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Insoluble and Flexible Silk Films Containing Glycerol Shenzhou Lu,†,‡ Xiaoqin Wang,‡ Qiang Lu,†,‡ Xiaohui Zhang,‡ Jonathan A. Kluge,‡ Neha Uppal,‡ Fiorenzo Omenetto,‡ and David L. Kaplan*,‡ National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215021, P.R. China, and Department of Biomedical Engineering, Bioengineering & Biotechnology Center, Tufts University, Medford, Massachusetts 02155 Received August 30, 2009; Revised Manuscript Received October 12, 2009
We directly prepared insoluble silk films by blending with glycerol and avoiding the use of organic solvents. The ability to blend a plasticizer like glycerol with a hydrophobic protein like silk and achieve stable material systems above a critical threshold of glycerol is an important new finding with importance for green chemistry approaches to new and more flexible silk-based biomaterials. The aqueous solubility, biocompatibility, and well-documented use of glycerol as a plasticizer with other biopolymers prompted its inclusion in silk fibroin solutions to assess impact on silk film behavior. Processing was performed in water rather than organic solvents to enhance the potential biocompatibility of these biomaterials. The films exhibited modified morphologies that could be controlled on the basis of the blend composition and also exhibited altered mechanical properties, such as improved elongation at break, when compared with pure silk fibroin films. Mechanistically, glycerol appears to replace water in silk fibroin chain hydration, resulting in the initial stabilization of helical structures in the films, as opposed to random coil or β-sheet structures. The use of glycerol in combination with silk fibroin in materials processing expands the functional features attainable with this fibrous protein, and in particular, in the formation of more flexible films with potential utility in a range of biomaterial and device applications.
Introduction Silk fibroin has excellent film-forming capabilities and is also compatible for use in the human body.1,2 Silk fibroin films have good dissolved oxygen permeability in the wet state, similar to that of human skin, which suggests potential applications for these films in wound dressing and artificial skin systems.3,4 However, films formed from silk fibroin are soluble in water because of dominating random coil structures. The structural features of the protein can be transformed from random coil to β-sheet form by treatment with heating,5 mechanical stretching,6 immersion in polar organic solvents,7 and curing in water vapor.8 This structural transition results in aqueous insolubility, thus providing options for the use of the material in a range of biomedical and other applications such as sensor platforms.9 However, these pure silk fibroin films tend to be stiff and brittle in the dry state over time, exhibiting impressive tensile strength but low elongation.8 Therefore, there remains a need to modify the physical and mechanical properties of silk films to control properties, mainly toward more flexible systems. Blending polymers with suitable plasticizers is a traditional approach to address the needs as outlined above. For example, prior studies have shown that silk film properties can be modified through blending with other synthetic or natural polymers, such as sodium alginate, polyallylamine, chitosan, cellulose, poly(caprolactone-co-D,L-lactide), S-carboxymethyl keratin, poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG) (300 or 8000 g/mol), and poly(ethylene oxide) (PEO) (900 000 g/mol).10-18 For example, we have reported blends of silk fibroin with PEO as a route to enhance rates of materials stabilization18,19 and the use of water as a plasticizer to improve film properties.8 * To whom correspondence should be addressed. E-mail: david.kaplan@ tufts.edu. † Soochow University. ‡ Tufts University.
However, in many cases, improved blends to control mechanical properties remain a challenge. In particular, avoiding additions of other polymers and generating systems that will stabilize properties for extended time frames remain a need. Therefore, in the present study, we sought to explore alternative plasticizer options and, in particular, glycerol. Glycerol has previously been used to improve silk film properties, including the use of 10% glycerol solutions during film formation.20 The glycerol was able to reduce phase separation between silk and PVA in the blend21 or to accelerate silk gelation.22 These earlier studies suggested that glycerol was at least partially compatible with silk and thus offered an important option for further study with respect to film properties in the absence of other polymers. In the present study, glycerol was blended with silk fibroin and then cast into films. These modified films were assessed for mechanical properties and structural features to better understand the interactions between the silk fibroin and glycerol. The data suggest specific interactions between these two components that provide important benefits to the film properties, primarily enacted by affecting silk fibroin crystallization behavior; the formation of the β sheets is the stabilizing physical cross-linker in the films.
Experimental Section Materials. Cocoons of Bombyx mori silkworm were purchased from the Institute of Sericulture, Tsukuba, Japan. All other chemicals used in the study were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water from the Milli-Q system (Millipore) was used throughout this research. Silk Fibroin Purification. Silk fibroin aqueous stock solutions were prepared as previously described.23 In brief, cocoons of Bombyx mori were boiled for 20 min in an aqueous solution of 0.02 M sodium carbonate and then rinsed thoroughly with pure water. After drying,
10.1021/bm900993n CCC: $40.75 2010 American Chemical Society Published on Web 11/18/2009
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the extracted silk fibroin was dissolved in 9.3 M LiBr solution at 60 °C for 4 h, yielding a 20% (w/v) solution. This solution was dialyzed against distilled water using Slide-a-Lyzer dialysis cassettes (MWCO 3500, Pierce) for 3 days to remove the salt. The solution was optically clear after dialysis and was centrifuged to remove the small amounts of silk aggregates that formed during the process, usually from environment contaminants that are present on the cocoons. The final concentration of silk fibroin aqueous solution was ∼6% (w/v). This concentration was determined by weighing the residual solid of a known volume of solution after drying. Preparation of Silk/Glycerol Blend Films. The purified silk fibroin solution was mixed with glycerol at weight ratios of 0, 5, 10, 20, 30, 40, and 50% (w/w). The mixed solutions were poured into a Petri dish and dried at room temperature in a laminar flow hood overnight. Unless otherwise stated, the “dry blend films” refer to the films prepared by directly casting and drying overnight, whereas the “wet blend films” refer to the same cast and dried films but subsequently extracted in ultrapure water at 37 °C for 1 h and then dried. For additional variables in the treatment groups, methanol treatments were used, and in these cases, the films (with and without glycerol blends) were immersed in 90% (v/v) methanol for 1 h and then dried in air. Dissolution. Blend films were cut into approximately 5 × 5 mm2 squares, and one square film was weighed and immersed in ultrapure water in a 2 mL tube to a concentration of 1% (weight of film/volume of water), and kept at 37 °C for 1 h and 1 day. After the incubation, the silk films were removed from the solution, dried in the air overnight, weighed, and compared with the mass of original film to obtain the residual mass (%). The remaining solution was subjected to UV absorbance measurement at 280 nm. The absorbance values were converted to the amount of silk solubilized in water using purified silk fibroin solution at various concentrations as standards. The amount of dissolved silk fibroin was then compared with the total silk fibroin mass in the film to obtain the percentage of the film dissolved silk fibroin in water. Fourier Transform Infrared (FTIR) Spectroscopy. The secondary structures present in the films, including random coil, R helices, β-pleated sheets, and turns were evaluated using Fourier self-deconvolution (FSD) of the infrared absorbance spectra. FTIR analysis of treated samples was performed with a Bruker Equinox 55/S FTIR spectrometer (Billerica, MA) equipped with a deuterated triglycine sulfate detector and a multiple-reflection horizontal MIRacle ATR attachment (with a Germanium (Ge) crystal, from Pike Tech (Madison, WI)). A 5 × 5 mm2 square-shaped silk film was placed in the Ge crystal cell and examined with the FTIR microscope in the reflection mode. Background measurements were taken with an empty cell and subtracted from the sample reading. For each measurement, 64 scans were recorded with a resolution of 4 cm-1, and the wavenumber ranged from 400 to 4000 cm-1. FSD of the infrared spectra covering the amide I region (1595-1705 cm-1) was performed by Opus 5.0 software, as previously described.24 Absorption bands in the frequency ranges of 1616-1637 and 1695-1705 cm-1 represented enriched β-sheet structure; those in the range of 1638-1655 cm-1 were ascribed to random coil structure, those in the range of 1656-1663 cm-1 were ascribed to R helices, and those in the range of 1663-1695 cm-1 were ascribed to turns.24 Mechanical Properties. Tensile tests were performed on an Instron 3366 testing frame equipped with a 10 N capacity load cell, Biopuls testing system including submersible pneumatic clamps, and video extensometer for measuring strain. Film samples were cast onto silicone slats that were punched out using a die-punch after ASTM standard D638-5-IMP, the size appropriate for clamping and gauge length necessary for video extensometry. For dry tests, films were loaded onto the tester and tested under ambient conditions (22 °C, 50% RH) after being peeled off of silicone slats. For wet testing, films were soaked in deionized ultrapure water for 1 h at 37 °C. Subsequently, samples for wet testing were loaded into the tester and allowed to soak in the bath (0.1 M PBS at 37 ( 0.3 °C) for 2 min before the test was initiated.
Lu et al. For both tests, a strain control rate of 0.1% s-1 was specified on the basis of the initial clamp-to-clamp length (nominal length ∼25 mm, elongation rate 25 µm/sec). Load and elongation data were captured at 20 Hz. Video extensometer strain data were recorded at the same rate on the basis of two fiducial painted markers placed at a nominal distance of ∼1 cm on the surface of the thinnest portion of each film. The original cross-sectional area was determined by measuring the film thickness using a thickness gauge (model no. 7309, Mitutoyo) and multiplying by the specimen gauge width (3.21 mm). The nominal tensile stress and strain were graphed on the basis of the original crosssectional area and length, respectively, and the stiffness, yield strength, strain to failure, and ultimate tensile strength (UTS) determined. UTS was determined as the highest stress value attained during the test. The stiffness, or elastic modulus, was calculated by using a leastsquares (LS) fitting between the points corresponding to 10 and 25% of the UTS. This was deemed to be sufficient to capture the linear portion of the stress/strain curve objectively for all samples tested. The yield strength was determined by offsetting the LS line by 0.5% strain and finding the data intercept. The strain to failure was determined as the last data point before any decrease in load (failure strain minus the strain corresponding to 10% UTS noted earlier). At least N ) 4 samples were used under every condition, ranging from 10 to 50% glycerol, and data were compared with previously published values of pure silk film mechanical properties.18 Scanning Electron Microscopy (SEM). Silk films were fractured in liquid nitrogen and sputtered with platinum. The cross section and surface morphologies of the different silk films were imaged using a Zeiss Supra 55 VP SEM (Jena, Germany). Culture of Fibroblasts. Fibroblast cells were expanded in a growth medium containing 90% DMEM, 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 1000 U/mL streptomycin. Cell cultures were maintained at 37 °C in an incubator with 95% air and 5% CO2. The cultures were replenished with fresh medium at 37 °C every 2 days. For adhesion, cells were seeded on silk films that were precast in 24 well plates with 50 000 cells per well in 1 mL of serum-containing medium. Empty wells with tissue culture plastic (TCP) and no silk served as controls. To evaluate cell attachment, after 3 h of cell seeding, we added 50 µL of alamar blue to the culture medium, and the medium fluorescence (Ex ) 560 nm, Em ) 590 nm) was determined after 6 h of culture. During the culture, cell proliferation was determined using alamar blue staining, and cell morphology was monitored by phase contrast light microscopy (Zeiss, Jena, Germany) Statistics. All experiments were performed with a minimum of N ) 3 for each data point. Statistical analysis was performed by oneway analysis of variance (ANOVA) and Student-Newman-Keuls multiple comparisons test. Differences were considered significant when p e 0.05 and very significant when p e 0.01.
Results and Discussion Silk and Glycerol Dissolution from Blend Films. Dissolution of silk fibroin protein from the blend films was determined in water based on UV absorbance because the silk fibroin protein has a significant content of tyrosines that absorb at 280 nm, whereas glycerol does not absorb at this wavelength. After a rapid initial weight loss at the 1 h time point, no further significant difference was found for the residual mass and dissolved silk content over time (Figure 1). When the glycerol content in the blend films was 2 and 5% (w/w), the films completely dissolved in water, similar to the control films that contained no glycerol (Figure 1). Therefore, glycerol at concentrations lower than 5% (w/w) did not significantly change silk film properties. When the glycerol content in the films was increased from 10 to 20% (w/w), the residual mass of the films that remained insoluble increased from about 10 to 75% (p < 0.01, Figure 1). Further increases in glycerol to 30% (w/w) further reduced
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Figure 1. Dissolution of silk and glycerol from blend films. *Significant differences between groups (P < 0.01). Data represent the av ( SD (n ) 4).
solubility, although the results were not statistically significant when compared with the 20% data. These results indicated that 20% (w/w) glycerol was a critical concentration in terms of inducing significant changes to silk film properties, resulting in insolubility of the material in water. When the glycerol content was 10% (w/w) (p < 0.01, Figure 2A, Figure 1 in the Supporting Information). These structural changes were distinguished from the changes observed in methanol-treated and water-annealed silk films in the absence of glycerol. The secondary structure content remained unchanged when the glycerol content in the films was increased from 10 to 20% (w/w). Therefore, stable R-helical structures dominated the glycerol-blended material. Three-fold helical crystal structure (silk III) has been previously reported for silk at air-water interfaces using the Langmuir-Blodgett technique, reflecting the amphiphilic features of silk,29 but not in glycerol-modified silk materials. The silk III structure can be transformed into
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more stable silk II if the compression force becomes >35 mN · m-1. The residue distribution along the helix and the orientation of the chain axis have been well characterized in those studies.29 Whether the R-helical structure we observed in the present study has similar features to the reported silk III structure still remains a question. For the glycerol-blended silk films, after methanol treatment, β-sheet structure content increased to 50-60%, whereas R-helical structure content decreased to ∼20%, regardless of the glycerol content in the film (Figure 2B). This response in terms of structural transitions induced by methanol is different from that observed with the water-annealed silk films, where no conformational transition from R-helical to β-sheet occurs upon methanol treatment.8 Furthermore, after the 20% (w/w) glycerol blended silk films were rinsed with water, R-helical structure content decreased, whereas β-sheet and β-turn structure content increased to approximately 45 and 20%, respectively (p < 0.01, Figure 2C). Therefore, for glycerol-blended silk films, stable silk II structures (crystalline, β sheets) can be obtained. Mechanistically, glycerol appears to alter the silk fibroin intraand intermolecular interactions and result in a conformational transition from random coil to R-helices, an unstable intermediate state toward stable β-sheet structure formation. Furthermore, the presence of glycerol appears to stabilize the R-helical structure, preventing its further transition toward β-sheet structure. Likely, the concentration of glycerol must reach a critical level to achieve this structural control. For 20 and 50% (w/w) glycerol blend films, the molar ratios between glycerol and silk fibroin are approximately 1000:1 and 4000:1, respectively. Dissolution of glycerol by water treatment released the structural restrictions so that the R-helical structure was able to transition to β-sheets. The silk molecules in the blend films were probably not highly packed so that there was sufficient chain mobility to enact this structural transition. Mechanical Properties of Blend Films. Every silk film tested displayed a qualitatively similar stress/strain behavior: a linearly increasing likely elastic portion (where the stiffness and yield strength were extracted), followed by a plateau region that extended until eventual failure (where UTS and strain to failure were extracted). A one-way ANOVA was conducted to assess the impact of glycerol content on the four metrics of mechanical performance in both the wet and dry states. There was a statistically significant difference (p < 0.01) in all dry testing metrics. Statistically significant differences were also observed along two of the testing metrics under the wet testing condition (p < 0.05): yield strengths and stiffness (Table 1, Figure 3B). These results suggest that mechanical properties of as-cast (dry) glycerol-blended films are highly dependent on the amount of glycerol added, whereas the wet films are dependent on the amount of glycerol added to a lesser degree. Compared with methanol-treated silk films without glycerol, in the dry or wet states, the glycerol-blended films had much higher ductility (Figure 3A), an important property for many applications. The ductility of the glycerol/silk films was also greater than that of water-annealed silk films because the latter (dry) only exhibited elongation at break of ∼6%,8 21 times lower than that of the 50% dry glycerol silk films reported in the present study. Free water content may influence the flexibility of silk films.20 It is important to note, however, that the testing equipment used in this study may have been limited in its ability to detect all changes in failure-related metrics with necessary sensitivity beyond ∼200% strain (Figure 3A) because of the size of the water bath used and the limited working range of the video extensometer.
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Figure 2. FTIR determination of silk secondary structures in blend films with different glycerol content: (A) blend films directly after film casting, (B) blend films after 90% (v/v) methanol treatment for 1 h, and (C) 20% (w/w) glycerol film with and without water treatment for 1 h. *Significant differences between groups (P < 0.01). Data represent the av ( SD (n ) 4). Table 1. Mechanical Properties Dependence on Glycerol Content in Silk Filmsa Dry
0 10 20 30 40 50
stiffness (GPa)
yield strength (MPa)
strain to failure (%)
ultimate strength (MPa)
3.500 ( 0.900 1.380 ( 0.376 0.135 ( 0.072 0.138 ( 0.031 0.043 ( 0.015 0.023 ( 0.004
N/A ( N/A 19.242 ( 2.599 8.939 ( 1.920 8.639 ( 1.297 3.509 ( 0.635 1.956 ( 0.210
2.100 ( 0.400 1.800 ( 0.638 15.175 ( 10.947 59.675 ( 27.114 106.200 ( 52.370 129.825 ( 44.392
58.800 ( 16.700b 20.390 ( 2.473 9.422 ( 1.610 11.584 ( 0.562 6.339 ( 1.226 4.143 ( 0.558
b
b
Wet
0 10 20 30 40 50 a
stiffness (MPa)
yield strength (MPa)
strain to failure (%)
ultimate strength (MPa)
16.722 ( 6.065c 5.246 ( 0.647 6.646 ( 1.732 5.409 ( 0.857 4.669 ( 0.399 7.431 ( 3.302
2.274 ( 0.620c 0.703 ( 0.099 0.829 ( 0.427 0.828 ( 0.078 0.848 ( 0.375 0.640 ( 0.120
127.800 ( 69.367c 155.920 ( 67.013 177.150 ( 53.810 203.260 ( 22.486 241.250 ( 38.433 168.675 ( 47.872
3.464 ( 0.709c 1.952 ( 0.529 2.702 ( 0.959 2.978 ( 0.468 2.917 ( 0.675 2.196 ( 0.372
N/A: data not published.
b
Data taken with permission from ref 18. c Data taken with permission from ref 26.
Posthoc analysis revealed specific differences among varying glycerol contents. In comparison with values previously published for methanol-treated films, glycerol-blended films were significantly softer in both the wet and dry states (p < 0.05, Figure 3B,C). Additionally, when the glycerol content increased from 10 to 20% (w/w), the stiffness significantly decreased from 1.38 to 0.13 GPa for dry tests (p < 0.01, Figure 3C); however, there was no significant differences in the stiffness of films tested wet in the range of 10-50% glycerol (Figure 3B). Likewise, in the dry state, the yield strength and UTS generally decreased with increasing glycerol content. For both metrics, there was
highly significant differences (p < 0.01) among the 10, 20-30, and 40-50% groups because yield and ultimate strength appeared tightly correlated (Figure 3D). During wet testing, there was a significant difference between the yield strengths of methanol-treated controls and all glycerol-blended films (p < 0.01) and between the UTS of methanol-treated control and 10% glycerol-blended films (p < 0.05). However, the differences between UTS and yield strengths for samples tested wet were much greater than those tested dry, which coincided with the large amount of permanent deformation that could be sustained in the wet state prior to eventual failure (Table 1).
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Figure 3. Mechanical properties of blend films with different glycerol content: (A) strain to failure of wet and dry blend films, (B) stiffness of wet blend films, (C) stiffness of dry blend films, and (D) strength of dry blend films. *Significant differences between groups (*P < 0.05, **P < 0.01). Data represent the av ( SD (n g 4).
Native silk’s response to water can be characterized by the disruption of interchain hydrogen bonds and the swelling of amorphous regions in the film structure,30 whereas the role of glycerol in altering the helical content of the silk fibroin may also change the mechanical behavior of the films. Additionally, subsequent to the 1 h hydration treatment of the wet films, one would expect decreases in mechanical properties because of glycerol leaching out of films in the range of 10-30% glycerol. These combined factors may have resulted in the differences observed across all conditions in the dry state and the leveling off of wet-state properties after 10% glycerol addition. Silk Nanostructures in Blend Films. To assess the impact of glycerol on film properties further, morphological characterization was conducted. Silk films were fractured in liquid nitrogen, and the cross sections of the films were examined by SEM. Silk fibroin protein formed globular nanostructures with diameters of 100-200 nm when the glycerol content was 10% (w/w) (Figure 4A). The globules, however, were not observed when 20% (w/w) glycerol was blended in the film; the films had relatively smooth morphologies by SEM (Figure 4B). These results indicate that a high content of glycerol (>20% w/w) influenced silk fibroin self-assembly and nanostructure features. Interestingly, when the 20% (w/w) glycerol silk films were treated with water to leach out the glycerol, the silk fibroin selfassembled into nanofilaments, similar to those in methanoltreated pure silk films (Figure 4C,D). This observation is consistent with the secondary structure transitions discussed earlier with β-sheet structure formation in both water-treated and methanol-treated glycerol silk films (Figure 2B,C). Therefore, the nanostructures formation in glycerol-blended films correlated with the structural features in the films and are likely influenced by silk secondary structural changes. The silk nanofilament structures formed in the 20% (w/w) glycerol films after water treatment were further studied by SEM
Figure 4. SEM images of blend films: (A) glycerol content 10% (w/ w); (B) glycerol content 20% (w/w); (C) glycerol content 20% (w/w), water treated for 1 h; and (D) glycerol content 0%, methanol treated for 1 h. Scale bar ) 200 nm.
(Figure 5A,D). The nanofilament structures were more clearly visible at higher magnification (Figure 5B,E) and in side view (Figure 5C). In different regions of the film, distinguished morphologies and organization of nanofilaments was observed (compare Figure 5A,B and 5D,E), probably because of inhomogeneous drying rates during silk film casting. The size of the nanofilaments, however, was consistently 10-20 nm throughout the film. Attachment and Proliferation of Fibroblasts on Blended Films. The silk blend films with glycerol achieve mechanical properties that are suitable for wound healing and artificial skin applications. Therefore, in preliminary studies, the attachment and proliferation of fibroblast cells on the 30% (w/
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Figure 5. Nanofilament structures in water-treated silk films containing 20% (w/w) glycerol: (A,D) different regions in the film, (B) high magnification of A, (C) side view of part A, and (E) high magnification of part D. Scale bar ) 200 nm in parts A, C, and D; 100 nm in parts B and E.
Figure 6. Attachment and proliferation of fibroblasts on different surfaces. (A) Microscopic images of cultured fibroblasts on 30% (w/w) glycerol/ silk film, pure silk film, and TCP. (B) Attachment of fibroblasts on different films. (C) proliferation of fibroblasts on different films. Data represent the av ( SD (n ) 6). Bar ) 100 µm.
w) glycerol/silk films were assessed because fibroblast cell growth is critical for wound healing and skin regeneration. Further studies with a range of different cell types would be instructive pending the specific biomaterial utility for the silk/ glycerol system described here. Initial cell attachment (3 h) on all three surfaces was similar (first row in Figure 6A), as quantified by Alamar Blue staining (Figure 6B). Cell proliferation in 14 days of culture, however, was different on the different surfaces. After 4 days of culture, fibroblasts on TCP grew faster than those on pure silk films and blend silk films, an observation consistent with our prior studies on pure silk films.23,31 After 14 days of culture, the number of cells on TCP was ∼1.8 times greater than that on the silk films, and there was no significant difference between the pure silk films and blend silk films, as determined by Alamar Blue staining (Figure 6C). The 30% (w/
w) glycerol silk film differed from the methanol-treated silk film for fibroblasts proliferation only in the time period from 6 to 11 days, in which cells grew faster on the methanol-treated film than on the glycerol film (p < 0.01, Figure 6C). The 30% glycerol silk film was characterized by extensive β-sheet structure and nanofilament network formation after hydration, similar to methanol-treated silk films (see above). Both films showed similar properties to support fibroblast attachment and proliferation, indicating that the material support for cell culture was determined by silk secondary structure features and self-assembled nanostructures. Compared with TCP, relatively rough and hydrophobic surfaces of silk films might be less favorable for fibroblast growth in the early stages. We have previously shown that RGD-modified silk films exhibited
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Figure 7. Schematic illustration of silk structural transitions in the glycerol-blended silk films.
excellent surface properties to promote rapid attachment and proliferation of fibroblasts, osteoblast-like cells, and humanbone-marrow-derived mesenchymal stem cells.32 There, similar strategies could be employed with the silk/glycerol blends. Mechanism. The glycerol content in blend films was important for the control of silk secondary structural transitions and influencing the mechanical properties of the films. After mixing glycerol with silk and casting films, glycerol molecules interacted with silk fibroin chains via intermolecular forces, most likely hydrogen bonds between hydroxyl groups of glycerol and amide groups of silk.21 This interaction likely altered the hydrophobic hydration state of protein chains because they are veryhydrophobicproteinsduetothehighcontentofglycine-alanine repeats33 and, therefore, induced silk secondary structural change from predominant random coils (silk in solution or as cast) to R helices (Figure 7). Furthermore, this interaction stabilized the helical stage of silk unless the film was treated by solvents, such as methanol. Upon treatment, glycerol molecules bound to the silk fibroin chains were released and diffused into the surrounding medium, allowing the silk fibroin chains to assume a more thermodynamically stable state in the form of β sheets. The process is similar to the previously reported mechanism of silk structural transitions based on the change in hydrophobic hydration state of the protein chains,34 but the water molecules associated with silk fibroin protein were replaced by glycerol molecules in the current case. Uniqueness. Glycerol has been previously used to modify silk films in previous studies,20,21 and the role glycerol was playing different from that in the present study. In the case of glycerol/PVA/silk blend films, glycerol functioned to reduce the phase separation between PVA and silk by making hydrogen bonding bridges between the two materials so that tensile strength and elongation at break of the blend film were largely improved as compared with the pure silk film. The tensile strength and elongation at break for the 5% glycerol blend PVA/ silk film were 426 kg/cm2 and 53%, respectively, much lower than those reported in the present study with 40% blend glycerol (6.3 MPa and 106% in dry environment). The improvement was attributed to the presence of PVA because the highest glycerol concentration in the blend film was 8%. In the other case of study, silk film was treated (immersed) in 10% w/v glycerol
solution for 10 min at 95 °C and subsequently conditioned in a humidity drier at 25 °C and 50% relative humidity. Glycerol functioned as a plasticizer to preserve high water content in this case so that the film flexibility was improved, as observed by the improvement in film self-thinning and self-expanding. No tensile strength and elongation at break were reported. Treatment of glycerol converted silk structure from silk I to silk II, distinguished from the present study in which the blend of high concentration of glycerol induced the characteristic R-helical structure, which was further converted to β-sheet structure (silk II) upon leaching of glycerol. Therefore, glycerol plays different roles in silk films, depending on the processing methods and concentrations used and, therefore, resulting in distinguished film properties. The glycerol blended silk films reported in the present study demonstrate unique features on diverse and controllable silk structure transitions, desired mechanical properties, and ease of fabrication (one step film casting without further treatments). These features will make the film useful in the future biomedical applications, such as wound healing.
Conclusions Silk films blended with glycerol (>10% w/w) are enriched in R-helical structure, which further transits to crystalline β-sheet structures upon the removal of glycerol by water treatments. The blend films rich in β-sheet structure were composed of characteristic nanofilaments, whereas those rich in R-helical structure did not exhibit these morphologies. The blend films, either in the dry or wet state, were more ductile than methanoltreated and water-annealed pure silk fibroin films, while offering some control of ultimate and yield strength properties. Both glycerol-blended (30% w/w) and methanol-treated silk films supported fibroblast attachment and growth. Mechanistically, the role of glycerol appears to mimic that of water in controlling the structural transitions of the silk fibroin chains, providing a new and useful control point in regulating the structure and thus material properties of silk-based biomaterials. Acknowledgment. We thank the AFOSR and the NIH for support of various aspects of this work.
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Supporting Information Available. FTIR spectra of silk films containing different contents of glycerol and treatments. This material is available free of charge via the Internet at http:// pubs.acs.org.
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