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Stabilization of natural antioxidants by silk biomaterials Tingting Luo, Lei yang, Jianbing Wu, Zhaozhu Zheng, Gang Li, Xiaoqin Wang, and David L Kaplan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01636 • Publication Date (Web): 03 May 2016 Downloaded from http://pubs.acs.org on May 4, 2016

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ACS Applied Materials & Interfaces

Stabilization of Natural Antioxidants by Silk Biomaterials

Tingting Luoa #, Lei Yanga#, Jianbing Wua, Zhaozhu Zhenga, Gang Lia, Xiaoqin Wanga*, David L. Kaplanb**

a National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, China 215123 b Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, USA 02155

* Corresponding author: Tel.: +86 512 65883371; fax: +86 512 65883371. 199 Renai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province, P.R. China 215123 E–mail addresses: [email protected] ** Corresponding author. Tel.: +1 617 627 3251; fax: +1 617 627 3231. Departments of Biomedical Engineering, Tufts University, Medford, Massachusetts, USA 02155 Email address: [email protected]

#

The first two authors have equal contributions

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ABSTRACT:

The

stabilities

of

natural

antioxidants,

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vitamin

C

(VC),

(−)-epigallocatechin gallate (EGCG), and curcumin in silk films were examined and mechanisms of stabilization elucidated. The antioxidants were physically incorporated into three types of silk films: as-cast, dried from hydrogels and methanol-treated. The films were stored at 4, 37 and 45°C for 30 days in PBS buffer, pH 7.4, along with controls consisting of free antioxidants. The incorporation of antioxidants did not significantly change film morphology or secondary structure. When stored at 4°C, all samples showed similar antioxidant activities (% scavenging) at different time points, determined by the colorimetric 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. At the higher temperatures, the VC in the as-cast film, the EGCG in the as-cast and dried hydrogel films and the curcumin in the methanol-treated films retained more than 50% scavenging activity after 14 days of storage, significantly higher than the other samples. The interaction between the antioxidants and silk, as well as the degradation of the antioxidants, were investigated by fast-performance liquid chromatography (FPLC) and high-pressure liquid chromatography (HPLC), with an aim of understanding the mechanisms of silk-based stabilization. Binding of antioxidant molecules to the hydrophobic or the hydrophilic/hydrophilic boundary regions of silk, depending on the chemical properties of the antioxidant, may account for the observed stabilization effects. The data can help guide further engineering of antioxidant-functionalized silk biomaterials.

KEYWORDS : silk, vitamin C, EGCG, curcumin, stabilization

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1.INTRODUCTION Aging is accompanied by a number of cardiovascular, brain and immune system diseases, which translate into high medical and societal costs. Various approaches have been investigated to deal with the challenge of aging, including managing oxidative stress. Oxidative stress has been implicated in various diseases as well as in the aging process, 1 thus natural antioxidants have been widely studied for anti-aging strategies. The human body generates many antioxidants, such as vitamin A,2 coenzyme Q10,3 uric acid

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and

glutathione.5 These antioxidants act as scavengers to remove free radicals, preventing cell damage in order to mediate the impact of aging processes. Many antioxidants, especially natural antioxidants from foods, are believed to be beneficial towards anti-aging and have been formulated into neutraceuticals and are used worldwide. 6

Vitamin C (2-(1,2-dihydroxyethyl)-4,5-dihydroxyfuran-3-one), also called ascorbic acid, is an antioxidant and one of the most important water-soluble vitamins, naturally present in fruits and vegetables, and widely used as a food additive.7 Vitamin C (VC) can scavenge superoxide anion radicals, hydrogen peroxide, hydroxyl radicals, singlet oxygen and reactive nitrogen oxide. 8 VC is also involved in the synthesis of red blood cells and collagen, the later being a key component of connective tissues, blood vessels, bone matrix, cartilage and tissue repairs.9 Catechins from tea leaves possess diverse biological properties, including anti-oxidant,10 anti-inflammatory,11 and anticarcinogenic activities,12 as well as protection against neurodegenerative13 and cardiovascular diseases.

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The

green tea catechins, primarily (−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epicatechin (EC), have been associated

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with a series of health benefits.15 EGCG can inhibit metalloproteinases, protein kinases, tumor proteasome and proteins involved in DNA replication.16 The polyphenol curcumin (1, 7-bis (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione), is one of the most active components of the Indian spice curry Turmeric (Curcuma longa Linn.).17 Curcumin features a wide range of therapeutic features such as anti-cancer,18,19 anti-oxidant,19,20 anti-arthritic,21 anti-amyloid,22 anti-ischaemic,23 and anti-inflammatory properties. 24 Curcumin has been considered a third generation cancer chemopreventive agent in the US and phase II clinical trials have been carried out in Germany. 25

The clinical effects of anti-oxidants are still under debate. For example, the clinical benefits of curcumin have been demonstrated in some studies, while other studies reported less significant or even no effects. Importantly, the effective doses used in these studies were quite different. 26 One possibility that caused this discrepancy may be due to the stability of the anti-oxidant during the storage and/or administration of the specific treatment in the study. Many natural antioxidants are very sensitive to temperature, pH and light. 27-30 For instance, the absorbance intensity of curcumin in sodium phosphate buffer, pH 7.0, decreased more than 80% when the samples were incubated at 25°C for 2 hrs.27 When orange juice containing VC was stored at 5°C for 7 days, the intensity of VC peak on HPLC decreased more than 80%.28 The concentration of endogenous phenolic acids in virgin olive oil decreased more than 80% within 20 days when the samples were stored at 50°C.30 Therefore, prevention of auto-oxidation of natural antioxidants to prolong their in vitro and in vivo functions is important in using these types of molecules as neutraceuticals or therapeutics.

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Silk fibroin protein, the main component in silkworm cocoon fibers, has evolved from a textile material to biodegradable protein biomaterials with multiple functions, attributed to the excellent mechanical properties, biocompatibility and biodegradability.31 Silk fibroin can be prepared in a variety of format, such as particles,32 films,33 porous scaffolds34 and gels, 35 via all-aqueous extraction and processing. Therefore, silk has been widely applied in biomedical fields, such as tissue engineering, biomedical devices and drug delivery devices. 36-38 Due to the high content of hydrophobic crystalline beta-sheet domains, silk biomaterials have been used to stabilize biologically active compounds, including enzymes and antibodies among others.39 Some enzymes maintained catalytic activity in silk films for more than 6 months at room temperature.39,40

Biomaterials, including silk fibroin, have been used in combination with antioxidants to improve their stability and bioavailability as neutraceuticals or therapeutics. In addition, anti-oxidants have been shown to modulate cell activities. We recently reported that curcumin, when physically bound to silk films, significantly enhanced the proliferation and differentiation of human mesenchymal stem cells when compared to free curcumin (in monomer or aggregate form) in cell culture medium.41 The goal of the current study was to compare different anti-oxidants for their binding affinities and stabilities in silk biomaterials, and to elucidate mechanisms of silk stabilization of anti-oxidants. A focus here is on the role of silk secondary structure, especially crystalline beta sheet content, in the stabilization process. The findings and mechanisms elucidated will be useful for developing more efficient anti-oxidants for anti-aging-related applications, but also for

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engineering functionalized biomaterials for cell culture and tissue repairs.

2. EXPERIMENTAL SECTION 2.1. Materials. Bombyx mori silk fibers were purchased from Xiehe Silk Incorporation, Zhengjiang, China. (-)-Epigallocatechin gallate was purchased from Weikeqi Biological technology company, Sichuan, China. Ascorbic acid (VC), curcumin, 2,2-Diphenyl-1picrylhydrazyl (DPPH) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

2.2. Preparation of silk fibroin solution. Silk fibers weighing 30 g were boiled in 12 L 0.02 M sodium carbonate solutions for 30 min, rinsed with ultrapure water three times, drained and dried in a fume hood overnight, following previously published protocols.31 The dried fibers were dissolved in 9.3 M lithium bromide solution to obtain a concentration of 20% (w/v), dialyzed against pure water for 36 hrs to remove the lithium bromide and then centrifuged to remove insoluble fibrous debris. 41 The final silk fibroin (hereafter termed silk) concentration was about 8% (w/v) after purification. The solution obtained was store at 4°C.

2.3. Fabrication of silk-antioxidant films. Curcumin was dissolved in ethanol at a concentration of 2.5 mg mL-1, mixed with 4% silk solution at a 1:4 volume ratio. VC and EGCG were dissolved in water at a concentration of 1.25 mg mL-1 and mixed with 8% silk solution at a 1:1 volume ratio. For materials characterization, the silk/antioxidant solution was added to 24-well plates with 200 µL in each well. For stability testing, the

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silk-antioxidant solution was added into 96-well plates with 50 µL in each well. Three types of films were fabricated: (1) As-cast films: silk-antioxidant solutions in the plates were dried in a fume hood overnight. (2) Dried hydrogel films: the plates containing silk-antioxidant solution were covered and left on the bench overnight until the solution formed non-flowable hydrogels. The covers were then removed to allow the gel to dry into films. (3) Methanol-treated films: the films were prepared as in (1). After drying, 90% methanol was added to each well (1 ml for 24 wells and 300 µl for 96 wells) and the plates were left in a fume hood overnight to let the methanol completely evaporate. To prepare control samples (no silk), water was used instead of silk solution.

2.4. Scanning electron microscopy (SEM).

The films obtained as described above

were immersed in liquid nitrogen for approximately 2 min, and then broken with tweezers. The films were mounted on the sample stubs with the broken sides (cross sections) facing up. After the samples were sputter-coated with Au for 90 s images were taken using a Hitachi Scanning Electron Microscope (S-4800, Tokyo, Japan) at 3.0 kV.

2.5. Fourier transform infrared spectroscopy (FTIR). The secondary structure of silk was determined using a FTIR spectrometer (Nicolet 5700, USA) for measurement in the reflection mode. The measurement range was 400-4000 cm-1.

Fourier

self-deconvolution (FSD) of the infrared spectra covering the amide I region (1595-1705cm-1) was performed using software (peakfit4.12) to estimate the content of silk secondary structures (random coils, alpha-helices, beta-sheets, beta-turns).

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2.6. In vitro degradation of silk films. For degradation tests, 1 mL of silk solution (4%) containing 20% ethanol was added into a 35 mm plastic dish, and the three types of films were prepared as mentioned above. The films were immersed in 2 mL solution of 5 U mL-1 protease XIV in PBS buffer, pH 7.4, and incubated at 37°C. The same samples immersed in PBS alone served as negative controls. At designated time points (4, 8, 12, 24 hrs), samples were rinsed with ultrapure water and the solutions, in some cases containing small silk fragments, were removed using syringes. The samples were dried at 60°C for 2 hrs and weighed. The percentage weight loss over time was determined.

2.7. Antioxidant stability. Silk films containing antioxidants prepared in 96-well plates were immersed in 200 µL PBS (pH 7.4, containing 0.05% NaN3). Free antioxidants in PBS were set as controls. The plates were tightly sealed with parafilm and stored at 4°C，37°C and 45°C. At designed time points (0, 1, 3, 7, 14, 30, 60 days) the plates were opened and dried in a fume hood overnight and the film samples were exposed to DPPH. Briefly, 100 µL DPPH solution (200 mM in 90% methanol) was added into each well containing a dried film. The samples were incubated at 37°C for 60 min, and 50 µL solution was removed for absorbance measurement at 517 nm using a microplate spectrophotometer (Bio-Tek synergy H1, USA). The amount of DPPH that was scavenged relative to the original DPPH (% scavenging) was calculated using the equation: (1 – measured absorbance/DPPH absorbance)*100. The structural stability of various antioxidants in solution and in the films was assessed by high-pressure liquid chromatography (HPLC). The dried film samples stored at 45°C were extracted with shaking for 30 min using 20 mM NH4H2PO4 containing 0.5% HPO3 (pH adjusted to 3.5

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with H3PO4) for the VC/silk films, 50% ethanol for the EGCG/silk films, and 90% methanol for the curcumin/silk films. Different solvents were used for extraction in order to achieve high concentrations of the three antioxidants in solution, as their solubilities in water are different.

The solutions were run through a 0.45 µm membrane filter after

extraction and a 10 µL aliquot was injected into the HPLC (Thermo Scientific TSQquantum AccessMAX, USA). The mobile phase used for VC was the same as the extraction solution (isocratic elution). The total running time was 5 min at a flow rate of 0.5 mL/min. UV detection was at 245 nm. The mobile phase A for EGCG and curcumin was a deaerated mixture of H2O containing 0.1% TFA. The mobile phase B was a deaerated mixture of acetonitrile containing 0.1% TFA. The gradient program was as follows: 0-5 min 5% B; 5-10 min 5-100% B; 10-12 min 100-5% B; 13-15 min 5% B. The flow rate was 0.5 mL/min. The detection wavelength was 280 nm for EGCG, and 425 nm for curcumin.

2.8. Association of antioxidants with silk. Fast-performance liquid chromatography (FPLC) and DPPH analyses were used to determine the association of the antioxidants with the silk. For FPLC analysis, the solutions collected from the freshly prepared dried hydrogel films (day 0) in the stability study were subjected to FPLC (AKTApurifier100, USA) analysis, equipped with a Superose 12 chromatographic column (10×300 mm, GE, USA). The mobile phase was 10 mM PBS, pH 7.4 and the flow rate was 1 mL/min. The sample injection volume was 100 µL. For the ultrafiltration experiment, VC and EGCG dissolved in water were mixed with 8% silk solution at a ratio of 1:1 (v/v). The final concentrations of VC and EGCG were 0.05, 0.1, 0.2 and 0.4 mg/mL. Silk solution (4%)

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in the absence of antioxidants was used as the negative control. VC and EGCG solution in the absence of silk served as positive controls. The mixed solutions were incubated at 37°C for 10 min and filtered through the Amicon Ultra 10K device (molecular weight cut off 10,000 Da, Millipore, USA) by centrifugation at 4,000 rpm for 1 hr. The filtered solutions were mixed with DPPH at volume ratio 1:1 and the % scavenging was determined as described above.

2.9. Release of antioxidants from silk films. To prepare samples for the release study, 200 µL silk/antioxidant solution was added into 24-well plates as described above. The final concentrations of the antioxidants in the mixtures were 0.1, 0.25, 0.5 and 1 mg/mL, and the silk concentration was 4%. Three types of films (as-casted, dried hydrogel, methanol-treated) were prepared using the same methods as for the stability tests. To start the release experiment, 2 ml release medium (PBS buffer, pH7.4, supplemented with 0.5% Tween80 and 3% methanol) was added to each well. The plates were sealed with parafilm and incubated at 37°C on a shaker at 80 rpm. At specific time points (1, 3, 6, 12, 20, 30, 46 hrs), 1 mL of the release medium was moved to an empty tube and 1 mL fresh medium was added into the well. Antioxidant concentration in the release medium was determined by measuring absorbance at 245 nm (VC), 280 nm (EGCG) and 425 nm (curcumin). Antioxidant concentration was calculated based on the standard curves measured under the same conditions. The percentage release was obtained by comparing the amount of antioxidant in the release medium with the initial loading in the film. Each test group contained four repeat samples.

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2.10. Statistics. Results were expressed as means ± standard deviations. Statistical differences between samples were evaluated in SPSS (16.0) using a one-way student’s t-test. Differences were considered significant when p < 0.05.

3. RESULTS AND DISCUSSION 3.1. Physical characterization of silk films. The cross-section of the as-cast silk films was smooth with no microstructures observed by SEM (data not shown). The hydrogel films showed microparticles (Figure 1 a-d), and the methanol-treated films contained fiber networks (Figure 1 e-h). Loading of VC, EGCG and curcumin did not significantly alter the silk film morphologies when compared to the plain silk films (data not shown).

Figure 1. SEM images of silk/antioxidant films.

a-d, dried hydrogel films; e-h,

methanol-treated films. a,e, plain silk films; b,f, silk/VC films; e,g, silk/EGCG films; d,h, silk/curcumin films.

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Table 1. Secondary structures in silk films based on FTIR analysis. Random

coil

β-sheet (%)

α-helix (%)

β-turn (%)

(%) As-cast

24.9±1.2

35.6±1.5

7.8±3.1

28.0±1.4

Dried hydrogel

45.7±0.3

25.3±2.4

7.9±1.6

17.8±1.9

Methanol-treated

47.0±1.4

26.5±4.1

7.7±1.6

12.9±3.0

The secondary structures of silk were characterized by FTIR. The characteristic absorbance peaks in Amide I region (1600-1700 cm-1) are shown in Figure 2. The as-cast films showed peaks around 1645 cm-1, indicating the films were dominated by random coil structures (Silk I, Figure 1A). The absorbance peaks of the dried hydrogel (Figure 2B) and methanol-treated films (Figure 2C) were around 1623 cm-1 and 1626 cm-1, respectively, indicating both types of films were dominated by beta-sheet structures (Silk II). 42 The Amide I peaks were deconvoluted to quantify secondary structures (Table 1) and tbeta-sheet content was approximately 45.7 and 47.0% in the dried hydrogel and methanol-treated films, respectively, higher than that in the as-cast film (24.9%). The as-cast films contained more random coil structures (35.6%) than the dried hydrogel (25.3%) and methanol-treated (26.5%) films. These results were consistent with the literature. 43

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Figure 2. FTIR spectra of silk films. a, as-cast films; b, dried hydrogels; c, methanol-treated. SF = silk

3.2. Proteolytic degradation. The stabilities of the various silk films in PBS buffer, pH 7.4, and protease XIV solution were evaluated (Figure 3). The as-cast silk films degraded rapidly (4-6 hrs) in both solutions. Except for an initial loss of less than 10% weight, likely due to the dissolution of loosely associated silk, the dried hydrogels and methanol-treated films were stable in PBS buffer for long periods of time (Figure 3A). The same films, when incubated in protease XIV, degraded rapidly, with the dried hydrogel films losing 45% and the methanol-treated film losing 30% weight after 8 hrs (Figure 3B). Thus, the beta-sheet-dominated silk films exhibited higher proteolytic

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resistance than the random coil-dominated silk films, consistent with the literature. 44 The crystalline beta-sheets formed in the methanol-treated films might be more compact than those formed in the dried-hydrogel, thus more resistant to the attack of protease XIV.

Figure 3. Weight loss of silk films during incubation in various solutions. A, PBS buffer, pH 7.4. B, PBS buffer, pH7.4 containing protease XIV.

3.3. VC stability. When stored at 4°C, the as-cast, dried hydrogel and methanol-treated films exhibited 77.9, 40.3 and 55.1% scavenging, respectively, significantly higher than the control of VC dissolved in PBS (11.6% scavenging) in the first 14 days (Figure 4A).

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Figure 4. VC stability in silk films. The films were stored at 4°C (A), 37°C (B) and 45°C (C) and the antioxidant activity (% scavenging) were analyzed by DPPH. D, HPLC analysis on the films stored at 45°C for 14 days. The arrow in D indicates the position (2.1 min) where the original VC peak was. Asterisks indicate significant difference between samples (p < 0.05). When stored at higher temperatures, the as-cast films were superior to the other films in maintaining the DPPH activity of VC. After 14 days of storage at 37°C and 45°C, the as-cast films retained 57.2% and 59.0% scavenging activity, respectively, significantly higher than the dried hydrogel and methanol-treated films stored at the same conditions (Figure 4 B,C). At 30 days, the % scavenging measured dropped to baseline for all the samples. The degradation of VC was studied using HPLC. The elution time for the VC

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extracted from the freshly prepared samples (day 0) was within the range of 1.8 - 2.1 min (Figure S1). After storage for 14 days, the peak for the control sample decreased more than 40-fold and the elution time shifted from 2.1 min (day 0) to 1.5 min, likely due to degradation of VC (Figure 4D), consistent with the results in the literature. 28 The three film samples (as-cast, dried hydrogel, methanol-treated) showed two peaks with elution times of 1.8 min (major) and 2.1 min, representing modified VC and original VC, respectively (Figure 4D). After 30 days, the as-cast film sample showed the peak at 1.5 min while the other two films showed peaks at 1.8 min (Figure S1). Thus, the elution time and peak heights by HPLC for the VC embedded in the silk films gradually changed within 30 days, along the same trend as the changes in antioxidant activities. The peak heights and positions of the three film samples were similar in the first 14 days, whereas the anti-oxidant activities were significantly different during this time period. Other mechanism(s) rather than structural changes of the compounds might account for these differences, such as the formation of VC aggregates or VC/silk complexes that retained antioxidant activity but behaved differently in HPLC. 3.4 EGCG stability. When the VC-loaded films were stored at 4°C, the antioxidant activity was maintained in all the samples, with approximately 40% scavenging retained after 30 days (Figure 5A). For the samples stored at 37°C, the as-cast and dried hydrogel films had approximately 62.8% scavenging retained after 14 days, significantly higher than those for the methanol-treated film (3.0%) and the control (21.1%). The same trend was seen for the samples stored at 45°C; approximately 48.2% and 60.8% scavenging remained for the as-cast and dried hydrogel films, respectively, significantly higher than those for the methanol-treated film and the control (13.6%) (Figure 5B,C). At 30 days, no

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significant difference was seen between films.

The solution-state EGCG (control) at

day 0 showed an elution peak at 6.9 min by HPLC (Figure 5D). The intensities of the EGCG peaks from three types of films were 3-4 times lower than that of the controls (Figure 5D). Since the oxidant activities were similar for all the samples at day 0, the results indicated that EGCG was not efficiently extracted from the three types of films. Interestingly, the EGCG peak at 6.9 min disappeared for all the samples stored at 45°C for 14 days (Figure S2), while the anti-oxidant activities still remained high in these samples (Figure 5C). Similar to VC, a mechanism other than a structural change of EGCG might be responsible for the discrepancy between the DPPH and HPLC data. One explanation is the strong binding of EGCG and VC to silk, which could not be dissociated by the solution used for extraction (20 mM NH4H2PO4 for VC and 50% ethanol for EGCG) but still retained antioxidant activity. This was investigated further by FPLC.

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Figure 5. EGCG stability in silk films. The films were stored at 4°C (A), 37°C (B) and 45°C (C) and the antioxidant activity (% scavenging) were analyzed by DPPH. D, HPLC analysis on the films stored at 45°C for 14 days. The arrow in D indicates the control EGCG peak. Asterisks indicate significant difference between samples (p < 0.05).

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Figure 6. Determination of silk/EGCG and silk/VC complex using FPLC and DPPH assay. A, FPLC determination on the release medium of silk/EGCG films; B, DPPH assay on silk/EGCG mixed solution after ultrafiltration. C, FPLC determination on the release medium of silk/VC films; D, DPPH assay on the silk/VC mixed solution after ultrafiltration. 3.5 Interaction of VC and EGCG with silk. The solution collected from the freshly prepared silk films (day 0) in the stability study was subjected to FPLC analysis (Figure 6A,C).

The silk/VC and silk/EGCG film samples showed peaks at early positions

(around 11 mL), likely silk or silk/antioxidant complexes, whereas the peaks of free VC and EGCG (around 18 mL) were not present, likely due to strong binding to silk. The results indicate that VC and EGCG might have bound to silk. To confirm the assumption, VC and EGCG were mixed with silk at different ratios, and the solutions were filtered 19



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using an ultrafiltration device (molecular weight cut off 10,000 Da) to remove the possible silk/antioxidant complexes, and the solution after ultrafiltration was subjected to DPPH assay. The control samples were the free VC and EGCG mixed with water. The increase of VC and EGCG concentrations in silk resulted in increased antioxidant activities determined in the flow-through solutions, until reaching the levels of the control samples (Figure 6B,D). The critical concentrations for the VC and EGCG to reach the highest antioxidant activities were 0.4 and 0.2 mg/ml, respectively. The mechanism for VC or EGCG associating with silk remains unclear. Although both molecules are water soluble the aromatic moieties present in their structures might interact or bind with the hydrophobic regions of silk, or with the hydrophobic/hydrophilic boundary regions in silk. 3.6 Curcumin stability. When stored at 4°C, the antioxidant activity of curcumin remained stable in the methanol-treated silk film (approximately 80% scavenging left after 14 days) but not for the other two samples (Figure 7A). For the samples stored at 37°C, the anti-oxidant activities were approximately 50% for the as-cast and methanol-treated films, while approximately 20% for the dried hydrogel films and controls after 14 days (Figure 7B). For the samples stored at 45°C, the % scavenging was more than 60% for the methanol-treated films, while approximately 20% for all the other samples after 14 days (Figure 7C). Compared to VC and EGCG, the curcumin peak on HPLC did not change position with time. The peak heights decreased 4-8 times and approx. 20 times at day 14 and day 30, respectively, compared to that at day 0 (Figure S3). At day 30, significant degradation peaks were found before and after the curcumin peak, and the degradation of the control samples was more pronounced than the film samples.

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Interestingly, when the curcumin was completely dissolved (0.1 mg/ml) in either water or PBS, pH 7.4, and the solution was incubated at 45°C for 3 and 7 days, it was found that there was more degradation in the PBS than in water (Figure 7D). Almost no curcumin could be determined in PBS after 7 days. In the above stability studies, the curcumin in the control sample was in powder form (suspension) at a higher concentration (0.5 mg/ml) in PBS. Thus, curcumin at low concentrations (free molecule form) was more sensitive to the environment of the PBS buffer. In contrast, curcumin in an aggregated form or an immobilized form in silk films can be stabilized for a long period of time.

Figure 7. Curcumin stability in silk films. The films were stored at 4°C (A), 37°C (B) and 45°C (C) and the antioxidant activity (% scavenging) were analyzed by DPPH. D, HPLC analysis on curcumin stored as a soluble form in various solutions.

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Figure 8. Release of antioxidants from silk dried-hydrogel films. A, VC; B, EGCG, C, curcumin. The film samples were prepared by drying silk hydrogels containing 0.1, 0.25, 0.5 and 1 mg/ml antioxidants. 3.7 Release of antioxidants from silk films. The release of VC and EGCG from all the dried hydrogel films plateaued within 10 hrs (Figure 8A,B). The as-cast films containing VC and EGCG quickly dissolved in the release medium, thus the release data could not be obtained. The methanol-treated VC and EGCG films showed similar release profiles to the dried hydrogel films (Figure S4 and S5, respectively). In the case of curcumin, 60-80% of the total curcumin in the dried hydrogel films was released within 48 hrs in PBS, pH7.4 containing surfactant and methanol (Figure 8C), compared to almost no curcumin release when the release medium was PBS buffer, pH7.4 (data not shown) but without the surfactant and methanol. For the as-cast films, the samples 22


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containing low concentrations of curcumin (0.1, 0.25 mg/ml) dissolved rapidly (< 5 hrs), while the samples containing high concentrations (0.5, 1 mg/ml) of curcumin showed similar release profiles to the dried hydrogel and the methanol-treated films (Figure S6). Thus, compared to VC and EGCG, curcumin was released more slowly from the dried hydrogel and methanol-treated silk films. As discussed below and previously reported,41 curcumin may bind to the beta-sheet regions in silk via hydrophobic interactions, and the strong hydrophobic interaction restricted its release from the silk matrix into the surrounding release medium. VC and EGCG are more hydrophilic so their interaction with silk was weaker compared with curcumin, resulting in relatively fast release. All three anti-oxidants are small compounds with molecular weights ranging from100 to 500 Da, so the differences seen in release kinetics should not be due to differences in diffusion rates.

Figure 9. Chemical properties of antioxidants and their relationship with silk binding/release profiles. 23



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3.8 Mechanisms of stabilization. Silk extracted from B. mori is composed of three structural components (heavy chain, light chain, P25). The heavy chain, the key component to determine material properties, consists of the hydrophilic, non-repetitive N-, C-terminus and spacer regions, and the hydrophobic, repeating, large hexapeptide (GAGAGS, GAGAGY) domains. Depending on processing, the repetitive hexapeptide domains can form different contents of crystalline beta-sheet regions, conferring tunable strength and stability to silk biomaterials. Because of the diversity in silk secondary structure, various compounds, from small molecules to peptides and proteins, can bind to silk via different intermolecular interactions, including hydrogen bonding, electrostatic interactions, and hydrophobicity. In the present study, three antioxidants with distinct chemical structures likely bound differently to the different regions of silk. Curcumin has two phenol groups, which are connected by two α,β-unsaturated carbonyl groups, which are good Michael acceptors and undergo nucleophilic addition (Figure 9). Curcumin is hydrophobic, with water solubility less than 0.1 mg/ml and logD (distribution coefficient between octanol and water) of 2.9 at pH7.4. Curcumin bound to silk mainly by hydrophobic interactions, likely to the hydrophobic, crystalline beta-sheet regions. The binding with silk molecules increased the stability of curcumin. The results are consistent with previous reports. VC (ascorbic acid) is a weak organic acid that is structurally 45

derived from the six-carbon sugar glucose (Figure 9). VC is highly water soluble (solubility 245 mg/ml and logD of -4.99 at pH7.4). EGCG is the ester of epigallocatechin and gallic acid with four rings (Figure 9), soluble in water (33.3-100 mg/ml) and slightly hydrophobic (logD of 1.62 at pH7.4). The mechanism for VC or EGCG associating with silk is not as clear. Compared to VC, more EGCG molecules bound to silk as shown in

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the ultrafiltration experiment (Figure 6B,D), which suggests that hydrophobicity might play a role in binding. The aromatic moieties present in the structure of curcumin and EGCG might have strongly interacted or bound with the hydrophobic regions, or with the hydrophobic/hydrophilic boundary regions of silk. Despite different binding regions, the antioxidant activities of all three antioxidants were protected by silk over time (>14 days) at high temperatures (37°C and 45°C). Silk biomaterials functionalized with antioxidants, especially curcumin, have been reported previously, but little was known about the mechanism underlying binding and functionality. Although still preliminary, the data in the present study suggested that antioxidants and silk may interact in both solution(random coil-rich) and solid- (beta-sheet rich) states, likely the mechanism underlying silk-based stabilization of antioxidants. 4. CONCLUSIONS Three antioxidants (VC, EGCG and curcumin) with distinct chemical properties were incorporated in silk films, which were treated in three different ways and stored in PBS buffer, pH 7.4, at 4, 37, and 45°C. VC and EGCG likely interacted with the hydrophobic residues of silk via the aromatic moieties. The strong silk-antioxidant interaction effectively stabilized the antioxidants, with activities preserved for 14 days at 37°C and 45°C compared to controls consisting of the free antioxidants in solution. Given the therapeutic effects of antioxidants in aging, inflammation and cancer treatments, the findings and the mechanisms elucidated in the present study may be useful toward the development of new silk-based techniques for the sequestration and delivery of these types of compounds.

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The authors declare no competing financial interest.

Supporting Information. Available experimental results for the HPLC analyses and release profiles for VC, EGCG and curcumin from silk films after storage at various temperatures.

ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of China grant (project no.

51273138 and 81372837), Start-up Fund of Soochow University (project no. 14317432), Natural Science Foundation of Suzhou City Jiangsu Province China (Grants No SYN201403) and US NIH P41 EB002520.

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