3,4-Dihydroxyphenylalanine (DOPA)-Containing Silk Fibroin: Its

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3,4-Dihydroxyphenylalanine (DOPA)-containing silk fibroin: its enzymatic synthesis and adhesion properties Hiromitsu Sogawa, Nao Ifuku, and Keiji Numata ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01309 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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ACS Biomaterials Science & Engineering

3,4-Dihydroxyphenylalanine

(DOPA)-containing

silk

fibroin: its enzymatic synthesis and adhesion properties Hiromitsu Sogawa1, Nao Ifuku1, and Keiji Numata1,* 1

Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1,

Hirosawa, Wako-shi, Saitama, 351-0198 *Corresponding Author. E-mail: [email protected]

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 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

ABSTRACT Silk fibroin (SF) is a fascinating natural biomaterial that exhibits remarkable mechanical properties and biocompatibility. Meanwhile, biological adhesive materials have gathered much attention as biomedical and eco-friendly material due to their characteristic properties. Herein, we report the excellent adhesive function of enzymatically modified SF. The tyrosine residues of SF were successfully converted to dihydroxy-L-phenylalanine (DOPA) unit using tyrosinase as a biocatalyst. The content of DOPA was evaluated by amino acid composition analysis. Adhesive functions of DOPA-modified SF (DOPA-SF) between several material surfaces including mica, paper, polypropylene, wood and silk film were elucidated by lap shear tests. Fourier transform infrared measurements demonstrated that the adhesion strength of DOPA-SF was not directly related to the b-sheet formation of silk molecules. This ecofriendly and facile method offers a new perspective to fabricate natural adhesive materials for various application areas.

KEYWORDS silk fibroin / adhesion / tyrosinase / DOPA / enzymatic modification


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INTRODUCTION Silk fibroin (SF) from the Bombyx mori silkworm is a fascinating natural biopolymer and is widely studied due to its outstanding features such as remarkable mechanical properties, biodegradability, and biocompatibility.1 The repetitive GAGAGX hexapeptide units (X = S, Y, or A), which form an antiparallel b-sheet structure, are a main component of SF and are essential for exhibiting high tensile strengths.2–5 Several attempts for the modification of SF have been reported to modulate its properties and to add novel functions. For example, coupling reactions using cyanuric chloride,6–9 carbodiimide,10–12 glutaraldehyde,13 alkoxysilane,14 and isocyanate15 have been demonstrated, whereas the diazonium coupling chemistry was also applied in terms of selective introduction of unnatural functional groups to the tyrosine residues.16,17 Enzymatic modification is also an attractive approach because highly selective modification of complicated compounds can be achieved under mild conditions.18–21 The introduction of poly(2,6-dimethylenephenylene ether) onto the side chain of SF via the grafting-from method using horseradish peroxidase as a catalyst has been developed in our group to impart hydrophobicity to silk materials,19 while Freddi and coworkers reported tyrosinase-catalyzed modification of SF to graft the polysaccharide chitosan.20,21 In addition, the fabrication of silk composites with other materials such as polymers22–25 and inorganics provided desired properties.26,27 The development of these approaches is significantly important due to a high potential for the practical use of silkbased materials.



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Meanwhile, adhesives are essential materials in our everyday life, and in particular, biobased adhesives are quite important as sustainable/eco-friendly biomedical materials. Recently,

we

have

synthesized

adhesive

polypeptides

consisting

of

tyrosine,

dihydroxyphenlyalanine (DOPA) and lysine units by chemoenzymatic reactions and revealed their outstanding adhesive properties, which were better than super glue.28 The catechol moiety of DOPA is known to play a crucial role in its adhesive function and to mediate strong adhesion to versatile surfaces.29–34 With a variety of adhesive catecholderived polymers,35–38 Kaplan and coworkers prepared catechol-functionalized SF and found an increase of adhesive strengths compared with SF lacking catechol functionalization.39 They conjugated the catechol moiety by a condensation reaction using dopamine hydrochloride after enrichment of the carboxyl group on SF because it contains only a few aspartic and glutamic acid units (0.5 and 0.6 mol%, respectively).2,4 Based on these previous studies, the low to moderate density of DOPA residues in the materials shows excellent adhesive function for a variety of surfaces. Thus, we focus on direct oxidative modification of the tyrosine residue in SF from B. mori because the appropriate percentage of tyrosine (5.3 mol%) was originally included in SF. The fabrication of silk-based biocompatible materials with excellent adhesive properties is expected by this environment-friendly and economical approach. Herein, we prepared DOPA-modified SF (DOPA-SF) via tyrosinasecatalyzed modification and evaluated the adhesive function of DOPA-SF by lap shear tests. Additionally, the relationship between the secondary structure of DOPA-SF and its adhesive


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properties were also evaluated by Fourier transform infrared (FT-IR) analysis.



EXPERIMENTAL SECTION Materials and measurements SF was obtained from silkworm cocoons of B. mori, and its aqueous solutions with different concentrations (10, 15, 20 and 25 g/L) were prepared by a previously reported protocol.1,23 Namely, silkworm cocoons were boiled for 30 min in a 0.02 M Na2CO3 solution, and subsequently washed with Milli-Q water. After drying for 24 h at 25 °C, extracted residues were dissolved in a 9.3 M LiBr solution at 60 °C for 2 h. It was further dialyzed with MilliQ water for 72 h using a dialysis membrane (Pierce Snake Skin MWCO 3500; Thermo Fisher Scientific, Waltham, MA, USA) to give SF aqueous solution. Tyrosinase from mushrooms (³1000 U/mg solid) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mica, paper, polypropylene resin (PP) and wood were purchased from Nisshin EM Co., Ltd., TOPPAN FORMS Co., Ltd., Artec Co., Ltd. and Lychee (Jinghua, China), respectively. Silk film was prepared from the aqueous SF solution according to a previously reported method.40 The other chemicals were used as received without purification unless otherwise noted.

Enzymatic modification of the tyrosine unit in SF to DOPA To the SF aqueous solution (1.0 mL), modified phosphate buffer solution (1.0 mL; 40 mM boronic acid, 0.3 M NaCl, 0.2 M ascorbic acid, 0.2 M phosphate buffer, pH 4.2) and tyrosinase (final concentration: 1300 U/mL) were added and stirred at 25°C for 24 hours in open-air conditions to produce the DOPA-SF solution at pH 4.2. The obtained solution was


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used for adhesive tests without further purification. The enzymatic reaction was conducted 3 times for each SF concentration. DOPA-SF solutions at different pH values (7.0, 8.0, 10 and 12) were made by adding 5.0 M NaOH aq. to this solution. The DOPA content was evaluated by amino acid composition analysis, namely, the ninhydrin method.41 Briefly, the hydrolyzed amino acids were characterized using High-Speed Amino Acid Analyzers, L-8900 and L8500A (Hitachi-HighTech, Tokyo, Japan). In addition to the natural amino acids, DOPA (Sigma-Aldrich) was used to calibrate the analyzer, and accordingly, the peak was assigned as DOPA (see Figure S1).

Evaluation of adhesion strength Twenty µL of DOPA-SF solutions were coated on the freshly cleaved mica sheet. After overlaying a second mica sheet, pasted sheets were clipped with a bulldog clip to ensure good contact and were allowed to stand for 24 hours at room temperature. The overlay was 10 mm width ´ 15 mm length and the total overlay area was 1.5 ´ 102 mm2. The adhesive function of the samples was evaluated by a lap shear test machine (EZ-Test, Shimadzu, Kyoto, Japan) with elongation rate of 10 mm/min. The initial distance between grips was set at 25 mm. The measurements were performed at approximately relative humidity 40% and 25°C. The breaking strain, adhesive strength, adhesive fracture energy and Young’s modulus were analyzed based on the stress-strain curves of the prepared samples. The experiments were conducted 5 times for each condition. The adhesive function between paper, PP and wood



was also characterized in the same conditions. The sample preparation of silk film was varied because pasted samples were ruptured at overlapping points by lap shear tests when samples were prepared in the same conditions as others. A silk film of 10 mm width ´ 10 mm length was first pasted on a flat wood sheet (10 mm width ´ 40 mm length) with a super glue, and 20 µL of DOPA-SF solution was added to the surface of the silk film. Curing conditions were maintained in the same manner as others, although the overlay was changed to 10 mm width ´ 5 mm length (total overlay area: 0.5 ´ 102 mm2). The initial distance between grips was also changed to 35 mm. The elongation rate was kept at 10 mm/min. Photographs for sample preparation and lap shear tests are shown in Figure 2 and Figure S2–S5.

ATR-FT-IR measurements Attenuated total reflection (ATR)-FT-IR was measured on an IRPrestigae-21 Fourier transform infrared spectrophotometer (Shimadzu Corporation, Kyoto, Japan) with a MIRacle A single reflection ATR unit using a Ge prism. Fifty µL of DOPA-SF solutions (15 g/L) at different pH values (4.2, 7.0, 8.0, 10, 12) were dropped on each surface and dried at room temperature for 16 hours to prepare the samples. The measurements were conducted from 3800 to 600 cm–1. The background spectra obtained in the same conditions were subtracted from the scan for each sample. SF with b-sheet structure was induced by methanol treatment for 24 hours,39 while SF with random structure was obtained from as-casted sample.


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Statistical analysis The significance of differences in the mechanical property studies was determined by paired t-tests with a two-tailed distribution and one-way analysis of variance (ANOVA) using Microsoft Excel. Differences were considered statically significant at p < 0.06 (*) and p < 0.01 (**).



RESULTS AND DISCUSSION Enzymatic modification of SF using tyrosinase As catechol interaction of DOPA with surfaces is essential for adhesive function,29–34 excessive oxidation of DOPA to 2,4,5-trihydroxyphenylalanine (TOPA) and dopaquinone should be prevented to maximize its adhesive properties. Meanwhile, we have found efficient and suitable conditions for tyrosinase-catalyzed oxidation to convert tyrosine residues of polypeptides to DOPA without further oxidation.28 Thus, this mild oxidation condition was also applied to SF modification in this study. After the treatment of SF solution with tyrosinase at 25°C for 24 hours, homogeneous SF solutions were slightly turbid (Figure 1a). This solution was directly used for adhesive tests without further purification in this study because the effect of tyrosinase itself for adhesion property would be neglectable. Park and coworkers reported that the adhesive strength of chitosan did not change in the presence and absence of tyrosinase.42 The content of DOPA after the reaction was characterized by amino acid composition analysis (Figure S1). It was estimated to contain approximately 1.3 mol% of DOPA and was almost constant for all the concentration ranges studied (Figure 1b). In addition, the conversion ratio of the modification reaction was estimated to be approximately 25% based on the amount of tyrosine units in the SF. Although solution NMR experiment was difficult because of low solubility of DOPA-SF toward common deuterium solvents after modification reaction, introduction of DOPA moiety was surely identified by increasing sample size up to 3 for each concentration.


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Figure 1. (a) Tyrosinase-catalyzed modification of SF to DOPA-SF at different concentrations, together with the photographs of solutions. The caps of grass sample bottles were opened during the reaction. (b) The DOPA content in DOPA-SF after modification.



Evaluation of adhesive function of DOPA-SF The adhesion function was evaluated by lap shear tests. Test samples were prepared by coating DOPA-modified SF solution (DOPA-SF) between two mica surfaces and tested on a lap shear test machine (Figure 2). Based on their stress-strain curves (Figure 3), the adhesion properties including breaking strain, adhesive strength, adhesive fracture energy, and Young’s modulus at different silk concentrations and pH levels are summarized in Figure 4. We maintained the measurement conditions at an approximate relative humidity of 40% and 25°C because the mechanical properties of SF are sensitive to humidity and temperature.43 It was obvious that both adhesive strength and fracture energy were improved by the introduction of DOPA at any silk concentration and pH level, whereas maximum values (0.97 MPa and 19.1 KJ/m3, respectively) were observed for 15 g/L of DOPA-SF at pH 10. The increase in the Young’s modulus of DOPA-SF compared to that of nonmodified SF was also observed probably because the original outstanding physical properties of silk has started to appear for DOPA-SF since the interface of mica and silk more strongly interact. These results suggest that the adhesive function of SF was improved by DOPA modification. When we looked at the data more precisely, higher adhesive properties appeared at higher pH levels. For example, the adhesive strength of DOPA-SF (15 g/L) at pH 10 was 1.4 times larger than that of pH 4.2. Marine adhesive proteins such as Mytilus edulis foot protein-5 (Mefp-5) are known to be rich in not only DOPA but also lysine and form networks involving the amine group of lysine and the catechol of DOPA.44 SF originally contained lysine units, although


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the content was not high (0.2 mol%).2 Therefore, the presence of both deprotonated lysine and DOPA at higher pH values enhanced the adhesion resulting from DOPA interactions in a similar manner to Mefp-5. In addition, adhesive function decreased by increasing the concentration of DOPA-SF to more than 15 g/L. This tendency is coincident with the report that the excess amounts of DOPA in artificial adhesive polypeptides do not contribute to its adhesive strength significantly.45 This is probably because efficient contact of DOPA moiety with mica surface was interrupted somehow. It also should be noted that a linear relationship between the concentrations of nonmodified SF and adhesive strength was confirmed, indicating that silk itself can bond mica substrates without a catechol moiety. Kaplan and coworkers reported similar features when they applied SF to the adhesion of titanium46,47 and aluminum sheets39, while other groups demonstrated biocompatibility and strong adhesive of SF for skin.48,49 Besides, we also investigated adhesive properties of SF toward chamois leather.50 Although these findings suggest the high potential of SF itself for adhesives, DOPA modification is important from the viewpoint of not only improving its adhesive function but also widening the variety of applicable surfaces.



Figure 2. Photographs of (a) sample preparation of mica sheets and (b) lap shear tests. Overlay area: 1.5 ´ 102 mm2 (10 mm width ´ 15 mm length).


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Figure 3. Stress-strain curves of pasted mica samples. The silk concentrations were (a) 10, (b) 15, (c) 20, and (d) 25 g/L, respectively. Elongation rate: 10 mm/min.



Figure 4. Adhesion properties of DOPA-SF. (a) Breaking strain, (b) adhesive strength, (c) adhesive fracture energy, and (d) Young’s modulus of DOPA-SF for mica substrate at different silk concentrations and pH levels. *Significant differences between groups at p < 0.06. **Significant differences between groups at p < 0.01.


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We next evaluated the adhesive properties of DOPA-SF for different surfaces including paper, PP and wood, and silk film. As mentioned above, SF solution itself exhibited good adhesion properties for several surfaces such as skin. Thus, several surfaces, whose homogeneity to SF seems to be low, were chosen to emphasize the significance of DOPAmodification. DOPA-SF solution (15 g/L) at different pH levels was coated on substrates. Figures 5 and 6 show stress-strain curves of pasted samples and a summary of adhesive function for these substrates, respectively. Note that the overlay area of silk film (0.5 ´ 102 mm2) was changed to three times smaller than that of others (1.5 ´ 102 mm2) because the rupture of samples took place at other adhesive points by lap shear tests when the samples were prepared under original conditions (Figures S2–S5). We could not complete lap shear tests with SF solution and DOPA-SF solutions at pH 4.2 and 7.0 for this modified condition due to the failure of sample preparation. DOPA-SF exhibited higher adhesive function than SF for not only mica but also for all tested substrates, especially at higher pH values. The highest adhesive strength was observed for silk film even though the overlay area was decreased. Meanwhile, the Young’s modulus determined with paper, PP and wood was almost constant for both SF and DOPA-SF in contrast to mica. This outcome occurred because DOPA-SF adhered to these surfaces relatively weakly, and cleavage on the surface took place before the modulus of silk itself had appeared in contrast to mica. Improvement in adhesive strengths was observed for these surfaces; however, it was not significant. Conversely, the Young’s modulus determined with silk film was approximately 200 MPa, which was the



highest for all the materials studied. Together with the results of adhesive strengths, it was concluded that DOPA-SF functioned against various surfaces, most strongly interacted with silk film and exhibited outstanding adhesiveness.


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Figure 5. Stress-strain curves of pasted samples of (a) paper, (b) PP, (c) wood, and (d) silk film. The silk concentration and the elongation rate were set to 15 g/L and 10 mm/min, respectively.



Figure 6. (a) Breaking strain, (b) adhesive strength, (c) adhesive fracture energy, and (d) Young’s modulus of DOPA-SF for mica, paper, PP, wood, and silk film at different pH levels. Silk concentration: 15 g/L. *Significant differences between groups at p < 0.06. **Significant differences between groups at p < 0.01.

Characterization of secondary structures of DOPA-SF on the surfaces To understand the secondary structures of DOPA-SF on the material surfaces, ATR-FT-IR measurements were performed (Figure 7).51 The samples were prepared by casting DOPASF solutions (15 g/L, 50 µL) on the surfaces and by drying at room temperature for 16 hours. SF with b-sheet structure was prepared by treating as casted sample with methanol for 24 hours as control.39,52 Consequently, DOPA-SF on all the tested surfaces adopted a mainly


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random-coil conformation, as evidenced by broad peaks at 1650 and 1540 cm–1 of amide I and amide II absorption peaks, respectively.40 Besides, b-sheet structure was enhanced at acidic condition preferably for every surfaces, whereas random conformation was maintained at higher pH level. This tendency is coincident with previous report.53 DOPA-modification of tyrosine moiety did not affect the ability of SF to form a b-sheet structure. The β-sheet formation at each condition was probably triggered by sample drying. As the increase of adhesive strength was observed by increasing pH levels, it was concluded that β-sheet structure was not directly related to adhesive strength. It seems to be more related to the values of Young’s modulus.



Figure 7. ATR-FT-IR spectra of DOPA-SF at different pH levels on (a) mica, (b) paper, (c) PP, (d) wood, (e) silk film, and (f) SF with random coil and b-sheet structures as control. Silk concentration: 15 g/L. Pink dotted lines represent amide I (1650 cm–1) and amide II (1540 cm–1) absorptions of random coil structure, while the brown dotted line represents amide I (1625 cm–1) absorption of b-sheet structure.


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CONCLUSIONS Tyrosinase-catalyzed modification of SF from the B. mori silkworm was successfully achieved to produce DOPA-modified SF. Amino acid composition analysis revealed 25% of tyrosine residues in SF were converted to DOPA resulting in a DOPA-SF solution with ca. 1.3 mol% of DOPA moiety. Although the content of DOPA was not high, significant improvement of the adhesive function of DOPA-SF for mica surface was confirmed by lap shear tests. A higher adhesion function was observed for higher pH levels. DOPA-SF exhibited excellent adhesion not only for mica but also for several types of substrates including paper, PP, wood and silk film. It was also found that the adhesion strength was not assisted by the formation of a b-sheet structure of DOPA-SF based on ATR-FT-IR measurements. This simple but powerful approach offers a new perspective to fabricate natural adhesive materials with an environmentally benign process.



CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was financially supported the Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT) of the Japan Science and Technology Corporation (JST), and RIKEN Engineering Network.


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Graphical Abstract


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3,4-Dihydroxyphenylalanine (DOPA)-Containing Silk Fibroin: Its

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