Biocompatible and Biodegradable Dual-Drug Release System Based

Apr 3, 2012 - The present silk-based system for dual-drug release also demonstrated ... have recently become attractive for the combined administratio...
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Biocompatible and Biodegradable Dual-Drug Release System Based on Silk Hydrogel Containing Silk Nanoparticles Keiji Numata,*,† Shoya Yamazaki,†,‡ and Naofumi Naga‡ †

Enzyme Research Team, RIKEN Biomass Engineering Program, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Department of Applied Chemistry, Materials Science Course, College of Engineering, Shibaura Institute of Technology, 3-7-5 Toyosu, Koto-ku, Tokyo 135-8548, Japan

‡

S Supporting Information *

ABSTRACT: We developed a facile and quick ethanol-based method for preparing silk nanoparticles and then fabricated a biodegradable and biocompatible dual-drug release system based on silk nanoparticles and the molecular networks of silk hydrogels. Model drugs incorporated in the silk nanoparticles and silk hydrogels showed fast and constant release, respectively, indicating successful dual-drug release from silk hydrogel containing silk nanoparticles. The release behaviors achieved by this dual-drug release system suggest to be regulated by physical properties (e.g., β-sheet contents and size of the silk nanoparticles and network size of the silk hydrogels), which is an important advantage for biomedical applications. The present silk-based system for dual-drug release also demonstrated no significant cytotoxicity against human mesenchymal stem cells (hMSCs), and thus, this silk-based dual-drug release system has potential as a versatile and useful new platform of polymeric materials for various types of dual delivery of bioactive molecules.

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INTRODUCTION Dual-drug delivery systems, which are capable of controlling the release behaviors of multiple drugs, have recently become attractive for the combined administration of different drugs and the optimization of therapeutic effects.1−3 One such dualdrug delivery system is based on hydrogel cross-linking with nanoparticles, which has undergone impressive progress in terms of both syntheses and applications, via polymeric networks (Figure 1).4,5 Hydrogels are three-dimensional macromolecular networks used to functionally demonstrate the controlled release of cells and bioactive molecules such as drugs, antibodies, proteins, peptides, and genes. The hydrogel containing nanoparticles of synthetic polymers, that is, poly-Nisopropylacrylamide and poly(lactide-co-glycolide), were reported and showed their potential as controlled drug-delivery systems.6−9 However, with current synthetic methods, it remains difficult to regulate hydrogel networks and the resulting bioactivity, which has hindered the design of new synthetic hydrogels for use as biomaterials.10,11 Polypeptide micelles incorporated into hydrogels have also been shown to exhibit release behavior regulated by polypeptide-based micelles.1 Self-assembled hydrogels of pullulan and fibrin containing nanoparticles also exhibited specific insulin and bone morphological protein delivery functionality.12,13 Biopolymers such as polysaccharides and proteins are therefore promising candidates for target-specific drug-delivery systems. Silk proteins have been used successfully for several decades as sutures. These proteins have also been explored as biomaterials for drug delivery systems; their excellent mechanical properties, versatility in processing, and low © 2012 American Chemical Society

cytotoxicity have earned them Food and Drug Administration approval for such expanded utility.14−16 The degradation products of silk proteins with β-sheet structures, when exposed to α-chymotrypsin, have recently been identified and shown to have no cytotoxicity to in vitro neuron cells.17,18 Kaplan and coworkers investigated silk-based hydrogels and found them to be biocompatible and have low-cytotoxicity; they also demonstrated silk solution gelation could be induced by pH changes, ultrasonication, or vortexing.19−23 Our group also developed a quick and easy method of preparing silk hydrogels using ethanol, and we then determined the effects of the state of water of silk hydrogels on various biological properties, such as cellular viability.24 In silk hydrogels, bound water appears to support cell-adhesion proteins in the cellular matrix, that is, proteins can interact with the surface of silk hydrogels; hence, silk hydrogels have been considered as candidate noncytotoxic biomaterials.24 As with silk hydrogels, several types of silk nanoparticles complexed with other biomacromolecules (e.g., chitosan, sericin, elastin, and genes) have been developed due to their excellent availability and biocompatibility.25−29 Silkbased nanoparticles used for gene delivery have recently been reported to provide biodegradability, biocompatibility, high transfection efficiency, and DNase resistance.30−32 The secondary structure of silk sequences can be used to regulate the enzymatic degradation rates of such delivery systems, and thereby, the secondary structure can be manipulated to control Received: January 17, 2012 Revised: March 6, 2012 Published: April 3, 2012 1383

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Figure 1. Schematic representation of formation of silk hydrogel containing silk nanoparticles for dual-drug release. Smaller particles denote drug molecules and larger particles indicate silk nanoparticles. Silk molecules in solution formed microfibrils and molecular networks by an addition of ethanol.

the release of bioactive molecules from silk nanoparticles.31 Silk-based nanoparticles are therefore potentially useful candidates as gene and drug carriers; however, the various properties (e.g., self-assembly, controllable degradation rates) of silk have never been successfully exploited for the development of completely silk-based nanoparticles. Furthermore, to the best of our knowledge, no fully silk-based biocompatible and slowly biodegradable dual-drug delivery system has been fabricated to date. In the present study, we developed an easy, quick method for preparing silk nanoparticles using ethanol. We also analyzed the size, zeta-potential, secondary structure, enzymatic degradability, drug-release behavior, and cell viability of the resulting nanoparticles. Silk nanoparticles were incorporated into silk hydrogels consisting of silk-microfibril networks, and then a dual-drug release system based on the silk nanoparticles, as well as on the molecular networks of silk hydrogels, was successfully fabricated. The relationship between the dual-release behaviors of model drugs and the structure of silk hydrogel composed of silk nanoparticles and silk fibrils was also examined.

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(final concentration of 0.1 g/L) into the silk solution before applying the heating treatment. The other steps were identical to those described above. The encapsulation efficiency of each fluorescent dye in the silk nanoparticles was determined by gravimetric analysis of each dye solution. Characterization of Silk Nanoparticles. The silk nanoparticles were characterized by zeta potentialmeter (Zetasizer Nano-ZS; Malvern Instruments, Ltd., Worcestershire, U.K.), Atomic Force Microscopy (AFM; Seiko Instruments, Inc., SPI3800/SPA 300HV, Chiba, Japan), and Fourier transform infrared spectroscope (IR Prestige-21; Shimadzu, Kyoto, Japan). Hydrodynamic diameters of the samples were determined using the dynamic light scattering (DLS) mode of the zeta potentialmeter. Zeta potential and zeta deviation of the samples were measured three times by zeta potentialmeter, and the average data were obtained using Dispersion Technology Software version 5.03 (Malvern Instruments, Ltd.). The silk nanoparticle solution was cast on cleaved mica, and observed in air at room temperature using a 400 μm long silicon cantilever with a spring constant of 1.5 N/m in tapping mode AFM. Calibration of the cantilever tip-convolution effect was carried out to obtain the true dimensions of objects according to previously reported methods.34−36 When the hemispherical cantilever tip interacts with the silk nanoparticle having a hemispherical cross section on the substrate, the true width of the particle (Wtrue) can be expressed as

MATERIALS AND METHODS

Wtrue = WAFM − 2wa

Preparation of Silk Solution. Silk solution was prepared by reference to a method reported previously.33 Silkworm cocoons of Bombyx mori were cut and boiled for 30 min in a 0.02 M NaCO3 solution and then washed with Milli-Q water to remove sericin proteins and wax. The extracted silk proteins were dried and dissolved in a 9.3 M LiBr solution at 60 °C for 2 h at a concentration of 20 wt %. The silk solution was dialyzed with Milli-Q for at least 4 days using a dialysis membrane (Pierce Snake Skin MWCO 3500; Thermo Fisher Scientific, Waltham, MA). The dialysis was completed when the conductivity of the dialysis solution was identical to that of Milli-Q. The silk solution of 1 mL was dried at 60 °C for 24 h, and then the resultant silk film was weighed to determine the concentration of the silk solution. The final concentration of the silk solution was approximately 60 g/L. Preparation of Silk Nanoparticles. Silk solutions with different concentrations were treated at 120 °C at 0.1 MPa for 20 min. The treated silk solution was centrifuged at 1500 g for 1 min to remove aggregations and its supernatant was mixed with ethanol for 2 h. The solvent was replaced with Milli-Q water, and then solutions containing silk particles were obtained. The absence of ethanol in the silk nanoparticle was confirmed by differential scanning calorimetry (DSC, Pyris 1; Perkin-Elmer, Waltham, MA). DSC measurements were performed using a Perkin-Elmer Pyris 1 (Waltham, MA) equipped with a cooling accessory. Silk nanoparticles containing fluorescent dyes, that is, Rhodamine B (RhB), Texas Red (TR), and fluorescein isothiocyanate (FITC), were prepared by adding each fluorescent dye

(1)

where WAFM is the width of the particle measured by AFM and wa is the width of artifacts. The spherical part of the tip is expected to be in contact with the spherical surface of the particle during scanning, because the thickness of particle (H) may be less than the apex radius of the spherical tip (R) in this study. Therefore, the value of wa is given as

wa = [(R + H )2 − R2]1/2 − H

(2)

The combination of eqs 1 and 2 gives the true width of the particle (Wtrue) in this study. On the basis of the geometry of a cantilever tip whose radius is about 10 nm, calibration was performed to calculate the true diameters of the particles. An attenuated total reflectance Fourier transform infrared spectroscope (ATR-FTIR; IR Prestige-21; Shimadzu) equipped with a multiple-reflection, horizontal MIRacle ATR attachment using a Ge crystal (Pike Tech, Madison, WI), and a DLATGS detector with temperature control for Middle/Far IR was used to evaluate the secondary structure of silk proteins forming the hydrogels. For each measurement, 128 scans were accumulated with a resolution of 4 cm−1, and the wavenumber ranged from 400 to 4000 cm−1. ATR-FTIR spectra in the amide I region were deconvoluted to determine the fraction of the β-strand structures formed during gelation with ethanol. The deconvolution and quantitative evaluation of the ATR-FTIR spectra, especially on the β-strand content, were carried out using IR solution 1.50 (Shimadzu) according to previous literatures.37−41 1384

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Preparation of Silk Hydrogel Containing Silk Nanoparticles. Silk hydrogel was prepared using a method reported previously.24 Briefly, the silk solution was mixed and slightly vortexed with ethanol solution containing the silk nanoparticles at 37 °C. The ratios of the silk solution/ethanol solution were 5/5, resulting in silk concentrations of 30 g/L. The silk gel with silk nanoparticles was washed in Milli-Q for 24 h, and silk hydrogel without ethanol was obtained. Silk hydrogels containing the fluorescent dye, RhB, were prepared by immersing the silk hydrogel in RhB solution (final concentration of 0.1 g/L) for overnight. The silk hydrogel containing RhB was washed with Milli-Q water for 2 h to remove excess dyes at the surface of the silk hydrogel. The load of RhB in the silk hydrogel was determined by gravimetric analysis of the RhB solution. Fluorescence Microscopy. Silk nanoparticles containing fluorescent dyes and the silk nanoparticle-containing silk hydrogel were cast on a slide glass with cover glass and then were used as samples. RhB, TR, and FITC were observed by fluorescence microscopy and differential interference contrast microscopy (Axio Observer Z1, 100× objective; Carl Zeiss, Oberkochen, Germany) at 550, 596, and 498 nm excitation and 570, 615, and 522 nm emission, respectively. Images were obtained using a CCD camera with AxioVision Rel 4.8 software (Carl Zeiss). Release Behavior of Model Drugs. The silk nanoparticles and silk hydrogels containing one of the fluorescent dyes were treated with a proteolytic enzyme, protease XIV (Sigma-Aldrich), in 0.1 M phosphate buffer solution (pH: 7.4) at 37 °C for different incubation times. The concentration of the enzyme solution was set at 300 μg/ mL. Release behaviors of RhB, TR, and FITC were characterized at excitation (550, 596, and 498 nm) and emission wavelengths (570, 615, and 522 nm) by a plate reader (SpectraMax M3; Molecular Devices, Sunnyvale, CA). At each time point (1, 2, 3, 6, 12, 15, 24, 30, 35, and 55 h), we measured fluorescence intensity of the supernatant and replaced the supernatant with fresh enzyme solution. The fluorescence intensity values at the each time point were determined by adding the intensity values from 1 h to the each time point. For instance, the intensity value at 3 h is sum of the intensity values measured using supernatant fractions collected at 1, 2, and 3 h. The silk nanoparticles and silk hydrogels lacking the dyes were used for background calibration. Cumulative release (%) was determined based on the 100% relase of dye from the silk nanoparticles using hexafluoro2-propanol (HFIP) instead of the protease solution. Cell Culture and In Vitro Cell Viability. Human mesenchymal stem cells (hMSCs) were purchased (Lonza Walkersville Inc., Walkerville, MD) and cultured in growth medium containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 1 ng/mL basic fibroblast growth factor (bFGF) in the presence of 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL fungizone at 37 °C in a 5% CO2 incubator. For cell viability, hMSCs (8000 cells/ well) were seeded into 96-well plates coated with silk hydrogel or deposited with silk nanoparticles and cultured for 48 h in the media (100 μL). The cell viability of the hMSCs on the samples was characterized by a standard 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, WI) according to the manufacturer’s instructions (n = 8). The cell viability was calculated as follows: [cell viability, %] = [absorbance at 490 nm of cell culture incubated on silk hydrogel]/ [absorbance at 490 nm of cell culture incubated on a 96-well cell culture plate (positive control)] × 100. Statistical Analysis. Statistical differences in cell viability were determined by unpaired t-test with a two-tailed distribution, and differences were considered statistically significant at p < 0.05. The data in the cell viability experiments are expressed as means ± standard deviation (n = 8).

and ethanol treatment. The diameters of the silk nano- and microparticles, determined by DLS, were found to decrease with decreases in the concentration of the starting material, namely, the silk solution (Figure 2A). Although ethanol

Figure 2. Diameters of silk particles prepared at different concentrations before and after ethanol (EtOH) treatment (A). AFM images of silk particles prepared at 1.0 g/mL before (B) and after (C) EtOH treatment.

treatment with the silk solution clearly led to an increase in the diameter of the silk particles, the silk nanoparticles were successfully obtained from silk solutions at a relatively low concentration (1.0 mg/L). Formation of silk nanoparticles from more dilute silk solution (less than 1.0 mg/L) was not homogeneous and efficient enough to be used in the present study. The diameter of the silk nanoparticles determined by DLS was approximately 175 ± 3 nm. The AFM observations of the silk nanoparticles before and after application of the ethanol treatment indicated aggregation of the silk nanoparticles prior to the ethanol treatment (Figure 2B), whereas after the ethanol treatment, the silk nanoparticles showed no aggregation and homogeneous size distribution (Figure 2C). After the ethanol treatment, the mean diameter and height of the silk nanoparticles, determined by AFM in air, were 72 ± 12 and 5.1 ± 0.1 nm, respectively. Size, as determined by AFM in air, was smaller than that determined by DLS, indicating that the silk nanoparticles were swollen in the aqueous environment. The zeta-potentials of the silk nano- and microparticles before and after ethanol treatment were also characterized (Figure 3A). The zeta-potentials before ethanol treatment increased with an increase in the concentration of the silk solution, while zeta-potentials after the ethanol treatment decreased with an increase in the concentration of the silk solution. The silk nanoparticles with a diameter of approximately 175 nm demonstrated a zeta-potential of −12.5 ± 0.8

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RESULTS AND DISCUSSION Characterization of Silk Nanoparticles. Silk nano- and microparticles were prepared from silk solution by the application of heat (120 °C) under high pressure (0.1 MPa) 1385

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Figure 4. Differential interference contrast microscopy (A−C) and fluorescence microscopy (D−F) images of silk nanoparticles incorporated with Rhodamine B (A, D), Texas Red (B, E), and FITC (C, F).

the silk nanoparticles with and without the dyes are listed in Table 1. The silk nanoparticles with TR and RhB were of similar sizes (approximately 175 nm) to those of the silk nanoparticles without dyes, whereas the silk nanoparticles with FITC exhibited a larger diameter, 426 ± 138 nm. The zetapotentials of the silk nanoparticles with and without the dyes were not significantly different. The release behavior of fluorescent dyes from the silk nanoparticles in the presence of proteases (300 μg/mL) at 37 °C was characterized at the appropriate wavelengths by a microplate reader (Figure 5). Cumulative release (%) was determined based on the 100% release of dye from the silk nanoparticles using HFIP instead of the protease solution. Protease XIV, which is known to degrade the β-sheet structures of silk proteins, was used as a model protease in this study.18 Without the protease, no significant release of RhB from the silk nanoparticles was detected, indicating that the release of dyes from the silk nanoparticles was due to the presence of the protease. None of the samples showed bursting of the dyes, indicating that the dyes were not attached at the surface but rather had been incorporated into the silk particles. Significant amounts of RhB and TR were released from the silk nanoparticles by enzymatic degradation, especially after 9 h of incubation time. The release behaviors of RhB and TR from the silk nanoparticles were identical, due to similar hydrophobicity and size. On the other hand, FITC, which is a more hydrophobic dye than RhB and TR, was associated with larger-sized silk nanoparticles (Table 1) and exhibited much slower release behavior from the silk nanoparticles for a duration of 24 h due to stronger hydrophobic interactions between the silk molecules and FITC. Further, the silk microparticles showed almost no significant release of any of the dyes within 12 h due to the slower enzymatic degradation rate compared to that of the silk nanoparticles. These results indicate that silk nanoparticles with a diameter of less than 500 nm are the appropriate candidates for incorporation into silk hydrogel for dual-drug release systems. Formation of Silk Hydrogel Containing Silk Nanoparticles. Ethanol solution containing the silk nanoparticles was prepared at 1.0 mg/L silk solution (Figure 6A-1), and this solution was mixed with the same amount of silk solution (60 g/L, Figure 6A-2). After approximately 5 s, a silk gel containing the silk nanoparticles was obtained. The silk gel was washed

Figure 3. Zeta-potentials of silk particles prepared at different concentrations before and after ethanol (EtOH) treatment (A). ATR-FTIR spectra of the silk particles prepared at 1.0 g/mL before (B) and after (C) EtOH treatment. Arrows (1) denote the original spectra of the silk nanoparticles, and arrows (2) denote deconvoluted results from the original spectra.

mV. These zeta-potentials differed between before and after ethanol treatment, which indicates that the surface of the silk particles became more negatively charged due to exposure of the hydrophobic sequences, namely, the β-sheet structures. To confirm this change in the secondary structure of the silk molecules, ATR-FTIR measurements of the silk nanoparticles were performed (Figure 3B,C, Figure S1). Arrows 1 and 2 in Figure 3B,C denote the amide I peaks of the silk nanoparticles and the deconvoluted peaks that originated from the β-strand structures, respectively. According to previous assignments,42,43 the peak areas revealed that the silk nanoparticles prior to ethanol treatment had a β-strand content of 39%, whereas those after ethanol treatment showed 46% β-strand content. This increase in β-strand content suggests that the ethanol treatment induced the formation of the β-sheet structure of silk molecules at the surface of the silk nanoparticles, which would be in agreement with the negatively charged surface of the silk nanoparticles postethanol treatment (Figure 3A). Release Behavior of Model Drugs from Silk Nanoparticles. To obtain silk nanoparticles containing model drugs, silk solutions (1.0 mg/L) containing three types of fluorescent dyes (RhB, TR, and FITC) were heated at 120 °C under high pressure (0.1 MPa) and were treated with ethanol, as described in the Materials and Methods. Three types of fluorescent dyes were used as model drugs with different hydrophobicities. The resultant silk nanoparticles in which the fluorescent dyes had been incorporated were observed by fluorescence microscopy to evaluate incorporation of the dyes in the silk nanoparticles (Figure 4). Each fluorescent dye was successfully observed in each silk nanoparticle, indicating the incorporation of the model drugs into silk nanoparticles, even though the dyes were hydrophilic. The silk nanoparticles showed encapsulation efficiency of approximately 35% for TR as well as that of around 55% for RhB and FITC. The sizes and zeta-potentials of 1386

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Table 1. Diameters and Zeta-Potentials of the Silk Nanoparticels with and without Fluorescent Dyes a

diameter, nm zeta-potential,a mV a

silk only

silk w/TRb

silk w/RhBb

silk w/FITCb

175 ± 3 −12.5 ± 0.8

163 ± 30 −7.4 ± 0.3

203 ± 28 −11.4 ± 0.8

426 ± 138 −11.2 ± 1.8

Determined by zeta-nanosizer. bTR, Texas Red; RhB, Rhodamine B; and FITC, fluorescein isothiocyanate.

constructed for the purpose of dual-drug release, as shown in Figure 1. Dual-Drug Release from Silk Hydrogel Containing Silk Nanoparticles. As mentioned above, RhB was added to the molecular networks of the silk hydrogels, whereas FITC was incorporated into silk nanoparticles in the silk hydrogel. The dual release of model drugs from the silk hydrogel containing the silk nanoparticles in the presence of protease XIV (300 μg/ mL) was characterized by fluorescence intensity at 37 °C (Figure 7). Burst release of RhB from the silk hydrogel Figure 5. Release behavior of fluorescent dyes, Rhodamine B (RhB), Texas Red (TR), and FITC, from silk nanoparticles in the presence of protease XIV at 37 °C. Control experiment denotes release behavior of RhB from silk nanoparticles in the absence of protease XIV at 37 °C.

Figure 7. Dual-release behaviors of FITC and Rhodamine B (RhB) from the silk nanoparticles (NP) and from the silk hydrogel in the presence of protease XIV at 37 °C.

Figure 6. Gelation behavior of silk solution. Ethanol containing silk nanoparticles with incorporated FITC (A1) and silk solution of 60 g/L (A2) were mixed, resulting in gelation of the silk solution (A3). Fluorescence microscopy image of silk nanoparticles containing FITC in the silk hydrogel (B) and of the silk hydrogel containing Rhodamine B in its molecular networks (C).

(approximately 90%) was observed within 1 h of incubation time (Figure 7, white circles). In contrast, slow and constant release of FITC from the silk nanoparticles was observed (Figure 7, black circles). After 5 days, FITC was completely released due to the enzymatic degradation of the silk hydrogel and the silk nanoparticles. These two release behaviors clearly differed, which successfully demonstrated the dual-drug release from silk hydrogels containing silk nanoparticles. Silk nanoparticles containing FITC in the silk hydrogel showed the same release behavior as the silk nanoparticles in the absence of hydrogels (Figure 5), which indicated that the enzymatic degradation of the silk nanoparticles was not influenced by the molecular networks of the silk hydrogel. The release of RhB from the silk hydrogel was relatively fast, because of fast degradation of noncrystalline silk molecules in the silk hydrogel by protease XIV, as reported previously.18 The size of the silk hydrogel network has been reported to range from 5 to 50 μm,24 which is large enough for proteases to degrade the silk molecules of nanoparticles and hydrogels. Furthermore, according to previous reports,24,31 enzymatic degradation rate of silk-based materials can be regulated by the physical properties, such as crystallinity and sizes. Therefore, the release behavior of drugs from the silk-based dual-drug release system has potential to be regulated by the physical properties of the silk nanoparticles and the nework sizes of the silk hydrogel. Silk Hydrogel and Silk Nanoparticles as Biomaterials. The cytotoxicity of the silk nanoparticles and the silk hydrogel containing the silk nanoparticles was determined using the standard MTS assay with hMSCs to evaluate the potential of those materials as practical biomaterials. Figure 8 shows the 48

with Milli-Q, and then a silk hydrogel containing the silk nanoparticles was obtained (Figure 6A-3). The method to prepare silk hydrogel by adding ethanol, which was established previously,24 was easy and rapid enough to construct silk hydrogel containing silk nanoparticles under relatively mild conditions. The molecular networks of silk hydrogel induced with ethanol have been reported to be composed of silk microfibrils.24 Two different model drugs, FITC and RhB, were added to the silk nanoparticles and the silk hydrogels, respectively, yielding silk hydrogel containing FITC in the silk nanoparticles and RhB in the silk hydrogel networks. This is because we targeted relatively slow and constant release of model drug from the silk nanoparticle as well as fast release from the silk hydrogel. The fluorescence microscopy images of the silk hydrogel showed both FITC and RhB signals (Figure 6B,C). The FITC signals were distributed as particles in the hydrogel (Figure 6B), indicating that the silk nanoparticles were successfully entrapped in the silk hydrogel. Additionally, the RhB signals were observed across the entire area of the hydrogel (Figure 6C), demonstrating that the molecular networks or the silk hydrogel were filled with RhB. Further, the silk hydrogel containing RhB was washed with Milli-Q for 2 h to remove excess RhB and load of RhB in the silk hydrogel was determined to be 3.3 wt %. These results confirmed that silk hydrogel containing silk nanoparticles had been successfully 1387

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-48-467-9525. Fax: +81-48-462-4664. E-mail: keiji. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Sumitomo Foundation (to K.N.).

Figure 8. Cell viability of hMSCs with silk nanoparticles (NP, white circles) seeded on silk hydrogels with (white squares) and without silk nanoparticles (black squares), as determined by absorbance at 490 nm and measured using cell cultures after 48 h incubation. Cell viability of 100% was calculated based on a positive control, namely, the cell culture seeded on a cell-culture plate after 48 h incubation. Error bars represent the standard deviation of samples (n = 8). *Significant difference between two groups at p < 0.05.

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h cell viability of the silk nanoparticles and the silk hydrogel with and without the silk nanoparticles. At a low concentration (90%).24 As the present silk-based system for dual-drug release demonstrated no significant cytotoxicity against hMSCs, and thus, it has noteworthy potential as a practical biomaterial.

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CONCLUSIONS After developing an easy, quick method of preparing silk nanoparticles using ethanol, our group fabricated a dual-drug release system based on these silk nanoparticles together with the molecular networks of silk hydrogels. The model drugs incorporated into the silk nanoparticles and silk hydrogels showed fast and slow release times, respectively, thereby indicating successful dual-drug release from silk hydrogels containing silk nanoparticles. Taking into account the results of previous studies,24,31 the release behaviors of model drugs from our dual-drug release system can be regulated by exploiting the physical properties (e.g., β-sheet contents, size) of silk nanoparticles and hydrogels, which provides an important advantage for biomedical applications. The findings of the present study also suggest that this silk-based dual-drug release system has potential as a versatile and useful new polymeric material platform for the dual delivery of various bioactive molecules.

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REFERENCES

(1) Wei, J.; Chen, F.; Shin, J. W.; Hong, H.; Dai, C.; Su, J.; Liu, C. Biomaterials 2009, 30, 1080−1088. (2) Richardson, T. P.; Peters, M. C.; Ennett, A. B.; Mooney, D. J. Nat. Biotechnol. 2001, 19, 1029−1034. (3) Lehar, J.; Krueger, A. S.; Avery, W.; Heilbut, A. M.; Johansen, L. M.; Price, E. R.; Rickles, R. J.; Short, G. F., 3rd; Staunton, J. E.; Jin, X.; Lee, M. S.; Zimmermann, G. R.; Borisy, A. A. Nat. Biotechnol. 2009, 27, 659−666. (4) Hennink, W. E.; van Nostrum, C. F. Adv. Drug Delivery Rev. 2002, 54, 13−36. (5) Glotzer, S. C.; Solomon, M. J. Nat. Mater. 2007, 6, 557−562. (6) Huang, G.; Gao, J.; Hu, Z.; St John, J. V.; Ponder, B. C.; Moro, D. J. Controlled Release 2004, 94, 303−311. (7) Muthu, M. S.; Rawat, M. K.; Mishra, A.; Singh, S. Nanomedicine 2009, 5, 323−333. (8) Wang, C.; Flynn, N. T.; Langer, R. Adv. Mater. 2004, 16, 1074−+. (9) Zhang, J. T.; Huang, S. W.; Xue, Y. N.; Zhuo, R. X. Macromol. Rapid Commun. 2005, 26, 1346−1350. (10) Ehrick, J. D.; Deo, S. K.; Browning, T. W.; Bachas, L. G.; Madou, M. J.; Daunert, S. Nat. Mater. 2005, 4, 298−302. (11) Kopecek, J. Nature 2002, 417 (388−389), 391. (12) Akiyoshi, K.; Kobayashi, S.; Shichibe, S.; Mix, D.; Baudys, M.; Kim, S. W.; Sunamoto, J. J. Controlled Release 1998, 54, 313−320. (13) Chung, Y. I.; Ahn, K. M.; Jeon, S. H.; Lee, S. Y.; Lee, J. H.; Tae, G. J. Controlled Release 2007, 121, 91−99. (14) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Biomaterials 2003, 24, 401−416. (15) Numata, K.; Kaplan, D. L. Adv. Drug Delivery Rev. 2010, 62, 1497−1508. (16) Wang, Y.; Kim, H. J.; Vunjak-Novakovic, G.; Kaplan, D. L. Biomaterials 2006, 27, 6064−6082. (17) Numata, K.; Kaplan, D. L. Macromol. Biosci. 2011, 11, 60−64. (18) Numata, K.; Cebe, P.; Kaplan, D. L. Biomaterials 2010, 31, 2926−2933. (19) Hu, X. A.; Lu, Q. A.; Sun, L.; Cebe, P.; Wang, X. Q.; Zhang, X. H.; Kaplan, D. L. Biomacromolecules 2010, 11, 3178−3188. (20) Yucel, T.; Cebe, P.; Kaplan, D. L. Biophys. J. 2009, 97, 2044− 2050. (21) Wang, X. Q.; Kluge, J. A.; Leisk, G. G.; Kaplan, D. L. Biomaterials 2008, 29, 1054−1064. (22) Matsumoto, A.; Chen, J.; Collette, A. L.; Kim, U. J.; Altman, G. H.; Cebe, P.; Kaplan, D. L. J. Phys. Chem. B 2006, 110, 21630−21638. (23) Kim, U. J.; Park, J. Y.; Li, C. M.; Jin, H. J.; Valluzzi, R.; Kaplan, D. L. Biomacromolecules 2004, 5, 786−792. (24) Numata, K.; Katashima, T.; Sakai, T. Biomacromolecules 2011, 12, 2137−2144. (25) Anumolu, R.; Gustafson, J. A.; Magda, J. J.; Cappello, J.; Ghandehari, H.; Pease, L. F. ACS Nano 2011, 5, 5374−5382. (26) Mandal, B. B.; Kundu, S. C. Nanotechnology 2009, 20. (27) Zhang, Y. Q.; Shen, W. D.; Xiang, R. L.; Zhuge, L. J.; Gao, W. J.; Wang, W. B. J. Nanopart. Res. 2007, 9, 885−900. (28) Zhang, Y. Q.; Wang, Y. J.; Wang, H. Y.; Zhu, L.; Zhou, Z. Z. Soft Matter 2011, 7, 9728−9736.

ASSOCIATED CONTENT

S Supporting Information *

Additional data (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org. 1388

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(29) Zhu, L.; Hu, R. P.; Wang, H. Y.; Wang, Y. J.; Zhang, Y. Q. J. Agric. Food Chem. 2011, 59, 10298−10302. (30) Numata, K.; Hamasaki, J.; Subramanian, B.; Kaplan, D. L. J. Controlled Release 2010, 146, 136−143. (31) Numata, K.; Kaplan, D. L. Biomacromolecules 2010, 11, 3189− 3195. (32) Numata, K.; Subramanian, B.; Currie, H. A.; Kaplan, D. L. Biomaterials 2009, 30, 5775−5784. (33) Jin, H. J.; Kaplan, D. L. Nature 2003, 424, 1057−1061. (34) Numata, K.; Hirota, T.; Kikkawa, Y.; Tsuge, T.; Iwata, T.; Abe, H.; Doi, Y. Biomacromolecules 2004, 5, 2186−2194. (35) Numata, K.; Kikkawa, Y.; Tsuge, T.; Iwata, T.; Doi, Y.; Abe, H. Macromol. Biosci. 2006, 6, 41−50. (36) Numata, K.; Kikkawa, Y.; Tsuge, T.; Iwata, T.; Doi, Y.; Abe, H. Biomacromolecules 2005, 6, 2008−2016. (37) Chen, H.; Hu, X.; Cebe, P. J. Therm. Anal. Calorim. 2008, 93, 201−206. (38) Hu, X.; Kaplan, D.; Cebe, P. Macromolecules 2008, 41, 3939− 3948. (39) Hu, X.; Kaplan, D.; Cebe, P. J. Therm. Anal. Calorim. 2009, 96, 827−834. (40) Lu, Q.; Hu, X.; Wang, X. Q.; Kluge, J. A.; Lu, S. Z.; Cebe, P.; Kaplan, D. L. Acta Biomater. 2010, 6, 1380−1387. (41) Teramoto, H.; Miyazawa, M. Biomacromolecules 2005, 6, 2049− 2057. (42) Hu, X.; Kaplan, D.; Cebe, P. Macromolecules 2006, 39, 6161− 6170. (43) Tretinnikov, O. N.; Tamada, Y. Langmuir 2001, 17, 7406−7413.

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dx.doi.org/10.1021/bm300089a | Biomacromolecules 2012, 13, 1383−1389