Spider and silkworm silk proteins (fibroins) have been investigated widely because of their toughness, light weight, biodegradability and biocompatibility [1
]. In addition to silk fibers and textiles, silk protein is processable and has been used to generate various biopolymer-based materials, including nanoparticles, porous materials, films, sponges, and hydrogels, for use in biomedical applications such as tissue engineering, drug/gene delivery and regenerative medicine [5
]. In nature, silk fiber is used as a structural material in spider webs, spider draglines and silkworm cocoons [8
]. However, silk protein has not yet been used as a structural material on a bulk scale. One of the limitations and drawbacks to the practical use of silk as a structural material is its water sensitivity, namely, silk protein-based materials are highly sensitive to water.
Water molecules in silk-based materials are categorized roughly into two types, namely, bound water and free water. Bound water molecules strongly interact with silk molecules and show different characteristics to those of bulk water; in contrast, free water means unbound water molecules that behave similarly to bulk water [10
]. The influences of bound water on the biological and physical properties of silk molecules have been widely reported by several groups. Asakura and coworkers reported that the hydration of Bombyx mori
silk molecules induces the stabilization of silk I forms based on solid-state NMR analysis [14
]. The secondary structure and dynamics of swollen B. mori
silk molecules were also characterized by 13
C- and 1
H-pulsed NMR [15
]. Asakura et al. have also reported that hydration does not affect the crystalline fraction of B. mori
silk fibers [17
]. It was reported that bound water influences the glass transition temperature (Tg
) of B. mori
silk molecules, that is, the Tg
decreases with an increase in the water content of the silk films [18
]. In addition to B. mori
silk, the storage modulus and loss tangent of Nephila edulis
spider dragline [21
] and the elastic modulus values of Antheraea pernyi
silkworm silk [23
] and Argiope trifasciata
spider silk [24
] are reported to be affected by bound water. Bound water is considered to disrupt the hydrogen bonds between silk molecules in amorphous phase, and hence to enhance the mobility of the silk molecules, as well as influence the glass transition behavior [18
]. Considering the biological properties of silk-based materials, the state of the water molecules in a silk hydrogel controls the effect of the hydrogel on cell viability, namely, human cell lines and cell-adhesion proteins in the extracellular matrix preferentially expand and adhere on silk molecules hydrated with more bound water [13
Macroscopic studies on the effect of water and relative humidity (RH) on silk materials have been reported by several groups. Cebe and coworkers reported that hot-water vapor annealing induces crystallization in silk films [26
]. In our previous studies, we demonstrated the effects of the water content in silk films and fibers on crystallization, bio- and thermal degradation [27
]. By using thermal gravimetric analysis and differential scanning calorimetry (DSC), the silk samples prepared at different RHs were analyzed in terms of the effects of the water content on thermal degradation, crystallization and transition of B. mori
silk materials. The hydration state and RH affected the mechanical properties of silk fibers [23
]. At a relatively high RH, approximately 97%, the toughness and degree of crystallinity of silk films increase dramatically, indicating that the appropriate hydration of silk molecules induces crystallization and plasticization simultaneously [28
]. The RH from 20% to 60% resulted in tough and strong silk materials by using various types of silk hydrogels. Dehydration did not negatively impact the biodegradability of the silk resins and hydrogels [27
]. Thus, the thermal stability, mechanical properties and other attributes of silk materials are regulated by their water content and crystallinity. To exploit silk and silk-based materials as practical structural materials for human use, stabilization against water molecules is necessary.
As introduced above, silk-based materials are water-sensitive and show different physical properties at different humidities. However, this instability of silk under wet conditions is detrimental to its use as a structural material. To overcome the water sensitivity of silk-based materials, in this study we developed a silk composite material with a fluoropolymer, which is famous for its hydrophobicity and waterproofness. Blend films of silk proteins and fluoropolymer showed enhanced surface hydrophobicity and vapor barrier properties. The coating of fluoropolymer on silk textiles was resistant to washing and shrinkage treatments. This material design, with a protein biopolymer and a fluoropolymer, will broaden the applicability of protein-based materials.
2. Materials and Methods
2.1. Preparation of Silk Powder
To obtain silk powder samples, silk fibroin solution was prepared according to a previously reported method [7
]. Briefly, B. mori
silkworm cocoons were cut and boiled for 30 min in a 0.02 M Na2
solution, and subsequently washed with MilliQ water to remove wax layers and sericin. The extracted silk fibroins were dried at 25 °C for 24 h, and dissolved in a 9.3 M LiBr solution at 60 °C for 2 h at a concentration of 200 g/L. The silk solution was dialyzed with MilliQ water for at least 4 days using a dialysis membrane (Pierce Snake Skin MWCO 3500; Thermo Fisher Scientific, Waltham, MA, USA). Dialysis was completed when the conductivity of the dialysis solution was identical to that of MilliQ water. The silk solution was lyophilized to yield the silk powder.
2.2. Film Preparation
Silk powder was dissolved into hexafluoroisopropanol (HFIP) to generate a silk HFIP solution (25 g/L). It took approximately 24 h to dissolve the silk powder in the HFIP at 25 °C. The silk HFIP solution was blended with a fluoropolymer, Lumiflon® LF600X (AGC Chemical Company, Tokyo, Japan). The solvents (xylene and ethylbenzene) in the Lumiflon® LF600X solution (50 wt % fluoropolymer, 26 wt % xylene, 24 wt % ethylbenzene) were evaporated, and the resulting solid fluoropolymer was dissolved into HFIP (25 g/L). To prepare the film samples, the silk HFIP solution and the fluoropolymer HFIP solution were mixed and casted on a Teflon Petri dish. After drying for 16 h, films with a thickness of approximately 50 μm were obtained.
2.3. Tensile Tests
The tensile tests of the film samples were performed by a mechanical testing apparatus (EZ-LX/TRAPEZIUM X, Shimadzu, Kyoto, Japan) [30
]. The initial length of the film sample was approximately 5 mm. The extension speed was 10 mm/min, and a 500 N load cell was used. The strength at break, Young’s modulus, elongation at break, and toughness were obtained based on the resultant stress–strain curves.
2.4. Thermal Analysis
DSC measurements were performed using a DSC 8500 (Perkin Elmer Inc. Waltham, MA, USA) to quantitatively characterize the thermal properties of the silk and fluoropolymer according to a previous report [30
]. Approximately 10 mg of the sample was transferred to a DSC aluminum pan which was first cooled to −60 °C, and then heated to 240 °C at a rate of 20 °C/min. The glass transition (Tg
) and water evaporation were determined from the DSC thermograms.
2.5. Scanning Electron Microscopy (SEM) Observations
The sample morphology was analyzed by SEM. Silk samples were sliced into small pieces approximately 2 mm × 2 mm × 1 mm. The sliced samples were mounted onto an aluminum stub, sputter-coated with gold, and imaged by SEM (JSM6330F, JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 5 kV. The cross-section of the samples was prepared using a microtome (RM2265, Leica Microsystems GmbH, Wetzlar, Germany) with a diamond blade.
2.6. Advancing Contact Angle of Water
The wettability of the cast film surface was estimated by the advancing contact angle (θadv
) measurement with distilled water, using a FACE Contact Angle Meter CA-X (Kyowa Interface Science, Saitama, Japan), according to a previous procedure [31
]. Cast films of the samples were prepared on glass substrates by solution casting from the silk and fluoropolymer HFIP solutions, as described above. The θadv
value was calculated as the average of ten data obtained at different points on the surface (n
2.7. Water Vapor Barrier Test
The water vapor permeability rates of the film samples were determined at 37 °C using a Mocon Permatran-W model 1/50G (Modern Controls, Minneapolis, MN, USA) under standard conditions (ASTM 3985) [32
]. Each measurement was continued until the water vapor permeability rate reached a stable value.
2.8. Shrinkage Test
B. mori silk textiles were kindly provided by Spiber Inc. (Tsuruoka, Japan). The silk textiles were immersed in Lumiflon® LF600X (50 wt % fluoropolymer, 26 wt % xylene, 24 wt % ethylbenzene) for 1 min, and then dried in air at 25 °C and an RH of approximately 40%. Square silk textile samples (approximately 50 mm × 50 mm) with and without a fluoropolymer coating were used for the shrinkage test. The samples were immersed in hot water (40 °C) for 10 min and were dried at 25 °C and an RH of 65% for 16 h. This washing and drying cycle was performed three times. To evaluate the shrinkage of the samples, the squares of the samples were measured, and the changes in the area of the samples were determined. The test was performed three times, and the results are expressed as mean values and standard deviations.
2.9. Biodegradation Test
The biochemical oxygen demand (BOD) test was performed to determine the biodegradability of the silk samples in activated sludge (Chemicals Evaluation and Research Institute, Tokyo, Japan) with an Oxitop IS-6 (WTW GmbH, Weilheim in Oberbayern, Germany), according to a previous procedure [33
]. A sample film (approximately 10 mg) was immersed in 100 mL of activated sludge at 25 °C for 30 days. The activated sludge was replaced with fresh sludge every 5 days. Before and after the biodegradation test, the morphologies and mechanical properties of the samples were characterized by the methods explained above.
In this study, we developed a silk composite material with a fluoropolymer to overcome the water sensitivity of silk-based materials. Blending and coating silk protein-based materials, such as films and textiles, with a fluoropolymer enhanced the surface hydrophobicity, water vapor barrier properties, and size stability during washing/drying cycles. However, the blending of the fluoropolymer with silk proteins cannot stabilize the silk materials perfectly. In the case of the silk films coated with the fluoropolymer, the water and biodegradation resistances of the silk materials were improved. Thus, fluoropolymer treatment is expected to broaden the applicability of silk materials as well as protein-based materials. The other technique to modify the surface property of silk materials is plasma treatment. However, plasma treatment sometimes digests silk molecules at the surface of the silk materials, resulting in more water-sensitive surface. The fluoropolymer coating also has a disadvantage, namely, the loss of the original texture of the silk materials. To resolve this issue, we plan to design and synthesize other fluoropolymers to realize a fluoro-coating layer with a thickness of molecular level. The combination of protein-based materials and fluoropolymers will open a door for many applications in biomaterial, structural material and apparel material fields.