Structure and Biodegradation Mechanism of Milled Bombyx mori Silk

Jun 29, 2012 - Megan K. DeBari , Rosalyn D. Abbott .... Rangam Rajkhowa , Abdullah Kafi , Qi Tony Zhou , Anett Kondor , David A.V. Morton , Xungai Wan...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Biomac

Structure and Biodegradation Mechanism of Milled Bombyx mori Silk Particles Rangam Rajkhowa,† Xiao Hu,‡ Takuya Tsuzuki,† David L. Kaplan,‡ and Xungai Wang*,†,§ †

Australian Future Fibers Research and Innovation Centre, Deakin University, Vic 3217, Australia Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States § School of Textile Science and Engineering, Wuhan Textile University, Wuhan, China ‡

S Supporting Information *

ABSTRACT: The aim of this study was to understand the structure and biodegradation relationships of silk particles intended for targeted biomedical applications. Such a study is also useful in understanding structural remodelling of silk debris that may be generated from silk-based implants. Ultrafine silk particles were prepared using a combination of efficient wet-milling and spraydrying processes with no addition of chemicals other than those used in degumming. Milling reduced the intermolecular stacking forces within the β-sheet crystallites without changing the intramolecular binding energy. Because of the rough morphology and the ultrafine size of the particles, degradation of silk particles by protease XIV was increased by about 3-fold compared to silk fibers. Upon biodegradation, the thermal degradation temperature of silk increased, which was attributed to the formation of tight aggregates by the hydrolyzed residual macromolecules. A model of the biodegradation mechanism of silk particles was developed based on the experimental data. The model explains the process of disintegration of β-sheets, supported by quantitative secondary structural analysis and microscopic images.

■

protease XIV to about 2 nm thick nanofilaments.9 A decrease in β-sheet content from about 50% to about 30% was found as a result of this enzymatic hydrolysis. However, despite the higher degradation rate expected for the non-β-sheet phases, the reasons for the proportional increase of such fractions remained elusive.9 Lu et al. reported that silk films containing a higher fraction of silk I phase structure (non β-sheet) degraded faster than those with higher fraction of silk II phase (β-sheet).10 On the other hand, stretched and stabilized films with substantial βsheet content degraded rapidly.11 Such results demonstrate that the degradation of silk is not always directly proportional to βsheet content and may depend on the architecture and distribution of different conformational phases in the fine structure of silk. The morphology and fine structures of silk particles are different from other forms of silk and could also vary depending on the fabrication methods. Therefore, biodegradation of silk particles requires further investigations. The objective of this work was to gain insight into the morphology and fine structure of silk particles and to correlate their secondary structure with enzymatic degradation. Protease

INTRODUCTION Silk fibroin produced by a Bombyx mori silkworm is a large protein consisting of a heavy (H) and a light (L) chain linked by a disulfide bond. The H chain (391 kDa) has hydrophobic repetitive modular domains of GAGAGS that pack into β-sheet crystals. The crystals are interrupted by other domains and hydrophilic groups in the sequence forming the amorphous part of silk.1 Silk fibroin-based biomaterials have been developed for a broad range of biomedical and biotechnological applications, leading to the transition of silk from traditional textile fibers to advanced materials.2 Among these, micro and nanosized silk particles have shown promising results as resorbable vehicles for drugs and growth factors for diagnostic and tissue engineering applications.3−5 Particles have also been used as fillers in composite scaffolds.6,7 In applications as carriers of therapeutic or diagnostic agents, understanding the biodegradation behavior of silk particles is important. In tissue engineering, the biodegradation rate of the scaffold material is also critical, as it should match the rate of tissue regeneration. Degradation rate is also important for controlling cellular metabolism and differentiation.8 In the past, kinetics and mechanisms of degradation of silk were studied using in vitro models. Numata et al. reported that 5 nm thick crystal-like structures could be fragmented by © 2012 American Chemical Society

Received: May 12, 2012 Revised: June 28, 2012 Published: June 29, 2012 2503

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

buffered saline (PBS) and were agitated inside the incubator at 37 °C. Enzyme concentration was 1 mg/mL in the slow degradation experiment in which the solution was replaced with fresh solution every 2 days to maintain the enzyme activity. In the rapid degradation experiment, 2 mg/mL solution was used, and the solution was replaced every day. Specimens were collected at 3 h and 2, 16, 30, and 56 days in the case of slow degradation and 3 h, 6 h, and 1, 3, 6, and 12 days in the case of rapid degradation tests. The number of specimens was 3 at each time point. At the end of degradation, particles were treated in 15 mL 2% acetic acid solution for 30 min at room temperature followed by rinsing in 15 mL dH2O at room temperature. Rinsing was repeated five times. During the change of enzyme solution and rinsing, samples were centrifuged at 4170 × g for 15 min (CT15 RT, EVISA), and there was no evidence of particles in the discarded supernatants. Control samples were treated only in a PBS solution without enzyme. Replacement of the PBS solution and rinsing of the control specimens were carried out in the same manner as to the enzyme-treated samples. At the end of rinsing, samples were put in liquid N2 and lyophilized (LABCONCO-FreeZone). Dry particles were conditioned again at 20 ± 2 °C and 65 ± 2% relative humidity for 24 h and weighed to determine the weight loss. Particle Size Distribution. Prior to freeze-drying, 20 mL of enzyme treated and rinsed particle suspension in water was collected for measurement of size distribution. To break the aggregates formed from repeated centrifugation during change of enzyme solutions and rinsing, the suspensions were sonicated for 30 s at 70% amplitude (Vibra-Cell, 130 W, 20 kHz). Particle size distribution was measured using a Mastersizer 2000 (Malvern Instruments, U.K.) fitted with Hydro 2000S. The dispersion medium was DI water. Data analysis was performed by the Malvern software using the following material parameters: refractive index of silk 1.561, absorbency of silk 0.1.17,18 The absorbency value was determined in our previous studies so as to give an average hydrodynamic particle size consistent with the results of a scanning electron microscopy (SEM) study.18 Each sample was measured four times. All results were presented according to a volumebased particle size distribution. Volume-based distribution is more relevant, as in the case of number-based size distribution, very large number of fine particles representing a small mass of measured particles would shift the d(0.5) to a much smaller value. d(0.5) represents the median of a volume-based distribution of the size of particles. Scanning Electron Microscopy. Morphology of the particles was observed under a scanning electron microscope (Carl Zeiss AG SUPRA 55VP) at 1−2 kV accelerated voltage and 1.5−4.5 mm working distance. Prior to SEM imaging, particles were made conductive by an approximately 1 nm-thick evaporated carbon coating. Fourier Transform Infrared (FTIR) Analysis. Infrared spectra of desiccated silk fiber and particles were recorded with an attenuated total reflectance (ATR) FTIR spectrophotometer (Vertex 70) (Bruker Biosciences Pty Ltd., Australia). Each spectrum was obtained in absorbance mode in the range of 4000−600 cm−1. The average of four measurements was used for analysis. To avoid dichroic interference in the spectrum, snippets prepared for milling were used to represent fibers. To measure different conformations, average spectrum in the amide I mode was deconvoluted and curve fitted (1595−1705 cm−1) using OPUS 5.5 software, adapting the procedure used previously after slight modifications.19 Briefly, deconvolution was carried out adapting a Lorentzian model using a bandwidth of 25 cm−1 and a noise reduction factor of 0.3. Baseline was corrected before and after deconvolution. Curve fitting was done using the Gaussian model by the autofit program using initially local least-squares followed by Levenberg−Marquardt algorithms. Fixed band widths (5.015) were initially selected based on the deconvoluted spectra and known conformational positions of amide I regions: 1619 cm−1 (intermolecular β-sheet), 1627 cm−1 (intramolecular β-sheet), 1647 cm−1 (random coil), 1666 cm−1 (β-turn), 1680 cm−1 (β-turn), 1696 cm−1 (tight β-turn), and 1700 cm−1 (strong β-turn). Finally, each individual spectrum was area normalized to obtain percentage conformations within the amide I region. The procedure was repeated four times with slight changes in positions of initial selection for each spectrum.

XIV was used due to its high activity toward silk β-sheet structure.9,12 Insight into the structure and degradation relationships of silk will assist in tuning its degradation behavior through optimization of the particle fabrication process and thereby will further assist in developing targeted biomedical applications with silk particles. In addition to degradation kinetics and structure-degradation relationships, this study examined the structures of enzyme hydrolyzed silk. The residues of biodegradation can be the major causes of inflammatory reactions impacting the success rate of a biomaterial. It is known that conformation of certain structural proteins such as prion protein, PrP, contributes to neuro-generative diseases.13 According to some preliminary studies, silk materials containing β-sheets do not show cytotoxicity.9 However, conformations of degraded products and associated influence on cytotoxicity have not been well investigated. As part of such investigations, this study examined secondary structures of in vitro degraded silk particles with the aim to understand structural remodelling during enzyme action. It is hoped that the study will help to predict the behavior of particulate debris generated from various forms of silk materials. Silk particles used in this study were fabricated by milling instead of a bottom-up approach via dissolution of fibers and then regeneration.14 In regenerated silk materials, the original microstructure of the fiber is difficult to reform.15,16 In addition, silk regeneration often relies on the use of chemicals such as lithium salts and organic solvents in addition to water. On the other hand, milling of silk fibers is an alternate and attractive approach to develop fine silk particles avoiding chemicals other than those used in degumming.17 Unlike the bottom-up approach, the top-down process utilizes only mechanical energy to produce particles.

■

EXPERIMENTAL SECTION

Materials. Stiffled B. mori silk cocoons were sourced from Fabric Plus Ltd. (India). All reagents, unless stated otherwise, were purchased from Sigma Aldrich Australia, Ltd. Silk Degumming. The silk cocoons were cut to remove pupae. To remove sericin, degumming was performed in a laboratory dyeing machine (Thies, USA) using 2 g/L sodium carbonate and 0.6 g/L sodium dodecyl sulfate (Sigma-Aldrich, Australia) at 100 °C for 20 min with a material mass (g) to liquor volume (mL) ratio of 1:25. After degumming, the cocoons were washed thoroughly with warm distilled water (dH2O) followed by cold dH2O to make sure that all the chemicals were washed out completely. Powder Production. Silk powder was produced using the method reported earlier.17 Briefly, degummed silk fibers were chopped in a cutter mill (Pulverisette 19 from Fritsch Gmbh, Germany) fitted with a 1 mm grid and operating at 2888 rpm. Chopped snippets were wet milled for 6 h in a stirred media mill (1S Attritor from Union Process, USA). Stirrer speed was set at 280 rpm and 20 kg yttrium doped zirconium oxide balls of 5 mm diameter were used as the milling media. Batch size was 200 g of snippets and 2 L of deionized (DI) water. Cooling water (approximately 18 °C) was circulated through the vessel jacket during milling. After wet milling, the slurry was spray dried (B-290 from Buchi Labortechnik AG, Switzerland) to produce silk powder using the following conditions: inlet temperature, 130 °C; pump setting, 25% (18−20 mL/min); and aspirator setting, 100% (42.5m3/h). Enzymatic Degradation. The degradation of the silk fibers and particles was evaluated using protease XIV from Streptomyces griseus (Sigma-Aldrich) with an activity of 3.5 U/mL. Powder specimens of about 100 mg were conditioned at 20 ± 2 °C and 65 ± 2% relative humidity for 24 h and weighed. Specimens were then treated with 10 mL of enzyme solution at pH 7.4 prepared using 0.1 M phosphate2504

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

Thermal Properties. Differential scanning calorimetry (DSC) measurements were made in a TA Instrument Q100 DSC (TA Instruments, New Castle, DE) under a dry N2 gas flow and equipped with a refrigerated cooling system. Desiccated specimens of about 5 mg were used for the measurements and were encapsulated in aluminum pans. The samples were heated at 5 °C/min from −30 to 350 °C. X-ray Scattering Analysis. Wide-angle X-ray scattering (WAXS) was performed on a diffractometer (PANalytical-Xpert PRO) with Cu Kα radiation (λ = 0.154 nm) operated at 40 kV and 30 mA. The scanning rate was 0.01°/s. A low-noise, single-crystal silicon specimen holder was used to avoid any interference of background scattering. Spectra were analyzed using the Xpert High Score Plus software. Statistical Analysis. Results were statistically analyzed using twotailed student’s t tests at 95% confidence level to compare the sample means wherever applicable. A statistically significant difference was considered if p < 0.05.

Figure 2. Change of mass of fiber and powder during enzyme degradation tests: slow tests (1 mg/mL protease XIV) and rapid degradation tests (2 mg/mL protease XIV). Data shown as mean ± SE, n = 3.

■

RESULTS Change in Size and Mass of Silk Fibers and Particles. B. mori silk fibers were chopped and wet-milled in an attritor for 6 h to get a stable suspension of fine particles in water with a volume d(0.5) of 6.4 μm. The suspension was then spray-dried to generate silk powders. There was no change in particle size during spray drying. Despite milling under circulating chilled water to avoid thermal degradation during milling, mechanical energy in the powdering process caused some decrease in molecular weight of fibroin (see Supporting Information Figure S1). To study biodegradation, the milled powder was treated in a PBS solution with or without enzyme. As illustrated in Figures 1 and 2, 12 days exposure without enzyme (pH 7.4, 37 °C)

degradation studies and every day in the case of rapid studies. As illustrated in Figure 2, total mass loss and rate of mass loss of the particles were significantly higher than the fibers. In the slow degradation study, particle mass dropped rapidly to less than 60% in about 6 days. The degradation rate became slower thereafter, and the remaining mass was about 40% at the end of 8 weeks. In the case of fibers, there was no indication of degradation during the first 2 weeks. Mass loss was about 20% in 30 days, and reduction thereafter to 8 weeks was only marginal (p > .05, student's t test). High rate of degradation of powders could be further accelerated in the rapid tests. More than 50% mass was removed by protease XIV within 3 days, although the degradation slowed down significantly thereafter. Concentration as well as enzyme replacement rate influence the kinetics of degradation, as protease is known to lose activity after about 1 day.20 Volume-based particle size distributions of B. mori silk particles at different degradation time points are presented in Figure 3. Median diameters of particles are shown in Figure 1. The d(0.5) value dropped from 6.4 to 1.8 μm at the end of 12 days, indicating a reduction in equivalent microsphere diameter by about 72%. SEM images of particles are shown in Figure 4.

Figure 1. Volume median particle size (d(0.5)) of enzyme-degraded silk powder at different time of rapid degradation tests; 12 d (PBS): 12 days PBS-only treated powder (protease XIV 2 mg/mL).

caused no change in d(0.5) and mass of the particles. Error bars can not be seen in Figure 1, as standard deviations were less than 1 × 10−3 due to homogeneous mixing during milling. The results confirm that despite much larger surface area and decrease in molecular weight compared to the fibers, the mechanically produced ultrafine silk particles were insoluble in PBS solution. The changes in particle size distribution and mass loss as a result of protease XIV hydrolysis were monitored at different time points. Concentrations of protease XIV were 1 mg/mL and 2 mg/mL, and corresponding experiments were termed as slow and rapid degradation, respectively. To maintain activity, enzyme solutions were replaced every 2 days in the slow

Figure 3. Particle size distribution at various times of rapid degradation test. 2505

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

Figure 4. SEM images of silk powder during degradation (protease XIV: 1 mg/mL).

positive correlation between a band in the region 1691−1700 cm−1 with the formation of β-turns.22 Considering the positive correlation of the band in this region with crystal formation in silk, we have distinguished it from other β-turns and define it as tight β-turns. Formation of these tight β-turns is reflected prominently in FTIR results of enzyme-treated samples, but they do not enhance WAXS reflection (to be discussed subsequently) as opposed to β-sheets that add to WAXS peaks. In Figure 6, amide I absorptions normalized using OPUS5.5 software are shown for degummed B. mori silk fiber, 30 min and

The upper two images in Figure 4 show the change in particle size due to prolonged enzyme action, and the lower three images show how the porous architecture changes with the increase in enzyme treatment time. Figure 5 illustrates the SEM images of silk fibers before and after enzyme treatment. The enzyme-treated fibers show

Figure 5. SEM images of silk fibers: (a) Fiber before degradation; (b) fracture of a degraded fiber in 12 d; (c) surface erosion of a degraded fiber in 12 d.

surface erosion at some places. Some portions of fibers also fractured during enzyme treatment. On the other hand, fibers before enzyme treatment do not show such features. The images thus suggest surface erosion and fracture of silk fibers, which could be due to increase in brittleness during the enzyme treatment. Change in Secondary Structure. FTIR amide I (1600− 1700 cm−1) region reflects CO vibration in the protein backbone. The band positions in the region change depending on the backbone conformation and therefore can be used to determine molecular conformation. The 1616−1637 cm−1 region represents the β-sheet, and the 1638−1663 cm−1 region is usually attributed to amorphous protein.19,21 Within the amorphous region, there is a general agreement to use the 1638−1655 cm−1 for assigning random coil and the remaining region to helical conformation. The 1665−1690 cm−1 is assigned to β-turn, whereas a strong band in the region 1691−1700 cm−1 is often used for β-sheet.19,21 However, using real-time FTIR during silk crystallization, Chen et al. found a

Figure 6. FTIR Amide I region (F0: Degummed fiber at start; P 12 d(B): 12 days PBS-only treated powder; P 12 d: 12 days enzymedegraded powder; P 30 min: 30 min enzyme degraded powder); protease XIV: 2 mg/mL.

12-day enzyme-treated powders, and the powder treated for 12 days in PBS. There was no difference in the patterns of powders after PBS treatment, and hence only the 12-day PBS treated plot is presented in Figure 6. After milling, there was a noticeable shift in the β-sheet peak from around 1619 cm−1 to around 1625 cm−1. On the other hand, protease XIV hydrolysis did not change the β-sheet position from around 1625 cm−1. As illustrated by the arrows, there were small but continuous shifts (decrease) in the amorphous region during enzyme treatment. The height and position of the tight β-turn peak around 1697 cm−1 remained unchanged, but due to the depression in the band in the amorphous region, sharpness of the peak increased after biodegradation. 2506

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

spectrum, and the dotted curves are the individual deconvoluted peaks. The percentage conformations derived from areanormalized data of fiber and powders at different time points of protease XIV treatment are presented in Figure 8. Standard errors from minor changes applied in band positions during curve fittings are very small and hence are not visible in Figure 8.

For improved resolution and estimation of different conformations, the broad amide I band was Fourier selfdeconvoluted (FSD) and curve fitted to yield a set of bands from which the conformation could be determined. Second derivatives of the original FTIR spectra were used to select band positions that were then allowed to shift using the autofit option in OPUS5.5. Based on surveyed literature, we previously reported on dividing the β-sheet region of 1616−1637 cm−1 into intermolecular (1616−1627 cm−1) and intramolecular (1627−1638 cm−1) subzones.19 In this definition, the forces in the hydrogen bonded planes of the fibroin β-sheet crystals are intramolecular, whereas van der Waals force are involved in crystal stacking as intermolecular forces. Therefore the initial band potions around 1619 cm−1 and 1630 cm−1 have been assigned to represent intermolecular and intramolecular β-sheet fractions, respectively. Examples of curve-fitted FSD for fiber, PBS-only, and 12 d protease XIV-treated powders are presented in Figure 7. The solid curve is the resultant

Figure 8. Percentage of different conformations; F0: Degummed fiber at the start; P 12 PBS: Day 12 PBS-only treated powder; P 30 min: 30 min enzyme-degraded powder; P 3 d: 3 days enzyme-degraded powder; P 12 d: 12 days enzyme degraded powder; data are from rapid degradation studies (protease XIV: 2 mg/mL).

Milling caused a substantial fall in β-sheet (intermolecular + intramolecular) from 57% to 44% and an associated increase in random coil from 17% to 28%. The reduction in β-sheet can be attributed to the decrease in intermolecular β-sheet content from 33% to 12% despite partial offset by some increase in the intramolecular fraction. β-sheet (intermolecular + intramolecular) in powder increased marginally from 44% to 48% at the end of 12 days of biodegradation. Enzyme action also caused noticeable increase in tight turn (from 11% in PBS control powder to 14% in 12-day enzyme-treated powder). These conformational changes caused by milling and enzyme hydrolysis were statistically significant ((p < 0.05, student's ttests). The combined rise in β-sheet and tight β-turn was matched by an equivalent fall in random coil conformation, which dropped from 28% (PBS control) to 20% (12 d enzyme treated powders). β-turn fraction remained nearly unchanged (not shown in Figure 8). The proportional change in amorphous contents, however, did not match the extensive mass loss (Figure 2), indicating that protease XIV did not remove proteins in the amorphous domain alone. Interestingly, compared to the initial period of degradation, a change in conformation was pronounced between 3-day and 12-day time points despite statistically insignificant mass loss in that period. Figure 9 presents normalized WAXS of fiber and powder with or without protease XIV. There was a shift of the center of the crystal peak from 20.7° to about 20.3° due to milling. The peak center slightly increased to 20.5°, and the peak intensity reduced after enzyme treatment. Change in Thermal Properties. Response to temperature by polymers including structural proteins varies depending on their molecular conformation and secondary structure. DSC

Figure 7. Curve-fitted FSD spectra of amide I regions (B-ter: intermolecular β-sheet, B-tra: intramolecular β-sheet, r: random coil, t: β-turn, tt: tight β-turns (protease XIV: 2 mg/mL). 2507

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

about 22% in 8 weeks in this study. A relatively higher degradation rate in that previous study can be attributed to more frequent replacement of enzyme and use of higher substrate-to-enzyme solution ratio (1:70 against 1:10 used in our study), which are known to be important determinants of degradation rate.23 In the case of particles, the mass loss was substantially higher than the fibers. A substantial shift of the left tail of particle distribution as shown in Figure 3, particularly during the later part of degradation, suggests generation of a large number of finer particles. In the past, a surface erosion mechanism was proposed to explain the degradation of silk materials by protease XIV.20 Considering such a surface erosion process, a mass loss of 60% occurring in the case of 12-day degraded particles (Figure 2), would have resulted in a reduction in sphere diameter by 23% only. Silk particles used in this study were not solid spheres but were highly porous, and the enzyme degradation occurred through pore surfaces leading to fracture into finer particles (Figure 4). Volume-based distribution was used to represent particle size in this study. Hence without particle fracture, any large number of ultrafine insoluble debris from surface erosion representing a small fraction of mass alone could not shift the size distribution substantially. As a result of particle fracture, much larger reduction in particle size occurred (about 72%) than what would have occurred from surface erosion alone. B. mori silk fibers are viscoelastic with about 15−20% elongation at break.2,24 Due to their fineness and viscoelasticity, silk fibers are highly pliable and can be subjected to stress and twists without breakage during yarn manufacturing. The flexibility of a viscoelastic silk material comes from the amorphous domains. The reduction of amorphous content as suggested by FTIR and DSC may reduce the ductility of such materials and increase their brittleness.24−27 Evidence of fiber fracture due to enzymatic hydrolysis is available in Figure 5. Although it is difficult to show the direct evidence of the fracture in particles, the proportional reduction of amorphous domains indicates that brittleness of particles increased resulting in fracture of particles during protease XIV treatment. The mass of the particles decreased only marginally toward the end of degradation tests (i.e., from 3 to 12 day time points, Figure 2). Thus the reduction of particle size at that stage as shown in Figure 3 can be attributed primarily to the fragmentation of particles. A mild ultrasonication was used to break the aggregates before the measurements of particle size distribution. There did not appear to be any influence of ultrasonication on particle fracture as there was no difference in particle size between the as-milled without ultrasonication and ultrasonicated 12 day PBS-treated samples. This was also verified by particle size measurement of enzyme-treated particles without sonication but by adjusting the pH of the solution to change the zeta potential to avoid particle aggregation. We therefore suggest that enzymatic hydrolysis was primarily responsible for particle fracture and size reduction. Fragmentation of brittle porous enzyme-degraded powder resulted in ultrafine particles as reflected in Figures 1, 3, and 4. Particle fracture and substantial reduction in size depend on the particle fabrication methods. The morphology of milled particles is significantly different compared to solid spheres of silk prepared from silk solution through various bottom-up approaches.5,28 The porous structure of milled fiber particles contributes to a higher surface area compared to solid spheres.

Figure 9. WAXS (F: Fiber; P 12 d(PBS): 12 days PBS only treated powder; P 12 d: 12 days enzyme degraded powder (protease XIV: 2 mg/mL).

studies were conducted on silk before and after milling and after enzyme treatment (Figure 10).

Figure 10. DSC curves (F: Fiber; P 12 d(B): 12 days PBS only treated powder; P 3 d: 3 days enzyme degraded powder; P 12 d: 12 days enzyme degraded powder (protease XIV:2 mg/mL).

The endothermic peak at around 65 °C was due to the release of bound water and is referred to as Tg(1).10 In the case of fiber, it was at 63.5 °C. The peak increased to 69.7 °C after milling into powder, indicating a stronger interaction of bound water and silk powder as a result of an increase in amorphous domain. Tg(1) in powders decreased with the enzyme treatment, dropping down to the level of fiber after 12 d treatment. The endothermic peak above 250 °C for both fibers and powders commenced at the same temperature. However, the peak was sharper and degradation temperature lower in powder (284 °C) compared to fibers (293 °C). The degradation temperature of powder slowly increased after enzyme treatment.

■

DISCUSSION Mass loss profile during enzyme hydrolysis in the case of fibers was similar to the results reported by Horan et al., although the scale of degradation was different.20 They reported about 50% mass reduction for B. mori silk fibers in about 6 weeks when exposed to a 1 mg/mL protease XIV solution, while it was 2508

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

powder indicates that enzyme hydrolysis could reduce silk crystallinity. Such a change was not reflected in the FTIR results because any change in intermolecular β-sheet was more than offset by the decrease in random coil contents due to enzymatic hydrolysis. A random short-range order due to interaction of short chains is known to produce a WAXS hallo with center close to the (210) plane.31,32 A slight shift of the peak center toward a higher angle (20.3° to 20.5°) reflects a decrease of the (200) plane component and an increase in short-range order due to chain rupture and short chain aggregation. The change in WAXS pattern of B. mori films due to protease XIV hydrolysis was studied by Li et al. who reported on reduction in β-sheet and development of new silk I crystal structure.12 In that study, the formation of silk I structure was proposed based on peaks at 11.7°,19.7°, and 24.7° corresponding to 7.5, 4.5, and 3.6 Å crystal spacing, respectively.12,34 We observed that protease XIV was not completely soluble in PBS at pH 7.4 and precipitates were mixed with the powder. Therefore a mild acid wash was needed to remove such residues or bound protease XIV from the silk powder (see Supporting Information Figure S2). We also found that after treatment at pH 7.4, 37 °C, protease XIV residual crystals showed WAXS peaks reflecting some similar crystal spacing assigned to silk I (see Supporting Information Figure S3). These crystal peaks, particularly those at 11.7° and 20.9°, disappeared after the acetic acid wash, providing clear evidence against conversion of silk β-sheet (silk II) to silk I structure due to protease XIV treatment. Washing by water alone after degradation tests could not remove the bound protease XIV from the particles. In such specimens, due to the presence of the strong protease XIV peaks, broad and low intensity diffraction peaks of degraded silk powder were not easily detectable. Further evidence of reduction in intermolecular force during milling and the formation of tight aggregates of degraded chains and reduction in amorphous domain due to protease XIV hydrolysis were obtained from DSC. Tg(1) in the case of powder was higher than fibers (Figure 10). It suggests a stronger interaction with water because of the reduced intermolecular β-sheet providing access to water binding sites and faster kinetics of water molecules. On the other hand, a fall in Tg(1) after enzyme treatment suggests reduction in moisture binding amorphous regions in enzyme hydrolyzed powder, which was also confirmed by FTIR results. The second endotherm in DSC starts above 250 °C and represents delamination of β-sheets and degradation of chains. Martel et al. confirmed that amorphous silk degrades at a temperature in which silk crystallites remain intact.32 In the crystalline region, thermal energy initially delaminates β-sheets followed by their degradation.32 Crystallites in silk have a wide variation in size.31 The broad degradation peak is due to such variation as seen in Figure 10. Overlapping of disintegration and degradation of amorphous and crystalline regions was also responsible for a broad second endotherm peak. The sharper peak with a lower degradation temperature in powder (284 °C) compared to fibers (293 °C) suggests that the crystal structure in powder was considerably opened up by the milling forces, and hence a lower thermal energy was required for their disintegration, whereas the compact crystallites in fibers required more thermal energy resulting in a higher degradation temperature. The combination of a large amorphous region (lower degradation temperature) and stronger crystals in fibers

For example, we reported earlier that wool powder produced by milling with a volume d(0.5) of 5 μm was equivalent to 200− 300 nm solid spheres due to their highly porous morphology.29 The morphology of the silk particles used in this study is similar to wool particles reported earlier, and we have verified that BET surface area falls in the same range (result is not shown). Surface area is an important contributor to the rate and extent of enzymatic degradation of silk fibroin as the mass loss happens through a surface erosion process.20 As a result of high surface area, as shown in Figure 2, the percentage loss of mass of silk powders at the last experimental time point with respect to the initial mass was 3-fold compared to silk fibers (60% vs 22%) . The first step of an enzyme degradation process is surface-binding, and the second step is enzymatic hydrolysis of the protein backbone.30 Hence both fineness of particles and porous architecture resulted in higher surface area per unit mass and contributed to the high scale and kinetics of degradation. Change in secondary structure of silk during milling and enzymatic hydrolysis was studied by FTIR, WAXS, and DSC. Reduction in intermolecular β-sheet fraction in powders as shown by FTIR (Figure 8) indicates that milling forces created defects in stacked β-sheets. FTIR results in Figure 8 also suggest that the relative portions of amorphous phase (random coil) decreased toward the later part of degradation during which the rate of mass loss was reduced (Figure 2). We therefore propose that, during the initial phase of degradation, enzyme penetrated into the amorphous as well as open β-sheet crystal defect zones and thus removed both fractions in equal proportions. Once the enzymes hydrolyzed these easily accessible regions, the enzyme could not degrade the remaining regions such as more compact β-sheet crystals or tight β-turn aggregates. As a result, there was a proportional increase in the β-sheet and β-turn fractions after enzyme treatment. Short chains created during enzymatic hydrolysis had good mobility to form strong aggregates, which also contributed to the increase in tight β-turn fractions (Figure 8). WAXS results in Figure 9 show the broad nature of the peaks in all samples. Such reflections come from the diffuse nature of reflections arising from the small size of crystallites superimposed by reflection from the amorphous hallo.31 The amorphous hallo is due to the short-range order related to chain−chain correlation constrained by interchain hydrogen bonding, and the center of the hallo is close to the (210) peak position from silk crystals. Thus the silk WAXS peak for the βcrystals is fairly broad.32 The peak centered around 20.3° is composed of two strong peaks at 18.9° and 20.8° from (200) and (210) crystal planes, respectively.31 Both these planes lie in the direction normal to crystal forming backbone GAGAGS sequence.31,33 In particular, the (200) plane diffraction is due to regular stacking of β-sheets. Therefore significant reduction in intensity and broadening of these peaks, as seen after milling, indicates increase disorder between β-sheets in powders. This happened because the milling forces could destabilize the weak intersheet van der Waals stacking forces in silk crystallites. The apparent shift of the center of the crystal peak from 20.7° to about 20.3° was also due to the increase in intermolecular sheet packing distance due to higher side chain mobility caused by mechanical energy. The results support reduction in FTIR intermolecular β-sheet reflection and provide further evidence of the destabilization of β-sheet stacking as a result of silk milling. A further reduction in XAWS peak intensity in 12-day protease XIV-treated powder compared to control PBS-treated 2509

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

Figure 11. Schematic diagrams of degradation mechanisms and SEM images of silk particles from slow degradation experiments.

(higher degradation temperature) was responsible for wider endothermic peaks in fibers. In the case of powder, the degradation happened quickly due to defects in crystallites, which resulted in a sharper endothermic peak. Further, onset of degradation at a higher temperature in enzyme-treated powder compared to fiber was due to reduced amorphous phase. Progressive reduction of the amorphous phase during enzyme treatment was also suggested by FTIR results. Enzyme could penetrate into the loose crystallites and produced short chain segments as reflected by the increase in tight β-turn fractions (Figure 8). We hypothesize that the tight turns and other short segments, due to their increased mobility, could form strong aggregates through hydrogen bonding and/ or cross-linking. Less amorphous domains and the presence of these strong aggregates increased the degradation temperature of enzyme-treated powder. The WAXS result, as discussed already, also supported the formation of such aggregates having a short-range order. Based on the results of this study, a model of the microstructure change in silk fibroin due to milling and enzymatic hydrolysis has been proposed (Figure 11). Crystallites in silk are formed by stacking of β-sheets, which in turn are formed by hydrophobic segments of silk fibroin H chains.35,36 Hydrogen bonds are responsible for intermolecular forces in the β-sheets. Relatively weak van der Waals interactions provide the stacking intermolecular forces between the sheets. On milling, the mechanical energy was used to fragment the fibers. The milling energy broke the weak van der Waals forces, disturbing the regular stacking order of the βsheets. The change in silk fibroin structure after milling was reflected in WAXS peak position and intensity (Figure 9) and

reduction in thermal degradation temperature and increase in Tg(1) (Figure 10). However, FTIR studies suggested that the intramolecular hydrogen bonds within the β-sheets were retained in the particles providing structural stability and hence silk powders remained insoluble. We earlier reported adequate stability of the milled silk particles with some partial solubility in hexafluoroisopropanol (HFIP), which could be advantageously used to develop porous silk−silk composite scaffolds.6,7,37 The stability against quick dissolution of the particles can be attributed to adequate intermolecular β-sheet forces, while partial solubility can be attributed to the reduced intermolecular β-sheet after milling. An open structure provided enzyme molecules better access to the silk micro structure. Such insight is important for the design of particles to obtain optimum mechanical and degradation properties of particle-based systems. The mechanism of particle degradation is also shown in Figure 11. Enzyme predominantly acted on the hydrophilic segments, reducing random coil proportion in the structure (Figure 8). Relatively compact β-sheets largely remained resistant to hydrolysis, but rupture of random coils linking the β-sheets led to a progressive removal of sheets from the structure (dotted circles in Figure 11). The reduction in β-sheet crystallites size during enzyme treatment caused a progressive drop in WAXS peak intensity with the increase in treatment time. The process of separation of β-sheet and random coils created holes in the structure. As the hydrolysis continued, large holes were created in the particle surface as evident in highly magnified SEM images in Figure 11 leading to surface erosion. According to this mechanism, depending on the rupture sites, the degradation fragments at any particular time could vary in 2510

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

tended for biomedical implant applications. It will also be useful in understanding the structural transformation of silk debris that may be generated from silk-based implants.

size. An earlier analysis of protease XIV degradation suggested the formation of a combination of small (less than 50 kD) and large (more than 200 kD) segments,38 which supports this model. Dissolved fractions from the particle structure were removed when enzyme solution was replenished with a fresh solution. The supernatant was clear without any turbidity, indicating that there were no large insoluble fragments in the solution and mass loss was due to separation of small dissolved fragments only. FTIR results of residual mass at the early part of the enzyme treatment showed only marginal change in the secondary structure despite significant loss of mass. Thus, both random coil and β-sheet fragments were dissolved and removed. The large fragments of freed segments remained insoluble. Earlier studies suggested that protease XIV could degrade soft segments within the β-sheets.9 We therefore propose that as the process continued, the large insoluble βsheets freed from the particles were further hydrolyzed by enzyme molecules leading to the generation of smaller fragments such as β-turns and bands. Depending on the nature, size, and conformation of β-sheet and their fragments, they were either dissolved or, due to their increased mobility, formed aggregates (tight β-turns) through hydrophobic interactions or formed cross-links. Similar cross-linking of short chain segments through −NH2 and −OH end groups due to chain breakage and associated increase in modulus of silk fibers during thermal treatment have been reported.32 Similarly, there are reports on oxidative cross-linking after protease cleavage of protein.39 Proportional increase in tight β-turn fractions at a later part of degradation was confirmed by FTIR results (Figure 8). Chen et al. provided evidence that β-turns could be formed easily from shorter mobile chain fragments and caused an increase in peak at 1693 cm−1.22 The aggregates and cross-linked structure proposed in this model increased the FTIR peak intensity around 1697 cm−1 leading to a proportional increase of tight β-turn fractions. Such β-turn fractions did not contribute to the WAXS peak, but together with reduced random coil fraction, were responsible for the higher thermal degradation temperature of enzyme-treated powder.

■

ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure 1: SDS-PAGE of degummed silk. Supplementary Figure 2: SEM image of protease XIV, silk particles after protease XIV with or without acetic acid prewash. Supplementary Figure 3: WAXS of protease XIV as received and treated with buffer and silk powder treated with protease XIV and washed without acid pretreatment. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We acknowledge the financial support from the Australian Research Council (discovery project Grant DP 1094979) and the NIH (P41 EB002520) for this work.

■

REFERENCES

(1) Zhou, C.-Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; Li, Z.-G. Nucleic Acids Res. 2000, 28 (12), 2413−2419. (2) Omenetto, F.; Kaplan, D. Science 2010, 329, 528−531. (3) Pritchard, E. M.; Kaplan, D. L. Expert Opin. Drug Delivery 2011, 8 (6), 797−811. (4) Shchepelina, O.; Drachuk, I.; Gupta, M. K.; Lin, J.; Tsukruk, V. V. Adv. Mater. 2011, 23 (40), 4655−5660. (5) Wang, X.; Yucel, T.; Lu, Q.; Hu, X.; Kaplan, D. L. Biomaterials 2010, 31 (6), 1025−1035. (6) Rajkhowa, R.; Gil, E. S.; Kludge, J. A.; Numata, K.; Wang, L.; Wang, X.; Kaplan, D. L. Macromol. Biosci. 2010, 10 (6), 599−611. (7) Rockwood, D. N.; Gil, E. S.; Park, S.-H.; Kluge, J. A.; Grayson, W.; Bhumiratana, S.; Rajkhowa, R.; Wang, X.; Kim, S. J.; VunjakNovakovic, G.; Kaplan, D. L. Acta Biomater. 2011, 7, 144−151. (8) Park, S.-H.; Gil, E. S.; Shi, H.; Kim, H. J.; Lee, K.; Kaplan, D. L. Biomaterials 2010, 31 (24), 6162−6172. (9) Numata, K.; Cebe, P.; Kaplan, D. L. Biomaterials 2010, 31 (10), 2926−2933. (10) Lu, Q.; Hu, X.; Wang, X.; Kluge, J. A.; Lu, S.; Cebe, P.; Kaplan, D. L. Acta Biomater. 2010, 6 (4), 1380−1387. (11) Lu, Q.; Zhang, B.; Li, M.; Zuo, B.; Kaplan, D. L.; Huang, Y.; Zhu, H. Biomacromolecules 2011, 12 (4), 1080−1086. (12) Li, M.; Ogiso, M.; Minoura, N. Biomaterials 2003, 24 (2), 357− 365. (13) Stockel, J.; Safar, J.; Wallace, A. C.; Cohen, F. E.; Prusiner, S. B. Biochemistry 1998, 37 (20), 7185−7193. (14) Rockwood, D. N.; Preda, R. C.; Yucel, T.; Wang, X.; Lovett, M. L.; Kaplan, D. L. Nat. Protoc. 2011, 6 (10), 1612−1631. (15) Zhou, G.; Shao, Z.; Knight, D. P.; Yan, J.; Chen, X. Adv. Mater. 2009, 21 (3), 366−370. (16) Rajkhowa, R.; Levin, B.; Redmond, S. L.; Wang, L.; Kanwar, J. R.; Atlas, M. D.; Wang, X. J. Biomed. Mater. Res., Part A 2011, 97A, 37−45. (17) Rajkhowa, R.; Wang, L.; Kanwar, J.; Wang, X. Powder Technol. 2009, 191 (1−2), 155−163. (18) Rajkhowa, R.; Wang, L.; Wang, X. Powder Technol. 2008, 185 (1), 87−95. (19) Hu, X.; Kaplan, D.; Cebe, P. Macromolecules 2006, 39 (18), 6161−6170.

■

CONCLUSIONS Mechanical energy in milling opened up the silk microstructure by overcoming weak intermolecular hydrophobic forces between the stacked β-sheets within the silk crystallites. The open microstructure resulted in higher Tg(1) and lower degradation temperature. High surface area, porous morphology, and reduced crystallinity of silk particles were responsible for nearly a 3-fold increase in mass loss at the end of the enzymatic hydrolysis period compared to silk fibers. Volumebased d(0.5) of particles reduced from 6.4 to 1.8 μm within the experimental biodegradation time, which was attributed to the fracture of porous and brittle particles. Experimental data on morphology and structure of fibers and particles could be used to design a model to reflect the mechanism of microstructural change during milling and biodegradation of silk particles. The model demonstrated enzyme action in the hydrophilic links between the crystallites, progressively removing the β-sheets. Enzyme degraded particles showed a higher resistance to thermal degradation and a sharper endothermic peak in DSC due to reduced proportion of the amorphous domain and the formation of tight aggregates and/or cross-links by β-sheet fragments. This study is expected to assist in understanding the structure−biodegradation relationships of silk particles in2511

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512

Biomacromolecules

Article

(20) Horan, R. L.; Antle, K.; Collette, A. L.; Wang, Y.; Huang, J.; Moreau, J. E.; Volloch, V.; Kaplan, D. L.; Altman, G. H. Biomaterials 2005, 26 (17), 3385−3393. (21) Mouro, C.; Jung, C.; Bondon, A.; Simonneaux, G. r. Biochemistry 1997, 36 (26), 8125−8134. (22) Chen, X.; Knight, D. P.; Shao, Z. Soft Matter 2009, 5, 2777− 2781. (23) Arai, T.; Freddi, G.; Innocenti, R.; Tsukada, M. J. Appl. Polym. Sci. 2004, 91 (4), 2383−2390. (24) Rajkhowa, R.; Gupta, V. B.; Kothari, V. K. J. Appl. Polym. Sci. 2000, 77 (11), 2418−2429. (25) Morton, W. E.; Hearle, J. W. S. Physical Properties of Textile Fibres, 3rd ed); Woodhead Publishing Limited: Cambridge, U.K., 1993. (26) Hearle, J. W. S.; Lomas, B.; Cooks, W. D. Atlas of Fibre Fracture and Damage to Textiles, 2nd ed.; Woodhead Publishing: Cambridge, U.K. (27) Kothari, V. K.; Rajkhowa, R.; Gupta, V. B. J. Appl. Polym. Sci. 2001, 82, 1147−1154. (28) Lammel, A. S.; Hu, X.; Park, S.-H.; Kaplan, D. L.; Scheibel, T. R. Biomaterials 2010, 31 (16), 4583−4591. (29) Rajkhowa, R.; Zhou, Q.; Tsuzuki, T.; Morton, D. A.; Wang, X. Powder Technol. 2012, 224, 183−188. (30) Nair, L. S.; Laurencin, C. T. Prog. Polym. Sci. 2007, 32 (8−9), 762−798. (31) Drummy, L. F.; Farmer, B. L.; Naik, R. R. Soft Matter 2007, 3 (7), 877−882. (32) Martel, A.; Burghammer, M.; Davies, R. J.; Riekel, C. Biomacromolecules 2007, 8 (11), 3548−3556. (33) Warwicker, J. O. J. Mol. Biol. 1960, 2, 350−362. (34) Lotz, B.; Keith, H. D. J. Mol. Biol. 1971, 61 (1), 201−202. (35) Ha, S.-W.; Gracz, H. S.; Tonelli, A. E.; Hudson, S. M. Biomacromolecules 2005, 6, 2563−2569. (36) Du, N.; Yang, Z.; Liu, X. Y.; Li, Y.; Xu, H. Y. Adv. Funct. Mater. 2011, 21, 772−778. (37) Gil, E. S.; Kluge, J. A.; Rockwood, D. N.; Rajkhowa, R.; Wang, L.; Wang, X.; Kaplan, D. L. J. Biomed. Mater. Res., Part A 2011, 99 (1), 16−28. (38) Numata, K.; Kaplan, D. L. Biochemistry 2010, 49 (15), 3254− 3260. (39) Kang, J. J. I.; Neidigh, J. W. Chem. Res. Toxicol. 2008, 21 (5), 1028−1038.

2512

dx.doi.org/10.1021/bm300736m | Biomacromolecules 2012, 13, 2503−2512