rapid solvent evaporation and the draft from 0.2 mm to 200 nm induced by the electrospinning process prevents the formation of β-sheet conformations. Thermal Behavior. Figure 7 shows the thermograms of regenerated pure fibroin and pure keratin processed as films and nanofibers. The regenerated fibroin film has an endothermic peak between 100 and 150 °C caused by water evaporation and a prominent endothermic peak at 280 °C attributed to the thermal decomposition of unoriented fibroin chains.41 On the DSC curve of fibroin nanofibers glass transition is 175 °C, the crystallization exothermic peak is 230 °C, and the endothermic peak is 280 °C. The latter is also true for the film.42 The presence of a glass transition followed by crystallization of amorphous fibroin chains supports the results obtained by IR analysis. Indeed, nanofibers are characterized predominantly by the presence of

Keratin/Fibroin Nanofibers

R-helix/random coil structures that crystallize in the form of β-sheet structures as a consequence of thermal treatments. In regard to regenerated keratin, the first endothermic peak in the range 100-150 °C due to water evaporation was followed by a peak in the 200-250 °C temperature range, related to denaturation of R-helix crystallites.43 It is worth noting that, compared with pure keratin film, pure keratin nanofibers have a helical denaturation peak at a lower temperature, suggesting the R-crystallites formed by electrospinning are thermally less stable. This behavior is probably due to the different evaporation rates of the formic acid. The high speed of formation of the nanofibers compared with that of the film limits the molecular rearrangement and crystallization of the keratin chains. The DSC curves of keratin/fibroin blend nanofibers are shown in Figure 8. The nanofibers richer in fibroin behaved like those of pure fibroin, confirming a negligible interaction between the proteins in these blends as can be inferred from the viscosity. On the other hand, the nanofibers richer in keratin and the nanofibers made of keratin/fibroin, 50/50 w/w, showed the helical denaturation peak at a temperature higher than that of the other blends, suggesting an enhancement of thermal stability of the R-crystallites, probably due to the already postulated interaction between keratin and fibroin.

Conclusions Keratin/fibroin blend solutions prepared in formic acid were both cast to obtain films and electrospun to produce nanofibers. All blend solutions were electrospun successfully except for pure keratin solution where nanofibers with bead defects were obtained. Morphological investigation showed that as the keratin amount in the blend increased, the nanofibers became thinner and more homogeneous. Conformational analysis performed by FT-IR and DSC evidenced that the electrospinning process promotes the formation of the R-helix/random coil secondary structures of the proteins. For blends with higher keratin percentage, the measured viscosity deviates positively from the theoretical values as a consequence of keratin/fibroin interactions, which promote the formation of network structures. Maximum synergism is observed for the 50/50 w/w keratin/ fibroin blend. The nanofibers obtained have potential application in the biomedical field, particularly in tissue engineering, due to their biocompatibility, good spinnability, and increased antithrombogenicity of blended proteins. Acknowledgment. The authors gratefully acknowledge the Regione Piemonte for supporting this research through the project HI-TEX (DGR No. 227-4715).

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