Silk Fibroin Thin Films with

Feb 16, 2010 - †School of Chemical Engineering, University of Campinas, UNICAMP, P.O. ... Brazil, and ‡Department of Materials Science and Enginee...
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Layer-by-Layer Deposited Chitosan/Silk Fibroin Thin Films with Anisotropic Nanofiber Alignment Grinia M. Nogueira,† Albert J. Swiston,‡ Marisa M. Beppu,† and Michael F. Rubner*,‡ †

School of Chemical Engineering, University of Campinas, UNICAMP, P.O. Box 6066, 13083-970 Campinas-SP, Brazil, and ‡Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received December 16, 2009. Revised Manuscript Received January 20, 2010 Chitosan/silk fibroin multilayer thin films were assembled using layer-by-layer deposition. The resultant multilayer films contained nanofibers aligned parallel to the dipping direction. Fiber deposition and orientation was enabled uniquely by a judicious choice of solvent and drying conditions and layer-by-layer assembly with chitosan. The deposition of oriented nanofibers was found to be the result of a unique combination of layer-by-layer and Langmuir-Blodgett type processing. Fiber orientation was confirmed by fast Fourier transform (FFT) analysis of optical micrographs and atomic force microscopy (AFM). Bidirectional fiber alignment was realized by rotating the substrate between multilayer deposition steps. Infrared spectroscopy revealed that the silk fibroin adopted the silk II secondary structure in the deposited films. We anticipate that these anisotropic films are able to combine the biocompatibility of a natural polymer system with the mechanical strength of SF, two properties useful in many biological applications including scaffolds suitable for guiding cell attachment and spreading.

Introduction The layer-by-layer (LbL) assembly method usually consists of the alternate deposition of oppositely charged polyelectrolytes.1 However, LbL assembly may be based on other interactions such as hydrogen bonds, covalent bonds,2 and hydrophobic interactions.3 LbL processing has been used by a number of groups to produce uniform, conformal thin films on several different types of substrates. These thin films have been explored for use in a number of technologies, including optical,4 wetting,5,6 antifogging,7 and biological applications.8,9 The applications considered to date, however, generally require uniform films over the entire substrate since the properties of interest do not rely on a specific spatial orientation of the constituent molecules in the plane of the film. Postdeposition modifications, such as stamping,10,11 have been used to introduce spatial property fluctuations, but to our knowledge there are no reports on controlling in-plane macroscopic orientation and properties during film deposition. LbL thin films with molecules adopting a specific and controllable orientation during deposition are highly desirable, as it would allow for anisotropic mechanical, electrical, and optical properties. *Corresponding author: Ph 617-253-4477; Fax 617-258-7874; e-mail [email protected]. (1) Decher, G.; Schlenoff, J. B. Mutilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Wiley-VCH: New York, 2003. (2) Zhang, Y.; Yang, S.; Guan, Y.; Cao, W.; Xu, J. Macromolecules 2003, 36, 4238–4240. (3) Kotov, N. A. Nanostruct. Mater. 1999, 12, 789–796. (4) Zhizhong Wu, D. L. M. R. R. C. Small 2007, 3, 1467. (5) Su, B.; Li, M.; Shi, Z.; Lu, Q. Langmuir 2009, 25, 3640–3645. (6) Zhai, L.; Berg, M. C.; Cebeci, F. C.; Kim, Y.; Milwid, J. M.; Rubner, M. F.; Cohen, R. E. Nano Lett. 2006, 6, 1213–1217. (7) Cebeci, F. C.; Wu, Z.; Zhai, L.; Cohen, R. E.; Rubner, M. F. Langmuir 2006, 22, 2856–2862. (8) Lee, D.; Cohen, R. E.; Rubner, M. F. Langmuir 2005, 21, 9651–9659. (9) Swiston, A. J.; Cheng, C.; Um, S. H.; Irvine, D. J.; Cohen, R. E.; Rubner, M. F. Nano Lett. 2008, 8, 4446–4453. (10) Berg, M. C.; Choi, J.; Hammond, P. T.; Rubner, M. F. Langmuir 2003, 19, 2231–2237. (11) Jiang, X.; Zheng, H.; Gourdin, S.; Hammond, P. T. Langmuir 2002, 18, 2607–2615.

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Most natural materials have directional mechanical and functional characteristics by preferentially presenting different phases. Examples of such systems include bone, nacre, and bamboo, all of which are extremely mechanically robust composites comprised of individually weak components. A great deal of research has focused on how to reproduce these eloquent natural behaviors in vitro using synthetic materials. Chitosan (CHI) is a polysaccharide obtained from the deacetylation of chitin, a biopolymer found in many crustacean shells. Soluble CHI is a polycation below the pKa of its primary amines (∼6.5); as the pH increases, deprotonation of these amines leads to a water-insoluble polymer and subsequent precipitation.12 LbL processing can be used to form CHI thin films based on hydrogen bonds or electrostatic interactions with another polymer. Studies have focused on using these films to improve surface biocompatibility or control cell interactions.13-16 Silk fibroin (SF) is a fibrous protein found in natural silk. SF molecules consist of a heavy (∼350 kDa) and light (∼25 kDa) chain connected by disulfide bonds.17 The heavy chain consists of repetitive Gly-Ala-Gly-Ala-Gly-Ser domains. While a considerable number of hydrophobic residues (Ala) limit water solubility, the heavy chain also contains hydroxyl residues (Ser and Tyr) that can promote water affinity. In addition, the heavy chain contains the ionizable residues Glu and Asp near the chain ends. The light chain does not have repetitive regions but is characterized by a high content of Glu and Asp residues.18 SF can be found in two (12) Payne, G. F.; Raghavan, S. R. Soft Matter. 2007, 3, 521–527. (13) Cai, K.; Hu, Y.; Jandt, K. D. J. Biomed. Mater. Res., Part A 2007, 82A, 927–935. (14) Croll, T. I.; O0 Connor, A. J.; Stevens, G. W.; Cooper-White, J. J. Biomacromolecules 2006, 7, 1610–1622. (15) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Langmuir 2004, 20, 448–458. (16) Sionkowska, A.; Wisniewski, M.; Skopinska, J.; Poggi, G. F.; Marsano, E.; Maxwell, C. A.; Wess, T. J. Polym. Degrad. Stab. 2006, 91, 3026–3032. (17) Tanaka, T.; Ohnishi, S.; Yamaura, K. Polym. Int. 1999, 48, 811–818. (18) Hossain, K. S.; Ohyama, E.; Ochi, A.; Magoshi, J.; Nemoto, N. J. Phys. Chem. B 2003, 107, 8066–8073.

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major secondary structures, identified as silk I and silk II. Silk I is a metastable conformation composed of random coils and R-helix structures.19,20 Silk II is primarily comprised of antiparallel β-sheets. Silk I can be converted to silk II by heating, mechanical deformation, or alcohol treatments.20,21 The LbL method can be used to assemble SF films with good mechanical properties and biocompatibility.13,22-25 The thin film assembly of SF by stepwise deposition was first investigated by Wang et al.25 SF films were built by successive deposition of SF on solid substrates; these films were stable at physiological conditions and supported adhesion and growth of human bone marrow stem cells, suggesting that they could be used to functionalize biomaterial surfaces. Cai et al.13 modified titanium surfaces with LbL-deposited SF and CHI films. The adherence and growth of osteoblast cells suggested this coating could significantly improve cellular compatibility of titanium surfaces. The mechanical properties of SF multilayer thin films were investigated by Jiang et al.22 These multilayer films presented a high elastic modulus of 6-8 GPa and ultimate tensile strengths up to 100 MPa. The authors claim that such films could be used in microscale biodevices, biocompatible implants, and coatings for artificial skin. The goal of this work was to create anisotropic CHI/SF LbL films on a solid substrate. The deposition method creates fibers aligned with the dipping direction, which can be changed by rotating the substrate during deposition, thus allowing for uni- to bidirectional fiber orientation. Cai et al.13 reported the deposition of CHI/SF films on titanium surfaces; however, these authors reported no fiber orientation. Usually, nonwoven SF fibers are made by electrospinning SF solutions onto a solid support.26,27 Recently, nonorientated nanofiber growth by self-assembly of a SF-like protein on mica substrates was reported by Hwang et al.28 These authors demonstrated that nanofiber formation is highly influenced by ionic strength, polymer concentration, and surface characteristics. Constructing controllably anisotropic thin films during LbL deposition offers new possibilities in the development of functional biomaterial interfaces. For instance, aligned fibers could act as a guide for adherent cell attachment, growth, and spreading, when the control of cell orientation (typical in biomimetic processes) is necessary to mimic the natural conditions where orientation determines tissue function.29-31 Anisotropic CHI/SF films could also be used as a biomaterial coating for bone regeneration. SF-derived materials show high (19) Hu, X.; Kaplan, D.; Cebe, P. Macromolecules 2006, 39, 6161–6170. (20) Yamada, K.; Tsuboi, Y.; Itaya, A. Thin Solid Films 2003, 440, 208–216. (21) Taketani, I.; Nakayama, S.; Nagare, S.; Senna, M. Appl. Surf. Sci. 2005, 244, 623–626. (22) Jiang, C. Y.; Wang, X. Y.; Gunawidjaja, R.; Lin, Y. H.; Gupta, M. K.; Kaplan, D. L.; Naik, R. R.; Tsukruk, V. V. Adv. Funct. Mater. 2007, 17, 2229– 2237. (23) Mandal, B. B.; Mann, J. K.; Kundu, S. C. Eur. J. Pharm. Sci. 2009, 37, 160– 171. (24) Wang, X. Y.; Hu, X.; Daley, A.; Rabotyagova, O.; Cebe, P.; Kaplan, D. L. J. Controlled Release 2007, 121, 190–199. (25) Wang, X. Y.; Kim, H. J.; Xu, P.; Matsumoto, A.; Kaplan, D. L. Langmuir 2005, 21, 11335–11341. (26) Ayutsede, J.; Gandhi, M.; Sukigara, S.; Micklus, M.; Chen, H. E.; Ko, F. Polymer 2005, 46, 1625–1634. (27) Jin, H. J.; Fridrikh, S. V.; Rutledge, G. C.; Kaplan, D. L. Biomacromolecules 2002, 3, 1233–1239. (28) Hwang, W.; Kim, B.-H.; Dandu, R.; Cappello, J.; Ghandehari, H.; Seog, J. Langmuir 2009, 25, 12682–12686. (29) Badami, A. S.; Kreke, M. R.; Thompson, M. S.; Riffle, J. S.; Goldstein, A. S. Biomaterials 2006, 27, 596–606. (30) Liang, D.; Hsiao, B. S.; Chu, B. Adv. Drug Delivery Rev. 2007, 59, 1392– 1412. (31) Ma, Z, K. M.; Inai, R.; Ramakrishna, S. Tissue Eng. 2005, 11, 101–109.

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potential to deposit calcium phosphate crystals during in vitro and in vivo experiments.32-34 These calcium phosphate deposits are precursors of hydroxyapatite, one of the main components of bone.35,36 A CHI/SF fiber coating could concurrently improve biocompatibility and encourage the controllable growth (via film surface chemistry and morphology) of inorganic crystals in a wellprescribed direction.

Materials and Methods Biopolymer Solutions. Silk fibers (Lot: BR08B) were obtained from Bratac S.A. (S~ao Paulo-Brazil). To remove sericin, fibers were degummed three times in an aqueous Na2CO3 (1 g/L) solution for 20 min at 80 °C, rinsed in Milli-Q water, and dried at 50 °C. Purified SF was dissolved in LiBr-EtOH-H2O (45:44:11 weight ratio) at 90 °C until total dissolution to a final concentration of 20 g/L.37 The SF solution was dialyzed for 3 days to remove salts and then diluted to 0.1 g/L in Milli-Q water. The pH was measured as 6.8 after dialysis and dilution of SF solution. Chitosan (Aldrich, low molecular weight) was dissolved in a 0.25 M sodium acetate and 0.25 M acetic acid solution to a concentration of 1 g/L and then filtered using a 0.45 μm membrane filter. The solution pH was adjusted to 6.0 or 4.0 using 1 M NaOH solution. Multilayer Deposition. Silicon substrates were immersed in CHI solutions for 10 min followed by two rinse steps in Milli-Q water under gentle agitation for 2 and 1 min. Substrates were then immersed in SF solutions for 10 min and rinsed as previously described. An optional 5 min drying step after each biopolymer deposition, using room temperature airflow, was applied to favor fibroin deposition.25 To build bidirectional fibers on Si substrates, two dipping processes were used: the first dipping process was done as described after which the substrates were rotated 90°, and a second dipping process was performed on the already-deposited film. For these bidirectional fiber experiments, pH 6.0 CHI was used exclusively. A room temperature drying step was performed between the two dipping processes. Figure 1 is a schematic of the dipping process used to construct bidirectional fibers. CHI/SF films with bidirectional fibers were built with 20 bilayers in the first dipping process and 10 or 20 bilayers in the second. Samples are labeled as (CHIa/SF)bc, where a is the pH of the chitosan solution, and b and c are the number of bilayers in the first and second dipping process, respectively. One bilayer is defined as a pair of CHI and SF layers after each cycle of film deposition. Film Characterization. Film thickness was measured by variable-angle spectroscopic ellipsometry operating at a 70° angle of incidence. Infrared spectroscopy on ZnSe substrates was used to determine silk fibroin’s secondary structure in a (CHI6.0/SF)20 film. Fiber alignment and orientation were observed using optical microscopy on a Zeiss Axioplan 2 upright microscope and confirmed using fast Fourier transform (FFT) of the micrographs. For FFT analyses, the Image-J software package was used. Atomic force microscopy (AFM) was performed using a Digital Instruments D3000 scanning probe microscope operating in tapping mode.

Results and Discussion CHI/SF Properties Depend on Solution pH and Drying between Layer Depositions. The physical and chemical (32) Fini, M.; Motta, A.; Torricelli, P.; Glavaresi, G.; Aldini, N. N.; Tschon, M.; Giardino, R.; Migliaresi, C. Biomaterials 2005, 26, 3527–3536. (33) Kong, X. D.; Cui, F. Z.; Wang, X. M.; Zhang, M.; Zhang, W. J. Cryst. Growth 2004, 270, 197–202. (34) Nogueira, G. M.; Aimoli, C. G.; Weska, R. F.; S., N. L.; Beppu, M. M. Key Eng. Mater. 2008, 361-363, 503–506. (35) Elliott, J. C. Structure and Chemistry of the Apatites and Other Calcium Orthophosphates; Elsevier Science: Amstersdam, 1994. (36) Forsen, S.; Kordel, J. Calcium in Biological Systems; John Wiley: New York, 1997. (37) Chen, X.; Knight, D. P.; Shao, Z. Z.; Vollrath, F. Polymer 2001, 42, 9969– 9974.

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Figure 2. Optical microscope images of (a) (CHI4.0/SF)20 and (b) (CHI6.0/SF)20 LbL films.

Figure 1. (a) Schematic of the CHI/SF multilayer film preparation process and (b) the two-step method used to control fiber alignment.

properties of polyelectrolyte multilayer films as a function of deposition conditions have been studied extensively.38,39 For weak polyelectrolytes, the pH of the deposition solution strongly influences film thickness, composition, and morphology. A processing parameter that has not been studied as much is the use of various drying conditions between polymer deposition cycles. Since it has been shown that drying SF during stepwise deposition favors the formation of β-sheet structures,25 we investigated how drying a multilayer film containing SF and CHI would change film properties. Film thickness and morphology as a function of the chitosan solution pH and a drying step between deposition cycles were analyzed. As anticipated, the pH of the chitosan solution influenced the thickness and morphology of the deposited films. Films deposited using CHI solutions at pH 4.0 were thicker than those deposited using CHI solutions at pH 6.0. However, biopolymer agglomerates were seen on films deposited using CHI at pH 4.0 (see Figure 2). At pH values well below CHI’s pKa, CHI is fully charged, thereby favoring film deposition since CHI and SF can interact both by hydrogen bonds and electrostatic interactions.13,40 Dialyzed SF fibroin solutions are metastable and form gels and aggregates, mainly under mechanical agitation and at high temperature.41 Low pH values favor the formation of these gels and aggregates,42,43 making multilayer assembly at pH 4.0 prone to the formation of inhomogeneous films. In sharp contrast, multilayer films assembled with the CHI solution at pH 6.0 exhibited a unique fibrous morphology with nanofibers oriented in the dipping direction. Optical microscope images for (CHI4.0/SF)20 and (CHI6.0/SF)20 multilayer films deposited on Si substrates are shown in Figure 2. Fiber orientation is present (38) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213–4219. (39) Zhu, T.; Fu, X.; Mu, T.; Wang, J.; Liu, Z. Langmuir 1999, 15, 5197–5199. (40) Chen, X.; Li, W. J.; Zhong, W.; Lu, Y. H.; Yu, T. Y. J. Appl. Polym. Sci. 1997, 65, 2257–2262. (41) Wang, H.; Zhang, Y. P.; Shao, H. L.; Hu, X. C. Int. J. Biol. Macromol. 2005, 36, 66–70. (42) Kim, U. J.; Park, J. Y.; Li, C. M.; Jin, H. J.; Valluzzi, R.; Kaplan, D. L. Biomacromolecules 2004, 5, 786–792. (43) Matsumoto, A.; Chen, J.; Collette, A. L.; Kim, U. J.; Altman, G. H.; Cebe, P.; Kaplan, D. L. J. Phys. Chem. B 2006, 110, 21630–21638.

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Figure 3. Thicknesses of (a) (CHI6.0/SF)20 with drying step, (b) (CHI6.0/SF)20 without drying step, (c) (CHI4.0/SF)20 with drying step, and (d) (CHI4.0/SF)20 without drying step on Si substrate as determined by ellipsometry. Inset shows the growth profile of the (CHI6.0/SF) film with a drying step.

only in (CHI6.0/SF)20 multilayers, whereas (CHI4.0/SF)20 films are comprised of nonaligned fibers and aggregates. Drying between deposition cycles significantly increased film thickness by ∼38% for (CHI6.0/SF)20 and ∼83% for (CHI4.0/ SF)20, as showed in Figure 3. Besides the increase in film thickness, the drying step seems to favor the deposition of SF nanofibers, since just a few isolated fibers were observed on (CHI6.0/SF)20 films with no drying step during the dipping process. The effect of drying during SF deposition is known to induce a β-sheet conformation, which stabilizes the film and discourages dissolution in subsequent deposition steps.25 A growth curve for the (CHI6.0/SF) multilayer fabricated with the drying step is shown in Figure 3 (inset). Film growth is linear between 10 and 40 bilayers. The refractive index for a 10-bilayer film was 1.53, a value similar to that obtained from other SF films.22 Because of the high heterogeneity of the (CHI4.0/SF) films and the unique aligned nanofiber morphology observed with (CHI6.0/SF) multilayers, further characterization was performed only for (CHI6.0/SF) films prepared with the drying step. The FTIR spectrum of a (CHI6.0/SF)20 multilayer film is shown in Figure 4. Both silk I and II structures were observed in the deposited films. The peak associated with aliphatic amino DOI: 10.1021/la904741h

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Figure 4. FTIR spectrum of (CHI6.0/SF)20 on a ZnSe substrate prepared with a drying step between layer depositions.

Figure 5. Optical microscope images of unidirectional fiber alignment in a (CHI6.0/SF) multilayer film deposited on Si with (a) 10, (b) 20, (c) 30, and (d) 40 bilayers.

groups is attributed to CHI. The presence of β-sheets (silk II) may be attributed to film dehydration during the drying step25 and/or hydrogen bonds between SF and CHI molecules.44 In general, postdeposition treatments are used to stabilize SF films by changing the secondary structure from silk I to silk II. Thermal or alcohol treatments are two common techniques used to induce this conformational transition. However, in this multilayered system, β-sheets are observed as deposited, thereby making postdeposition treatments to stabilize the films unnecessary. SF Fiber Deposition and Orientation. A unique characteristic of the (CHI6.0/SF) multilayer system is the presence of nanofibers on the substrate, oriented parallel to the dipping direction (see Figure 5). Typical fibril diameters are about 190 nm. Although many authors have investigated the multilayering of SF films, the LbL deposition of oriented SF fibers has not been reported. Comparing our experimental procedure with those reported by Wang et al.,25 Jiang et al.,22 and Cai et al.,13 we note that there are differences in the solvent system used to dissolve the SF and in the film assembling method. Wang et al. and Jiang et al., for instance, dissolved SF with a similar solvent system but did not use CHI to facilitate film deposition. In contrast, Cai et al. used a different solvent for SF (a CaCl2 ternary solvent), and CHI was used to build the multilayer films. From these observations, we suggest that both the SF solvent (44) Chen, X.; Li, W.; Yu, T. J. Polym. Sci., Part B: Polym. Phys. 1997, 35, 2293– 2296.

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Figure 6. Optical microscope images and FFT patterns (inset) of (a) (CHI6.0/SF)200, (b) (CHI6.0/SF)2010, and (c) (CHI6.0/SF)2020 films on Si, where t is the film thickness as determined by ellipsometry.

system and the use of CHI in the multilayer assembly process strongly influence the formation and deposition of SF fibers during film assembly. Optical microscope images of unidirectionally deposited (CHI6.0/SF) films made with intermediate drying steps are shown in Figure 5. The observed density of fibers increases with the number of deposited bilayers. In the lower limit of 10 bilayers, fiber deposition is observed, but the surface is not uniformly covered by these fibers. Aligned fibers are observed in multilayer films containing up to 40 bilayers. We hypothesize that chitosan is critical for the creation of multilayers with oriented SF fibers without the need for any postdeposition modifications. Indeed, when silk fibroin was deposited on Si under identical deposition conditions but without alternating layers of chitosan, no fibers were observed. It is known that SF can be deposited via a process similar to LbL but without the use of a cationic partner.25 In this case, a drying process, addition of salts to a SF solution, or rinsing with a methanol/water solution was utilized to favor the multilayer film deposition. Such SF films were ∼20 nm thick when 6 layers were deposited using a drying step and without adding salts to the SF solution. Addition of salts, the use of a drying step, and methanol/ water rinsing favored the deposition of SF; however, the authors did not report the presence and orientation of fibers in any of the conditions analyzed. This reinforces our conclusion that multilayer assembly with chitosan is needed to create thin films with aligned nanofibers. Langmuir 2010, 26(11), 8953–8958

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Figure 7. AFM tapping-mode height images of (a) (Chi6.0/SF)200, (b) (Chi6.0/SF)2010, and (c) (Chi6.0/SF)2020 films on Si.

In our case, no fibers were found when a CHI/SF film was deposited using a different ternary solvent (CaCl2-EtOH-H2O 1:2:8 molar ratio) for SF. Moreover, film thicknesses were greater (∼40% higher) for this CaCl2 ternary solvent as compared to the LiBr ternary solvent. These results show that the solvent system strongly influences SF fiber formation and deposition. Bivalent ions, such as calcium, are more effective in dissolving SF molecules based on the “salting-in” effect;45 therefore, it is expected that bivalent cationic solutions induce a higher solvation of SF fibers. Intermolecular hydrogen bonds between SF and CHI may be also encouraging the creation and/or deposition of these fiber structures, and the drying process would induce film dehydration and consequently β-sheet formation.25 Hydrogen bonds between CHI and SF molecules were reported by Chen et al.40 These authors observed by infrared spectroscopy that hydrogen bonds occur mainly between the amino group in CHI and the amide group in SF and that these interactions are sensitive to pH, breaking down in acidic conditions. In another paper, Chen et al.44 described the conformational transition of SF induced by blending with CHI. A mechanism of the conformation transition was suggested: SF chains use the rigid CHI chains as a template to form a β-sheet structure according to the strong hydrogen bonding between CHI and SF. In the β-sheet structure, hydrogen bonds are formed between adjacent segments of the SF polypeptide chains that are aligned side by side in a parallel or antiparallel direction.46 Having dissolved molecules or fibrils in solution depends on the solvation strength of the medium. In the LiBr ternary solvent system, fine, partially solvated fibrils have been found to pre-exist in the solution and may precipitate on the solution surface by forming a thin solid film.47 These fibrils may assemble onto the CHI surface during deposition and become further stabilized during rinsing and drying steps. In contrast, SF molecules are strongly solvated in the CaCl2 ternary solvent, precluding the formation of fibrils in the dialyzed solution. It has also been reported by other authors47-49 that thin monolayer films can be formed at the air-water interface of an aqueous SF solution, including films with fibrils. The molecular structure of such Langmuir-Blodgett type monolayers was determined to be a silk III structure;a hexagonal packing of silk molecules in a left-handed 3-fold helical chain conformation, as described by Valluzzi et al.49 This conformation separates the Ser (hydrophilic) and Ala (hydrophobic) residues to the opposite (45) Sashina, E. S.; Bochek, A. M.; Novoselov, N. P.; Kirichenko, D. A. Russ. J. Appl. Chem. 2006, 79, 869–876. (46) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry; Worth Publishers: New York, 2000; Vol. 3. (47) Happey, F.; Hyde, A. J.; B, M. Biopolymers 1967, 5, 749–756. (48) Valluzzi, R.; Gido, S. P. Biopolymers 1997, 42, 705–717. (49) Valluzzi, R.; Gido, S. P.; Muller, W.; Kaplan, D. L. Int. J. Biol. Macromol. 1999, 24, 6.

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side of the helical axis. The silk III molecular structure was determined in the uncompressed state of a Langmuir-Blodgett monolayer; however, this structure can reorient to form the silk II structure when high pressure is applied.49 Muller et al.50 described the formation of SF monolayers at the air-water interface of a Langmuir-Blodgett trough when using SF dissolved in a LiBr solvent. The authors observed a dominance of the silk II molecular structure and attributed this to the drying and mechanical forces present during the LB film compression and transfer process. However, no fiber deposition and/or orientation were reported. Shulha et al.51 reported spontaneous nanofibril formation and agglomeration of SF adsorbed on Si substrates. The fibrils and aggregates were observed over time (1-21 days) by AFM. These results could indicate that in our case fibrils may also be formed on the substrate during dipping. However, we believe that most of the fibers adsorbed on the substrate surface were already in solution, since the contact time of the substrate with the SF solution is relatively short. All of these prior results suggest that in our case monolayer-like films of SF nanofibers are formed at the air-water interface of the SF dipping solutions. These surface supported films can be clearly observed on our assembly solutions made from LiBr. During layer-by-layer assembly, these monolayer films are transferred onto the solid substrate in the form of oriented nanofibers. The previously deposited chitosan molecules play the critically important role of stabilizing and attracting these nanofibers that become aligned by the forces present when the substrate is withdrawn from the dipping solution. Thus, this is a unique combination of layer-by-layer and Langmuir-Blodgett type deposition. Bidirectional Nanofiber Alignment. Bidirectional fiber alignment can be realized by rotating the substrate between multilayer deposition steps (see Figure 1). Optical microscope images for bidirectionally orientated fibers fabricated from (CHI6.0/SF) films are shown in Figure 6. The respective fast Fourier transforms (FFT’s) are found on the top right side of each image. Here, we compare fibers deposited in one direction to fibers bidirectionally aligned. The samples are labeled as (CHI6.0/SF)ab, where a corresponds to the number of bilayers of the first dipping process and b corresponds to number of bilayers of the second dipping process;bidirectional orientation induced by rotating the substrate 90° from the original position (see Figure 1b). The FFT’s clearly display a crosslike pattern that confirms bidirectional fiber orientation in multilayer films of (CHI6.0/SF)1020 and (CHI6.0/SF)2020 when compared to the unidirectional orientation observed in (CHI6.0/SF)200. The bidirectional orientation (50) Muller, W. S.; Samuelson, L. A.; Fossey, S. A.; Kaplan, D. L. Langmuir 2002, 9, 1857–1861. (51) Shulha, H.; Po Foo, C. W.; Kaplan, D. L.; Tsukruk, V. V. Polymer 2006, 47, 5821–5830.

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was also confirmed by an azimuthal average of the FFT images (see Supporting Information). Fiber orientation was also observed via AFM. Figure 7a shows unidirectional-deposited fibers, and Figure 7b shows the bidirectional fibers of (CHI6.0/SF)2010. Figure 7c shows AFM images of (CHI6.0/SF)2020, where fibers oriented in the second dipping direction are mostly observed, most likely due to the nature of AFM probing only the surface. Only one rotation angle was studied in this work, but any angle could be used to create complex crosshatched composites with a customized level of anisotropy.

Conclusion CHI/SF films with oriented nanofibers can be prepared using the LbL method. By adjusting the substrate orientation during deposition, it is possible to control fiber orientation on the film surface. The inherent biocompatibility of these two biopolymers, combined with their excellent mechanical and physical properties, makes the ability to form and control these fibers an attractive candidate for surface functionalization in several biomaterials applications. Such applications may include biomaterial coatings

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to improve biocompatibility of implanted devices, biotemplates for orientation of cell adhesion and growth, and for oriented nucleation and growth of inorganic crystals (calcium phosphates). Mechanical properties and cell compatibility of these films are the subject of ongoing work. Acknowledgment. The authors thank Prof. Robert Cohen, Dr. Adam Nolte, Erik Williamson, and Zekeriyya Gemici for valuable discussions. We acknowledge CAPES-Brazil for funding. This work was supported in part by the MRSEC Program of the National Science Foundation under Award DMR-0819762. This material is based upon work supported under a National Science Foundation Graduate Research Fellowship. Supporting Information Available: Comparison between tapping mode AFM images for CHI/SF films prepared using CaCl2 and LiBr ternary solvent system for SF and the azimuthal angle obtained by FFT of optical microscope images of CHI6.0/SF films. This material is available free of charge via the Internet at http://pubs.acs.org.

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