Influence of Casting Temperature on the Near-Surface Structure and

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Influence of Casting Temperature on the Near-Surface Structure and Wettability of Cast Silk Fibroin Films Oleg N. Tretinnikov*,† B. I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk 220072, Belarus and Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

Yasushi Tamada*,‡ National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan Received May 29, 2001. In Final Form: August 6, 2001 The near-surface structure and the wettability of silk fibroin films cast from aqueous solutions on hydrophobic polystyrene substrates at various temperatures is investigated by Fourier transform infrared attenuated total reflection spectroscopy (FTIR-ATR) and measurement of contact angle. The FTIR data reveal that the near-surface region of the films is enriched in random coil conformations of the protein at the expense of a reduced fraction of R-helix and β-sheet conformations. The relative random coil/β-sheet content shows a marked dependence on the casting temperature, displaying a minimum at 50 °C. The minimum occurs concurrently with a maximum in the wettability of film surfaces by polar liquids. In the lower wettability region, the film surfaces of this hydrophilic protein are hydrophobic, whereas in the enhanced wettability range they are slightly hydrophilic. The experimental data indicate that during formation of fibron films, R-helix and β-sheet structures are rejected by the interface because of their non-surface-active character, whereas random coils are energetically favored because at the interface they convert into a surface-active conformation which effectively minimizes the interfacial free energy and renders the polymer surface hydrophobic. In the narrow range of casting temperatures centered at 50 °C, the effect of the interface is overweighed by the bulk thermodynamics favoring the β-sheet crystallization of fibroin. Though the interfacial conformation is not accessible by FTIR-ATR, its surface-active character in combination with the unique composition and amino acid sequence of fibroin allows one to conclude that the possible chain structure is one that separates the hydrophobic alanine and hydrophilic serine residues to opposite sides of the plane passing through the chain axis.

Introduction Silk fibroin is the protein that forms the filaments of silkworm silk and gives silk its high mechanical strength, elasticity, and softness. In addition to the outstanding mechanical properties, silk fibroin displays biological compatibility, stability to most solvents, and specific structural and functional characteristics.1 Because of that, this natural polymer, in addition to its traditional use as a textile fiber, has recently attracted considerable interest as a material suitable for biomedical and biotechnological applications. Representative examples are the use of silk fibroin films as oxygen- and drug-permeable membranes,2 supports for enzyme immobilization,3,4 and substrates for cell culture.5,6 In the above applications, fibroin films are prepared by casting from aqueous solutions on low-energy substrates (e.g., polyethylene or polystyrene). The bulk structure of †

E-mail: [email protected] or [email protected]. ‡ E-mail: [email protected]. (1) Silk Polymers: Materials Science and Biotechnology; Kaplan, D., Adams, W. W., Farmer, B., Viney, C., Eds.; ACS Symposium Series 544; American Chemical Society: Washington, DC, 1994. (2) (a) Minoura, N.; Tsukada, M.; Nagura, M. Polymer 1990, 31, 265. (b) Chen, J.; Minoura, N.; Tanioka, A. Polymer 1994, 35, 2853. (3) Miyairi, S.; Sugiura, M.; Fukui, S. Agric. Biol. Chem. 1978, 42, 1661. (4) Demura, M.; Asakura, T. Biotechnol. Bioeng. 1989, 33, 598. (5) Inoue, K.; Kurokawa, M.; Nishikawa, S.; Tsukada, M. J. Biochem. Biophys. Methods 1998, 37, 159. (6) Higuchi, A.; Yoshida, M.; Ohno, T.; Asakura, T.; Hara, M. Cytotechnology 2000, 34, 165.

the cast films has been a subject of intensive studies over the last 15 years.7-11 The high glycine content of silk fibroins allows great conformational variability, while their highly repetitive amino acid sequence gives rise to very regular conformations. As a result, in the bulk of cast films, silk fibroin can assume coexisting R-helix, β-sheet, and random coil conformations. The proportion of these structural forms changes markedly with film casting conditions such as the initial concentration of protein, drying rate, and casting temperature. This, in turn, has a strong impact on the mechanical and thermal properties of the films. While the bulk structure and properties of cast fibroin films are of significant importance, the surface characteristics appear to be equally important, especially in connection with the growing interest in biomedical applications of the films. The surface properties of a material are the key factors controlling the interactions that occur (7) Magoshi, J.; Nakamura, S. J. Polym. Sci., Polym. Phys. Ed. 1985, 23, 227. (8) (a) Tsukada, M. J. Polym. Sci., Polym. Phys. Ed. 1986, 24, 457. (b) Tsukada, M. J. Polym. Sci., Polym. Phys. Ed. 1986, 24, 1227. (c) Tsukada, M.; Freddi, G.; Gotoh, Y.; Kasai, N. J. Polym. Sci., Part B: Polym. Phys. 1994, 32, 1407. (d) Tsukada, M.; Freddi, G.; Monti, P.; Bertoluzza, A. J. Polym. Sci., Part B: Polym. Phys. 1995, 33, 1995. (e) Freddi, G.; Monti, P.; Nagura, M.; Gotoh, Y.; Tsukada, M. J. Polym. Sci., Part B: Polym. Phys. 1997, 35, 841. (9) Ishida, M.; Asakura, T.; Yokoi, M.; Saito, H. Macromolecules 1990, 23, 88. (10) Mathur, A. B.; Tonelli, A.; Rathke, T.; Hudson, S. Biopolymers 1997, 42, 61. (11) (a) Sonoyama, M.; Miyazawa, M.; Katagiri, G.; Ishida, H. Appl. Spectrosc. 1997, 51, 545. (b) Chen, X.; Shao, Z.; Marinkovic, N. S.; Miller, L. M.; Zhou, P.; Chance, M. R. Biophys. Chem. 2001, 89, 25.

10.1021/la010791y CCC: $20.00 © 2001 American Chemical Society Published on Web 10/19/2001

Structure and Wettability of Silk Fibroin Films

when it is exposed to biological environments. Understanding of the surface features of the cast fibroin films could therefore provide new opportunities for these materials in biomedical fields. However, reports on the surface characterization of silk fibroin in the form of cast films are practically lacking. Hsu et al. analyzed thin silk fibroin films cast on poly(tetrafluoroethylene) (PTFE) and glass substrates by external reflection infrared spectroscopy and found that the films cast on the highly oriented PTFE surface exhibit more β-sheet structure than those cast on unoriented PTFE or on glass.12 Kaplan and Gido studied silk fibroin films prepared by the LangmuirBlodgett (LB) technique at the air-water interface.13,14 They found that in the LB films, fibroin adopts a surfaceactive, 3-fold helical structure which is distinctly different from the known secondary structures of the protein. In this paper, we report vibrational spectroscopic and contact angle measurements made on thick silk fibroin films cast from aqueous solutions on hydrophobic polystyrene substrates at various temperatures. We use Fourier transform infrared attenuated total reflection spectroscopy (FTIR-ATR) to investigate the chain conformation in the near-surface region of the cast films as a function of casting temperature. We also follow the wettability behavior of the film surfaces by the measurement of contact angle and use the contact angle data to determine the surface energy characteristics of the films. As will be shown, the near-surface region of the cast fibroin films differs markedly from the bulk in the conformational structure and its dependence on the casting temperature. Moreover, the casting temperature appears to have a marked influence on the surface wettability and energetics of the films. Importantly, the temperature-induced changes in the surface properties correlate closely with the corresponding changes in the surface structure. Experimental Section Preparation of Silk Fibroin Films. Aqueous solutions of silk fibroin (2.5-2.8% w/v) were obtained by dissolving degummed silk fibers from Bombyx mori with 9.0 M LiBr solution, followed by dialysis against water (Milli-Q quality). Fibroin films about 35 µm thick were cast on bacteriological grade polystyrene Petri dishes (Iuchi Co., Japan) in the following way. The fibroin solution in the amount of 10 mL was applied on the bottom of dish and forced to spread uniformly over the whole surface area by gently shaking and tilting the dish. After that, the dish was covered to ensure slow evaporation of solvent and placed in a clean oven at a given temperature. All the films thus prepared were first removed from the substrates and stored at room temperature and 65% relative humidity for several days and then subjected to FTIR-ATR and contact angle measurements. In one case, a thinner (∼6 µm) film was prepared at 22 °C using the same procedures, except that the amount of casting solution was proportionally lower. The films were colorless, homogeneously transparent, and optically smooth (on both sides) in appearance. The two sides of the films will be referred to as the air surface (air side) and substrate surface (substrate side). FTIR-ATR Measurements. The FTIR-ATR analysis was performed with a Jasco FTIR-350 spectrophotometer equipped with a nitrogen-cooled mercury-cadmium-telluride (MCT) detector and a multiple reflection horizontal ATR attachment. Each spectrum was recorded at a resolution of 4 cm-1, and the number of scans was 100. The internal reflection elements (IRE) used were Ge and KRS-5 prisms with an angle of incidence of 45°. (12) Chen, C. C.; Riou, S.; Hsu, S. L.; Stidham, H. D. Langmuir 1996, 12, 1035. (13) Muller, W. S.; Samuelson, L. A.; Fossey, S. A.; Kaplan, D. L. Langmuir 1993, 9, 1857. (14) (a) Valluzzi, R.; Gido, S. P.; Zhang, W.; Muller, W. S.; Kaplan, D. L. Macromolecules 1996, 29, 8606. (b) Valluzzi, R.; Gido, S. P. Biopolymers 1997, 42, 701. (c) Valluzzi, R.; He, S. J.; Gido, S. P.; Kaplan, D. L. Int. J. Biol. Macromol. 1999, 24, 227.

Langmuir, Vol. 17, No. 23, 2001 7407 Under these conditions, the effective penetration depth of the IR beam into the polymer film is approximately 1/15 and 1/5 the wavelength (or 0.4 and 1.2 µm at 1650 cm-1) for Ge and KRS-5 IRE, respectively.15 Curve fitting of the amide I band was performed using the Origin 6.0 (Microcal) software and assuming Lorentzian line shapes for the component bands. No smoothing of spectra was used, except three-point smoothing in the taking of second derivatives. Contact Angle Measurements. The liquids used for contact angle measurements were water (Milli-Q grade), glycerol (Sigma, 99.8%), ethylene glycol (Wako, 99.5%), methylene iodide (Aldrich, 99%), and R-bromonaphthalene (Aldrich, 98%). Static contact angles of fibroin films against these liquids were measured by the sessile drop method using an Erma G-1 contact angle goniometer (Erma Inc., Tokyo, Japan) at 22 °C and about 65% relative humidity. These were determined 10-20 s after application of the liquid drop. The contact angle of water diminished after this time as the result of the gradual penetration of liquid into the polymer film. Contact angles exhibited by glycerol and ethylene glycol changed very slowly, and those for methylene iodide and R-bromonaphthalene were constant for several minutes. The liquid drops (5 µL) were applied with a microliter syringe. To avoid contamination of one liquid by another and to speed up the measurements, an individual syringe was used for each liquid. All reported contact angle values are the average of at least 10 measurements taken at different locations of the film surface. The standard deviation was about (2° for water contact angles and less than or equal to (1° for those of other liquids. Determination of Surface Energy. The surface energy was determined for the free and substrate surfaces of the fibroin films from the contact angle data using the Lifshitz-van der Waals/acid-base (LW/AB) approach developed by van Oss, Chaudhury, and Good.16 This method yields the solid surface energy (γs) as the sum of the two components γsLW and γsAB (i.e., γs ) γsLW + γsAB), associated with electrodynamic Lifshitz-van der Waals (LW) interactions and (Lewis) acid-base interactions (AB), respectively. The component γsAB is a combination of the electron-acceptor (γs+) and the electron-donor (γs-) parameters of the surface energy: γsAB ) 2(γs+γs-)1/2. In the absence of spreading pressure (which is generally the case for low-energy solid surfaces when the contact angle exceeds 20°),17 the relationship between the equilibrium contact angle (θ) and the surface tension components of liquids and solids is given by the modified Young-Dupre´ equation,

γL(1 + cos θ) ) 2xγSLWγLLW + 2xγS+γL- + 2xγS-γL+ (1) where addition of the subscript “L” denotes the surface energy components and parameters of the test liquids. Thus, from contact angle measurements with three different liquids of known surface energy components and parameters, the solid surface energy can be determined by solving a system of three equations in the form of eq 1 with respect to the surface energy components and parameters of the solid.

Results and Discussion FTIR-ATR Study. To determine the conformational structure of the fibroin films from their IR spectra, we closely observed the amide I region between 1600 and 1700 cm-1 which is the most useful for the IR structural analysis of proteins.18 The amide I band represents primarily the CdO stretching vibration of the amide group. The frequency of this vibration depends on the strength of hydrogen bonding between the CdO and N-H groups, (15) Harrick, N. J. Internal Reflection Spectroscopy; Interscience: New York, 1967. (16) (a) van Oss, C. J.; Chaudhury, M. K.; Good, R. J. Adv. Colloid Interface Sci. 1987, 28, 35. (b) van Oss, C. J.; Chaudhury, M. K.; Good, R. J. Chem. Rev. 1988, 88, 927. (b) van Oss, C. J.; Good, R. J. J. Macromol. Sci., Chem. 1989, 26, 1183. (17) (a) Good, R. J. J. Colloid Interface Sci. 1975, 52, 308. (b) Johnson, R. E., Jr.; Dettre, R. H. In Wettability; Berg, J. C., Ed.; Dekker: New York, 1993; p 1. (18) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 269.

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Figure 1. FTIR-ATR spectra of silk fibroin films cast at different temperatures on polystyrene substrates. The spectra were obtained with the use of (a) Ge-45° IRE and (b) KRS-5-45° IRE on the air (solid lines) and substrate sides (dotted lines) of films.

which in turn is determined by the particular conformational structure of the protein backbone. In general, the amide I mode associated with the R-helical conformation occurs at 1650-1660 cm-1, the random coil conformations give bands in the range of 1640-1650 cm-1, and the β-sheet conformation results in IR bands between 1620 and 1640 cm-1.18-20 The amide I region of the FTIR-ATR spectra of the silk fibroin films cast from 2.5-2.8% aqueous solutions at various temperatures on PS substrates are shown in Figure 1a. The spectra were obtained using Ge IRE at an incidence angle of 45°. For each casting temperature, the spectrum measured on the air side of the film was practically identical to that of the substrate side. The spectra of films cast at 22 and 30 °C exhibited a nearly symmetric amide I band centered at 1651 cm-1. This spectral pattern is characteristic to silk fibroin films consisting of R-helixes mixed with random coil structures and is commonly observed on casting the films at ambient temperatures.8a,b When the casting temperature was raised to 40 °C, a new strong band at 1626 cm-1 appeared in the spectra in addition to the main peak at 1651 cm-1. On increasing the casting temperature up to 50 °C, the 1626 cm-1 band became dominant due to a drastic increase in its intensity, while the peak at 1651 cm-1 turned into a shoulder. As the absorbance at 1626 cm-1 is assigned to the β-sheet conformation of silk fibroin,9,21 the evolution of the amide I band reflects an abrupt increase in the β-sheet content with the increasing temperature of film casting. This tendency of silk fibroin to attain the β-sheet conformation on casting at elevated temperatures has been well documented.7,8a Moreover, it has been shown that the formation of β-sheets is favored up to the casting temperature of 100 °C, provided the drying rate is kept low.8a However, when in the present study the casting temperature was raised further from 50 to 70 °C, a notable decrease in the intensity of the 1626 cm-1 band was observed indicating that the β-sheet content went down. This unexpected behavior could not be due to the effect of drying rate, because the films studied were cast under conditions of slow evaporation of the solvent. On the other hand, the FTIR-ATR spectra under consideration reflect (19) Surewicz, W. K.; Mantsh, H. H. Biochim. Biophys. Acta 1988, 952, 115. (20) Haris, P. I.; Chapman, D. Biopolymers 1995, 37, 251. (21) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712.

the structure of a thin (ca. 0.4 µm) near-surface region of the films, whereas the literature refers solely to the bulk structure. Therefore, it seemed reasonable to ascribe the inconsistency with the literature data to an effect of the interface on the near-surface structure. The above finding prompted us to increase the probing depth of the FTIR-ATR measurements in order to get insights on the bulk structure of the films. This was realized using a KRS-5 IRE with a 45° incidence angle instead of Ge IRE. The probing depth was about 1.2 µm in the amide I region, and the corresponding spectra are shown in Figure 1b. In comparison to the spectra obtained with Ge IRE (Figure 1a), the amide I bands shown in Figure 1b are somewhat shifted and widened toward lower wavenumbers. This should be explained by spectral distortions associated with the fact that the employed angle of incidence (45°) is close to the critical angle of internal reflection for the KRS-5 IRE-protein film system (42°).15 Nevertheless, in the range of casting temperatures from 22 to 50 °C, the temperature dependence of the amide I band was qualitatively similar to that observed with the use of Ge IRE. That is, the β-sheet peak was not seen in the spectra of films cast at 22 and 30 °C, while at 40 °C it appeared as a highly intensive band which increased in the intensity further at 50 °C. At the same time, unlike the spectra obtained with Ge IRE, the intensity of the β-sheet peak did not decrease but remained practically constant on increasing the temperature up to 70 °C, in full accord with the respective literature data for the bulk of fibroin films.7,8a Besides, in the spectra measured with KRS-5 IRE, the intensity of the β-sheet peak was systematically higher than in the corresponding spectra obtained with Ge IRE, in the whole range of casting temperatures. These results support the above conclusion that the near-surface structure of fibroin films studied is different from the bulk and this structural specificity is manifested in the spectra obtained with the use of Ge IRE. The reduced intensity of the β-sheet peak in these spectra indicates that the β-crystallization of silk fibroin was hampered in the vicinity of the film surface. To quantify the surface content of various conformational forms of the protein, the FTIR-ATR spectra obtained with Ge IRE were curve fit. Since no principal difference in the results between the air and substrate surfaces could be expected, taking into account the established similarity of the corresponding spectra for each casting temperature,


Langmuir, Vol. 17, No. 23, 2001 7409 Table 1. Frequencies and Structural Assignments of Amide I Component Bands as well as the Fractional Band Areas in the FTIR-ATR Spectra of Fibroin Films Cast at 22 and 50 °C (Ge-45° IRE, Air Side) frequency (cm-1) 1611 1619 1626 1631 1641 1649 1659 1668 1674 1682 1691 1699

Figure 2. Second derivative FTIR-ATR spectra of silk fibroin films cast at (a) 22 °C and (b) 50 °C (Ge-45° IRE, air side).

only the spectra of the air surfaces were analyzed. The number and location of individual component bands used in curve fitting were obtained from the second derivative of the original spectra. As an example, Figure 2 shows the second derivative FTIR-ATR (Ge-45°) spectra of fibroin films cast at 22 and 50 °C. They reveal 12 component bands. Each band is positioned in both spectra at the same frequency (within (1 cm-1).22 Figure 3 shows the amide I contour with the fitted component bands in the FTIRATR (Ge-45°) spectra of fibroin films cast at different temperatures. All the spectra differ one from another in the relative intensity of components, and the difference is most striking between the spectra of films cast at 22 and 50 °C. Thus, the 22 °C spectrum is completely dominated by bands at 1641, 1649, and 1659 cm-1, whereas in the 50 °C spectrum these bands are counterbalanced with a triplet consisting of 1619, 1625, and 1631 cm-1 (22) With the use of curve-fitting procedures, there always exists a danger of producing artificial peaks. The fact that no spectrum-tospectrum variation in the number and position of component bands was observed, although the global band shape changed markedly, indicates that all the bands resolved by curve fitting are real peaks associated with the vibrational modes of protein.

relative band area (%) 22 °C

50 °C

assignment

0.8 2.5 4.3 3.8 20.8 22.9 17.8 8.4 3.8 5.1 8.6 1.1

2.0 9.5 12.3 12.7 12.9 13.9 13.5 7.1 3.2 4.6 5.9 2.4

β-sheet β-sheet β-sheet β-sheet random coil random coil R-helix turns turns and bends turns and bends turns and bends β-sheet

peaks. The latter three bands, taking into account their location in the “β-frequency” range18-20 and the established β-crystallization of fibroin on casting at 50 °C, can be only assigned to β-sheet structures. The band at 1659 cm-1 is due solely to R-helixes.8c,8e The bands at 1641 and 1649 cm-1 can be assigned to random coil structures.23 The frequencies of these peaks fall into the region of random coil modes,18-20 and furthermore, as can be seen in Figure 3, their intensities change with the casting temperature in a similar manner which is different from that displayed by R-helix or β-sheet bands. The remaining components include a very weak band at 1611 cm-1 and five somewhat more intensive bands in the region of 1660-1700 cm-1. The high-frequency bands are believed to represent turns and bends,18-20 though contribution of β-sheets may not be excluded, in particular for the 1699 cm-1 band,20,21 the intensity of which changes with the casting temperature similarly to that of the β-sheet triplet. β-Sheets may also be associated with the peak at 1611 cm-1. This uncertainty in the assignments of the last six components is not critical to the current study. Temperature variations, if any, in the area (integrated intensity) of these bands are insignificant on the scale of the total amide I band area. As a result, the spectroscopic picture of structural alterations in the films studied appears entirely represented in the intensity change of structural bands between 1615 and 1660 cm-1. This point is supported further in Table 1 which summarizes the frequencies and structural assignments of the amide I component bands as well as the fractional band areas for 22 and 50 °C casting temperatures. The near-surface fractions of R-helixes, β-sheets, and random coils in the studied films were estimated by adding the areas of all bands assigned to each of these structures and expressing the sum as a fraction of the total amide I band area. The data thus obtained are plotted against casting temperature in Figure 4.24 It can be concluded that the near-surface regions of films cast at 22 and 30 (23) Nonordered protein conformations usually give a broad band centering at ∼1645 cm-1 which represents a composite of closely spaced unresolved components (refs 18 and 19). The splitting of the “amorphous” amide I mode in the silk fibroin films into two well-resolved components may indicate the presence of two distinctively different patterns of hydrogen bonding in nonordered structures. A recently proposed assignment of the 1648 cm-1 component to the 3-fold helical structure of fibroin (Wilson, D.; Valluzzi, R.; Kaplan, D. Biophys. J. 2000, 78, 2690.) might also be considered, but this assignment is still to be proved. (24) It should be emphasized that the data in Figure 4 represent estimates of the content of structural forms and not the absolute values. This is because the corresponding calculations assume equal absorption coefficients for all the amide I components, and the validity of this assumption remains to be tested. Nevertheless, this approach has been shown to be of particular value in testing the nature and extent of conformational changes in proteins induced by various treatments (refs 18 and 19).

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Figure 3. The amide I contour with the fitted component bands in the FTIR-ATR spectra of silk fibroin films cast at different temperatures (Ge-45° IRE, air side).

Figure 4. The near-surface fractions of R-helixes (R), β-sheets (β), and random coils (r) in the cast fibroin films as a function of casting temperature.

°C have the same structural composition characterized by a predominance of random coils. The range 30-50 °C reveals a steep rise in the β-sheet content, accompanied with an equally abrupt lowering of the random coil fraction. However, at higher temperatures, the content of β-sheets drops down and that of random coils increases. The fraction of R-helixes is nearly independent of the casting temperature, except a relatively slight decrease at 50 °C. Thus, the quantitative data confirm the conclusion of the preceding analysis that the surface β-sheet crystallization is maximum at the casting temperature of 50 °C. At the same time, a new feature is revealed. The transformation to the β-sheet structure occurs due mostly to random coil f β-sheet conformational transition, whereas the fraction

of R-helixes changes only slightly. Hence, the maximum in the β-sheet content is accompanied with a minimum in the content of random coils. Returning to the beginning of this section, we recall that the comparison of FTIR-ATR spectra obtained with Ge IRE (Figure 1a) and KRS-5 IRE (Figure 1b) indicated clearly that the surface content of β-sheets was systematically lower than their bulk content. However, no conclusion could be made with regard to the surfaceversus-bulk fraction of R-helixes because of the spectral distortion of the amide I band measured with KRS-5 IRE. Therefore, the bulk structure was analyzed additionally by transmission FTIR. To this end, a thinner fibroin film (∼6 µm, i.e., still in the thick film limit) was cast at 22 °C to allow for reliable transmission FTIR measurements in the amide I region. The FTIR-ATR spectra of this film (data not shown) were similar to those of the thick film cast at the same temperature, indicating no effect of the reduced film thickness on the surface and bulk structure. The original and curve-fitted transmission FTIR spectra of the 6 µm thick film are shown in Figure 5. Table 2 compares the bulk fractions of R-helixes, β-sheets, and random coils derived from the transmission spectrum with the surface fractions obtained from the corresponding ATR (Ge-45°) spectrum. It is seen that the surface fraction of not only β-sheets but also R-helixes is lower than the corresponding bulk fraction. Contact Angle Study. Table 3 summarizes the surface free energy components and parameters for the test liquids used in the present study. The acid-base parameters shown are based on the acid-base scale γL+/γL- ) 1 for water as a reference liquid. The data for polar liquids (water, glycerol, and ethylene glycol) are ones that have been commonly used in the solid surface energy determination. The data for methylene iodide and R-bromonaphthalene, unlike those used in the absolute majority


Langmuir, Vol. 17, No. 23, 2001 7411

Figure 5. Original (upper trace) and curve-fitted transmission FTIR spectra of 6 µm thick fibroin film cast at 22 °C. Table 2. Near-Surface and Bulk Fractions of r-Helix, β-Sheet, and Random Coil Structures in the Silk Fibroin Film Cast at 22 °C near-surface fraction bulk fraction

R-helix

β-sheet

random coil

0.18 0.23

0.13 0.22

0.44 0.31

Figure 6. Contact angles of test liquids on the air (open symbols) and substrate surfaces (filled symbols) of cast fibroin films as a function of casting temperature.

Table 3. Surface Tension Components and Lewis Acid-Base Parameters (in mJ/m2) of Liquids Used for Contact Angle Measurements liquid

γL+

γL-

γLAB

γLLW

γL

water (W) glycerol (GL) ethylene glycol (EG) methylene iodide (MI) R-bromonaphthalene (BN)

25.5 3.92 2.8 0.72 0.39

25.5 57.4 30.1 0 0.48

51.0 30.0 18.2 0 0.60

21.8 34.0 29.8 50.8 44.0

72.8 64.0 48.0 50.8 44.6

of previous works on the subject, take into account the slight polar character of these liquids, as reflected in the nonzero values of acid-base parameters.25 The generally accepted strategy of neglecting the polar nature of halogenated hydrocarbons may lead to erroneous and inconsistent results in the surface energy determination of polar solids.26 In Figure 6, the contact angles for the test liquids observed on the free and substrate surfaces of solutioncast fibroin films are plotted versus casting temperature. It can be seen that increasing the casting temperature resulted in a minimum in the contact angle for all the polar liquids used. The minimum was relatively deep for highly polar water and rather shallow for less polar glycerol and ethylene glycol. Importantly, it occurred in the range 40-50 °C, that is, at the casting temperatures where the minimum in the surface content of random coil structures and the maximum in the β-sheet content were observed in the FTIR-ATR study (cf. Figure 4). Furthermore, the contact angles of polar liquids on the air surfaces were always slightly lower than those on the substrate surfaces. The contact angles of virtually nonpolar methylene iodide and R-bromonaphthalene did not change with the casting temperature and, also, did not display any significant difference in the values between the air and substrate surfaces. Surface Energy from LW/AB Method. The contact angle values for water, glycerol, and methylene iodide were used to calculate the surface energy characteristics, (25) Janczuk, B.; Wojcik, W.; Zdziennicka, A. J. Colloid Interface Sci. 1993, 157, 384. (26) Tretinnikov, O. N. J. Colloid Interface Sci. 2000, 229, 644.

Table 4. Surface Free Energy Components and Parameters (in mJ/m2) for the Air Surface of Fibroin Films Cast at 22 and 50 °C, Calculated Using the Contact Angles for Water, Glycerol or Ethylene Glycol, and Methylene Iodide casting temp (°C) 22 50

liquids

γS+

γS-

γSAB

γSLW

γSTOT

W-GL-MI W-EG-MI W-GL-MI W-EG-MI

1.9 2.0 1.7 1.4

12.0 11.8 19.6 21.4

9.5 9.6 11.6 11.0

30.0 30.1 30.8 30.5

39.5 39.7 42.4 41.5

γSLW, γS+, and γS-, of the film surfaces. No appreciable variation in the calculated values was observed when glycerol was replaced with ethylene glycol in the liquid trio. This is exemplified in the respective data for the air surface of films cast at 22 and 50 °C in Table 4. The data also reveal that the basic parameter γS- is more sensitive to the surface changes introduced by the change of casting temperature than the acid-base component γSAB. Thus, although the difference in the values of γS- between the two surfaces is 80%, the corresponding values of γSAB differ only by 20%. This is because the γSAB component is influenced by the values of both the acidic and basic parameters and the former (γS+) shows no appreciable change with casting temperature. The calculated values of γSLW, γS+, and γS- for the air and substrate surfaces of fibroin films are plotted versus casting temperature in Figure 7. Within the experimental accuracy, the γSLW component was similar for both surfaces and independent of the casting temperature. The γSLW values were in the range of 30.2 ( 0.8 mJ/m2. The temperature dependence of the basic parameter γSdisplayed a pronounced maximum at 40-50 °C for both the air and substrate surfaces. Besides, the γS- values of the free surface were systematically higher than those of the substrate surface. The acidic parameter γS+ somewhat varied with the casting temperature and the contacting medium; however, the variations were in the limits of experimental error. The obtained values of γS+ (1.6 ( 0.4 mJ/m2) turned out to be by an order of magnitude lower than those of the basic parameter (∼10-20 mJ/m2, depending on the casting temperature). This predominant

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Figure 7. Surface free energy components for the air (open symbols) and substrate surfaces (filled symbols) of cast fibroin films as a function of casting temperature.

basicity of fibroin surfaces cannot be explained on the basis of known acid-base properties of individual amino acids constituting the protein.27 Most likely, we deal here with an imperfection of the LW/AB method which is known to overestimate the surface basicity at the expense of underestimating the acidic component.28 This feature of the method does not allow the acidic and basic components of a given surface to be compared directly. However, it allows one to compare the acidic or basic components of different surfaces. It implies also that the observed variation of the basic parameter should be regarded as a change in the surface polarity in the whole rather than just in the surface basicity. Molecular Interpretation. The contact angle results of the present study revealed that the surface wettability of cast fibroin films was affected by the temperature of film casting. That is, increasing the casting temperature resulted in a minimum in the contact angle (a maximum in wettability) for the polar liquids used, which occurred in the temperature range 40-50 °C (Figure 6). The highest amplitude of the contact angle variation was observed with water and amounted to ∼10°. Since the magnitude is apparently small, some might have supposed that the effect of casting temperature on wettability is not worthy of discussion. However, the wettability change is associated with a nearly 2-fold increase in the surface polarity (as reflected in the variation of the γS- parameter in Figure 7), which is certainly significant. Furthermore, the region of contact angles is also important. Specifically, a water contact angle (θW) of 65° has recently been shown to represent a boundary dividing the solid surfaces into hydrophobic and hydrophilic ones based on their water wettability.29 Then, one can see immediately in Figure 6 that the surfaces of fibroin films cast at 40-50 °C are hydrophilic (θW ) 60-64° < 65°), whereas those of the films cast at temperatures outside the 40-50 °C region are hydrophobic (θW ) 68-71° > 65°). (27) Spange, S.; Schmidt, C.; Kricheldorf, H. R. Langmuir 2001, 17, 856. (28) Berg, J. C. In Wettability; Berg, J. C., Ed.; Dekker: New York, 1993; p 75. (29) Vogler, A. V. Adv. Colloid Interface Sci. 1998, 74, 69.


The wettability change of a multifunctional polymer is known to be associated primarily with the change in surface functional-group composition and/or surface hydrogen-bonding interactions, resulting from the structural reorganizations of macromolecules in the vicinity of the polymer surface.30,31 The fact that the pattern of wettability change in the films studied (Figure 6) resembles closely that of structural alterations in the near-surface region of the films (Figure 4) clearly indicates that the above mechanism is indeed operative in this system. The fact that the hydrophilic protein produces hydrophobic film surfaces (on casting at ambient temperatures) indicates that during film formation the fibroin macromolecules in the vicinity of the surface take on a surfaceactive conformation which allows them to maximize the surface exposure of hydrophobic groups by surface orientation and/or to minimize the surface content of dangling (non-hydrogen-bonded) polar groups by optimizing the intra- and interchain hydrogen-bonding interactions, in a drive to minimize the free energy at the polymer-air and polymer-hydrophobic substrate interfaces. To adopt this specific structure, the protein macromolecule must possess a high degree of conformational freedom on the level of neighboring amino acid residues. The latter is intrinsic to the fibroin random coils. On the contrary, the conformation of peptide sequences involved in R-helixes or β-sheets is fixed. Furthermore, R-helix and β-sheet structures of silk fibroin, formed in the bulk crystallization, do not exhibit surfactancy; that is, they cannot separate the hydrophilic and hydrophobic residues to opposite sides of the interface.14 Therefore, the surface-active conformation of the fibroin macromolecule is achievable only via the conformational reorganization of random coils. Accordingly, random coils must be energetically favored at the interface and, consequently, enriched in the nearsurface region of cast films. Indeed, the surface enrichment of random coil structures was observed by FTIR spectroscopy (Table 2). On the basis of the same arguments, the abrupt transition from the hydrophobic to hydrophilic surface state, occurring between 30 and 40 °C (Figure 6), can be readily explained by the abrupt decrease in the surface content of (potentially) surface-active random coil structures due to the random coil f β-sheet transition taking place at the same temperatures (Figure 4). The FTIR-ATR and contact angle data indicate that the β-sheet crystallization originating from the bulk has a profound influence on the surface structure and, hence, on the surface wettability when the casting temperature is in the range 40-50 °C. However, higher temperatures of casting render the film surfaces again hydrophobic, as is evident from the contact angle results for the casting at 70 °C. This reversal of wettability occurs concurrently with a marked increase in the surface content of random coils and, thus, is in full accordance with the surface structure-wettability relationship introduced above. We believe that at casting temperatures above 50 °C the bulk thermodynamic force driving the formation of β-sheet crystals weakens and the effect of the interface favoring the random coil structures becomes dominant. It is also likely that the surface equilibrium between the two structural forms is much less stable than the bulk equilibrium. This could account for the fact that the formation of β-sheets at the surface becomes thermodynamically hindered at 70 °C while in the bulk it is still as high as at lower temperatures (Figure 1). Generally speaking, the wettability change of the cast fibroin film (30) (a) Tretinnikov, O. N. Langmuir 1997, 13, 2988 and references therein. (b) Tretinnikov, O. N.; Ikada, Y. Langmuir 1994, 10, 1606. (31) Tretinnikov, O. N.; Ikada, Y. Macromolecules 1997, 30, 1086.


with the casting temperature indicates the importance of bulk thermodynamics in determining the equilibrium surface state in this system and, as such, represents an example of the integration of bulk and interfacial properties in polymeric materials.32 In the conclusion of this section, we briefly consider the systematically higher hydrophobic content (higher contact angle of polar liquids) on the substrate surfaces as compared to the air surfaces of the films (Figure 6).33 The surface exposure of hydrophobic groups at the proteinair interface occurs due solely to the missing neighbor effect;34,35 polar (hydrophilic) groups tend to escape into the bulk, since they are energetically more penalized for the lack of neighbors at the surface in comparison with nonpolar (hydrophobic) groups. At the protein-substrate interface, there is still a missing neighbor effect driving the polar groups away from the surface and, additionally, an attraction force between the hydrophobic PS substrate and the hydrophobic moieties of protein, driving them to segregate at the surface. Thus, it is the hydrophobic attraction imposed by the substrate that renders the contacting polymer surface more hydrophobic than the free (air) surface. Possible Surface Conformation of Fibroin. According to the above molecular considerations, the experimental data of this study indicate that during formation of fibron films, R-helix and β-sheet structures are rejected by the interface because of their non-surfaceactive character, whereas random coils are favored because at the interface they convert into a surface-active conformation which effectively minimizes the interfacial free energy and renders the polymer surface hydrophobic. Though with the use of ATR spectroscopy we could observe the accumulation of the “precursory” conformation in the near-surface region, it is clearly impossible to identify the (32) (a) Carey, D. H.; Grunzinger, S. J.; Ferguson, G. S. Macromolecules 2000, 33, 8802. (b) Khongtong, S.; Ferguson, G. S. J. Am. Chem. Soc. 2001, 123, 3588. (33) Surface roughness could not be a source of the observed difference in the wettability of opposite film surfaces by polar liquids, because the surfaces were microscopically smooth and, furthermore, no such a difference was observed with the nonpolar liquids (Figure 6). (34) Mu¨ller, M.; Binder, K. Macromolecules 1998, 31, 8323. (35) Budkowski, A.; Rysz, J.; Scheffold, F.; Klein, J.; Fetters, L. J. J. Polym. Sci., Part B: Polym. Phys. 1998, 36, 2691.

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protein conformation at the surface because of the limited surface sensitivity of the method. Nevertheless, the unique composition and amino acid sequence of fibroin allows some reasonable speculations on this matter. Silk fibroin is composed predominantly of glycine, alanine, and serine (∼85% in total) in a rough 3:2:1 ratio, and the rest is represented by amino acids with large side chains. The structure is dominated by highly repetitive, crystallizable [Gly-Ala-Gly-Ala-Gly-Ser]n sequences, with corresponding side groups of H, CH3, H, CH3, H, and CH2OH, which are broken by less regular sequences of the bulky amino acids and Gly-Ala or Gly-Ser repeats.1,14,36 From this structure and taking into account that the content of amino acids with bulky hydrophobic side chains (Pro, Phe, Leu, Val) is rather low (

Influence of Casting Temperature on the Near-Surface Structure and

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