Fabrication of Protein Films from Genetically Engineered Silk-Elastin

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Fabrication of Protein Films from Genetically Engineered Silk-Elastin-Like Proteins by Controlled Cross-Linking Liang Chen, Ming-Liang Zhou, Zhigang Qian, David L Kaplan, and Xiaoxia Xia ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00794 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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ACS Biomaterials Science & Engineering

Submitted to ACS Biomaterials Science & Engineering as an article

Fabrication of Protein Films from Genetically Engineered Silk-Elastin-Like Proteins by Controlled Cross-Linking

Liang Chen,† Ming-Liang Zhou,† Zhi-Gang Qian,† David L. Kaplan,‡ and Xiao-Xia Xia*,†

†

State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of

Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ‡

Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford,

Massachusetts 02155, United States

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Xiao-Xia Xia: 0000-0001-8375-1616 Notes The authors declare no competing financial interest.

1 ACS Paragon Plus Environment


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ABSTRACT Protein films are an important class of materials for applications in biomedicine and biotechnology. The rational design of protein polymer sequence and selection of customized cross-linking offers unique opportunities to engineer desirable functionalities into these materials. Here we report the fabrication of a series of films with tunable physiochemical properties from genetically engineered silk-elastin-like proteins (SELPs). The SELPs were recombinantly biosynthesized with different ratios of silk-to-elastin blocks and periodic cysteine residues incorporated in the elastin blocks. A disulfide cross-linking method was developed for the preparation of the SELP films under mild oxidative conditions with a low concentration of hydrogen peroxide, in comparison with the physical cross-linking method used with the organic solvent methanol. Film properties were characterized for solubility, water absorption, hydrophilicity, surface roughness, and cyto-compatibility. The results indicated that customized cross-linking supported the fabrication of films from the SELP proteins with tunable features, including smooth, water stable film materials with cyto-compatibility. Interestingly, hydrogen peroxide oxidation was a preferred cross-linking method for the cysteine-containing SELPs with a low ratio of the silk-to-elastin blocks, while methanol treatment was suitable for fabricating films from the SELPs with a high ratio of silk-to-elastin blocks into stable films with rougher surfaces. We anticipate that an appropriate combination of polymer design and cross-linking might be a useful strategy for the preparation of protein films for diverse applications. KEYWORDS: silk-elastin-like protein polymers, protein films, disulfide cross-linking, genetic engineering, methanol treatment


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INTRODUCTION Nature has evolved a wide variety of remarkable protein materials, such as collagen, elastin, resilin, and silk. These complex yet highly functional materials are composed of peptide sequences with well defined structures, such as α-helixes, β-turns, β-sheets, and β-spirals.1,2 With the rapid development of molecular biology and genetic engineering, the above unique peptide sequences have been employed as consensus motifs for the design and biosynthesis of artificial protein polymers.2,3 These genetically engineered protein polymers often inherit from their parent proteins certain remarkable structural and functional characteristics.4-6 In addition, individual peptide sequences that exhibit different chemical, physical or biological properties have been recombined at the genetic level for the recombinant synthesis of multiblock protein polymers7,8 By doing so, materials scientists have now been able to genetically engineer protein polymers with a high level of control over material sequences, structures, properties, and functions.1,2 Silk-elastin-like protein polymers (SELPs) are a family of such genetically engineered protein copolymers, consisting of tandem repeats of silk-like (GAGAGS) peptide blocks derived from natural silk proteins produced by Bombyx mori and elastin-like (GXGVP) peptide blocks from tropoelastin in mammals, where X in the elastin block can be substituted by any amino acid except proline to affect the coacervation process of elastin.9 Silk-like peptide blocks provide thermal and chemical stability as well as physical cross-linking sites, while the elastin blocks offer the copolymer stimuli-responsiveness, that is, render the SELPs structural transitions upon external stimuli such as increased temperature or changed pH.10 Through tuning the ratio of silkto-elastin blocks, or replacing or modifying the amino acid in the X position of the elastin block, a wide variety of SELPs with distinct responsive properties were produced.11 For example,



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replacement of the amino acid in the X position of the elastin block with cysteine resulted in SELPs endowed with both thermal and redox responsive properties.12 Because of the advantages of the genetic machinery providing exquisite control of chemical and physical properties at the molecular level, these recently developed SELPs have demonstrated potential as materials for broad applications in tissue engineering, controlled drug delivery, and gene therapy.13,14 SELPs have attracted much attention due to the versatile processing approaches available for fabricating them into diverse structures with desirable properties.15 For example, SELPs have been fabricated into material formats such as nanoparticles,16-18 fibers,19 films,20,21 scaffolds,22 and hydrogels.23 Among these material formats, SELP films are rarely studied although such protein films have shown potential for diverse applications. In recent studies, solutions of silk fibroins have been solvent cast for the preparation of microfluidic devices,24 insulating materials for bioelectronics,25,26 and lithographic nanoarchitectures.27 A lysine-containing SELP (SELP47K) was cast into optically transparent films and the potential applications in the ophthalmic drug delivery was assessed.20,28 More recently, an alanine-containing SELP (SELP-59A) was cast into films using water or formic acid as solvents, and the mechanical and electrical properties were reported.21 The above studies demonstrated the potential of SELP films in a variety of applications. In addition, methanol treatment was demonstrated in these studies as a suitable method for physical cross-linking of SELP proteins into films, enabling aqueous insolubility and good mechanical properties due to the dehydration-induced physical cross-linking resulting in crystallization. However, this method might not be applicable to SELPs with a low ratio of silk blocks because of the intrinsic cross-linking mechanism by inducing silk blocks to form aggregated strands. Furthermore, methanol treatment might not be compatible with the encapsulation of some sensitive drugs, proteins or cells in these protein matrices.29 Therefore,


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there is a need for the development of alternative cross-linking methods for film preparation from diverse SELPs with varying protein sequences and structures. Inspired by disulfide cross-linking for the formation of SELP hydrogels with tunable mechanical properties and redox stimuli responsiveness in our recent study,12 we were interested in whether insoluble SELP films with tunable properties could be prepared via disulfide crosslinking. Therefore, three SELP proteins with periodic cysteine residues incorporated at the X position of the elastin block and various ratios of the silk-to-elastin blocks (1:8, 1:4, and 1:2) were studied for film formation under mild oxidative conditions with the addition of a low concentration of H2O2. The prepared films were then characterized with respect to water stability, swelling, surface morphology and roughness, structural conformation, and cytotoxicity in vitro, in comparison with films prepared from the same SELPs but with methanol treatment.

MATERIALS AND METHODS Preparation of SELPs. SE8C,

S2E8C,

and

S4E8C,

which

[(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)],

consisted

of

12

[(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)2],

and

12

repeats

repeats 11

repeats

of of of

[(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)4], respectively, were used in this study. Their recombinant expression and purification were described in our recent study.12 Briefly, the SELPs were recombinantly expressed with an N-terminal decahistidine tag in bacterium Escherichia coli by shake flask cultivation and purified using immobilized-metal-affinity chromatography with Ni-NTA agarose. Following elution from the Ni-NTA agarose column, the proteins of interest were added with 10 mM dithiothreitol (a reducing agent), and then dialyzed at 4 °C



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against deionized water. Following dialysis, the protein solutions were concentrated to approximately 50 mg mL−1, and the freshly prepared samples were used for protein film preparation. Film Formation. Three types of protein films, non-, H2O2-, and methanol-treated films were prepared from each of the three SELPs. Briefly, 50 µL of each SELP solution at a final protein concentration of 4.0% (w/v) was cast on polydimethylsiloxane (PDMS), followed by solubility, equilibrium swelling study, thermal characterization, and secondary structure assessments. In addition, the protein solutions were also cast on mica discs (9.9 mm diameter; Ted Pella, Inc., Redding, CA) to form films for contact angle analysis, surface roughness and morphology analysis. For cell culture studies, the proteins and other components were first filtered through a sterile, 0.22-µm membrane filter (RephiLe Bioscience, Ltd., Shanghai, China), and then cast into wells of a flat bottom 96-well cell culture plate (Nest Biotechnology Co., Ltd, Wuxi, China). The non-treated films were prepared by casting the protein solutions and then dried in a clean bench laminar flow at room temperature for two days at 60% relative humidity. The H2O2-treated films were prepared as described above for the non-treated films, except that the casting solutions were obtained by mixing each SELP protein with H2O2 at a low, final concentration of 0.03% (w/v). After initial drying, some non-treated films were submerged in 99.9% methanol for 4 hours, and subsequently allowed to dry, to obtain the methanol-treated films. Solubility. The film samples were weighed and transferred to wells of 24-well cell culture plates (Costar 3524, Corning Inc., Corning, NY). One milliliter of 0.01% (w/v) potassium sorbate, to prevent microbial contamination, was added into each well. At the indicated time points, a 20 µL aliquot


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of the well-mixed solution was sampled, and an equal volume of deionized water was added back to each well. Protein concentration in the sampled solutions was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Rockford, IL). The amount of dissolved protein was compared with the initial mass of the film, which revealed the percentage of the mass loss of the film in water. For each type of film samples, three independent measurements were performed and averaged. Equilibrium Swelling. Before swelling analysis, the film samples were first placed in a vacuum oven and dried overnight at 80 °C. After equilibration to room temperature in a desiccator, the dry films were weighed, and immediately soaked in 1 mL of 0.01% (w/v) potassium sorbate on a 24-well cell culture plate at room temperature (ca. 25 °C). After each time interval, the swollen films were taken out and then weighed after removing excess water with filter paper. The swelling ratio was calculated from Equation (1): where Ws was the mass of a swollen SELP sample and Wd was the initial mass of the dried film sample. = s/d (1) Contact Angle. To evaluate the surface hydrophilicity of the films, contact angle measurements were performed at room temperature using the DSA30 drop shape analyzer (Kruss, Hamburg, Germany). The contact angles were detected by depositing 3 µL of deionized water drops on the sample surface and analyzing the optical images of droplets using the sessile drop fitting algorithm with the accessory ADVANCE software (Kruss). Measurements were performed on five or more replicates of each type of SELP films and the average contact angle and standard deviation were calculated.



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Surface Roughness. The surface roughness of the film samples prepared on mica discs were estimated using a multimode AFM (Bruker, Germany) with a Nanoscope IIIa scanning probe controller (Digital Instruments, Santa Barbara, CA) and the associated Nanoscope Analysis software version 1.40. Images were recorded under tapping mode with a scanning area of 10 µm x 10 µm in triplex and a scan rate of 1.0 Hz. The mean surface roughness (Ra) of each film was taken from the software analysis, and mean values and standard deviations were calculated accordingly. Secondary Structure Analysis by FTIR. The secondary structures of the SELP films were analyzed using attenuated total reflectionFourier transform infrared (ATR-FTIR) spectroscopy at room temperature. Briefly, 64 scans were conducted for each film sample with a resolution of 4 cm-1 over the wavelength range of 400-4000 cm-1 using a Nicolet 6700 spectrometer (Thermo Scientific, Madison, WI) in ATR mode, and the backgrounds due to atmospheric H2O and CO2 were deducted automatically using the affiliated OMNIC software (Thermo Scientific). Then the FTIR spectra were linearly baselined and smoothed to determine maximal absorption peak in the amide I zone (1600-1700 cm-1) for each film sample, using the 7-point quadratic Savitsky-Golay smoothing algorithm. Following this, the OriginPro v9.2 software (OriginLab Corp., Northampton, MA) was used for baseline correction and subsequent iterative curve fitting of the spectra using Gaussian algorithm with R2 more than 0.999, in which the second derivatives of the spectra were used for peak positioning guidance. The contents of individual secondary structural components were calculated by integrating the area of each fitted band under the curve divided by the total area of the amide I band, according to a previously established protocol.21


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Cytotoxicity. For cytotoxicity assessments, the H2O2-, and methanol-treated films were rinsed twice with sterile phosphate buffered saline (HyClone Laboratories, GE Healthcare Life Sciences, South Logan, UT). Prior to cell seeding on the films, mouse pre-osteoblast cells (MC3T3-E1; Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured on 96-well plates in α-Modified Eagle’s Medium (GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies Corp., Carlsbad, CA), 1% penicillin-streptomycin, and 1% (w/v) glutamine at 37 °C with 5% CO2. The cells were trypsinized in the logarithmic growth state, and then evenly seeded onto the films at a density of 5×103 cells per well. A fluorescencebased live/dead staining method was performed at 72 h after seeding to assess cell viability on the films using LIVE/DEAD® Viability/Cytotoxicity Kit (Invitrogen). Statistical Analysis. The single factor analysis and student t-tests were carried out at α = 0.05 to determine whether differences were statistically significant between tests. All studies except contact angle analysis (in quintuplicate) were performed in triplicate.

RESULTS AND DISCUSSION Preparation of SELP Films. Our recent study demonstrated that cysteine-containing SELPs were oxidized with mild H2O2 at physiologically relevant temperatures to form disulfide crosslinks, resulting in the formation of hydrogels with tunable mechanical properties.12 Inspired by this observation, we hypothesized that these cysteine-containing SELPs could be potentially oxidized to form protein films under mild, physiologically relevant conditions. To test this hypothesis, the three SELPs that were



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genetically engineered with varying ratios of silk-to-elastin blocks at 1:8, 1:4, and 1:2, and a cysteine residue at the X position of the central elastin block (GVGVP)4(GXGVP)(GVGVP)3 were examined (Figure 1A). The three SELPs, abbreviated as SE8C, S2E8C and S4E8C, were recombinantly

expressed

with

an

N-terminal

tag

(MGHHHHHHHHHHSSGHIDDDDKHMGAGAGS) in E. coli and purified using immobilizedmetal-affinity chromatography as previously described.12 Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis confirmed that the protein polymers had a purity greater than 95%, which was suitable for film formation and characterization.

Figure 1. (A) Constructs of recombinant SELPs that contain cysteine residues in the elastin blocks and varying ratios of silk-to-elastin blocks in each monomer repeat. (B) Photographs of the SELP films treated with H2O2 or methanol. The blue dots in the photograph were crystals of Nickel(II) sulfate hexahydrate used to support the films for improved photographic contrast. The diameters of the films were ~8 mm.

To prepare the SELP films, the purified SE8C, S2E8C and S4E8C proteins in aqueous 10 ACS Paragon Plus Environment

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solution with and without the addition of low concentration of H2O2 at 0.03% (w/v) were cast on PDMS surfaces to allow film formation upon drying. Notably, the three SELPs contained silk blocks, which were prone to crystallize with the formation of beta-sheets in a process that can be accelerated by solvents such as methanol. Therefore, some control films (without H2O2 treatment) were subsequently treated with pure methanol for 4 h prior to air-drying again. The methanol-treated films were thus obtained. As shown in Figure 1B, the H2O2-treated films were generally smooth and transparent, while the methanol-treated films were more wrinkled. Wetting Properties of the SELP Films. Having prepared the SELP films, we next explored their wetting properties. First, solubility tests were performed by incubating the three types of SELP films in deionized water at room temperature. The control, non-treated films derived from any one of the three SELPs dissolved within minutes (data not shown), whereas the H2O2- and methanol-treated films were stable under these conditions. Therefore, the cumulative solubility of the H2O2- and methanol-treated films of SE8C, S2E8C and S4E8C were monitored over a period of 48 h. As shown in Figure 2A, the H2O2-treated SE8C films were quite stable in aqueous solution, with only approximately 6% of the protein film dissolved in the supernatant after an extended time of 48 h. However, significantly higher levels of the H2O2-treated S2E8C (~44 %) and S4E8C (~75%) films were dissolved during the same period of time, and dissolution of these two films occurred within the first four hours of incubation. The inferior solution stability of these two films may be due to lower degrees of disulfide cross-linking in the cysteine-containing SELPs with higher ratios of silk-to-elastin blocks. As shown in our recent study, dynamic light scattering analysis for solutions of the SELPs revealed that the existence of more silk blocks in S2E8C and S4E8C led to preassembly of the proteins into micellar-like particles, which might entrap considerable



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proportion of the free thiol groups of the cysteine residues. This lack of accessibility would possibly lead to weaker disulfide cross-linking and hence inferior stability for the H2O2-treated S2E8C and S4E8C films.

Figure 2. Cumulative solubility of H2O2-treated (A) and methanol-treated SELPs films (B). Surprisingly, the methanol-treated S4E8C films were very stable, with negligible protein (0.4%) dissolved in solution even after 48 h (Figure 2B). On the other hand, the methanol-treated SE8C and S2E8C films were moderately stable in solution, with approximately 25-35% of the proteins dissolved. A possible explanation was that the methanol-treated films derived from 12 ACS Paragon Plus Environment

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S4E8C with the highest content of silk blocks might contain sufficient amounts of insoluble βstructures and thus exhibit longer term stability in aqueous solution, because methanol treatment has been demonstrated to trigger dehydration of the silk blocks to form highly-packed insoluble structures such as β-sheets.30 However, for the SELPs with lower ratio of the silk blocks, methanol treatment might not be enough to physically cross-link the polymer chains into the stable β-sheet structures, and covalent disulfide cross-links among the cysteine-containing elastin blocks offered an alternative means to fabricate water-insoluble protein films. Taken together, these results indicate that the SELPs with varying ratios of silk-to-elastin blocks could be tailored via cross-linking option (chemical disulfide vs physical β-sheet) to achieve stability.

Figure 3. Swelling ratio of the H2O2-treated SE8C and methanol-treated S4E8C films. Next, we evaluated the swelling properties for the H2O2-treated SE8C and methanol-treated S4E8C films, while the films with inferior stability in hydrated conditions were not tested. As depicted in Figure 3, both films rapidly swelled, reaching maximum water uptake capacity within four hours. The methanol-treated S4E8C films exhibited a significantly lower swelling



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ratio of 2.00 ± 0.37 than that of the H2O2-treated SE8C films at 4.96 ± 0.40. This difference might be due to methanol treatment enabling the formation of highly compacted water insoluble β-sheet structures, which contributes to a tighter cross-linked network than the disulfide crosslinking. The swelling ratios of the SE8C and S4E8C films were comparable and even lower than the values determined for other protein films in earlier studies. For example, a range of swelling ratios (~10–25) were reported for the α-elastin films crosslinked with ethylene glycol diglycidyl ether.31 This earlier study also demonstrated that the degree of crosslinking was negatively correlated with the swelling ratio of the protein films.31 Furthermore, the surface hydrophilicity of these two films was evaluated by contact angle measurements. The results showed the contact angle of the H2O2-treated SE8C films was 74.96 ± 2.74°, while it was 71.23 ± 2.39° for the methanol-treated S4E8C films. As the contact angles of these two SELP films were less than 90°, it was deduced that the surface of the films were hydrophilic, which might facilitate the attachment and growth of living cells on the films. Surface Morphology and Roughness of SELPs Films. For more detailed characterization of the SELP films, we examined surface morphology and roughness of the films because these properties can significantly influence the process of the attachment of living cells and other biomedical or biotechnological applications. Therefore, the non-, methanol-, and H2O2-treated films were prepared on mica discs for the atomic force microscopy (AFM) analysis. As shown in Figure 4A, the formation of globules was observed on the different types of S2E8C and S4E8C films, but not on the SE8C films. A closer inspection of the S2E8C and S4E8C films revealed that the H2O2 treatment did not noticeably change the film surface morphology, while methanol treatment induced the formation of larger aggregates compared with the respective non-treated films. These results indicated that the silk blocks might play a role in the formation of globules because a higher ratio of the silk-to-elastin blocks led to 14 ACS Paragon Plus Environment

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the formation of more particles. This observation was in good agreement with our previous study on the fundamental self-assembly mechanism of SELPs, in which more particles were formed with an increase in the ratio of the silk blocks.7

Figure 4. Surface morphology and roughness analysis. (A) AFM images of the films derived from SE8C, S2E8C and S4E8C with and without treatments. Each image is 10 µm x 10 µm, and the height bar on the right is applicable to all the images. (B) Surface roughness of the SELP films determined from the AFM images.

Following analysis of the surface morphology, the surface roughness values were calculated 15 ACS Paragon Plus Environment


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from the AFM images of the films (Figure 4B). A link between surface roughness and the ratio of silk-to-elastin blocks was identified, as an increase in the ratio resulted in a rougher film surface. For example, all three types of SE8C films were remarkably smooth, exhibiting a surface roughness at the 1 nm scale. On the other hand, the S2E8C and S4E8C films were approximately three- to six-fold rougher than the SE8C films. One possible explanation was that a low ratio of the silk-to-elastin blocks compromised the formation of highly compacted β-sheet structures that would require more silk blocks interacting with each other. Furthermore, methanol treatment resulted in much rougher surfaces for the S2E8C and S4E8C films compared with their respective non-treated counterparts, whereas H2O2 treatment did not appreciably impact the surface roughness. Together, these results indicated that cross-linking treatment and the ratio of silk-to-elastin blocks, one of the key design parameters for the SELPs, were critical in modulating the surface morphology and roughness of the resulting films. Structural Characterization. Next, the changes in the secondary structures of the SELP films upon H2O2 and methanol treatments were examined by FTIR (Figure 5). The control, non-treated films of the three SELPs showed IR signals centered at approximately 1626 cm-1, indicative of anti-parallel β-sheet conformation. The methanol-treated films possessed amide I absorption bands at ~1618 cm-1, which indicated the formation of aggregated strands upon methanol treatment. Surprisingly, H2O2 treatment exerted different effects on the shift of absorption peak for the SELP films. For the films of SE8C and S2E8C, the absorption peak shifted from 1626 cm-1 to 1621 cm-1, while the shift was from 1626 cm-1 to 1633 cm-1 for the S4E8C film, indicating a lower amount of aggregated strands formed in this scenario.


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Figure 5. FTIR absorbance spectra of the SELP films.

To further characterize conformational changes upon H2O2 or methanol treatment, we quantified the contents of individual secondary structural components in the SELP films by deconvolution of the FTIR spectra (Figure S1). Aggregated strands, β-sheet, β-turn, and unordered structure were assigned for each fitted band using a method established earlier.30,32,33 It has been demonstrated that the β-structures including β-sheets and aggregated strands could be clearly differentiated to distinguish the structural changes among diverse treatments of the films 17 ACS Paragon Plus Environment


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based on a lysine-containing SELP polymer.20

Figure 6. The contents of individual secondary structural components in the non-, H2O2- and methanol-treated SELP films. Structural content was determined by curve fitting the amide I band of the SE8C films (A), S2E8C films (B), and S4E8C (C) films according to the second derivative spectra of each sample.

As shown in Figure 6, the overall contents of the β-structures including aggregated strands and β-sheets were comparable for the three non-treated films. In addition, methanol treatment 18 ACS Paragon Plus Environment

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significantly increased β-strand aggregation in both S2E8C and S4E8C films, but not in the SE8C film, which might be due to the lower content of silk blocks in the latter protein polymer. On the other hand, H2O2 treatment did not dramatically change the overall content of β-structures for the three SELP protein films, which was in good agreement with the observation that H2O2treated films were as smooth as the non-treated films, as shown in Figure 4. Cytotoxicity. Cytotoxicity assessments of the SELP films were used to explore potential applications in tissue culture. Here, only the H2O2-treated SE8C and methanol-treated S4E8C films, which exhibited appreciable stability in solution, were tested using the mouse pre-osteoblast cells (MC3T3-E1) to evaluate in vitro cyto-compatibility. Live/Dead staining was performed at 72 h after seeding the cells on the films and images were taken using a fluorescence microscope. The results showed that cells seeded on both films were viable for at least 72 h and took on a stretched morphology (Figure 7), indicative of good cyto-compatibility of the SELP films. In another study of Morgan et al., the mouse MC3T3-E1 cells were planted on films prepared from regenerated cocoon silk fibroin, and osteoblast attachment was observed.34 These observations were interesting and may inspire mechanistic studies on cell attachment to protein films without any RGD cell adhesive sequences. In addition, for both types of the SELP films, no observable difference existed before and after the cell culture, indicating appreciable stability of the SELP films. It would be interesting to examine whether these chemically or physically cross-linked SELP films are suitable for supporting growth of other cell lines and for a longer culture period in future studies.



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Figure 7. Live/Dead staining of cell viability for the mouse pre-osteoblast MC3T3 cells seeded on TCP (A), H2O2-treated SE8C (B), and methanol-treated S4E8C films (C) for 72 h. Living cells were fluorescent in green and dead cells in red, and the scale bars are 200 µm.

CONCLUSIONS Three recombinant SELP proteins genetically engineered with varying ratios of the silk-to-elastin blocks and cysteine residues in the elastin blocks were fabricated into protein films through chemical and physical cross-linking. Depending on molecular feature of the SELPs and crosslinking methods, protein films with modulated characteristics such as wetting, surface morphology and surface roughness were obtained. For the cysteine-containing SELPs with a low ratio of the silk-to-elastin blocks (SE8C), H2O2 oxidation was a preferred cross-linking method without the use of any organic solvents, resulting in smooth, water stable films. For the SELPs with a high ratio of silk-to-elastin blocks (S4E8C), methanol treatment induced the formation of aggregated strands and thus enabled the formation of very stable films with rougher surfaces. Additionally, the H2O2-treated SE8C and methanol-treated S4E8C films, which were crosslinked either chemically through disulfide bonds or physically through silk crystallization, demonstrated in vitro cyto-compatibility based on the ability to support the growth of a mouse pre-osteoblast cell line. These types of SELP films are anticipated to find broader applications in tissue engineering and drug delivery. Furthermore, the chemical cross-linking approach with a low concentration of mild H2O2 may be useful for the fabrication of films from other protein 20 ACS Paragon Plus Environment

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polymers with an appropriate content of the cysteine residues.

ASSOCIATED CONTENT Supporting Information Curve fitted second derivative spectra of the SELP films (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS This project was supported by the National Natural Science Foundation of China (31470216, 21674061). Support from the National Institutes of Health (P41EB002520 and U01EB014976) is also greatly appreciated. The authors would also like to acknowledge Instrumental Analysis Center of Shanghai Jiao Tong University.

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Fabrication of Protein Films from Genetically Engineered Silk-Elastin-Like Proteins by Controlled Cross-Linking

Liang Chen, Ming-Liang Zhou, Zhi-Gang Qian, David L. Kaplan, and Xiao-Xia Xia

Table of Contents Graphic.


Fabrication of Protein Films from Genetically Engineered Silk-Elastin

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