biomolecules

Article Effects of Ultraviolet Light Irradiation on Silk Fibroin Films Prepared under Different Conditions

Sora Lee, Soo Hyun Kim, You-Young Jo, Wan-Taek Ju, Hyun-Bok Kim and HaeYong Kweon *

Sericultural and Apicultural Materials Division, National Institute of Agricultural Sciences, RDA, Wanju-gun 55365, Korea; [email protected] (S.L.); [email protected] (S.H.K.); [email protected] (Y.-Y.J.); [email protected] (W.-T.J.); [email protected] (H.-B.K.) * Correspondence: [email protected]

Abstract: Silk fibroin (SF)-based materials are exposed to both natural and artificial ultraviolet (UV) light during preparation or administration. However, the effects of UV irradiation on SF films prepared under different conditions have not yet been described in detail. In this study, four SF films with different molecular weight (MW) distribution were fabricated using SF solutions, which were prepared by dissolving degummed SF for 0.5–24 h. We observed UV (365 nm) irradiation on SF films induced the increase of yellowness and absorbance at 310 nm of SF films, indicating the formation of new photo-products and di-tyrosine bonds by photo-oxidation. Due to di-tyrosine cross-links between SF chains, UV-irradiated SF films were not fully dissociated in urea solution. In addition to formation of new products, UV reduced the crystallinity of SF films by breaking hydrogen bonds of β-sheet conformation. Unlike the UV-induced decomposition of physical interactions, UV did not affect the covalent bonds (i.e., peptide bonds). Through these experiments, we could expect that SF with higher MW was more susceptible and SF with lower MW was more resistant to UV-induced photo-oxidation and photo-degradation. These results provide useful information about UV-induced aging of SF-based materials under natural sunlight and UV irradiating conditions.



 Keywords: silk fibroin film; ultraviolet aging; photo-oxidation; photo-degradation Citation: Lee, S.; Kim, S.H.; Jo, Y.-Y.; Ju, W.-T.; Kim, H.-B.; Kweon, H. Effects of Ultraviolet Light Irradiation on Silk Fibroin Films Prepared under 1. Introduction Different Conditions. Biomolecules 2021, 11, 70. https://doi.org/ Silk fibroin (SF) is one of well-known natural protein materials obtained from silk- 10.3390/biom11010070 worms. The primary structure of SF is highly unique and composed of repetitive glycine- alanine-glycine-alanine-glycine-serine (GAGAGS) [1]. These repeating sequences form the Received: 30 October 2020 crystalline region with β-sheet conformation maintained by various molecular interactions Accepted: 4 January 2021 such as hydrogen bonds, van der Waals interaction, and hydrophobic interaction. The Published: 7 January 2021 crystalline regions contribute to the strength of SF. In contrast, amorphous regions of relatively hydrophilic blocks composed of random/α-helix conformations contribute to Publisher’s Note: MDPI stays neu- the elasticity of SF. tral with regard to jurisdictional clai- Because of the high crystallinity of the degummed SF, SF has been generally regarded ms in published maps and institutio- as water-insoluble polymer. Instead of pure water, several chaotropic solvents [2] have been nal affiliations. used for solubilization and application of SF in a variety of forms such as films, sponges, hydrogels, or coating materials [3]. During the solubilization process of degummed SF, the structure of SF can be affected by various conditions such as the type of solvent, dissolution time, and temperature, resulting in differences in physical and chemical features of SF. For Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. example, different solvent systems that have different ionic forces can affect the molecular This article is an open access article weight (MW) and structure of SF [4]. Wang and colleagues compared the MW of SF distributed under the terms and con- solutions dissolved in 9.3 M LiBr and Ajisawa’s reagent (CaCl2/H2O/EtOH (molar ratio ditions of the Creative Commons At- 1/8/2)) by using Rouse-like rheological behavior, which enables the determination of tribution (CC BY) license (https:// molecular weight of SF [2]. MW of LiBr-dissolved SF was 179 kDa, whereas MW of Ajisawa’s creativecommons.org/licenses/by/ reagent-dissolved SF was 128 kDa, indicating that LiBr solvent system was milder. In fact, 4.0/). Yamada et al. also found that Ajisawa’s reagent caused degradation of the heavy chain of

Biomolecules 2021, 11, 70. https://doi.org/10.3390/biom11010070 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 70 2 of 11

SF [5]. In addition, the fast protein liquid chromatogram of Ajisawa’s reagent-dissolved SF showed a strong peak indicating 450 kDa and weak shoulder peak indicating 150 kDa. The peak of 150 kDa increased with the increase of dissolution time. Moreover, peak shift from 150 kDa to 110 kDa and new peak formation indicating 16 kDa were observed when dissolution time was 180 min [6]. Such a different MW distribution resulted in different particle size, rheological properties, and degree of transparency. Meanwhile, according to the literature, the gelling property of SFs triggered by ultrasonic treatment was poor in Ajisawa’s reagent system compared to LiBr system, although the similar MW and distribution of SFs were acquired by the control of dissolution time [7]. It can be expected that these two solvent systems might affect not only MW distribution of SF but also its structure of hydrophobic segments. Hence, the dissolution conditions must be set carefully considering the target application fields. In addition to various dissolution conditions, there are various external parameters that can affect the physicochemical properties of SF, such as heat, oxygen, volatile organic compounds, light, or absolute humidity [8]. Among them, ultraviolet (UV) light is a highly important parameter because during preparation and administration, SF-based fibers, medical devices, and additives for foods and cosmetics are generally exposed to natural or artificial UV light. Especially, UV sterilization of SF-based biomaterials is the required procedure before implantation [9]. Therefore, understanding the physicochemical stabilities of SF-based material under UV light is essential for a wide range of applications. For several decades, many researchers have tried to understand the UV-induced photo-aging process of silk fabrics, solutions, and films. The accumulated results have demonstrated that SF fiber is susceptible to UV-induced changes in color and bright- ness [10,11] because SF molecules contain numerous aromatic amino acids such as tyrosine (5.1%), tryptophan (0.1%), and phenylalanine (0.4%) [8]. In addition, it is revealed that photo-destruction of the ordered structures by UV irradiation makes silk fibers more brittle than those that are nontreated [12]. To reduce UV-induced weakening effect on mechan- ical properties, researchers fabricated silk fibers coated with TiO2 as UV-absorbers and confirmed more stable tensile properties [13]. Sionkowska and colleagues also found that thin films from a mixture of chitosan and silk fibroin were affected by UV light with a wavelength of 254 nm, showing the decrease of surface roughness that was detected by atomic force microscopy [14]. Although photo-induced oxidation and degradation are highly important reactions for using SF-based materials, to the best of our knowledge, the effect of dissolution time and the resultant different MW distribution on the resistance of SF-based materials against the photo-induced reaction have not been investigated in detail. Therefore, in this study, four types of SF solutions were prepared by dissolving degummed SF fibers for different times (0.5–24 h). Subsequently, SF films were fabricated and irradiated by UV light. In addition, spectroscopic and structural properties of UV-irradiated SF films were investigated.

2. Materials and Methods 2.1. Preparation of SF Aqueous Solutions Degumming of Bombyx mori cocoons was performed twice with 0.5% o.w.f. (on the weight of fiber) of sodium oleate (C18H33NaO2, Sigma Aldrich, MO, USA) and 0.3% o.w.f. ◦ of solution sodium carbonate (Na2CO3, Sigma Aldrich) with a bath ratio of 1:50 at 100 C for 1 h. The silk fibers were rinsed in warm water and air-dried at 50 ◦C. The degummed silk was dissolved in Ajisawa’s reagent solution containing calcium chloride (CaCl2, Showa, Japan), distilled water, and ethanol with molar ratio of 1:8:2 at 87 ◦C for 0.5 h, 2 h, 8 h, and 24 h. In 0.5 h-dissolution group, some undissolved fibers remained. However, no residual fibers were observed in the other groups. Next, SF solutions were dialyzed using a cellulose acetate tube (MWCO: 12–14 kDa) for 3 days. To remove insoluble components, filtration processes were performed before and after dialysis. The final concentration of aqueous SF solution was measured by weighing the remaining solid after drying at 130 ◦C. Biomolecules 2021, 11, 70 3 of 11

Six milliliters of 1.6 wt.% regenerated SF solutions were poured into 6-well plates and dried in fume hoods to prepare regenerated SF films.

2.2. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Silver Staining Protein concentration of each SF solution was measured using Bradford assay (Bio-Rad laboratories, Hercules, CA, USA) and the solutions were mixed with 2X Laemmle buffer (Bio-Rad) and 2.5% β-mercaptoethanol. Denatured SF samples at 95 ◦C for 2 min were loaded into the lanes of acrylamide gel (Bio-Rad) and electrophoresis was performed. Next, the gel was stained using a silver staining kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guide. Using the analyze line graph tool of the image processing software (ImageJ), the gray value profile was created along each lane of the gel image. The representative profile of three replicates was selected and shown herein.

2.3. UV Irradiation on SF Films SF films were irradiated in air at room temperature using a UV lamp (LF215LM, UVITEC, Cambridge, UK) which emits light at a wavelength of 365 nm. The average intensity of radiation was 2.37 ± 2.12 mW/cm2 at 5 cm from the light source. The dose of radiation during 1 h exposure was 8538 ± 453 mJ/cm2. The intensity of the incident light was measured using an UV light meter (YK-35UV, Lutron, Taiwan). Irradiation experiments were carried out on a well-plate and the location of films were rotated every 15 min for the uniform exposure of light.

2.4. Color Measurement Color change of SF films was measured using a spectrophotometer (Color i7, X-Rite, Grand Rapids, MI, USA). A white standard color plate (L = 94.27, a = 0.13, and b = 2.80) was used as a background and a standard for color measurements. Hunter color values (L: lightness; a: redness; b: yellowness) were averaged from more than 10 measurements from each sample. The total color differences (∆E) were calculated as follows:

∆E = [(∆L)2 + (∆a)2 + (∆b)2 ]0.5 (1)

where ∆L, ∆a, and ∆b are the difference between each color values of film specimen and the standard.

2.5. UV–Vis Absorbance and Fluorescence Spectroscopy The UV–vis absorption spectra of SF film before and after UV-irradiation were recorded with a Multiskan GO plate reader (Thermo Scientific, Waltham, MA, USA) in the range of 270 to 360 nm. Films were fixed inside of the cuvette cell. Absorbance of SF molecules released from SF films during immersion in 6 M urea solution at 10 mg/mL was also collected. The fluorescence intensity (excitation: 360/40 nm; emission: 460/40 nm) of the SF molecules in 6 M urea solution was measured by microplate reader (HT- Synergy, Bio-Tek, Winooski, VT, USA). For film and solution samples, the measured values of empty cuvette and the same volume of 6 M urea solution were used for blank subtraction, respectively.

2.6. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy ATR-FTIR experiments were carried out and the spectra of silk films were recorded with a Fourier transform spectrometer (S100, Perkin Elmer, Waltham, MA, USA) in the range of 1000 to 4000 cm−1. Thirty-two scans were averaged with resolution 4 cm−1 and the experiments were repeated more than 6 times. Amide I and III crystallinity index were determined by calculating the absorbance intensity ratios of 1620 cm−1/1652 cm−1 for amide I and 1264 cm−1/1230 cm−1 for amide III crystallinity index. Biomolecules 2021, 11, x FOR PEER REVIEW 4 of 11

−1 −1 Biomolecules 2021, 11, 70 determined by calculating the absorbance intensity ratios of 1620 cm /1652 cm for amide4 of 11 I and 1264 cm−1/1230 cm−1 for amide III crystallinity index.

2.7. Assay of 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS) 2.7. Assay of 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS) To determine the relative content of primary amines in SF film, TNBS assay was per- formedTo determineaccording to the the relative established content protocol of primary with minor amines modification in SF film, TNBS[15]. SF assay films was (2.5 performedmg) were mixed according with to250 the μL established of 200 mM protocolsodium phosphate with minor buffer modification (pH 8.2) [and15]. 250 SF filmsμL of (2.50.5% mg) of wereTNBS mixed solution. with The 250 µreactionL of 200 mixture mM sodium was phosphateheated at 40 buffer °C for (pH 4 8.2)h in and a shaking 250 µL ◦ ofincubator. 0.5% of TNBSTo stop solution. the reaction, The reaction 250 μL of mixture 6 N HCl was was heated added at into 40 Cthe for mixture. 4 h in a Next, shaking the incubator. To stop the reaction, 250 µL of 6 N HCl was added into the mixture. Next, mixture was autoclaved at 120 °C for◦ 30 min to dissolve any insoluble matter. The absorb- theance mixture of 100 μ wasL of autoclaved mixture was at read 120 atC 340 for 30nm min using to dissolvea microplate any reader insoluble (Multiskan matter. TheGO) µ absorbanceand all values of 100wereL subtracted of mixture by was the read value at 340of blank nm using solution a microplate prepared reader with same (Multiskan proce- GO)dure andwithout all values SF film. were subtracted by the value of blank solution prepared with same procedure without SF film. 3. Results and Discussion 3. Results and Discussion 3.1.3.1. CharacterizationCharacterization ofof MolecularMolecular WeightWeight DistributionDistribution ofof SFSF DissolvedDissolved forfor DifferentDifferent TimeTime AlthoughAlthough Ajisawa’sAjisawa’s reagentreagent hashas beenbeen usedused forfor dissolvingdissolving thethe degummeddegummed SFSF fibersfibers withwith highhigh utility, utility, it it can can make make damages damages in in the the intrinsic intrinsic structure structure of SF,of SF, resulting resulting in molecular in molec- scissionsular scissions [5,6]. [5,6]. As expected, As expected, SDS-PAGE SDS-PAGE results results shown shown in Figure in Figure1A indicate 1A indicate 0.5 h 0.5 and h 2and h SF2 h groups SF groups that that were were dissolved dissolved for for a relativelya relatively short short time time showed showed strong strong band band intensityintensity atat highhigh MWMW regionregion (>100(>100 kDa).kDa). UnlikeUnlike thethe lowlow bandband intensityintensity ofof 0.50.5 hh SFSF groupgroup betweenbetween 100100 andand 3535 kDa,kDa, thethe bandband intensityintensity forfor thethe 22 hh SFSF groupgroup waswas highhigh betweenbetween thethe samesame range,range, indicatingindicating thethe widewide distributiondistribution ofof MWMW forfor thethe 22 hh SFSF group.group. InIn thethe casecase ofof thethe 88 hh SFSF group,group, bandband intensity was gradually increased increased in in the the range range from from 180 180 kDa kDa to to the the bottom bottom of ofthe the gel. gel. For For the the 24 24 h hSF SF group, group, which which was was dissolved dissolved for for the the longest time, aa strongstrong bandband waswas shownshown onlyonly atat thethe lowerlower MWMW regionregion (<35(<35 kDa).kDa). MolecularMolecular distributionsdistributions ofof SFSF groupsgroups dissolveddissolved forfor differentdifferent timestimes werewere alsoalso observedobserved inin thethe graygray valuevalue profileprofile ofof eacheach lanelane ofof SDS-PAGESDS-PAGE gel gel (Figure (Figure1 B).1B). Considering Considering SF SF had had a a heavy heavy chain chain of of 391 391 kDa kDa and and a a light light chain chain ofof 2626 kDa [[16],16], we could expect expect the the bands’ bands’ upper upper region region of of 245 245 kDa kDa and and near near region region of of25 25kDa kDa were were contributed contributed by by the the heavy heavy and and light light chain chain of of SF, respectively.respectively. Therefore,Therefore, thethe bandsbands fromfrom 245245 kDakDa toto 2525 kDakDa andand belowbelow 2525 kDakDa werewere thoughtthought toto bebe thethe degradeddegraded heavyheavy andand lightlight chain,chain, respectively. respectively. According According to to the the results results showing showing the the weak weak band band intensities intensities of theof the 8 and 8 and 24 h 24 groups, h groups, we could we could conclude conclude that dissolution that dissolution for longer for longer than 8 hthan could 8 h almost could criticallyalmost critically degrade degrade the heavy the chains heavy of chains SF fibers. of SF fibers.

FigureFigure 1.1. SDS-PAGESDS-PAGE resultsresults ofof fourfour silksilk fibroinfibroin (SF) (SF) solutions. solutions. ( A(A)) Silver-stained Silver-stained gel gel image image showing show- theing differencethe difference of SFs of SFs in molecularin molecular weight weight distribution. distribution. The The lane lane onon thethe right side side indicates indicates the the molecular weight marker. The other four lanes represent SF with increasing dissolution time, from left to right. (B) The gray value profiles of protein bands on the SDS-PAGE gel. The set of intensity values is taken from each lane, from the top to the bottom, and plotted in the chart. Biomolecules 2021, 11, x FOR PEER REVIEW 5 of 11

molecular weight marker. The other four lanes represent SF with increasing dissolution time, from left to right. (B) The gray value profiles of protein bands on the SDS-PAGE gel. The set of intensity values is taken from each lane, from the top to the bottom, and plotted in the chart.

3.2. Effect of UV Irradiation on Color Change of SF Films The difference in MW distribution of SF can generally affect molecular structure and Biomolecules 2021, 11, 70 physicochemical properties. Given the SDS-PAGE results, we hypothesized5 of 11 that SF groups dissolved for different times would show different susceptibility to UV irradiation. To confirm this, dose-dependent color changes were evaluated. Total color differences (∆3.2.E) Effectcalculated of UV Irradiation from values on Color of ChangeHunter of L SF (lightness), Films a (redness), and b (yellowness) for the SF filmsThe differencewere plotted in MW with distribution yellowness of SF canindex generally (b) as affect a function molecular of structure UV irradiation and time. As depictedphysicochemical in Figure properties. 2, ∆E was Given not the affected SDS-PAGE by results, UV irradiatio we hypothesizedn time, that regardless SF groups of dissolution dissolved for different times would show different susceptibility to UV irradiation. To time.confirm In this,contrast, dose-dependent yellowness color index changes tended were to evaluated. increase Total with color UV differences irradiation. (∆E) Especially, the yellowingcalculated fromeffect values on the of Hunter 0.5 SF Lgroup (lightness), was astro (redness),nger than and b other (yellowness) groups, for showing the SF more than afilms 2-fold were increase plotted of with yellowness yellowness index index after (b) as 4 ah functionof UV irradiation. of UV irradiation In contrast, time. As the yellowness indexesdepicted of in Figurethe other2, ∆E groups was not were affected lower by UV than irradiation the 0.5 time, h group regardless even of dissolutionafter 4 h of irradiation. Yellowingtime. In contrast, is considered yellowness as index one tendedof the to most increase important with UV photo-oxidation irradiation. Especially, markers in pro- the yellowing effect on the 0.5 SF group was stronger than other groups, showing more teins.than aThe 2-fold development increase of yellowness of yellow index color after of SF 4 hfilms of UV might irradiation. be because In contrast, aromatic the amino acids producedyellowness new indexes products of the other containing groups were chromoph lower thanores the 0.5under h group UV even light after [17]. 4 h Utilizing of a qua- siproteomicirradiation. Yellowing approach, is considered Dyer and as onecolleagues of the most id importantentified photo-oxidationchromophoric markers photo-products de- rivedin proteins. from Thetyrosine development and tryptophan, of yellow color and of SF pr filmsoposed might bethe because photo-oxidation aromatic amino pathways [18]. acids produced new products containing chromophores under UV light [17]. Utilizing a Therefore,quasiproteomic these approach, color measurement Dyer and colleagues results identified indicate chromophoric the MW of photo-products SF can affect the suscepti- bilityderived to fromUV-induced tyrosine and production tryptophan, of and chromophores. proposed the photo-oxidation We also expected pathways that [18 UV]. irradiation canTherefore, be an theseecosustainable color measurement technique results for indicate modifying the MW color of SF properties can affect the of suscepti- SF film without us- ingbility chemical to UV-induced reagents. production Similarly, of chromophores. Sagnella an Wed colleagues also expected modified that UV irradiation the color properties of silkcan fiber be an ecosustainableand silk fibroin technique by doping for modifying RhoB color in the properties artificial of SF diet film withoutduring using silkworm raising chemical reagents. Similarly, Sagnella and colleagues modified the color properties of silk [19].fiber With and silk the fibroin increase by doping of feeding RhoB in time the artificial of RhoB diet-added during silkworm diet from raising 0 to [7219]. h, With chromaticity (C) increasedthe increase and of feeding hue angle time of(h RhoB-added°) decreased, diet indicating from 0 to 72 the h, chromaticity color change (C) increasedfrom between bluish- greenand hue and angle yellow (h◦) decreased, components indicating to an the almost color change red-purple from between component. bluish-green and yellow components to an almost red-purple component.

A.0.5 h B. 2 h 3 3 3 3 Yellowness Yellowness ΔE ΔE 2 2 2 2 Δ Δ E E

1 1 1 1 Yellowness index Yellowness Yellowness index Yellowness (normalized by std.) (normalized by std.) 0 0 0 0 0 1 2 3 4 0 1 2 3 4 UV irradiation time (h) UV irradiation time (h)

C.8 h D. 24 h 3 3 3 3 Yellowness Yellowness ΔE ΔE 2 2 2 2 Δ Δ E E

1 1 1 1 Yellowness index Yellowness Yellowness index Yellowness (normalized by std.) (normalized by std.) 0 0 0 0 0 1 2 3 4 0 1 2 3 4 UV irradiation time (h) UV irradiation time (h)

Figure 2. Yellowness and total color differences (∆E) of UV-irradiated SF films (1–4 h) prepared with Figurefour SF 2. solutions Yellowness that were and dissolved total color for differences different times (Δ (AE): 0.5of h;UV-irradiatedB: 2 h; C: 8 h; DSF: 24 films h) (n (1–4 > 10; h) prepared withmean four± SEM). SF solutions that were dissolved for different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h) (n > 10; mean ± SEM). Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 11

Biomolecules 2021, 11, 70 6 of 11 3.3. Effect of UV Irradiation on UV–Vis and Fluorescence Spectroscopic Properties of SF Films Changes of UV–vis absorbance spectra of SF films can detect the formation of new photo-products. As3.3. shown Effect in of UVFigure Irradiation 3A–D, on UV–Visthe main and peak Fluorescence around Spectroscopic 280 nm is Properties attributed of SF Filmsto aromatic amino acids thatChanges are present of UV–vis in absorbanceSF molecular spectra chains of SF such films as can tyrosine detect the (5.1%), formation phe- of new nylalanine (0.4%), andphoto-products. tryptophan As (0.1%) shown [1,8]. in Figure After3A–D, UV the irradiation, main peak aroundlight absorbance 280 nm is attributed at 310 nm tended to increaseto aromatic for amino all SF acids groups. that are These present results in SF molecular are probably chains suchdue asto tyrosine the for- (5.1%), phenylalanine (0.4%), and tryptophan (0.1%) [1,8]. After UV irradiation, light absorbance mation of new photo-productsat 310 nm tended by UV to increase irradiation for all such SF groups. as new These cross-links results are and/or probably an dueoxi- to the dized form of aromaticformation amino of newacids. photo-products Similarly, Sionkowska by UV irradiation and suchcolleagues as new cross-linksalso observed and/or an the changes in absorbanceoxidized formof SF of solutions aromatic amino at around acids. Similarly, 300–310 Sionkowska nm and they and colleagues explained also that observed 3,4-dihydroxylphenylalaninethe changes in(DOPA) absorbance might of SF solutionsbe formed at around by photo-oxidation 300–310 nm and they reaction explained be- that 3,4- tween phenylalaninedihydroxylphenylalanine and tyrosine, affecting (DOPA) the mightabsorbance be formed property by photo-oxidation [1]. reaction between phenylalanine and tyrosine, affecting the absorbance property [1].

A.0.5 h B. 2 h 1.2 UV1.2 UV 0.20 0 h 0.20 0 h 1.0 0.15 1 h 1.0 0.15 1 h 0.8 0.10 2 h 0.8 0.10 2 h 0.05 3 h 0.05 3 h 0.6 4 h 0.6 4 h 0.00 0.00 300 305 310 315 320 300 305 310 315 320 0.4 0.4

0.2 0.2

OD (normalized by A280) 0.0 OD (normalized by A280) 0.0 270 290 310 330 350 270 290 310 330 350 Wavelength (nm) Wavelength (nm)

C.8 h D. 24 h UV UV 1.2 0.20 1.2 0.20 0 h 0 h 1.0 0.15 1 h 1.0 0.15 1 h 0.10 0.10 0.8 2 h 0.8 2 h 0.05 3 h 0.05 3 h 0.6 0.6 0.00 4 h 0.00 4 h 300 305 310 315 320 300 305 310 315 320 0.4 0.4

0.2 0.2

OD (normalized by A280) 0.0 OD (normalized by A280) 0.0 270 290 310 330 350 270 290 310 330 350 Wavelength (nm) Wavelength (nm)

Figure 3. Absorbance spectra of UV-irradiated SF films (1–4 h) prepared with four SF solutions that were dissolved for Figure 3. Absorbance spectra of UV-irradiated SF films (1–4 h) prepared with four SF solutions different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h). that were dissolved for different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h). To know the presence of newly formed cross-links between SF chains, we immersed To know the presenceSF films in of 6 Mnewly urea solutionformed at cross-links 10 mg/mL and between wait for SF 24 h.chains, Because we it is immersed well known that SF films in 6 M ureaconcentrated solution at urea 10 mg/mL solution and can easily wait breakfor 24 hydrogen h. Because bonds it is in well SF, we known expected that SF films would be dissolved. In fact, intact SF films were almost dissolved, remaining few solid- concentrated urea solutionstate components, can easily as shownbreak inhydrogen Figure4A. However,bonds in withSF, we the increaseexpected of UVSF irradiationfilms would be dissolved.time, In fact, larger intact amounts SF offilms undissolved were almost and swollen dissolved, films were remaining observed few for all solid- SF groups, state components, asindicating shown SF in chains Figure were 4A. photo-cross-linked However, with by chemicalthe increase bonds insteadof UV ofirradiation hydrogen bonds. time, larger amounts of undissolved and swollen films were observed for all SF groups, indicating SF chains were photo-cross-linked by chemical bonds instead of hydrogen bonds. The chemical bonds formed by UV irradiation are thought to be di-tyrosine bonds. To confirm the formation of di-tyrosine bonds in released SF molecules, fluorescence in- tensity at 440–480 nm was measured at the excitation wavelength of 320–380 nm. Accord- ing to the literature, for UV irradiated insulin, the progressive formation of a peak around Biomolecules 2021, 11, 70 7 of 11 Biomolecules 2021, 11, x FOR PEER REVIEW 7 of 11

The chemical bonds formed by UV irradiation are thought to be di-tyrosine bonds. To confirm the formation of di-tyrosine bonds in released SF molecules, fluorescence intensity 405 nm was detectedat 440–480when samples nm was measured were excit at theed excitation at 320 nm wavelength due to di-tyrosine of 320–380 nm. bonds According [20]. to the Similarly, for all SFliterature, samples, for the UV fluorescence irradiated insulin, was the increased progressive with formation the increase of a peak of aroundUV irra- 405 nm diation time, indicatingwas detected the formation when samples of di-tyrosine were excited bonds at 320 nm (Figure due to di-tyrosine4B). These bonds di-tyrosine [20]. Similarly, bonds might be formedfor all upon SF samples, tyrosyl the radical fluorescence isomerization was increased and with enolization the increase reaction of UV irradiation [21]. time, indicating the formation of di-tyrosine bonds (Figure4B). These di-tyrosine bonds It is especially worthmight nothing be formed that upon SF groups tyrosyl radicalwith higher isomerization MW showed and enolization a higher reaction increase [21]. It is of fluorescence uponespecially increase worth of UV nothing irradiation that SF groups time. withThis higher fluorescence MW showed profile a higher is similar increase of to the color measurementfluorescence result upon in increaseFigure of2A–D UV irradiation and film time. morphologies This fluorescence shown profile in Figure is similar to 4A. Therefore, we thecan color conclude measurement the MW result of in SF Figure can2 A–Dinfluence and film the morphologies susceptibility shown to in UV Figure in 4A. Therefore, we can conclude the MW of SF can influence the susceptibility to UV in terms terms of formation ofof formation new photo-products. of new photo-products. These MW These effects MW effects might might be berelated related to to the the contentcon- of tent of aromatic aminoaromatic acids amino because acids longer because heat longer treatment heat treatment time time could could damage damage more more aro- aromatic matic residues duringresidues dissol duringution dissolution of degummed of degummed SF fibers. SF fibers.

FigureFigure 4. Morphologies 4. Morphologies of the remainingof the remaining four SF films four (A SF) and films fluorescence (A) and intensityfluorescence of the releasedintensity SF of molecules the re- from B ± SFleased films ( SF) after molecules immersion from of films SF infilms 6 M urea(B) after solution immersion for 24 h (ex. of 360/40; films in em. 6 460/40)M urea (n solution = 3; mean for SD;24 h 1-fold: (ex. 0 h UV irradiation). 360/40; em. 460/40) (n = 3; mean ± SD; 1-fold: 0 h UV irradiation). 3.4. Effect of UV Irradiation on Structural Stability of SF Films 3.4. Effect of UV Irradiation on Structural Stability of SF Films In addition to formation of new chemical bonds by UV, the high energy of UV can In addition to induceformation photo-degradation of new chemical of hydrogen bonds bonds by and UV, disulfide the high bonds. energy Therefore, of UV we evaluatedcan induce photo-degradationby FT-IR theof structuralhydrogen stability bonds of fourand differentdisulfide SF filmsbonds. after Therefore, exposure to UV.we FT-IRevalu- is very useful tool for understanding the chemical and physical structure of silk proteins. In the ated by FT-IR the structural stability of four different SF films after exposure to UV. FT- literature, a semiquantitative approach was chosen by calculating the relative intensities IR is very useful toolof amide for understanding bands to know the the extent chemical of SF degradation and physical [8]. Thestructure ratios of of two silk amide pro- band teins. In the literature,intensities a semiquantitative that indicate random/ approachα-helix was and chosenβ-sheet by conformations calculating havethe relative been used as intensities of amideone bands of degradation to know markers the extent by comparing of SF degradation the crystallinity. [8]. Therefore, The ratios we of acquired two FT- −1 −1 amide band intensitiesIR spectra that indicate and calculated random/ theα ratio-helix of 1620and cmβ-sheet(β-sheet) conformations to 1652 cm have(random been coil) for amide I crystallinity index (CI) and 1264 cm−1 (β-sheet) to 1230 cm−1 (random coil) used as one of degradationfor amide III markers CI. The FT-IR by comp spectraaring of nonirradiated the crystallinity. and 4 h irradiated Therefore, SF filmswe ac- showed quired FT-IR spectradifferent and calculated absorbance the intensities ratio of at the1620 specific cm−1 wavenumbers(β-sheet) to 1652 (cm−1 cm), which−1 (random indicated β- coil) for amide I crystallinitysheet and random index coil(CI) structures and 1264 (Figure cm−15 ().β In-sheet) the case to of1230 SF films cm−1 prepared (random by coil) a shorter −1 for amide III CI. Thedissolution FT-IR spectra time (0.5 of h andnoni 2rradiated h SF groups), and the 4 absorbance h irradiated values SF at films 1652 andshowed 1230 cm indicated random coil structure increased after 4 h UV irradiation, whereas the values at different absorbance intensities at the specific wavenumbers (cm−1), which indicated β- 1620 and 1264 cm−1 indicated β-sheet structure decreased or increased slightly. On the sheet and random coilother structures hand, in the (Figure case of the5). 8In h andthe 24case h SF of groups, SF films there prepared were no significantby a shorter changes dissolution time (0.5 h and 2 h SF groups), the absorbance values at 1652 and 1230 cm−1 indicated random coil structure increased after 4 h UV irradiation, whereas the values at 1620 and 1264 cm−1 indicated β-sheet structure decreased or increased slightly. On the other hand, in the case of the 8 h and 24 h SF groups, there were no significant changes in the absorbance values, thus indicating both structures. Especially, both values at 1264 and 1230 cm−1 of the 24 h SF group increased similarly after UV irradiation, meaning there were less compositional changes of β-sheet and random coil that occurred with UV irra- diation. Moreover, as shown in Figure 6, we could compare the calculated CI values upon the irradiation time. Like the tendency for formation of photo-products, the 0.5 SF group showed a strong decrease of both amide I and III CI. The destruction of crystalline struc- Biomolecules 2021, 11, x FOR PEER REVIEW 8 of 11

ture was thought to be affected by breakage of hydrogen bonds involved in β-sheet con- formation (Figure 6A). In addition, this result can explain the previously reported weak- Biomolecules 2021, 11, 70 ened mechanical properties of aged silk fibers under UV exposure [12,13].8 of Meanwhile, 11 the 2 h SF group showed a slight increase of amide I CI by 1 h UV irradiation and a gradual decrease after 1 h (Figure 6B). The newly formed di-tyrosine cross-links could dominantly contributein the absorbance to an increase values, thus of indicatingCI. However, both structures. when the Especially, dose of UV both light values was at 1264accumulated, the −1 decompositionand 1230 cm of effects the 24 of h SFUV group might increased reduce similarly the crystallinity after UV irradiation, by breaki meaningng hydrogen bonds, there were less compositional changes of β-sheet and random coil that occurred with UV likeirradiation. the 0.5 Moreover,SF group. as For shown the in 8 Figureh and6, 24 we SF could groups, compare the the CI calculated values were CI values not dramatically changedupon the (Figure irradiation 6C,D). time. LikeWe could the tendency expect for that formation SF chains of photo-products, with higher theMW 0.5 were SF extensively affectedgroup showed by UV a strongenergy decrease and the of bothphysical amide intera I and IIIctions CI. The were destruction easily ofbroken crystalline because a larger numberstructure of was hydrogen thought to bonds be affected were by involved breakage ofin hydrogen long SF bondschains involved than in in shorterβ-sheet chains. conformationFrom the (Figure TNBS6 A).assay In addition,results, we this could result cannot explainobserve the any previously remarkable reported differences in the weakened mechanical properties of aged silk fibers under UV exposure [12,13]. Meanwhile, contentthe 2 h SF of group ε-amine showed group a slight or increase newly of formed amide I CI N-terminal by 1 h UV irradiation amine groups and a gradual (Figure 7). These resultsdecrease indicate after 1 h that (Figure UV6B). did The not newly induce formed chain di-tyrosine scission cross-links and decrease could dominantly MW for all SF groups. Becausecontribute peptide to an increase bonds of are CI. However,covalent, when unlike the the dose crystallinity of UV light was that accumulated, is maintained the by physical moleculardecomposition interactions, effects of UV stronger might reduce energy the may crystallinity be needed by breaking to break hydrogen the peptide bonds, bonds. Con- like the 0.5 SF group. For the 8 h and 24 SF groups, the CI values were not dramatically sideringchanged (Figurethat several6C,D). Weother could studies expect thatreported SF chains the with molecular higher MW degradation were extensively by UV [22], light withaffected higher by UV energy energy or and longer the physical irradiation interactions timewere than easily the light broken used because for aour larger study might in- ducenumber chain of hydrogen scissions bonds and weredecrease involved the in MW long of SF SF. chains than in shorter chains.

A.Amide I region B. Amide III region 1652 1620 0 h 1264 1230 0 h 4 h 4 h 0.5 h 0.5

1680 1660 1640 1620 1600 1280 1260 1240 1220 1200 1652 1620 0 h 1264 1230 0 h 4 h 4 h 2 h 2

1680 1660 1640 1620 1600 1280 1260 1240 1220 1200 1652 1620 0 h 1264 1230 0 h 4 h 4 h 8 h 8

1680 1660 1640 1620 1600 1280 1260 1240 1220 1200 1652 1620 0 h 1264 1230 0 h 4 h 4 h 24 h 24

1680 1660 1640 1620 1600 1280 1260 1240 1220 1200 Wavenumber (cm−1) Wavenumber (cm−1)

FigureFigure 5. FT-IR spectra spectra of SFof filmsSF films nonirradiated nonirradiated and irradiated and irradiated with UV light with for UV 4 h; light (A) amide for 4 Ih; (A) amide I −1 −1 regionregion (1680–1600 cm cm),−1 (),B )(B amide) amide III region III region (1280–1200 (1280–1200 cm ). cm−1). Biomolecules Biomolecules2021, 11, x2021 FOR, 11 PEER, 70 REVIEW 9 of 11 9 of 11

Figure 6. AmideFigure 6. IAmide and III I and crystallinity III crystallinity index index calculated calculated fromfrom FT-IR FT-IR spectra spectra of UV-irradiated of UV-irradiated SF films preparedSF films with prepared four SF with four SF solutionssolutions that were that were diss dissolvedolved for for different different timestimes (A (:A 0.5: 0.5 h; B h;: 2 B h;: C2: h; 8 h;CD: 8: 24h; h) D (n: 24 > 6;h) mean (n > ±6;SEM). mean ± SEM). From the TNBS assay results, we could not observe any remarkable differences in the content of ε-amine group or newly formed N-terminal amine groups (Figure7). These A.results indicate0.5 that h UV did not induce B. chain scission2 andh decrease MW for all SF groups. Because0.8 peptide bonds are covalent, unlike0.8 the crystallinity that is maintained by physical molecular interactions, stronger energy may be needed to break the peptide bonds. Consid- ering0.6 that several other studies reported0.6 the molecular degradation by UV [22], light with higher energy or longer irradiation time than the light used for our study might induce chain0.4 scissions and decrease the MW of0.4 SF.

0.2 0.2 Absorbance at 340 nm Absorbance at 340 nm 0.0 0.0 0 1 2 3 4 0 1 2 3 4 UV irradiation time (h) UV irradiation time (h)

C.8 h D. 24 h 0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2 Absorbance at 340 nm Absorbance at 340 nm 0.0 0.0 0 1 2 3 4 0 1 2 3 4 UV irradiation time (h) UV irradiation time (h)

Figure 7. Absorbance at 340 nm obtained by 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay with UV-irradiated SF films prepared with four SF solutions. The SFs were dissolved for different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h) (n = 3; mean ± SD). Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 11

FigureBiomolecules 6. Amide2021, 11 ,I 70 and III crystallinity index calculated from FT-IR spectra of UV-irradiated SF10 offilms 11 prepared with four SF solutions that were dissolved for different times (A: 0.5 h; B: 2 h; C: 8 h; D: 24 h) (n > 6; mean ± SEM).

A.0.5 h B. 2 h 0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2 Absorbance at 340 nm Absorbance at 340 nm 0.0 0.0 0 1 2 3 4 0 1 2 3 4 UV irradiation time (h) UV irradiation time (h)

C.8 h D. 24 h 0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2 Absorbance at 340 nm Absorbance at 340 nm 0.0 0.0 0 1 2 3 4 0 1 2 3 4 UV irradiation time (h) UV irradiation time (h)

Figure 7. Absorbance at 340 nm obtained by 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay with FigureUV-irradiated 7. Absorbance SF films prepared at with340 four nm SF obtained solutions. The by SFs 2,4,6-trin were dissolveditrobenzene for different sulfonic times acid (TNBS) assay with UV-irradiated(A: 0.5 h; B: 2 h; C: 8 SF h; D films: 24 h) (nprepared = 3; mean ± withSD). four SF solutions. The SFs were dissolved for different times (4.A Conclusions: 0.5 h; B: 2 h; C: 8 h; D: 24 h) (n = 3; mean ± SD). In this study, investigation on the effect of UV irradiation on the physicochemical properties of SF films prepared under different conditions was carried out. UV irradiation on SF films not only formed of new bonds by photo-oxidation, but also damaged crys- tallinity of SF by photo-degradation. These UV-induced photo-reactions were confirmed by measuring color, UV–vis absorbance spectra, fluorescence, or FT-IR spectra. In addition, we could observe that SF with higher MW was more susceptible to these UV irradiation effects. To explain the exact mechanism about MW effect on photo-resistance of SF films, further studies in the molecular levels may be required. In conclusion, we expect that our study would provide useful information for preparing and applying SF-based materials under natural sunlight or UV irradiating conditions.

Author Contributions: S.L., conceptualization, formal analysis, investigation, visualization, and writing—original draft preparation; S.H.K., resources; Y.-Y.J., methodology and validation; W.-T.J. and H.-B.K., supervision, validation, and writing—review and editing; H.K., conceptualization, supervision, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript. Funding: This work was carried out with the support of “Research Program for Agriculture Sci- ence and Technology Development (Project No. PJ01502201)” Rural Development Administration, Republic of Korea. Biomolecules 2021, 11, 70 11 of 11

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

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