Curcumin-functionalized silk biomaterials for anti-aging utility

Journal of Colloid and Interface Science 496 (2017) 66–77

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Regular Article

Curcumin-functionalized silk biomaterials for anti-aging utility Lei Yang a,1, Zhaozhu Zheng a,1, Cheng Qian b, Jianbing Wu a, Yawen Liu a, Shaozhe Guo a, Gang Li a, Meng Liu d, Xiaoqin Wang a,⇑, David L. Kaplan c,⇑⇑ a

National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, People’s Republic of China Department of Breast Surgery, Harbin Medical University, Cancer Hospital, Harbin 150040, People’s Republic of China c Department of Biomedical Engineering, 4 Colby Street, Tufts University, Medford, MA 02155, USA d The Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, Soochow University, Suzhou, People’s Republic of China b

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 26 December 2016 Revised 30 January 2017 Accepted 31 January 2017 Available online 2 February 2017 Keywords: Curcumin Silk fibroin Rat bone marrow mesenchymal stem cells (rBMSCs) Anti-aging

a b s t r a c t Curcumin is a natural antioxidant that is isolated from turmeric (Curcuma longa) and exhibits strong free radical scavenging activity, thus functional for anti-aging. However, poor stability and low solubility of curcumin in aqueous conditions limit its biomedical applications. Previous studies have shown that the anti-oxidation activity of curcumin embedded in silk fibroin films could be well preserved, resulting in the promoted adipogenesis from human mesenchymal stem cells (hMSCs) cultured on the surface of the films. In the present study, curcumin was encapsulated in both silk fibroin films (silk/cur films) and nanoparticles (silk/cur NPs), and their anti-aging effects were compared with free curcumin in solution, with an aim to elucidate the mechanism of anti-aging of silk-associated curcumin and to better serve biomedical applications in the future. The morphology and structure of silk/cur film and silk/cur NP were characterized using SEM, FTIR and DSC, indicating characteristic stable beta-sheet structure formation in the materials. Strong binding of curcumin molecules to the beta-sheet domains of silk fibroin resulted in the slow release of curcumin with well-preserved activity from the materials. For cell aging studies, rat bone marrow mesenchymal stem cells (rBMSCs) were cultured in the presence of free curcumin (FC), silk/ cur film and silk/cur NP, and cell proliferation and markers of aging (P53, P16, HSP70 gene expression and b-Galactosidase activity) were examined. The results indicated that cell aging was retarded in all FC, silk/ cur NP and silk/cur film samples, with the silk-associated curcumin superior to the FC. Ó 2017 Elsevier Inc. All rights reserved.

⇑ Corresponding author. ⇑⇑ Corresponding author. 1

E-mail addresses: [email protected] (X. Wang), [email protected] (D.L. Kaplan). The first two authors contributed equally to this paper.

http://dx.doi.org/10.1016/j.jcis.2017.01.115 0021-9797/Ó 2017 Elsevier Inc. All rights reserved.

L. Yang et al. / Journal of Colloid and Interface Science 496 (2017) 66–77

1. Introduction Aging is a universal physiological process featured by progressive impairment in functional capacity of an organism [1]. At present, there are several assumptions concerning aging, with the most popular based on the free radical theory initially proposed by Harman [2,3]. The excessive generation of reactive oxygen species (ROS) could cause significant senescence, apoptotic or necrotic cell death. Aging cells lose their ability to replicate, yet remain metabolically active. The cells are enlarged and express agingrelated markers, including alkaline b-galactosidase, telomerase, 70 kilodalton heat shock proteins (hsp70), cyclin-dependent kinase inhibitor 2A (p16), and tumor protein p53 (p53) [4,5]. Antioxidants can delay or prevent the oxidation of cellular substances that can be otherwise oxidized. Herbs, spices, extracts of phenolic compounds such as gallic acid, carnosic acid catechin, eugenol [6], green tea extract, polyphenol constituents (e.g., epigallocatechin gallate (EGCG)) [7] and fruit extracts (e.g., ascorbic acid (Vitamin C), a-tochopherol (Vitamin E) and b-carotene) [8] all have antioxidant activity. Curcumin, also called curcuminoids, is a natural antioxidant that is isolated from turmeric (Curcuma longa) [9]. Curcumin has attracted attention as a food ingredient and also in drugs and nutraceuticals. Curcumin up-regulates the expression of cell proliferation-related genes, thus improving cell viability and reversing telomere shortening by increasing telomerase activity. Curcumin also inhibited the expression of cancer-related genes, such as P53 and P21 [10], reduced cell damage caused by oxidative stress, increased the expression of HSP70 [11], inhibited protein oxidation and lipid peroxidation during cell mitosis [12], induced apoptosis of cancer cells while had almost no damage to normal cells [13], and effectively alleviated oxidative damage and inflammatory reactions induced by chronic aging [14]. However, the instability of curcumin in neutral and alkaline conditions, where the compound undergoes hydrolytic degradation to feruloyl methane, ferulic acid and vanillin, hampers its utility [15]. Curcumin lost more than 90% of its anti-oxidative activity within 30 min in 0.1 M phosphate buffer and serum-free medium, while it was more stable in cell culture medium containing 10% fetal calf serum and in human blood; less than 20% of curcumin decomposed within 1 h, and approximately 50% of the curcumin remained after incubation for 8 h [16]. Therefore, curcumin in solution did not protect cells from ROS for an extended period of time. Modes to prevent curcumin from physical and chemical damage have become important issues when curcumin is to be utilized for many applications. Incorporation of curcumin in solid or semi-solid biomaterial forms is a promising approach; including encapsulationbased systems such as micro/nanoparticles [17], films [18], and hydrogels [19]. Silk is a high molecular weight structural protein derived from the Bombyx mori silkworm and has been widely used as a building block to fabricate biomaterials for tissue engineering and drug delivery. Silk materials, due to their hydrophobic nature and crystallization, are inherently more resilient against changes in temperature, moisture and pH than most other natural or synthetic polymers [20]. The entrapment and release of bioactive molecules from silk has been widely studied [21–25]. Curcumin has been loaded in different silk material formats, such as nanofibers [26], hydrogels [27], sponges [28], and thin films [29]. When curcumin was incorporated into methanol-treated (crystallized) silk films, and samples were stored at 37 °C in PBS at pH 7.4, more than 80% of the oxygen scavenging activity of curcumin remained after 14 days [29]. Furthermore, curcumin-incorporated silk films promoted adipogenesis from human mesenchymal stem cells (hMSCs), demonstrating retention of bioactivity upon processing

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into these films [30]. Since the level of curcumin released from the silk films was very low due to the strong binding of curcumin to hydrophobic silk beta-sheet domains, pro-adipogenesis was likely due surface exposed curcumin rather than the free curcumin in solution accessible to the stem cells. In the present study, the focus was on the anti-aging effect of silk-stabilized curcumin. Two silk material forms, i.e., film and nanoparticles, were studied and compared with free curcumin, using aging-related markers, including HSP70, P16 and P53 gene expression and b-galactosidase activity [31]. Inclusion of silkcurcumin nanoparticles helped to elucidate the mechanism of anti-aging of silk-associated curcumin and also provided data to support future applications of silk-stabilized curcumin, such as oral-delivered silk/curcumin nanoparticles. Silk-curcumin nanoparticles have been previously reported in the literature [32,33], however, the present studies focused more on the biological responses of different types of silk associated curcumin and the mechanisms underlying the responses. 2. Experimental section 2.1. Materials Partially degummed silk fibers were purchased from Xiehe Silk Corporation Ltd. (Hangzhou, China). Lithium bromide (LiBr) was purchased from Aladdin (Shanghai, China). Dialysis tubing (3500 MWCO) was purchased from Thermo Fisher (Shanghai, China). Curcumin (Cat#C7727, >80% pure) and 2,2-diphenyl-1picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Shanghai, China). Ethanol, polyethylene glycol 1000, 400 and other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Dulbecco’s modified eagle’s medium (HG-DMEM), Dulbecco’s modified phosphate-buffered saline (DPBS), Fetal bovine serum (FBS) (fatty-acid free), 1% penicillin– streptomycin and 0.05% trypsin–0.5 mM EDTA were obtained from GIBCO (Thermo Fisher, Shanghai, China). Quant-It PicoGreen dsDNA reagents were purchased from Life Technologies (Thermo Fisher, Shanghai, China). qPCR reagents were purchased from Roche Applied Science (Shanghai, China). Cell counting CCK-8 Kit and senescence b-Galactosidase staining Kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). E.Z.N.A. Total RNA Kit was purchased from OMEGA (Georgia, USA). 2.2. Preparation of silk fibroin solution Silk fibroin aqueous solutions were prepared as previously described [34]. Briefly, 30 g silk fibers were boiled in 12 L 0.02 M sodium carbonate (NaCO3) solutions for 30 min, rinsed with ultrapure water three times, drained and dried in a fume hood overnight. Dried silk fibers weighing 5 g were dissolved in 20 mL 9.3 M lithium bromide (LiBr) solution at 60 °C for 4 h. The dissolving solution was dialyzed against DI water for 48 h, followed by changing DI water at least 5 times to remove the lithium bromide. The solution obtained was centrifuged to remove insoluble fibrous debris. The silk fibroin solution concentration was
8% (w/v) after purification. The solution obtained was stored at 4 °C. 2.3. Fabrication of silk/cur films A stock solution was prepared by dissolved curcumin in ethanol at 2.5 mg/mL. The solution was mixed with 4% (w/v) silk at a volume ratio of 1:4. The final concentrations of curcumin in the blend solution were 0.125 and 0.05 mg/mL. To cast films, an aliquot of the mixed silk/curcumin solution was pipetted to cell culture plates (96-well plates: 30 lL, 24-well plates: 200 lL, 6-well plates:

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960 lL). The plates were covered and sealed with parafilm until the solution gelled. The hydrogels were air dried, forming a transparent thin film at the bottom of the plate. The prepared films are termed silk/cur film and distinguished by the original curcumin concentrations in solution (0.125 and 0.05 mg/mL). 2.4. Fabrication of silk/cur nanoparticles The silk/cur NPs suspensions were prepared by the PEG blending method previously described [35]. Briefly, a stock solution was prepared by dissolving curcumin in PEG (MW 10K) at a concentration of 5 mg/mL. The curcumin-PEG solution was mixed with 2.25% silk fibroin at a volume ratio 10/1, and then incubated at room temperature overnight. The resulting solution was diluted 10 times with DI water and centrifuged (Sorvall Legend Micro21R, Thermo Scientific, Germany) at room temperature for 20 min, and the pellet was washed 3 times with water by centrifugation and then resuspended in water. The suspension was frozen at 20 °C and lyophilized for 24 h using a lyophilizer (CHRIST Alpha 2–4 LSCplus, Martin Christ Gefriert-rocknungsanlagen GmbH, Osterode am Harz, Germany). To determine the encapsulation efficiency of curcumin in silk/ cur NPs, silk/cur NPs weighing 5 g were added into glass vials containing 5 mL of methanol. Samples were kept at 37 °C in a rotary incubator at 200 rpm for 3 h. The extraction solution was centrifuged (Sorvall Legend Micro21R, Thermo Scientific, Germany) at 12,000 rpm, room temperature for 20 min, and 200 lL of the supernatant was placed into 96 well plates. The fluorescence intensity of the supernatants was determined using a synergy H1 microplate reader (Bio-Tek, USA) at an excitation wavelength of 425 nm and an emission wavelength of 530 nm. All release samples were prepared in triplicate. Curcumin standard curves were prepared by dissolving 25 mg of curcumin in methanol to obtain a concentration of 2.5 mg/mL, followed by a serial 2-fold dilution. 2.5. Scanning electron microscopy (SEM) Silk/cur films were dipped in liquid nitrogen for approximately 3 min and then broken into small pieces with tweezers. The pieces were mounted on sample stubs, with the cross sections faced to the top. The samples were sputter-coated with Au (Hitachi E1045 Ion Sputter, Tokyo, Japan) for 90 s before taking images using a Hitachi Scanning Electron Microscope (SEM, S-4800, Tokyo, Japan) at 3.0 kV. For silk/cur NPs, an aliquot of particle suspension was dropped on a silicon wafer, which was fixed on a SEM sample stage and dried in a fume hood. The samples were subjected to the sputter-coating and SEM analysis as described above. 2.6. Fourier transform infrared spectroscopy (FTIR) The dried silk/cur films or nanoparticles were ground into powder together with potassium bromide (KBr), and pressed into discs for FTIR measurements using a Nicolet 5700 spectrophotometer (Thermo, USA). For each measurement, 24 scans were recorded with a resolution of 4 cm1 and a wavenumber range of 400– 4000 cm1. 2.7. Particle size distribution The silk/cur NPs obtained as above were suspended in water at 0.1 mg/mL, dispersed by ultrasonification (JY92-II DX, SCIENTZ, China) with 30% ultrasonic power, and then added in a disposable polystyrene cuvette (Cuvette acrylic, Thermo fisher FL America). The cuvette was loaded in a photocorrelation spectroscopy system (Nano ZS90, Malvern,Worcestershire, England) set at 25 °C under a

fixed angle of 90° to measure the average particle size (z-average size) and polydispersity index (PI) of nanoparticles. 2.8. Differential Scanning Calorimetry (DSC) The silk/cur films or nanoparticles obtained as above were ground into powder for the analysis. The Thermo Gravimetric Analyzer-Differential scanning calorimeter (TGA-DSC) studies were carried out using a SDT Q600 V20.4 Build 14 system (TA Instruments, New Castle, DE, USA). The scan rate was 2 °C/min in the temperature range 25–600 °C. 2.9. Laser confocal scanning microscopy (LCSM) The distribution of curcumin in silk/cur films was investigated by confocal microscopy. A piece of film (
5  5 mm) with an initial curcumin concentration of 0.25 mg/mL was imaged on an Olympus microscope (FV 1000, Japan) at an excitation wavelength of 488 nm. Single xy scans were collected with an optical slice of 1 lm along the z-direction. 2.10. Determination of curcumin release from silk materials Release of curcumin from silk/cur films and silk/cur NPs was determined by following the fluorescence of curcumin in the release medium. Silk/cur films were prepared as described above. One mL cell culture medium (DMEM medium containing 10% FBS) or PBS containing 0.5% Tween-80 and 3% methanol was added to each well. The samples were incubated at 37 °C with gentle shaking. At designated time points (0.5, 1, 1.5, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 25 days for cell culture medium, 0.5, 1, 1.5, 2, 3, 5, 7, 9, 11 and 13 days for PBS), the release medium was removed, and the same volume of fresh release medium was added. The fluorescence intensity of the release medium was determined using a synergy H1 microplate reader (Bio-Tek, USA) at an excitation wavelength of 425 nm and an emission wavelength of 530 nm. All release samples were prepared in triplicate. For silk/cur NPs, 1.5 mL aliquots of nanosphere suspension (containing 200 or 400 lM curcumin) in cell culture medium were incubated at 37 °C with gentle shaking. At designated time points (1, 3, 6, 10, 20, 26, 32, 44, 56, 70, 82, 94 and 118 h) the samples were centrifuged and the supernatants were subjected to fluorescence determination as described above. 2.11. Stability of curcumin in cell culture medium Silk/cur films were prepared in 24-well plates as described above, and 1 mL cell culture medium was applied to each well. The samples were incubated at 37 °C with shaking. The release medium was replenished every 48 h. At designated time points (0, 1, 3, 5, 7, 9, 11 and 15 days), a portion of film samples were dried in a fume hood for the DPPH (2,2-diphenyl-1picrylhydrazyl) free radical scavenging assay, based on a published method [36] with slight modification. Briefly, 1.5 mL of 1 mM methanolic solution of DPPH was added to each well containing dried films. The 24-well plates were then covered and incubated in the dark at 37 °C for 1 h. The absorbance was measured at 517 nm. The percentage of DPPH free radical scavenging activity was determined using the following equation:

DPPH scavenging effectð%Þ ¼ ðAbsDPPH  Abssilk=cur film Þ=AbsDPPH  100

ð1Þ

where AbsDPPH is the absorbance value determined at 517 nm for the blank DPPH solution and Abssilk/cur film is the absorbance value determined at 517 nm for the sample extracts. At least 3 film samples were assayed for averaging and statistical analyses.

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2.12. Rat bone marrow mesenchymal stem cells (rBMSCs) rBMSCs were isolated from 3- to 4-week-old male Sprague– Dawley (SD) rats (SLRC laboratory animal Co. Ltd. Shanghai, China). SD rats cells were used following approval by the Soochow University human ethics review committee and animal care and use committee. Briefly, the animals were sacrificed by cervical dislocation, and the femur and tibia were collected. The bone marrow was flushed out, and the collected cell suspension was added to DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic in a 6-well plastic plate. Adherent cells were then maintained in a humidified, 95% air/5% CO2, 37 °C incubator by refreshing the medium every two or three days. When grown to 85–90% confluence, the cells were rinsed with DPBS, and then were detached using 0.25% trypsin-EDTA. This procedure was repeated three or four times to collect rBMSCs at
90% purity. P-4 rBMSCs were seeded at 10,000 cells/cm2 on silk/cur films. The medium was replenished every three days. For free curcumin and silk/cur NPs, P-4 rBMSCs were seeded at 10,000 cells/cm2 on TCP and silk films. After 24 h of culture, the medium in each well was replaced with 1 mL culture medium containing 2 lM and 10 lM free curcumin or silk/cur NPs. The medium was replenished with the cell culture medium containing the same amount of free curcumin and silk/cur NPs every three days. After 3, 6 and 9 days of culture, the cells were washed with DPBS and subjected to total DNA quantification using a Quant-iT Picogreen dsDNA assay kit (Life Technologies). Picogreen staining reagent was prepared and added to the 100 lL cell medium according to the manufacturer’s protocol. The fluorescence generated was read using a multi-mode plate reader (BioTek instrument, USA), with an excitation wavelength at 480 nm and emission at 520 nm. The parallel samples were subjected to cell viability assay using CCK-8 kit assay (Dojindo, Shanghai, China). Briefly, the cells were washed with D-PBS, and incubated in fresh cell culture medium containing 1/10 CCK-8 solution for 2 h. Thereafter, 100 lL of cell culture medium was transfer to a new 96-well plate. The absorbance was measured at 450 nm using a microplate reader (BioTEK instrument, USA). 2.13. Aging gene expression analysis using real-time PCR At designated time points, the cells were rinsed with D-PBS, detached using 0.25% trypsin-EDTA, and lysed in 0.35 mL TRK Lysis Buffer. mRNA was isolated using an E.Z.N.ATM Total RNA Kit (OMEGA R6834). cDNA was synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche) and Veriti 96-well Thermal Cycler system (Applied Biosysterms), following manufacturers’ protocol. The cDNA samples were analyzed for gene (HSP70, P53 and P16) expression relative to the GAPDH housekeeping gene using a real-time PCR system (Applied Biosystems). The primers and probes for GAPDH, HSP70, P53 and P16 are shown in Table 1. For each sample, the Ct value was defined as the cycle number at which the amplification of each target gene was in the linear range of the reaction. Relative expression levels of each gene were calculated by normalizing to the Ct value of the housekeeping gene GAPDH (2DCt, Perkin Elmer User Bulletin #2). Data from three separate cultures of each type were averaged. 2.14. Expression of senescence-associated beta-galactosidase (SA-b-Gal) b-Gal staining on aging cells was performed using the Senescence gal actosidase staining kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s

Table 1 Primers and probes for GAPDH, HSP70, P53 and P16. Genes GAPDH HSP70 P53 P16

Primers sequence Forward primer Reverse primer Forward primer Reverse primer Forward primer Reverse primer Forward primer Reverse primer

50 -ATG TAT CCG TTG TGG ATC TGA C-30 50 - CCT GCT TCA CCA CCT TCT TG-30 50 - TGGCCCATTAAATAAGAACCAA-30 50 - CGAAGGCATAGAGATTCCAG-30 50 - AGAGAGCACTGCCCACCA-30 50 - AACATCTCGAAGCGCTCAC-30 50 -TCCTCCGCTGGGAACGT-30 50 - GGCGTG CTTGAGCAG AAGTT-30

protocol. b-Gal-positive cells were counted in three different fields and presented as percentage of total number of counted cells. 2.15. Statistics One-way and two-way analyses of variance were carried out using the SPSSÒ computer programme (SPSS Inc., Chicago, IL, USA). Differences in pairs of mean values were evaluated by the Tukey test for a confidence interval of 95%. Data are represented as means ± standard deviation. 3. Results and discussion 3.1. Physical characterization of silk films and nanoparticles 3.1.1. Silk nanoparticles Curcumin loaded nanoparticles (silk/cur NPs) had a size range of 150–700 nm, smaller than that of plain silk (no curcumin) nanoparticles (700–1200 nm) (Fig. 1A). This was consistent with the SEM observations, which showed a relatively homogeneous size distribution for the silk-curcumin materials (Fig. 1B). When observed under larger magnification, silk/cur NPs showed nonspherical shapes with some aggregates (Fig. 1B inset). The secondary structures of silk in silk/cur NP were analyzed by FTIR. As shown in Fig. 1C, both the control nanoparticles (a) and silk/cur NPs (b) showed a characteristic peak at 1625 cm1, indicating the formation of beta-sheet structure [29]. The blank nanoparticles also showed a weak absorbance peak at 1655 cm1, attributed to alpha-helical structure, which disappeared in the silk/cur NPs, suggesting the incorporation of curcumin in silk nanoparticles promoted the structural transition of the silk from alpha-helices. In the DSC analysis (Fig. 1D), the characteristic silk peak for silk/cur NPs (b) shifted slightly to higher temperature when compared to that of the control silk nanospheres (a), indicating that the incorporation of curcumin further stabilized the silk structure [37]. Curcumin can bind to silk via hydrophobic interactions between the phenol groups in curcumin and beta-sheet crystals in silk. Binding increased the stability of both curcumin and silk [4,8]. 3.1.2. Silk films Silk films fabricated by ethanol-induced gelation and air-drying showed a characteristic absorbance peak at 1625 cm1, similar to the films containing 0.05, 0.125 mg/mL curcumin (silk/cur film) (Fig. 2A). These results indicate that the films were dominated by silk beta-sheet structure. Incorporation of curcumin did not significantly alter the silk secondary structure in the films. All three silk/ cur films (0, 0.5, 0.125 mg/mL) showed a similar endothermic peak of silk around 287 °C (Fig. 2B). For the films containing curcumin (b&c), the endothermic peak of curcumin around 180 °C (a) did not appear but a new peak around 287 °C appeared, suggesting curcumin might have bound to silk forming a more thermally stable structure [4,8,37]. Similar to the blank silk film, the cross-section of the Cur/silk films showed a coarse texture full of

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Fig. 1. Characterization of silk/cur NPs. A, particle size distribution determined by DLS; B, particle size distribution and shape determined by SEM; C, silk secondary structural change determined by FTIR ((a) silk nanoparticles, (b) silk/cur NPs); D, thermodynamics determined by DSC ((a) silk/cur NPs, (b) silk NPs and (c) Curcumin).

Fig. 2. Characterization of silk/cur films. A, silk secondary structural change determined by FTIR ((a) 0.125 mg/ml silk/cur film, (b) 0.05 mg/ml silk/cur film, (c) silk film); B, thermodynamics determined by DSC ((a) curcumin, (b) 0.05 mg/ml silk/cur film, (c) silk film, (d) 0.25 mg/ml silk/cur film); C, film morphology determined by SEM; D, curcumin distribution in the films determined by laser confocal scanning microscopy (LCSM) with a Z stack (1 lm interval).

particles under SEM (Fig. 2C). No large curcumin crystals were identified. Thus, incorporation of curcumin did not significantly change the microstructure of silk films. Confocal microscopy indicated a gradient distribution of the green fluorescent curcumin

molecules along the vertical direction (top–bottom) in the film, with more molecules located in the middle of the film than near the surface, while the distribution along the horizontal direction of the film was more homogeneous (Fig. 2D).

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3.2. Curcumin stability and release from silk films /nanoparticles In cell culture conditions, a significant amount of free curcumin added to the culture medium was bound to silk films, as indicated in a separate experiment (Fig. S1). When DPPH scavenging activity was assayed for the free curcumin in the medium, the compound had lost its activity, only 8% and 20% of the original activity remained after 12 days for the 2 µM and 10 µM sample, respectively. This was in contrast with 30% and 55% of remaining activity for the film-associated curcumin at the same concentrations (Fig. S2). The loading efficiency of curcumin into silk films was theoretically 100% (no curcumin lost during material processing), while that of the nanoparticles was experimentally determined to be 17% based on the method described in Materials and Methods. Percent release (%) was then determined by comparing the amount of curcumin released to the medium to the total loading, under conditions of PBS, pH 7.4, supplemented with 3% methanol and 0.5% Tween 80 (accelerated medium) and cell culture medium with gentle stirring at 37 °C. When accelerated medium was used, the cumulative release of curcumin from the 0.125 mg/mL film samples was
75%, while that in the 0.05 mg/mL films was
55% at day 13 (Fig. 3A). The curves leveled off thereafter. This was consistent with our previous study [8]. When cell culture medium was used instead of the accelerated release medium, the release of curcumin was slower, with less than 40% total curcumin released over 25 days, and the release was more linear (less burst) than in the accelerated release medium (Fig. 3B). The difference in the results might be caused by different components in the two release media, as the methanol and surfactant (Tween 80) added to the PBS facilitated curcumin dissolution. The high concentration (10%) of fetal bovine serum (FBS) in the cell culture medium may also facilitate dissolution of curcumin, but to an extent less than the methanol and Tween 80. Linear and faster release of curcumin was obtained for the silk/cur NPs (200 lM) incubated in cell culture medium, with
50% of total curcumin released at 120 h (5 days) (Fig. 3C). Incomplete curcumin release from silk materials, especially at higher curcumin loading, was likely due to some of the curcumin molecules becoming tightly bound to silk beta-sheet structural domains. The curcumin molecules embedded in the silk films maintained DPPH free radical scavenging activity (
25% for the 0.05 mg/mL and
45% for the 0.125 mg/mL films) in the cell culture medium over 13 days (Fig. 3D). 3.3. Effect of curcumin on rBMSC proliferation and viability The growth of rBMSC on silk films with and without incorporation of curcumin was significantly slower than that on TCP (p < 0.05, Fig. S3), likely due to the rough surface morphology and hydrophobicity of silk films. When free curcumin (2 and 10 lM) was added to the blank silk film samples, the rBMSCs counted at day 3 was lower than that without addition (0 lM) (second row of Fig. S3), suggesting that the free curcumin inhibited cell proliferation in this case. Cell proliferation was quantitatively determined by DNA content. The addition of 10 µM free curcumin to the culture medium significantly inhibited cell proliferation on TCP and blank silk films during the first 3 days (p < 0.05, Fig. 4 A&B). Upon cell proliferation, the negative effect of free curcumin became less significant. For example, the samples supplemented with 2 µM free curcumin supported cell proliferation. At day 9, the cells grown on TCP were over-confluent, while the numbers on the blank silk films did not significantly change when compared to day 6 (Fig. 4 A&B). Silk/cur NPs did not significantly influence the proliferation of cells grown on TCP over 6 days (Fig. 4C), whereas they inhibited proliferation of the cells grown on the blank silk films only at the

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start (day 3) (Fig. 4D). Cell proliferation on the 0.05 and 0.125 mg/mL silk/cur films was significantly inhibited when compared to the control (blank film) over 9 days (p < 0.05, Fig. 4E), and inhibition was more pronounced for the higher loading (0.125 mg/mL) than the lower concentration samples. To compare cell viability between samples, the values determined by Cell Counting Kit (CCK-8, cell viability) staining were normalized by Picogreen fluorescence (DNA content). For TCP samples, cell viability was significantly improved at day 3 (p < 0.05) with the addition of free curcumin (2 and 10 µM), but decreased to a similar level at day 6 (Fig. 5A). For the silk film samples, the addition of free curcumin improved cell viability at day 3 and 6, especially for the curcumin concentration at 10 µM, and the cell viability levels did not change too much at day 9 (Fig. 5B). The addition of silk and silk/cur NPs to the culture medium did not affect the viability of the cells on TCP (Fig. 5C). For silk film samples, the addition of silk/cur NPs significantly improved cell viability at day 3 when compared to the blank control and silk nanoparticles alone, but inhibited cell viability at day 6 (p < 0.05, Fig. 5D). The cell viability of all four samples returned to a similar level by day 9, likely due to the adsorption and degradation of the silk nanoparticles. For silk/ cur film samples (0.05 and 0.125 mg/mL), cell viability was significantly improved when compared to the control (blank silk films) from day 3 to day 9, with the highest level of viability determined at day 6 (p < 0.05, Fig. 5E). Comparing the 0.05 and 0.125 mg/mL samples, the promotion effect of the later lasted longer than the former, likely due to the higher concentration of curcumin molecules that remained active with time when the loading was higher. 3.4. Effect of curcumin on rBMSC gene expression HSP70 gene is down-regulated with the progression of cell aging [31]. Compared to the controls, HSP70 expression was significantly lower at day 3 and 6 when free curcumin and silk/cur NPs (10 µM) were added to the TCP samples (p < 0.05, Fig. 6A and C). When free curcumin and silk/cur NPs were added to the silk films, HSP70 expression was also lower, but not as significantly as that on the TCP samples (Fig. 6B and D). For the silk films samples with silk/cur NPs, HSP70 expression was higher at day 12 when compared with blank silk films and silk nanoparticles, suggesting an inhibition of cell aging. This is similar to the silk/cur film samples, for which the HSP70 expression slightly decreased at beginning when compared to the controls, but significantly increased at day 12, especially for the 0.125 mg/mL samples (p < 0.05, Fig. 6E). In contrast to HSP70, the P53 gene is up-regulated with cell aging [38]. Addition of free curcumin and silk/cur NPs to the TCP samples did not significantly change P53 expression on day 3 and 6 (Fig. 7A and C). Compared to the blank silk films, P53 expression was significantly lower at day 6 for free curcumin (2 and 10 µM) on silk films, silk nanoparticles and silk/cur NPs on silk films, and silk/cur films (0.125 mg/mL) (p < 0.05, Fig. 7B, D, and E). Thus, the aging process of cells cultured under these conditions was inhibited, and the effect lasted at least 6 days. Similar to P53, the expression of P16 is associated with cell aging [39]. Addition of free curcumin (10 lM) on TCP, free curcumin (2 lM) on silk films, and silk/cur NPs (0, 10 lM) significantly inhibited the expression of P16 at day 6, in some cases at day 3 (p < 0.05, Fig. 8A–C). The addition of silk/cur nanoparticles (silk NP control and 10 lM silk/cur NPs) to the silk films inhibited P16 expression at day 3 but not at day 6 and 12 (Fig. 8D). For silk/cur film samples, P16 gene expression did not change significantly from day 3 to 12 (Fig. 8E). 3.5. Promotion of rBMSCs beta-Gal expression by silk/cur materials The aging of cells cultured on TCP (control), silk films with silk/ cur NP supplemented in the culture medium, and silk/cur films

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Fig. 3. Release kinetics and scavenging activity of curcumin from silk. A, release kinetics of curcumin from silk film in methanol-tween 20 release medium; B, release kinetics of curcumin from silk film in cell culture medium; C, release kinetics of curcumin from silk nanoparticles in cell culture medium; D, scavenging activity change determined for silk/cur films. Data presented are means ± SD (n = 4).

Fig. 4. Cytotoxicity of free curcumin and silk/cur materials determined by Pico green assay. A, free curcumin on TCP; B, free curcumin on silk films; C, silk/cur NP on TCP. D, Cur/silk NP on silk films; E, silk/cur film. Values are means ± SD with n = 3. * p < 0.05.

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Fig. 5. Normalized cell viability. A, free curcumin on TCP; B, free curcumin on silk films; C, silk/cur NP on TCP. D, silk/cur NP on silk films; E, silk/cur film. Cell viability values obtained by CCK-8 assays were normalized by the values from Pico green assay (DNA content). Values are means ± SD with n = 3. * p < 0.05.

were estimated by bata-Gal staining. The number of cells stained blue on TCP was higher than on the samples containing curcumin, indicating that the presence of curcumin inhibited cell aging (Fig. 9). Comparing the two silk/cur film samples (0.05 vs 0.125 mg/mL), the cells stained blue on the later were fewer than those on the former, suggesting the anti-aging effect of 0.125 mg/mL silk/cur film was greater than that of 0.05 mg/mL, consistent with the results from gene analyses.

4. Discussion Curcumin has been incorporated into silk films and its longterm stability and release profiles have been studied previously [29]. For example, in our prior studies, the adipogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on curcumin-incorporated silk films was reported [30]. Compared to the curcumin that was added directly to the cell culture medium, the silk film-associated curcumin significantly promoted adipogenic differentiation of hMSCs, suggesting the curcumin on the film surface might interact with hMSCs at the cell-material interface and impact intracellular signaling pathways in a different process from free curcumin that mainly functioned as a radical scavenger inside and outside of the cells. Curcumin is known for its anti-aging effects, likely due to its strong radical scavenging capability. Previous studies were usually conducted using free curcumin or curcumin nanoparticles, which had been internalized

into cells or adsorbed into the body. Few studies have focused on understanding how cells behave at the interface of curcuminincorporated silk films or other biomaterial interfaces. The findings and mechanisms elucidated would be useful for engineering biomaterials with functions to prevent or slow cell aging and could be used in a variety of biomedical applications, such as tissue engineering and tissue repair, along with basic cell propagation matrices during expansion and use; such as stem cells. Curcumin was first incorporated in silk nanoparticles and films using methods that have been previously published by our group [30,35]. These previous studies showed that the curcumin incorporated in silk films likely bound to the silk crystalline beta-sheet domains, which improved curcumin stability in solution as well as decreased release rates [29]. Incorporation of curcumin in silk films did not significantly impact the secondary structure of silk and there were no large aggregates of curcumin formed in the films. However, curcumin was not distributed evenly in the film matrix, with more curcumin located in the middle regions. This outcome was likely due to silk hydrogelation and air-drying processes, with more hydrophobic beta-sheet domains stacking inside the film matrix, thus more curcumin molecules were entrapped in the middle of the film after binding to the beta-sheet domains. The size of curcumin-loaded silk nanoparticles was smaller than that of the blank nanoparticles. This observation was likely due to the incorporation of curcumin in the presence of PEG, which slightly enhanced silk beta-sheet formation, as evident by FTIR. This effect would result in more condensed particles than those without

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Fig. 6. HSP70 gene expression in free curcumin and silk/cur materials. A, free curcumin; B, free curcumin on silk films; C, silk/cur NP; D, Cur/silk NP on silk films; E, silk/cur film. Values are means ± SD with n = 3. * p < 0.05.

loading of curcumin. For the curcumin-loaded silk films, near linear release of curcumin was observed when placed in accelerated medium (methanol/Tween-90/PBS), while almost no release occurred in pure PBS buffer, consistent with previous studies [29]. Interestingly, when cell culture medium was used for the release study, it curcumin was slowly released into the medium, with less than 40% release over 25 days, likely due to the binding of hydrophobic curcumin to the albumin molecules in the cell culture medium. It should be noted that our cell culture studies lasted 12 days, within which only a small amount curcumin (0.37 µM for the 0.05 mg/mL silk/cur film, 0.68 µM for the 0.125 mg/mL silk/cur film) was released from the films, much less than the amount of free curcumin (2 and 10 µM) added to the culture medium. Thus, when cells were cultured on top of the silk/cur films, curcumin exposed on the film surface played a more important role in regulating cell responses than that released into the medium, in line with our previous findings [30]. For silk/cur nanoparticles, the release was faster than that of silk/cur films due to larger surface areas. In this case, cells ‘sensed’ both free curcumin in the medium and silk/cur NPs over the 12 days. For both silk/cur films and silk/ cur NPs incubated in cell culture medium, curcumin maintained anti-oxidation activity (DPPH scavenging activity). In our previous studies we demonstrated that curcumin lost its natural structure and anti-oxidation activity within a few hours in aqueous solution [29].

We first tested free curcumin (2 and 10 µM) added to cells cultured on either TCP or blank silk films. In the latter case, most of the curcumin added to the culture medium adsorbed to the surface of silk films. The addition of curcumin significantly increased cell viability in the two samples in the first 3 days of culture. However, for the TCP samples, cell viabilities largely decreased after 6 days, while for the silk film samples the viability remained at a similar level, especially for the higher concentration (10 µM) samples. When the cell aging-related genes (HSP70, P53, P16) were checked, the expression of P53 and P16 in the silk film samples supplemented with 2 and 10 µM curcumin were more down-regulated when compared to those in the curcumin-supplemented TCP samples, indicating the curcumin molecules, likely those bound to the surface of silk films rather than those dissolved in the medium, inhibited cell aging (Fig. 6–8). There was some discrepancy in gene expression between the 2 and 10 µM curcumin-supplemented silk film samples. This might be due to the amount and distribution of curcumin bound to the films. In contrast to our expectation, the HSP70 gene was down-regulated rather than up-regulated in the curcumin-supplemented silk film samples. From the literature, it seems that the dose and time for curcumin to be present in the medium are important for regulating HSP70 gene expression [40]. Although the mechanism that caused down-regulation of HSP70 gene expression is not clear in the present study, the timing

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Fig. 7. P53 gene expression in free curcumin and silk/cur materials. A, free curcumin; B, free curcumin on silk films; C, silk/cur NP; D, silk/cur NP on silk films; E, silk/cur film. Values are means ± SD with n = 3. * p < 0.05.

might be the most important in light of the silk/cur NP and silk/cur films studies. Compared to blank silk nanoparticles and silk films, silk/cur NP on silk films and silk/cur film samples significantly inhibited cell aging after long time frames (6–12 days), as HSP70 gene expression was up-regulated while the P53 gene was down-regulated, even though the P16 gene expression was not significantly changed. These results were confirmed by the beta-Gal staining assay, a commonly used method to evaluate cell aging process. As discussed above, curcumin could release from silk/cur NP and the free curcumin molecules in the culture medium could bind to the blank silk films. Therefore, similar to the silk/cur film samples, curcumin molecules exposed on the film surface interacted with cell membranes, resulting in the up-regulation or down-regulation of aging-related genes. Although the mechanism of aging gene regulation is not yet clear in this case, it is likely different from that of the free curcumin, which acts more as radical scavengers rather than a gene expression regulator. This will need to be investigated in future studies. The present study used rat mesenchymal stem cells (rBMSCs) for anti-aging studies, as stem cells possess the potential to differentiate into many cell lineages, and are more sensitive to environmental changes than fully differentiated cell types. The findings from the present study could be directly used for stem cell-based tissue engineering, such as for the regeneration of bone, cartilage, skin and other tissues, and for developing curcuminfunctionalized material matrices to preserve stem cell viability

for longer periods of time. The same findings might also be applied to other natural anti-oxidants, such as vitamin C and epigallocatechin gallate (EGCG) from green tea. Our previous studies have found these anti-oxidants can also be functionally stabilized in silk materials [29]. Furthermore, anti-oxidants have been used as additives in the food industry to keep food fresh [41,42], in the cosmetic industry to prevent oxidation of many active ingredients, and to reduce the loss of collagen and other proteins in the skin [43]. Due to their free radical scavenging capabilities and other biological properties, anti-oxidants have also been utilized as nutraceuticals for anti-aging purposes, such as vitamin C and E, carotenoid, quercetin and curcumin, [6,44]. The data presented in this study should benefit future developments of more efficient anti-aging products by using silk biomaterials as stabilizing and delivery carriers.

5. Conclusions Curcumin has been successfully incorporated into silk films and silk nanoparticles without significantly affecting the structure and morphology of the materials. The strong association of curcumin with silk, likely binding to the hydrophobic beta-sheet domains on silk, retarded its release while preserving free radical scavenging activity. Long-time exposures of functional curcumin on silk surfaces helped maintain the viability of stem cells that were cultured on silk/cur films, as well as silk films with absorbed free cur-

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Fig. 8. P16 gene expression in free curcumin and silk/cur materials. A, free curcumin; B, free curcumin on silk films; C, silk/cur NP; D, silk/cur NP on silk films; E, silk/cur film. Values are means ± SD with n = 3. * p < 0.05.

Fig. 9. b-Gal staining of cells cultured on different samples. A, TCP; B, 0.05 mg/ml silk/cur film; C, 0.0125 mg/ml silk/cur film; D,10 lM silk/cur NP on silk films; E,10 lM silk/ cur NP on silk films. Bar: 200 lm. Red arrows indicate senescent cells. Cell numbers were counted and are indicated on the pictures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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cumin and silk/cur NP. The interaction between the surfaceexposed curcumin with stem cells significantly inhibited cell aging, as indicated by the down-regulation of P53 and P16 genes, and by beta-Gal staining. Curcumin-functionalized silk biomaterials should provide useful systems for a variety of stem cell and tissue regeneration applications as well as for in food and cosmetic industries related to anti-aging applications. Conflict of interest The Authors declare that there is no conflict of interest. Acknowledgements This work was supported by Natural Science Foundation of China grant (project no. 51273138, GZ1094 and 81301844), Start-up Fund of Soochow University (project no. 14317432), Natural Science Foundation of Suzhou City Jiangsu Province, China (Grants No SYN201403), and The Natural Science Foundation of Jiangsu Province, China (Grants No BK20150371). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2017.01.115. References [1] S. Govindan et al., Antioxidant and anti-aging activities of polysaccharides from calocybe indica var APK2, Exp. Toxicol. Pathol. 68 (6) (2016) 329–334. [2] D. Harman, Aging: a theory based on free radical and radiation chemistry, J. Gerontol. 11 (1956) 298–300. [3] D.B. Kim et al., Antioxidant and anti-ageing activities of citrus-based juice mixture, Food Chem. 194 (2016) 920–927. [4] C.R. Giordano, S.R. Terlecky, Peroxisomes, cell senescence, and rates of aging, BBA-Mol. Basis of Dis. 1822 (9) (2012) 1358–1362. [5] M.V. Blagosklonny, An anti-aging drug today: from senescence-promoting genes to anti-aging pill, Drug. Discov. Today. 12 (5–6) (2007) 218–224. [6] M.S. Brewer, Natural antioxidants: sources, compounds, mechanisms of action, and potential applications, Compr. Rev. Food Sci. Food Safety 10 (4) (2011) 221–247. [7] W.E. Bronner, G.R. Beecher, Method for determining the content of catechins in tea infusions by high-performance liquid chromatography, J. Chromatogr. A 805 (1–2) (1998) 137–142. [8] Z. Balogh et al., Formation and inhibition of heterocyclic aromatic amines in fried ground beef patties, Food Chem. Toxicol. 38 (5) (2000) 395–401. [9] O.P. Sharma, Antioxidant activity of curcumin and related compounds, Biochem. Pharmacol. 25 (15) (1976) 1811–1812. [10] J.H. Kim et al., Curcumin stimulates proliferation, stemness acting signals and migration of 3T3-L1 preadipocytes, Int. J. Mol. Med. 28 (3) (2011) 429–435. [11] Y.D. Hsuuw et al., Curcumin prevents methylglyoxal-induced oxidative stress and apoptosis in mouse embryonic stem cells and blastocysts, J. Cell. Physiol. 205 (3) (2005) 379–386. [12] Q.Y. Wei et al., Inhibition of lipid peroxidation and protein oxidation in rat liver mitochondria by curcumin and its analogues, BBA-Gen. Subjects 1760 (1) (2006) 70–77. [13] G.H. Altman et al., Silk-based biomaterials, Biomaterials 24 (3) (2003) 401– 416. [14] G.M. Cole et al., Prevention of Alzheimer’s disease: Omega-3 fatty acid and phenolic anti-oxidant interventions, Neurobiol. Aging 26 (2005) S133–S136. [15] H.H. Tonnesen, M. Masson, T. Loftsson, Studies of curcumin and curcuminoids. XXVII. cyclodextrin complexation: solubility, chemical and photochemical stability, Int. J. Pharm. 244 (1–2) (2002) 127–135.

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