Azide-Incorporated Clickable Silk Fibroin Materials with the Ability to

We demonstrated that azide groups in AzPhe incorporated into silk fibroin can be .... Raw silk fiber was reeled from six cooked cocoons on average usi...
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Azide-Incorporated Clickable Silk Fibroin Materials with the Ability to Photopattern Hidetoshi Teramoto,* Ken-ichi Nakajima, and Katsura Kojima Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences (NIAS), 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan S Supporting Information *

ABSTRACT: Incorporation of unnatural amino acids (UAAs) bearing bioorthogonal reactive groups into proteins could be a powerful tool for developing novel protein-based biomaterials with innovative and controlled performance. Bombyx mori silk fibroin is one of naturally derived protein materials extensively studied for biomaterials development due to its mechanical strength and biocompatibility. We recently reported the in vivo incorporation of UAAs, 4-substituted analogues of phenylalanine including 4-azidophenylalanine (AzPhe), into silk fibroin by expanding the repertoire of amino acids for protein biosynthesis in silk glands of B. mori using transgenic techniques. We demonstrated that azide groups in AzPhe incorporated into silk fibroin can be selectively modified by bioorthogonal azide−alkyne cycloaddition reactions (click chemistry). However, the incorporation of AzPhe into silk fibroin required a special feeding condition, which led to the limited production of silk fibroin. Here we report more efficient production of an AzPhe-incorporated silk fibroin (termed AzidoSilk) and its modification by click chemistry in varied material forms (thread, film, and porous sponge). Using this methodology, photolithographic micropatterning of fluorescent molecules directly onto silk fibroin film was achieved and should further expand the availability of silk-based biomaterials for cell culture substrates, drug delivery, tissue scaffolds, implantable devices, and so on. KEYWORDS: azide, click chemistry, phenylalanyl-tRNA synthetase, photopatterning, silk fibroin, unnatural amino acid

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phenylalanyl-tRNA synthetase α-subunit (BmPheRS-α) in the posterior silk gland (PSG), where silk fibroin proteins are synthesized. Upon the oral administration of UAAs, 4-chloro-, 4-bromo-, and 4-azido-substituted analogues of phenylalanine (Phe), to H01 larvae, they were successfully incorporated into silk fibroin in place of Phe residues in a residue-specific manner.38 In other studies we focused particularly on the incorporation of 4-azidophenylalanine (AzPhe), because it possesses a phenyl azide group available for bioorthogonal modification27 or photoinduced cross-linking.39 In fact, we have succeeded the incorporation of AzPhe into silk fibroin and verified that phenyl azide groups in AzPhe incorporated into silk fibroin worked as selective chemical handles for bioorthogonal azide−alkyne cycloaddition reactions (click chemistry).38 However, AzidoSilk could not be obtained by simply rearing H01 larvae on a commercially available standard diet, because Phe is preferentially incorporated into proteins during biosynthesis in competition with AzPhe, probably due to the relatively weak recognition of AzPhe by the A450G mutant of BmPheRSα. Therefore, for the production of AzidoSilk with the H01 line, a specially prepared synthetic diet with controlled Phe content

INTRODUCTION Silk fibroin produced by the domesticated silkworm, Bombyx mori, has long been used for biomaterials development because of its excellent mechanical strength and biocompatibility.1−3 Silk fibroin is regarded as a heterodimer of fibroin heavy chain protein (FibH; ∼ 390 kDa) and fibroin light chain protein (FibL; ∼26 kDa)4 (Figure S1). Silk fibroin can be processed into various material forms,5 and various types of silk-based materials have been investigated for a wide range of biomedical applications such as cell culture substrates,6−8 drug delivery,9−11 tissue scaffolds,12−14 implantable devices,15−17 and so on.1,2 To enhance the utility of silk fibroin for biomedical applications, a number of chemically-9,18−21 or genetically-22−25 modified silk fibroins have been developed and characterized. Bioorthogonal modification of proteins via the incorporation of unnatural amino acids (UAAs) has been attracting much attention as a powerful tool to expand the scope of proteins both in vitro and in vivo. 26,27 The methodology of bioorthogonal modification has been expanded to the development of novel protein-based biomaterials with innovative and controlled performance.28−31 A similar methodology using bioorthogonal reactions has been extensively studied for the fabrication of various kinds of polymer material utilizing proteins, polysaccharides, and synthetic polymers as building blocks.32−37 We previously generated a transgenic B. mori line (designated H01) expressing the A450G mutant of B. mori © XXXX American Chemical Society

Received: November 6, 2015 Accepted: January 10, 2016

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DOI: 10.1021/acsbiomaterials.5b00469 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

precipitates and stored at 4 °C until use. When needed, the silk fibroin solution was concentrated to the desired concentrations by a VC-36S centrifugal concentrator (TAITEC, Saitama, Japan). For the preparation of films, the silk fibroin solution (10−20 mg/mL) was cast onto silicon rubber (0.2 mL/cm2) and dried at 25 °C. The films were stored at room temperature until use. The films were insolubilized by immersing in 70% ethanol for ca. 30 min at room temperature followed by drying at 25 °C before use. For the preparation of porous sponges,40 the silk fibroin solution (40 mg/mL) was mixed with 1 vol % of dimethyl sulfoxide (DMSO), frozen overnight at ca. −20 °C, and then freeze-dried with a FDU-830 freezedryer (EYELA, Tokyo, Japan). The porous sponges were stored at room temperature until use. Cu-Catalyzed Azide−Alkyne Cycloaddition (CuAAC). Silk fibroin threads, films, or porous sponges (±AzPhe) were reacted overnight with 25 μM carboxyrhodamine 110 alkyne at room temperature in 50 mM Tris-HCl (pH 8), 1 mM CuSO4, 0.1 mM TBTA, and 1 mM sodium ascorbate. After reaction, they were washed with DMSO at room temperature or at elevated temperatures such as 50 °C followed by rinsing with deionized water. Fluorescence on threads and films was observed with a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan). Fluorescence on porous sponges was observed with a MZ16 FA fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany). Strain-Promoted Azide−Alkyne Cycloaddition (SPAAC). Thin silk fibroin films (±AzPhe) were formed on the bottom of a 96-well plate by casting 0.1 wt % aqueous solution of silk fibroin (±AzPhe) followed by drying at 50 °C. The films were insolubilized by immersing in 70% ethanol for ca. 30 min at room temperature followed by drying at room temperature. The films were reacted overnight with 0, 1, 5, and 25 μM sulfo-DBCO-biotin at room temperature in 50 mM Tris-HCl (pH 8). The wells were washed with DMSO and Tris-buffered saline with 0.05% tween 20 (TBS-T). The films were incubated with HRP-streptavidin (Nacalai Tesque) (1/ 10000 in TBS-T) for 1 h at room temperature followed by thorough washing with TBS-T. HRP activities on the films were detected using SureBlue TMB substrate (KPL, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The absorbance at 450 nm in each well was measured using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Silk fibroin threads, films, or porous sponges (±AzPhe) were reacted overnight with 5 μM sulforhodamine B DBCO at room temperature in 50 mM Tris-HCl (pH 8). After reaction, they were washed with DMSO at room temperature or at elevated temperatures such as 50 °C followed by rinsing with deionized water. Fluorescence was observed as described above. Photolithographic Patterning on AzidoSilk Threads and Films. AzidoSilk threads aligned horizontally on a slide glass were irradiated by 254 nm UV light for 3 min through a photomask (affixed with a decal reading “SILK”) using a TL-2000 ultraviolet translinker (UVP, Upland, CA, USA) equipped with five G8T5 bulbs. The irradiated threads were reacted overnight with 25 μM carboxyrhodamine 110 alkyne at room temperature in 50 mM Tris-HCl (pH 8), 1 mM CuSO4, 0.1 mM TBTA, and 1 mM sodium ascorbate. The reacted threads were washed thoroughly with DMSO at room temperature or at elevated temperatures such as 50 °C followed by rinsing with deionized water. Fluorescence was observed with a BZ-X710 fluorescent microscope (Keyence, Osaka, Japan). AzidoSilk films were irradiated by 352 nm UV light for 60 min through glass photomasks using a light box (SunHayato, BOX-W10) equipped with six FL15BL bulbs. The irradiated films were treated with 70% ethanol for ca. 30 min for insolubilization followed by drying at room temperature. The treated films were then reacted overnight at room temperature with 25 μM carboxyrhodamine 110 alkyne in 50 mM Tris-HCl (pH 8), 1 mM CuSO4, 0.1 mM TBTA, and 1 mM sodium ascorbate or 5 μM sulforhodamine B DBCO in 50 mM TrisHCl (pH 8). The reacted films were washed thoroughly with DMSO at room temperature or at elevated temperatures such as 50 °C followed by rinsing with deionized water. Fluorescence on the films was observed with a BZ-X710 fluorescent microscope (Keyence).

was required. Under this special feeding condition, we could only obtain thin cocoons because of the reduction of fibroin production (ca. 1/4 of that under the normal rearing condition).38 In this study, more efficient production of AzidoSilk using a commercially available standard diet was achieved by generating a new transgenic line expressing another mutant of BmPheRS-α. AzidoSilk was further processed into varied material forms and subjected to functionalization by click chemistry. Photopatterning of fluorescent molecules on AzidoSilk films by UV-induced decomposition of azide groups was also demonstrated.

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EXPERIMENTAL SECTION

Materials and Animals. All chemicals used in this study were of reagent grade and used as received. AzPhe was from Watanabe Chemical Industries (Hiroshima, Japan). Cellulose powder was from Nacalai Tesque (Kyoto, Japan). Tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl]amine (TBTA) and sulfo-dibenzocyclooctyne-biotin conjugate (sulfo-DBCO-biotin) were from Sigma-Aldrich (St. Louis, MO, USA). Biotin-PEG4-alkyne, carboxyrhodamine 110 alkyne, sulforhodamine B DBCO, and DBCO-PEG4-NHS ester were from Click Chemistry Tools (Scottsdale, AZ, USA). Green fluorescent protein (GFP) was from EMD Millipore (Billerica, MA, USA). Synthetic oligonucleotides were from Hokkaido System Science (Sapporo, Japan) and Sigma-Aldrich. A B. mori strain, MCS601, which has been maintained in the Transgenic Silkworm Research Unit at NIAS, was used for germline transformation. B. mori larvae were reared on an artificial diet (SilkMate PS) (Nosan Corporation, Yokohama, Japan) at 22−26 °C. Administration of AzPhe with the Specially-Prepared Synthetic Diet. AzPhe was mixed into the synthetic diet and administered to fifth-instar male larvae as described previously.38 Administration of AzPhe with the Commercially-Available Standard Diet. AzPhe was mixed with SilkMate PM (dried form) (Nosan Corporation) at a ratio of 0.1, 0.3, 0.5, 0.75, or 1.0% (w/w). Cellulose powder was added to normalize the dry weight among different conditions. Deionized water of 2.6 times volume per unit weight of dry diet was added and heated for 1 h at 50 °C with gentle shaking to make uniform slurries. The slurries were cooked for 5 min at 95 °C in an autoclave and stored in a refrigerator until use. The AzPhe-mixed diet was administered to male larvae from the third day of fifth instar. When the average body weights of larvae stopped increasing, each larva was individually transferred into a handmade paper box to allow it to start cocooning (at the seventh day in most cases). Silk Reeling. Cocoons were cooked for ca. 10 min using a VP-type vacuum cocoon cooking machine (HARADA, Okaya, Japan). Raw silk fiber was reeled from six cooked cocoons on average using a handmade reeling machine at a reeling speed of 30 m/min at 40 °C. The raw silk threads were twisted at a rate of 900 turns/m using a PAK-10UMH twisting machine (TSUDAKOMA, Kanazawa, Japan) followed by heating in an autoclave for 10 min at a thermal setting at 110 °C. After twisting, the raw silk threads were degummed in 0.5% Na2CO3 at 95 °C for 20 min with gentle stirring followed by washing with water. Tensile Test. The degummed silk threads prepared as above were equilibrated at 20 °C and 65% RH for over 24 h before testing. Tensile tests were performed with a gauge length of 100 mm and a strain rate of 150 mm/min using a RTA-100 tensile testing machine (Orientec, Tokyo, Japan) at 20 °C and 65% RH. Materials Fabrication. Cocoons were cut into small pieces and degummed with 0.02 M Na2CO3 according to the previous paper.5 The degummed cocoons were dissolved in 8 M LiBr solution at 35 °C at a concentration of 50 mg/mL and then mixed with a 1/4 volume of 0.5 M Gly-NaOH buffer (pH 9). The mixture was dialyzed using a membrane (Spectra/Por MWCO 12 000−14 000; Spectrum Laboratories, Rancho Dominguez, CA, USA) against deionized water, and then against 0.1 mM sodium carbonate buffer (pH ∼ 9). The dialyzed silk fibroin solution was centrifuged (9,500 × g, 4 °C, 1 h) to remove B

DOI: 10.1021/acsbiomaterials.5b00469 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION Generation of H03 Transgenic Line Expressing the T407A Mutant of BmPheRS-α in PSG. To achieve more efficient production of AzidoSilk using a commercially available standard diet, a new transgenic line named H03 (Figure 1) was

Figure 2. Comparison of AzidoSilk production between the H01 and H03 transgenic lines reared on a synthetic diet. (a) Detection of AzPhe incorporation into silk fibroin by MALDI-TOF-MS of the Phecontaining peptide fragment derived from FibL (SGNFAGFR, [M + H]+ = 855.41 Da). The peak intensities at ca. 855 Da were standardized in all spectra. A newly emerged peak at the +15 Da position (ca. 870 Da) (enlarged in the inset) was attributed to the replacement of one of the Phe residues in the peptide to AzPhe based on the previous finding that the azide groups in AzPhe are reduced to amino groups during analysis.38,42 (b) Fibroin production by H01 or H03 larvae fed a synthetic diet containing AzPhe (fixed to a 0.5 molar equiv of Phe in the diet) and the incorporation efficiency of AzPhe into silk fibroin. Bar plots represent the amounts of fibroin. Open and closed circles represent the ratio of peak intensities (870 Da/855 Da) in MALDI-TOF-MS analysis as an index of AzPhe incorporation. The plots from the control experiments without AzPhe administration show the levels of background signals. All values in the plots are the average of three independent experiments, where each experiment employed three male larvae. The error bars represent standard deviations. (c) Detection of azide groups in silk fibroin by CuAAC with biotin-PEG4-alkyne. Biotins attached to silk fibroin were detected by Western blotting. The signals assigned to FibH and FibL are shown with CBB-stained bands. Raw data without trimming are shown in Figure S4.

Figure 1. Generation of a new transgenic line, H03, for efficient production of an AzPhe-incorporated silk fibroin (termed AzidoSilk). The piggyBac plasmid vector containing the gene encoding the T407A mutant of BmPheRS-α with a PSG-specific FibL promoter was used for germline transformation of B. mori. Oral administration of AzPhe mixed in a commercially available standard diet to the fifth-instar H03 larvae led to efficient production of AzidoSilk as shown in the experimental data in this paper.

generated using the same procedure as previously reported for generation of the H01 line.38 The new H03 line expresses the T407A mutant of BmPheRS-α specifically in the PSG (Figure S2a). As with the A450G mutant, the T407A mutant exhibited aminoacylation activity toward AzPhe in vitro.41 In addition, the amino acid recognition of the T407A mutant was more relaxed than that of the A450G mutant judging from the inhibitory effect on protein biosynthesis observed in B. mori cultured cells under a Phe-restricted culture condition.41 Under a normal culture condition, such an inhibitory effect on protein biosynthesis was not observed by expressing the T407A mutant. It was assumed that noncognate amino acids were misaminoacylated to tRNAPhe when the cognate amino acid, Phe, was depleted in the cells. In contrast, expression of the A450G mutant did not cause the inhibitory effect on protein biosynthesis even under a Phe-restricted culture condition. These observations indicated that the T407A mutant has more relaxed amino acid specificity than the A450G mutant. We thus anticipated that the T407A mutant might exhibit better recognition toward AzPhe in the PSG of transgenic larvae. Production of AzidoSilk by H03 Transgenic Larvae. We first compared the efficiency of incorporation of AzPhe into silk fibroin in larvae fed a synthetic diet containing a fixed molar amount of AzPhe (0.5 molar equiv. to Phe in the diet) between the H01 and H03 lines. Upon the administration of AzPhe, larval growth was not clearly impeded for either line (Figure S3a), but diet ingestion was slightly smaller in H03 than H01 (Figure S3b). The production of silk fibroin decreased for both H01 and H03, with the reduction being more remarkable for H03 (Figure 2b, bar plots). This decrease in silk production would have resulted from the incorporation of AzPhe into proteins during biosynthesis, as observed in the case of other UAAs.38 The efficiency of incorporation of AzPhe into silk fibroin was estimated from the ratio of MALDI-TOF-MS peaks

assigned to the Phe-containing peptide fragment (SGNFAGFR) derived from FibL (ca. 855 Da) and its counterpart (ca. 870 Da), in which one of the two Phe residues in the peptide was replaced with AzPhe (Figure 2a). Based on this estimation, there was almost no incorporation of AzPhe into silk fibroin in the H01 larvae, as reported previously,38 whereas a much larger amount of AzPhe was incorporated into silk fibroin in the H03 line, even when the larvae were fed a diet containing a standard amount of Phe (Figure 2b, open and closed circles). The presence of larger amounts of azide groups in H03-derived silk fibroin was further confirmed by click reactions with biotin-PEG4-alkyne (Figure 2c). We inferred that the largely different incorporation of AzPhe into silk fibroin between H01 and H03 would have arisen from the different amino acid recognition capacity of the A450G and T407A mutants of BmPheRS-α.41 Alternatively, if the expression of the mutant genes in PSG is considerably different between the two lines, this could have affected the incorporation of AzPhe into silk fibroin. We therefore roughly compared the expression levels of the mutant genes in PSG of C

DOI: 10.1021/acsbiomaterials.5b00469 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Table 1. AzidoSilk Production by the Previous H01 Line and the Newly Generated H03 Line under the Respective Optimized Conditions line H01 H03

transgene PheRS-α A450G PheRS-α T407A

diet c

synthetic commerciald

Phe (w/w)a

AzPhe (w/w)a

AzidoSilk/larva (mg)

ratio of mass peaksb

ref

0.35 ∼1.0e

0.22 0.5

26.7 ± 9.4 87.3 ± 9.3

0.031 ± 0.011 0.051 ± 0.011

38 this study

a Amino acid contents in dry diet. bRatio of 870 Da peak (AzPhe-incorporated)/855 Da peak (parental) in MALDI-TOF-MS analysis (refer to Figure 2a). cThe composition of the synthetic diet was reported elsewhere.38 dSilkMate PM (Nosan Corporation) was used. eEstimated by amino acid composition analysis.

Fabrication of AzidoSilk Materials and Their Click Modifications. Using the H03 transgenic line newly established in this study, we considered that it would now be possible to produce high quality cocoons of AzidoSilk, which would be available for mechanical reeling of AzidoSilk threads. Indeed, using standard mechanical reeling techniques with subsequent twisting and degumming processes, AzidoSilk threads (Figure 4a) were successfully obtained from fresh cocoons. Figure 3 shows the comparison of the tensile

H01 and H03 larvae by RT-PCR. The results showed that the expression of the T407A mutant in H03 was comparable to or even weaker than the expression of the A450G mutant in H01 (Figure S2b). Because the DNA sequences coding the A450G and T407A mutants of BmPheRS-α differ by only two nucleotides, their translation efficiencies would be the same. We thus concluded that the largely increased incorporation of AzPhe into silk fibroin in the H03 line results from the intrinsic characteristics of the T407A mutant of BmPheRS-α. As reported previously, when the Phe content in the synthetic diet was lowered to 30% of the standard amount or less, H01 larvae were able to incorporate a greater amount of AzPhe into their silk fibroin.38 However, the larvae reared on such a Phe-restricted synthetic diet exhibited impeded growth and spun smaller amounts of silk fibroin. As shown in Figure 2, H03 larvae were able to incorporate much more AzPhe than the H01 larvae even when consuming a synthetic diet with a standard amount of Phe. It was thus expected that AzidoSilk could be more effectively produced by rearing H03 larvae on a commercially available, mulberry leaf-based standard diet, which is more appropriate for silk production and much cheaper than a synthetic diet. To test the availability of a commercially available standard diet for the production of AzidoSilk, varied amounts of AzPhe (0, 0.1, 0.3, 0.5, 0.75, and 1.0% of a dry diet) were mixed with the standard diet and then administered to fifth-instar male larvae of H03. Phe content in the standard diet was ca. 1% of the dry diet based on amino acid analysis (data not shown). The larvae grew normally even upon addition of 1.0% AzPhe with a modest decrease of diet intake (Figure S5a, b). As expected, a greater amount of silk fibroin was produced on the standard diet (Figure S5c). Incorporation of AzPhe into silk fibroin was investigated by MALDI-TOF-MS analysis (Figure S5c), and the results showed that the AzPhe incorporation increased with the amounts of AzPhe mixed in the diet (Figure S6). With respect to fibroin production, however, increased amounts of AzPhe in the diet had a negative effect (Figure S5c). We chose a rearing condition with a standard diet containing 0.5% AzPhe for the following experiments. Under this condition, an approximately 3-fold increase was observed in the amount of AzidoSilk produced by H03 along with the improved incorporation efficiency (the efficiency was increased ca. 1.6 times based on MALDI-TOF-MS analysis) compared with the previously optimized production condition using the synthetic diet for H01 (0.15 equiv of AzPhe with Phe ×0.3) (Table 1).38 Assuming that the ionization efficiencies of the two mass peaks in the MALDI-TOF-MS analysis (870 Da for the AzPhe-incorporated and 855 Da for the parental peptide peak) were the same, it was estimated that approximately one AzPhe residue was introduced into one silk fibroin heterodimer (FibH + FibL) possessing 35 Phe residues (Figure S1).4 These results demonstrated that H03 is much more suitable than H01 for large-scale production of AzidoSilk.

Figure 3. Tensile properties of AzidoSilk threads compared with control threads. Average maximum strength, strain at maximum strength, and Young’s modulus from three independent experiments are shown with error bars of standard deviations. The sizes of AzidoSilk and control threads were respectively 11.6 ± 2.3 and 11.6 ± 1.1 denier. In each independent experiment, data were collected for 50 threads and averaged. N.S. denotes not significant with P > 0.05 by t test.

properties of AzidoSilk and control threads. There were no significant differences between the two threads in either maximum strength, strain at maximum strength, or the Young’s modulus. Similar experimental results were previously obtained on 4-chlorophenylalanine (ClPhe)-incorporated silk threads.43 Since Phe residues exist only in the noncrystalline regions of silk fibroin4 (Figure S1) and the replacement to AzPhe occurs with low probability, it was not surprising that AzPhe incorporation had no adverse effects on the mechanical properties of silk threads. Chemical introduction of azide functionalities into silk fibroin was previously reported by several groups.21,44−46 In those reports, much larger amounts of azide groups than in our method were chemically introduced into silk fibroin (up to ca. 210 azide groups per one silk fibroin molecule21), which were then subjected to click reactions to obtain functionalized silk fibroin materials. In contrast, our method enables direct production of AzidoSilk in its native fibrous form, although much smaller amounts of azide groups (ca. one azide group per one silk fibroin molecule) were introduced. The chemical methods mainly target tyrosine residues existing both in the D

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Figure 4. Fabrication of materials from AzidoSilk and their modifications by CuAAC and SPAAC. BF and FL respectively denote bright field and fluorescence images. (a) AzidoSilk threads were modified with carboxyrhodamine 110 alkyne by CuAAC or sulforhodamine B DBCO by SPAAC. (b) AzidoSilk films were modified with sulfo-DBCO-biotin by SPAAC followed by the quantitative detection of biotins attached on the films with HRP-streptavidin. AzidoSilk films were also modified with carboxyrhodamine 110 alkyne by CuAAC or with sulforhodamine B DBCO by SPAAC. (c) AzidoSilk sponges were modified with carboxyrhodamine 110 alkyne by CuAAC or with sulforhodamine B DBCO and DBCO-modified GFP by SPAAC.

binding of fluorescent molecules. Transparent AzidoSilk films were prepared via a conventional procedure,5 then subjected to SPAAC with sulfo-DBCO-biotin (Figure 4b). Quantitative detection of the biotins attached to film demonstrated that SPAAC was quite selective for AzidoSilk film, although a low level of background signals was observed probably due to nonspecific binding of the reagents. AzidoSilk films could also be modified with carboxyrhodamine 110 alkyne and sulforhodamine B DBCO in a highly azide-selective manner (Figure 4b). Porous sponge materials derived from silk fibroin have been extensively studied as three-dimensional scaffolds for tissue construction and regeneration.12,13,25,47 In the present study, therefore, we prepared AzidoSilk sponges by a conventional method40 and subjected them to the same reactions as above (Figure 4c). The strong fluorescence on AzidoSilk sponges evidenced the excellent selectivity toward azide groups, although weak background fluorescence was still observed for the control sponges. To demonstrate the possibility of

crystalline and noncrystalline regions of silk fibroin for the introduction of azide groups, whereas our method restricts the introduction of azide groups only at the positions of Phe residues localized in the noncrystalline regions. We expected that such limitation of the positions where azide groups are introduced would not affect the mechanical strength of silk fibroin. In fact, it was confirmed that the AzidoSilk threads produced in this study retained the same mechanical strength with control threads not containing AzPhe (Figure 3). This feature would be quite important for the further application of AzidoSilk as a biomaterial. To assess the selectivity of azide groups for chemical functionalizations, AzidoSilk threads were subjected to CuAAC with carboxyrhodamine 110 alkyne and SPAAC with sulforhodamine B DBCO at room temperature. After thorough washing, green and red fluorescence was observed selectively on AzidoSilk threads (Figure 4a). Weak fluorescence was observed on control threads, which would have been due to nonspecific E

DOI: 10.1021/acsbiomaterials.5b00469 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 5. Photolithographic patterning of fluorescent molecules on AzidoSilk materials. (a) AzidoSilk threads were aligned horizontally on a slide glass and affixed with decals reading “SILK,” then irradiated with 254 nm UV light for 3 min. The irradiated AzidoSilk threads were clicked with carboxyrhodamine 110 alkyne followed by washing with DMSO and deionized water. (b) AzidoSilk films (7 × 7 mm2) were irradiated with 352 nm UV light for 60 min through glass photomasks whose patterns are shown on the right. The irradiated AzidoSilk films were clicked with sulforhodamine B DBCO (red fluorescence) or carboxyrhodamine 110 alkyne (green fluorescence) followed by washing with DMSO and deionized water.

fluorescence, probably because nonspecific binding was slightly increased in the irradiated areas because of morphological or chemical changes (Figure S8).

modifying AzidoSilk sponges with active proteins by click chemistry, green fluorescent protein (GFP), modified with DBCO-PEG4-NHS ester beforehand, was reacted with AzidoSilk sponge. Figure 4c shows that the GFP selectively attached to the AzidoSilk sponge, demonstrating that active proteins such as growth factors could be used to modify AzidoSilk sponge to enhance its function as a biomaterial. Photolithographic Patterning of Fluorescent Molecules on AzidoSilk Materials. Phenyl azides are known to exhibit photolysis upon UV irradiation.48 We verified that the click reactivity of azide groups in AzidoSilk threads almost disappeared after the irradiation of 254 nm UV light over 1.5 min (Figure S7a). AzidoSilk threads were aligned on a slide glass, irradiated with 254 nm UV light for 3 min through a photomask (with decals depicting the word “SILK”), and reacted with green fluorescent reagents (Figure 5a). As expected, only the masked parts of the threads became fluorescent. Next, we tried to transfer micropatterns onto AzidoSilk film using glass photomasks. We conducted control experiments to determine whether the azide groups in AzidoSilk film lost their click reactivity by UV irradiation through glass substrate. To ensure the light permeability through glass substrate, we employed longer wavelength (352 nm) UV light. We found that the click reactivity of azide groups in AzidoSilk films almost disappeared after the irradiation of 352 nm UV light over 45 min (Figure S7b). AzidoSilk films were thus irradiated with 352 nm UV light for 60 min through glass photomasks bearing micropatterns and then reacted with red and green fluorescent reagents (Figure 5b). The patterns on the photomasks were successfully transferred to AzidoSilk films on a micrometer scale. When the control film was used instead, a faint pattern was observed in the weak background

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CONCLUSIONS In conclusion, we succeeded in generating a new transgenic B. mori line suitable for efficient production of AzidoSilk, a silk fibroin in which phenyl azide groups are introduced as selective chemical handles without impairing the native mechanical strength of the fiber. AzidoSilk could be processed into varied material forms (thread, film, and porous sponge) with conventional techniques. All these types of AzidoSilk material are readily modified by click reactions (CuAAC and SPAAC), enabling reliable and easy modification of silk-based biomaterials under modest and bioorthogonal conditions. Photolysis of phenyl azides enabled photolithographic patterning of fluorescent molecules directly onto AzidoSilk films on a micrometer scale. Such precise spatial control of functionalization would largely enhance the utility of silk-based biomaterials for applications such as cell culture substrates, drug delivery, tissue scaffolds, or implantable devices. We preliminarily investigated whether we could change the adhesion of cells to a film of AzidoSilk, which was obtained from the previous H01 line, by attaching hydrophilic poly(ethylene glycol) (PEG) chains or a cell adhesive RGD motif by click reactions. Reduced cell adhesion was observed when the PEG chains were modified, whereas modification of the RGD motif did not achieve an increase in cell adhesion.49 Improved procedures including photolithographic patterning are now under investigation using AzidoSilk derived from the newly generated H03 line for the application of AzidoSilk as a F

DOI: 10.1021/acsbiomaterials.5b00469 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

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cell culture substrate with spatially controlled cell adhesion properties.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.5b00469. Additional experimental protocols and supporting data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: teramoto@affrc.go.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Transgenic Silkworm Research Unit at NIAS for providing silkworm eggs. We thank Dr. J. Ishibashi for help with the mass analysis and Dr. Q. Zhang for her technical assistance. This work was supported by JSPS KAKENHI Grant 24688008 and 15K07800.

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REFERENCES

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DOI: 10.1021/acsbiomaterials.5b00469 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.5b00469 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX