Factors Controlling the Deposition of Silk Fibroin Nanofibrils during

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Factors Controlling the Deposition of Silk Fibroin Nano-Fibrils During Layer-by-Layer Assembly Mariana Agostini de Moraes, Thomas Crouzier, Michael F. Rubner, and Marisa Masumi Beppu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5012135 • Publication Date (Web): 03 Dec 2014 Downloaded from http://pubs.acs.org on December 4, 2014

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Factors Controlling the Deposition of Silk Fibroin Nano-Fibrils During Layer-by-Layer Assembly

Mariana Agostini de Moraes1,2,3, Thomas Crouzier4, Michael Rubner3 and Marisa Masumi Beppu1*

1

School of Chemical Engineering, University of Campinas, UNICAMP, 13083-852, Campinas – SP, Brazil

2

Department of Exact and Earth Sciences, Federal University of São Paulo, UNIFESP, Diadema – SP, Brazil

3

Department of Materials Science and Engineering, Massachusetts Institute of Technology, MIT, 02139, Cambridge – MA, USA

4

Department of Biological Engineering, Massachusetts Institute of Technology, MIT, 02139, Cambridge – MA, USA

* Corresponding author: [email protected]

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ABSTRACT

The layer-by-layer technique has been used as a powerful method to produce multilayer thin films with tunable properties. When natural polymers are employed, complicated phenomena such as self-aggregation and fibrilogenesis can occur, making characterization and the obtainment of high quality films more difficult. The weak acid and base character of such materials provide multilayer systems that may differ from those found with synthetic polymers due to strong self-organization effects. Specifically, LbL films prepared with chitosan and silk fibroin (SF) often involve the deposition of fibroin fibrils, which can influence the assembly process, surface properties and overall film functionality. In this case, one has the intriguing possibility of realizing multilayer thin films with aligned nanofibers. In this paper, we propose a strategy to control fibroin fibril formation, by adjusting the assembly partner. Aligned fibroin fibrils were formed when chitosan was used as counterpart, while no fibrils were observed when poly(allylamine hydrochloride) (PAH) was used. Charge density, which is higher in PAH, apparently stabilizes SF aggregates at the nanometer scale, thereby preventing their organization into fibrils. The drying step between each layer deposition was also crucial for film formation as it stabilizes the SF molecules. Preliminary cell studies with optimized multilayers indicated that cell viability of NIH-3T3 fibroblasts remained between 90-100% after surface seeding, showing the potential application of the films in the biomedical area, as coatings and functional surfaces.

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1. Introduction The LbL technique is a versatile, low cost process that can be used to prepare ultrathin films with specific nano-architectures. The typical process consists of alternate deposition of oppositely charged polyelectrolytes and often involves electrostatic attraction as the main driving force for adsorption and multilayer buildup. However, interactions such as hydrogen bonds 1, hydrophobic interactions

2

and covalent bonds

3

can also play a major role in the

assembly process. One of main advantages of the LbL technique is the possibility of forming complex, multilayered heterostructures incorporating several different materials, as well as a rigorous control over the composition, properties and functionality of the resultant thin films 4

. Film properties can be controlled systematically by changing the polymer partners used in

the assembly process as well as by using the same two partners and varying the assembly conditions, such as solution pH, ionic strength, deposition time and polymer concentration. Silk fibroin (SF) is a protein fiber found in silkworm cocoons that can be used in the layer-by-layer (LbL) assembly process along with a companion polycation; the result being the formation of thin films assembled with a complex intermolecular binding. The stepwise deposition of all-SF films was first reported by Wang and co-authors 5. They verified an increase in film thickness with increasing salt concentration of the SF solutions and with decreasing pH. They also noted the need to dry the film after each layer deposition to stabilize the SF assemblies by inter- and intra-molecular interactions thereby allowing deposition of subsequent layers. Spin coating can also be used as a method to produce LbL thin films of SF with outstanding mechanical properties, such as an elastic modulus of 6.5 GPa and ultimate strength of 100 MPa 6. The LbL method may also be used to prepare SF microcapsules 7 with controlled thickness and permeability for drug delivery systems, and SF microcontainers with various shapes 8. LbL assembly of SF with the natural polymer chitosan


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(CHI), was first reported by Cai and co-authors

9

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for the coating of titanium surfaces with

improved osteointegration. Previous studies from our group 10 have shown that LbL films made with CHI and SF present SF nano-fibrils that can be aligned according to the dipping direction. To the best of our knowledge, this was the first and only report in the literature so far about SF fibrils observed in LbL films. These LbL thin films with specific and controllable topographic patterns can display anisotropic mechanical properties and could prove useful for the culture of certain cell types, known to respond to such topographic features

11, 12

. Indeed nature’s

most exquisite materials such as wood and spider silk owe part of their exceptional properties to the careful organization of fibers. Clearly, the presence of nanofibrils will affect the surface properties and functionality of the LbL thin films. Thus, it is desirable to develop simple processing techniques to control the structure of SF in thin films of controlled thickness and uniformity. Some authors have proposed several strategies for a better understanding of the molecular and supramolecular structure of SF and the processing of hierarchical assemblies of SF into fibers, but the whole process is still not well understood 13, 14. Studies indicate that regenerated SF (in aqueous solution) forms micelles, with hydrophilic blocks on the surface, in contact with water, and hydrophobic blocks inside the micelle

15

. One proposed

mechanism for SF fibril formation suggests that these micelles, depending on factors such as protein concentration and charge can aggregate to form nanofibrils 13. The stretching of the hydrophobic blocks presented inside SF micelles during the conformation transition to βsheets is also suggested as the responsible for SF nanofibrils formation 16, 17. The formation of SF nanofibrils in the context of LbL films is also poorly understood. Indeed, the pairing of SF with polyelectrolytes could affect fibril formation, and conversely,


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the formation of SF nanofibrils is likely to affect the properties of SF-based LbL films. Thus a better understanding of the parameters modulating fibril formation is of great interest and could lead to the design of well-controlled SF-based materials. In the present work, we investigate the role that the assembly partner plays in facilitating the formation of thin films containing SF fibrils. Two polymers were chosen as counterparts for SF LbL assembly: chitosan (CHI) and poly(allylamine hydrochloride) (PAH). It has already been shown in our previous work that SF nanofibrils are observed in LbL thin films assembled with CHI 10. CHI is a cationic polysaccharide found in crustacean shells. CHI has primary amines with a solution of pKa ≈ 6.5 that can be used for chemical modifications and electrostatic interactions 18, while PAH is a weak cationic polyelectrolyte that has a high charge density at neutral pH (solution pKa ≈ 8.5

19

). We show importantly that SF fibril formation can be

controlled by changing the assembly partner. The impact of the presence of SF fibrils on the chemical, mechanical and biological properties of the resultant films was characterized.

2. Materials and Methods 2.1 Solution preparation Silk cocoons of Bombyx mori silkworm (Bratac-Brazil) were degummed three times by soaking the cocoons in 1 g/L Na2CO3 solution at 85 °C for 30 min, to remove the sericin of the cocoons, and then rinsing in distilled water. The SF fibers were dried at room temperature (25°C) prior to dissolution. SF fibers were dissolved in a solution of LiBr:CH3CH2OH:H2O (45:44:11 weight ratio) to a final concentration of 20 g/L. The SF salt solution was dialyzed in distilled water for three days at ca. 10 ºC with a Slide-a-Lyzer dialysis cassette (MWCO 3500, Pierce), to remove the salts of the solvent. The final concentration of SF aqueous solution was 8 g/L, and was diluted in ultrapure water to 0.15


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g/L. The concentration of SF dialyzed solution was determined by casting SF solution in a Petri dish and weighing the dried mass after solvent evaporation. Low molecular weight chitosan (50000 g/mol, CHI, Sigma Aldrich, USA), extracted from crab shells, and minimum deacetylation degree of 85% was used. CHI was dissolved in 0.25 M acetic acid, to a final concentration of 1 g/L. The solution pH was adjusted to 6 using 5 M NaOH and then filtered using a 0.45 µm membrane filter. Poly(allylamine hydrochloride) (56000 g/mol, PAH, Sigma Aldrich) was dissolved in ultrapure water to a final concentration of 1 g/L. The solution pH was adjusted to 6 with 1 M NaOH solution and filtered using a 0.45 µm membrane filter. 2.2 Multilayer deposition We used silicon wafers and glass slides as substrates for multilayer buildup. The substrates

were

cleaned

and

received

a

pre-layer

of

15

bilayers

of

poly(diallyldimethylammonium chloride)/poly(sodium 4-styrenesulfonate) (PDAC/SPS), both at 0.01 mol/L in 100 mmol/L NaCl solution, according to previously published procedure 20, in order to make available a larger amount of negative charges on the substrate. Quartz slides were used for circular dichroism (CD) measurements and ZnSe was used for FTIR measurements, but without the PDAC/SPS pre-layer, that could influence the CD and FTIR results. The substrates were immersed in the CHI or PAH solution for 10 min followed by three rinse steps in ultrapure water for 2, 1 and 1 minutes under vertical agitation. Then the substrates were dried for 5 minutes at room conditions and without airflow. After the drying step, the substrates were immersed in the SF solution for 10 minutes, followed by the same rinse and drying steps previously described for CHI or PAH deposition. Then, we have the


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formation of one bilayer film. We are going to use the notation (CHI/SF)n or (PAH/SF)n, where n is the number of bilayers deposited. In addition, we analyzed the influence of the drying step between each layer deposition on film formation. For that, two different tests with the CHI/SF system were performed. In the first one, an all-wet LbL dipping process was performed. The same solutions and same deposition times described in the above paragraph were used, but without the drying step. In the second one, a drying step was performed, involving 10 minutes with airflow at 25°C. 2.3 Characterization 2.3.1 Ellipsometry Dry film thickness was measured by ellipsometry (Gaertner Scientific 3), with a wavelength of 633 nm, and incidence angle of 70°. Swelling of the films was determined by correlating the dry and wet film thickness as described previously

21, 22

. Film was placed on a quartz cell and the dry thickness was

measured. Then the film was soaked in ultrapure water and the film thickness after 5 minutes of immersion in water (equilibrium) was measured. Thickness was measured using a J.A. Woollam XLS-100 spectroscopic ellipsometer, from 400 to 1000 nm, at 70° incident angle and analyzed with WVASE32 software, using the Cauchy model to fit the data. The refractive index (λ = 633 nm) for the (PAH/SF)20 dry and wet films were 1.55 and 1.44, respectively, and for the (CHI/SF)20 dry and wet films were 1.54 and 1.43, respectively. 2.3.2 Quartz crystal microbalance with dissipation monitoring (QCM-D) Multilayers buildup was also monitored by quartz crystal microbalance with dissipation monitoring (QCM-D, Q-Sense AB, Sweden) using an open chamber at 25 °C and a quartz crystal coated with SiO2. QCMD measures the changes in frequency and dissipation 7 ACS Paragon Plus Environment

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at each layer deposition. These changes are associated with mass being adsorbed to the crystal and can be related to the thickness of the film being deposited, in the wet state, by applying a viscoelastic model. The same procedure used to build the multilayers films was done using the QCM-D. Briefly, 2 mL of CHI or PAH were added with a pipette to the crystal and adsorbed for 10 minutes. Then the solution was removed and 2 mL of ultrapure water was added and kept in the chamber for 2 minutes. Two other rinses were done for 1 minute each. Subsequently, the water was removed and the chamber was opened and dried at room conditions, without airflow, for 5 minutes. The same steps were repeated with the SF solution, building 1 bilayer. For QCMD analysis 5 bilayers were built. Data were modeled using the QTools software, applying Voigt viscoelastic model. 2.3.3 Contact Angle Contact angle measurements were done using a Video Contact Angle System VCA 2000

(AST

Inc.).

Ultrapure

water

drop

was

used

to

evaluate

the

hydrophobicity/hydrophilicity of the surface of the LbL films. The contact angle formed between the drop and the film surface was measured using the software VCAOptima. 2.3.4 Imaging Bright field images of the films were taken using an Axioplan 2 (Zeiss), to observe the SF fibrils on the films. Atomic force microscopy (AFM) was performed using a Veeco/Digital Instruments Nanoscope IV Dimension 3100 scanned probe microscope in tapping mode. The diameter of SF fibrils observed by the bright field and AFM images was measured by using ImageJ software.


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2.3.5 Chemical characterization Fourier Transformed Infrared Spectroscopy (FTIR) was performed on a Nicolet 6700 FTIR Spectrometer (Thermo Scientific) on 20 bilayer films deposited on ZnSe crystal. Each spectrum was acquired in transmittance mode by accumulation of 68 scans, resolution of 4 cm-1, in the range 400-4000 cm-1. Circular dichroism (CD) measurements were performed on a Aviv Circular Dichroism Spectrometer Model 202 purged with N2 gas. The spectra were recorded from 250 to 195 nm, with a bandwidth of 1 nm, step of 0.5 nm and accumulation of 3 scans. (CHI/SF)20 and (PAH/SF)20 LbL films were deposited on quartz slides for CD analysis. Also, SF solution at 0.15 g/L was analyzed on a quartz cuvette with 0.1 cm path length and the mean residue ellipticity values Θ were calculated using Eq 1 23: Θ = Θ௢௕௦

78 10݈ܿ

(1)

where Θ is mean residue ellipticity (degree.cm2/dmol), Θobs is the observed ellipticity value (degrees), 78 is the mean residue molecular mass of silk fibroin, l is the optical path length (cm) and c is the protein concentration (g/mL). 2.3.6 Mechanical characterization Mechanical characterization of the LbL films was performed by indentation in atomic force microscopy (Agilent 5500). An AFM tip of silicon model NSG03/Au (NT-MDT) with spring constant of 0.27 N/m (determined experimentally) was used. Force-displacement curves were recorded at indentation rate of 7.7 µm/s. 15 curves were recorded in 3 distinct regions of each sample, deposited in silicon wafer. Three types of LbL films were analyzed: 1) (PAH/SF)20; 2) (CHI/SF)20 and 3) (CHI/SF)10x10,where 10 bi-layers were deposited, then the substrate was rotated 90° and more 10 bi-layers were deposited, creating a bidirectional


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orientation of SF fibrils, as reported in our previous paper

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10

. For the (CHI/SF)20 films, the

force-displacement curves were recorded at points of the sample where SF fibrils were present. For the (CHI/SF)10x10 films, the force-displacement curves were recorded at points of SF fibril intersection. 2.3.7 Cell viability Cell viability when in contact with the LbL films was analyzed in the first, third and seventh day of culture. For that, the films deposited on a glass slide, containing SF or its cationic partner on top, were cut in pieces of approximately 25 mm x 10 mm, placed on a 6well Petri dish and seeded with NIH-3T3 fibroblasts at a concentration of 10,000 cells/cm². For cell viability the Live/Dead (Invitrogen) assay was used. Films with cells were incubated for 45 min in 1 x 10-6 mol/L calcein and 4 x 10-6 mol/L ethidium homodimer-1 in serum free DMEM. The staining was then visualized using an Observer Z1 inverted fluorescent microscope (Zeiss, Oberkochen, Germany) with a 10x 0.3 NA objective. Red cells indicated dead cells while green cells indicate live cells. The cell viability was calculated by counting the observed live or dead cells on three different images for each sample. 3. Results and Discussion We firstly investigated the growth conditions for both CHI/SF and PAH/SF films assembled via the layer-by-layer process. Literature reports the need of a drying step between each SF layer deposition for all-SF LbL films, to control the stability of the SF molecules and induce β-sheet formation 5. Our previous work also demonstrated the importance of a drying step with CHI/SF films, significantly increasing the film thickness 10. Here, we also observed that no film formation was possible when CHI/SF films were not dried between each layer deposition. In sharp contrast, with the drying process, a film deposited and SF fibrils were observed in the film (see Supporting Information). Thus, it is possible to conclude that the


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drying step is crucial for SF stabilization and film growth. A 5 minutes drying time at room conditions (without airflow) was chosen for subsequent films preparation because of the low uniformity of the films when airflow was applied. The 5 minutes drying time did not dry the layers completely. When the films were dried between depositions, the PAH/SF and CHI/SF films exhibited a linear-type growth behavior between 10 and 40 bilayers, as shown in Figure 1a. Multilayer films deposited with PAH were thicker than films assembled with CHI. To correlate the dry thickness (measured by ellipsometry) with the hydrated thickness (measured by QMC-D) of the films, the swelling capacity of 20 bilayer films was measured. The swelling of the (PAH/SF)20 and (CHI/SF)20 multilayers was 74 ± 4 % and 78 ± 3 %, respectively. The relatively high swelling level of the SF containing multilayers is related to the hydrophilic nature of the molecules used and the high water content of the films. Additionally, the films were stable in water, with a thickness variation of less than 1% in the dry state after immersion in ultrapure water for 7 days (data not shown). Figure 1b shows QCM-D results of the hydrated thickness of each deposited layer of multilayer films containing 0.5 to 5 bilayers (1 to 10 layers), after rinsing steps. It was observed by QCM-D that a decrease in hydrated thickness occurs after the deposition of each PAH or CHI layer (decimal numbers in Figure 1b). This same behavior has already been observed in LbL films assembled with CHI

24, 25

and has been related to the redissolution of

polymer complexes from the film. It was proposed in LbL films with linear growth of CHI/mucin

25

that mucin only forms stable complexes with the outer chitosan layer. The

other complexes formed are loosely bound and dissolve during the subsequent addition of chitosan. The same mechanism is probable in this study, where SF molecules are deposited on the outer CHI or PAH layer, and also form complexes that are not well attached to the surface and are removed during the subsequent CHI or PAH addition.


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Moreover, film thickness obtained by QCM-D reflects the wet, swollen state, after a rinsing step, but prior to the drying step of the multilayer buildup process. In addition, SF has a random coil conformation in solution, while after drying SF is stabilized and assumes a βsheet conformation 5. We speculate that this conformational change can also influence film thickness.

Figure 1: (a) Growth curves of PAH/SF and CHI/SF multilayers in the dry state; (b) Hydrated thickness after rinsing versus the number of deposited bilayers obtained by QCM-D. Connecting lines were added for clarity. Decimal numbers (.5) indicate deposition of the cationic partner (CHI or PAH) and integer numbers indicate deposition of SF.

To further analyze the influence of the polymer partner on the behavior of LbL films with SF, we performed water droplet contact angle measurements on films with 20 bilayers (SF on top) or 20.5 bilayers (CHI or PAH on top). All of the films exhibited a contact angle between 71-81° (Table 1), which is in accordance with the literature for dense SF membranes with β-sheet conformations

26, 27

. The films did not show any significant difference, which

means that the wettability of these films is in the same range and does not change 12 ACS Paragon Plus Environment

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significantly with the cationic partner or the outermost layer. These results are suggestive of interpenetration between the deposited polymer layers: a common occurrence in the LbL process. Cai and co-authors

9

observed a different behavior with CHI/SF LbL films. They

observe contact angles of 65° when CHI was on the top of the films, while films with SF on top had a contact angle of 36°. This difference is probably related to the molecular weight of chitosan and the procedure for film preparation, since they used higher molecular weight chitosan as well as a step of immersion in acetic acid after each chitosan layer deposition, which would limit the inter-diffusion of the film’s components and allowed for the formation of well-defined layers. Also note that the nature of the supporting substrate (glass versus silicon) does not alter significantly the wettability of the multilayer films, indicating similar growth behavior on these different substrates.

Table 1: Advancing water droplet contact angle measurements of SF LbL films deposited on silicon wafers and glass slides.

Silicon wafer Glass Substrate

26.6 ± 0.1

29.1 ± 1.1

(CHI/SF)20

77.6 ± 2.1

71.4 ± 5.9

(CHI/SF)20.5

76.2 ± 4.6

73.2 ± 3.4

(PAH/SF)20

76.8 ± 1.5

77.7 ± 1.8

(PAH/SF)20.5 80.7 ± 6.2

81.9 ± 1.8

An important question of this study is how the nature of the polymer partner influences the ability of SF to form fibrils in LbL assembled multilayer films. SF nano-fibrils 13 ACS Paragon Plus Environment

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(diameter ranging from ca. 200 nm to ca. 8 µm) are clearly present in LbL films assembled with CHI (Figure 2a). The SF fibrils seen in Figure 2a are probably the fibrils present on the last deposited layers and not in all the SF layers in the film, since the thickness of the film (ca. 140 nm) made it difficult to visualize fibrils in the inner layers. In sharp contrast however, when PAH was used for LbL film preparation, no fibrils were observed (or observed in a very small quantity). This shows that fibril formation is highly influenced by the polycationic pair used to assemble SF into multilayers. It is important to recall that (PAH/SF) films were somewhat thicker than (CHI/SF) films, which means that SF is successfully deposited with PAH, but not in the form of fibrils.

Figure 2: Optical microscope images of (a) (CHI/SF)20 and (b) (PAH/SF)20 deposited on silicon wafer substrates.

AFM images of the (CHI/SF)20 and (PAH/SF)20 multilayers also show that fibrils are present in multilayers assembled with CHI but not when PAH is used in the multilayer assembly (Figure 3). A bundle of SF fibrils (diameter of ca. 600 nm) is observed with the (CHI/SF)20 system in Figure 3a. These fibrils are probably located in the inner layers of the film, resulting in a small change in the final topography of the film. The surface roughness (2


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x 2 µm) was 5.17 nm for (CHI/SF)20 and 1.96 nm for (PAH/SF)20 films, indicating a smoother and more uniform surface of films with PAH.

Figure 3: AFM tapping mode height images of (a,b) (CHI/SF)20 and (c) (PAH/SF)20.

The influence of the substrate on SF fibril formation was evaluated by using a more hydrophobic substrate (contact angle = 81° ± 3°) of ZnSe to deposit the films. SF fibril deposition was again only observed on the films deposited with CHI, while no fibrils were observed on films with PAH (Supporting Information file). These results indicate that SF fibril formation is primarily influenced by the polymer partner (CHI or PAH) and not influenced by the hydrophobicity/hydrophilicity of the supporting substrate. To analyze if SF fibrils could be formed in an all-SF film, a (SF/SF)20 film was prepared. SF fibrils were observed in this film (diameter of ca. 200 nm) (Figure 4) similar to the fibrils in the LbL films with CHI, but the total film thickness was only about 29 ± 6 nm


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and probably just a few layers were deposited, indicating that the presence of a polycationic partner in the LbL process is essential for robust film growth.

Figure 4: A (SF/SF)20 film deposited on Si: (a) optical microscope image and (b) AFM tapping mode image.

AFM images of the (SF/SF)20 film confirm the presence of SF fibrils in the film (Figure 4b). The better resolution of fibrils seen in Figure 4b when compared to Figure 3, is related to the fact that the fibrils are essentially on the surface of a very thin (SF/SF)20 film, while in the case of the (CHI/SF)20 films, the fibrils are contained within the inner layers, which complicates their mapping, since AFM evaluates just the surface of the material. The fibrils observed in the LbL films are similar to the SF fibrils obtained by spin-coating a SF solution extracted from B. mori silkworm glands on mica substrates (diameter ranging from 25 to 250 nm), as reported by Greving and co-authors 14. Lee and co-authors observed the influence of intermolecular interactions on fibrillation of κ-casein/poly(acrylic acid) (PAA) in LbL films 28. It was verified that κ-casein fibril formation in LbL films could be triggered by controlling the intermolecular interactions between the polyelectrolytes. When the interactions between κ-casein and PAA were strong


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they hinder the mobility of κ-casein to form fibrils and no fibrils formation was observed. On the contrary, when the interactions were weak, fibrils were observed. The results indicate that κ-casein fibrillation in LbL films is a competitive process, since fibrillation just occurs when the intermolecular interactions between κ-casein and the polyelectrolyte are weaker than the hydrogen bonds between κ-casein β-sheets. We observe this same behavior in the LbL films with SF. The SF isoelectric pH is ca. 4.2 and, at neutral aqueous solution (as used in this study), SF is negatively charged 13, which favors the interaction with cationic polymers. PAH is a cationic polymer with pKa in the range between 8.0-9.0

19

, while the CHI pKa is reported in the range between 6.0-7.0

18

.

Thus, at pH 6.0 (the pH used for CHI and PAH solutions), CHI has just a few amine groups protonated while PAH is still highly protonated. In the LbL films in which SF could form more electrostatic interactions (PAH) no fibrils were observed. However, in the films with less capability of intermolecular interactions (CHI), fibrils were observed and are probably related to the assumption that SF-SF interactions are stronger than SF-CHI interactions in the studied conditions in the LbL films. These results indicate that the intermolecular interactions between SF and the polymeric pair will define the fibril formation in LbL films. Lu and co-authors

13

propose that SF molecules form aggregates, called “micelles”,

with hydrophilic blocks on the surface, in contact with water, and hydrophobic blocks on the interior of the micelles. The micelles are capable of aggregating and forming fibrils due to a combination of solution concentration and electrostatic charge. In dilute solutions, the repulsion between negative charges results in stable micelles, well dispersed in the solution. However, in concentrated solutions, micelle aggregation to form fibrils is the preferred path to reduce the repulsive forces. This can explain the tendency of SF to form fibrils instead of big agglomerates of micelles.


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In the (CHI/SF) films, the weak interactions between polymers will not prevent fibril formation. Then, probably during the drying step of multilayer buildup, the SF solution starts to concentrate and the SF micelles associate to form fibrils. The influence of solution concentration in morphological transitions is also observed in dipeptides

29

. In dilute solution the dipeptides self-assemble in the form of vesicle-like

structures, while in concentrated solutions these structures align and form tubular nanostructures throughout intermolecular interactions. Another approach suggests that SF fibrils are formed during the conformational transition of SF molecules from random coils to an organized β-sheet structure 16, 17, 30. In this case, the stretching of the hydrophobic blocks that are inside SF micelles during β-sheet formation would be the responsible for fibril formation 16. To explore this further, the secondary structure of SF was determined by FTIR (Figure 5a). Similar FTIR spectra were obtained for both (CHI/SF) and (PAH/SF) films, thus indicating that the polymeric pair did not influence the presence of functional groups or SF conformation as detected by FTIR. The presence of amide I (1620 cm-1) and amide II (1530 cm-1) peaks associated with the silk II conformation, and amide III (1230 cm-1) peaks associated with the silk I conformation indicate the co-existence of both conformations, typical of SF-based materials 31, 32, with predominance of a stable β-sheet structure. A stable SF structure (silk II) was also observed by other authors in SF LbL thin films 5, 10

and it was mainly attributed to the drying steps between layers, that stabilize the SF

molecules (formation of β-sheet) and allow for the next layer deposition. This stabilization during the drying step was also observed in our study, where LbL films with SF present a silk II conformation. Also, the fact that films with SF were not formed without the drying between each layer deposition confirms that SF is not stable without drying. The transition


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from silk I (present in SF solutions) to silk II is usually done by post-treatments with organic solvents (methanol, ethanol) annealing)

35

33

, or by exposure to high temperatures

or by shear-stress

34

or humidity (water

15

. However, in the LbL films with SF, the conformational

transition to silk II was achieved by drying the film between each layer deposition, a more simple and environmental friendly option.

(a) 100

Transmittance

(PAH/SF)20

(CHI/SF)20

1230

1530 1620

50

4000

3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm ) (b)

(c)

1500000

(PAH/SF)20

1000000

0

deg cm /dmol)

(CHI/SF)20 500000

θ obs

+

2

00 -500000

-3

-1000000

[θ ] (10

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-1500000

-2000000

-2 -4 -6 -8 -10 -12

-2500000

200

210

220

230

Wavelength (nm)

240

250

200

210

220

230

240

250

Wavelength (nm)

Figure 5: FTIR spectra of (PAH/SF)20 and (CHI/SF)20 films deposited on ZnSe substrates (a), and CD spectra of (PAH/SF)20 and (CHI/SF)20 films deposited on quartz slides (b) and a SF 0.15 g/L solution (c).


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Analysis of circular dichroism on the SF solution and also on the deposited films with CHI and PAH (Figures 5b and c) confirm this hypothesis. The SF solutions after dialysis showed CD spectra typical of a random coil

23

, while the 20 bilayer films showed spectra

typical for β-sheet conformations, with a negative ellipticity band at 213 nm. This result is in accordance with the FTIR results and with our hypothesis that the drying steps and the confinement of SF molecules in the thin film (at the nanometer scale) stabilizes SF in a silk II structure.

The presence of aligned SF fibrils in films of nanometric thickness can also increase the mechanical properties by acting as nano-structured reinforcing agents. The mechanical properties of the films were probed by AFM indentation (Figure 6), to analyze the influence of uni- and bi-directionally oriented SF fibrils on the mechanical properties of the films. All of the films exhibited an elasto-plastic behavior. The (PAH/SF)20 and the (CHI/SF)20 films had a similar mechanical behavior in all the analyzed curves. A higher resistance to probe penetration was only observed in the films with bi-directionally (see experimental section) oriented SF fibrils (CHI/SF)10x10. These results indicate that the presence of unidirectional oriented fibrils is not capable of providing stronger mechanical resistance in the LbL films. However, when the fibrils are bi-directionally oriented within the LbL film, an improvement in mechanical resistance is observed.


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1.2

(PAH/SF)20

1.0

(CHI/SF)20 0.8

Force (µ N)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(CHI/SF)10x10

0.6

Unload

0.4 Load 0.2 0.0 -0.2 -1.5

-1.0

-0.5

0.0

0.5

1.0

Distance (nm)

Figure 6: Typical force-distance curves obtained by nanoindentation in an AFM with LbL films with SF. The subscript 10x10 means that 10 bilayers were deposited in one direction, then the substrate was rotated 90° and more 10 bilayers were deposited, creating a bidirectional orientation of SF fibrils.

LbL films containing SF fibrils can be used as a matrix for cell growth. Indeed, nanostructured materials have shown to elicit interesting cellular responses such as tissue organization and cellular differentiation

11

. To examine the biocompatibility of the SF

containing films, CHI/SF and PAH/SF films were incubated with NIH3T3 fibroblasts and tested for their influence on cell viability using a membrane integrity dye (Figure 7). Fluorescence microscopy images of the cells at day-seven show a slightly less spread morphology when compared to glass. Cell density on SF-based films also remained lower than glass and could be improved in future studies by the adsorption of cell adhesive proteins such as fibronectin. However, cell viability remained around 90-100% during the 7 days of 21 ACS Paragon Plus Environment

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incubation. Also, the biocompatibility of the multilayers does not seem to be influenced by the last deposited layer, since similar results were obtained with CHI, PAH or SF as top layers. This result is related to the interpenetration between layers as observed by contact angle measurements. This first assessment of biocompatibility of the multilayers containing SF indicates that these films hold promise for biomaterial applications.

Figure 7: Cell viability of (CHI/SF) and (PAH/SF) films with SF or its counterpart on top of the film (20 or 20.5 bilayers) and fluorescence microscopy images of the cells with a live/dead stain. Live cells are in green, membrane compromised cells are in red.

4. Conclusion It was possible to prepare LbL thin films by successive deposition of CHI or PAH and SF and to control the formation of SF aligned fibrils within the films. The assembly partner, the chemical interactions and the drying step during deposition are the key factors controlling


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film deposition and fibril formation. The films were not toxic to cells and can be used as coatings for biomedical devices, drug delivery and biosensors with specific target molecules.

Supporting Information Available. Influence of the drying step between each layer deposition, the solvent used to dissolve fibroin and the substrate on the ability to deposit fibrils. QCMD data and fit of Voigt viscoelastic model. This information is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgment. We would like to thank Prof. Monica Cotta (LNB/IFGW/UNICAMP) for AFM nanoindentation experiments and Grinia Nogueira and Siddarth Srinivasan for valuable discussions. This work was supported by CNPq-Brazil and CAPES-Brazil. This work was supported in part by the MRSEC Program of the National Science Foundation, award number DMR – 0819762.

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For Table of Contents Use Only

Factors Controlling the Deposition of Silk Fibroin Nano-Fibrils During Layer-by-Layer Assembly

Mariana Agostini de Moraes, Thomas Crouzier, Michael Rubner and Marisa Masumi Beppu


Factors Controlling the Deposition of Silk Fibroin Nanofibrils during

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