Controllable transition of silk fibroin nanostructures: An insight into in vitro silk self-assembly process

Accepted Manuscript Controllable transition of silk fibroin nanostructures: an insight into in vitro silk self-assembly process Shumeng Bai, Shanshan Liu, Cencen Zhang, Weian Xu, Qiang Lu, Hongyan Han, David L. Kaplan, Hesun Zhu PII: DOI: Reference:

S1742-7061(13)00214-6 http://dx.doi.org/10.1016/j.actbio.2013.04.033 ACTBIO 2701

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

25 February 2013 15 April 2013 18 April 2013

Please cite this article as: Bai, S., Liu, S., Zhang, C., Xu, W., Lu, Q., Han, H., Kaplan, D.L., Zhu, H., Controllable transition of silk fibroin nanostructures: an insight into in vitro silk self-assembly process, Acta Biomaterialia (2013), doi: http://dx.doi.org/10.1016/j.actbio.2013.04.033

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Controllable transition of silk fibroin nanostructures: an insight into in vitro silk self-assembly process Shumeng Baia, b, Shanshan Liua, Cencen Zhang a, Weian Xub, Qiang Lua, *, Hongyan Hanb, *, David L Kaplanc, Hesun Zhud, a

National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, People’s Republic of China b

School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215123, People’s Republic of China

c

Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA

d

Research Center of Materials Science, Beijing Institute of Technology, Beijing 100081, People’s Republic of China

Corresponding authors: *Qiang Lu, Tel: (+86)-512-67061649; E-mail: [email protected]

Co-Corresponding authors: *Hongyan Han, Tel: (+86)-512-65880279; E-mail: [email protected]

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Abstract Silk fiber is one of the strongest and toughest biological materials with hierarchical structures where nanofibril with size of below 20 nm is critical factor in determining the excellent mechanical properties. Although silk nanofibrils have been found in natural and regenerated silk solutions, there is no way to actively control nanofibril formation in aqueous solution. Here we show a simple but effective method to prepare silk nanofibrils by regulating silk self-assembly process. Through a repeated drying-dissolving process, silk fibroin solution composed of metastable nanoparticles was firstly prepared and then used to reassemble nanofibrils with different sizes and secondary conformations under various temperatures and concentrations. These nanofibrils have similar size with that in natural fibers, providing a suitable unit to further assemble hierarchical structure in vitro. Several important issues such as the relationships between silk nanofibrils, secondary conformations and viscosity are also investigated, giving a new insight into the self-assembly process. In summary, besides rebuilding silk nanofibrils in aqueous solution, our study provides an important model to further understand silk structures, properties as well as forming mechanism, making it possible to regenerate silk materials with the exceptional properties in future. Keywords: Silk, Self-assembly, Nanofiber, Biomaterials

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1. Introduction Silk is one of nature’s most highly engineered materials featuring a rare combination of high strength and high breaking strain [1-2]. A great appeal of exploring silk still lies in the amazing mechanical properties that emerge from simple building blocks [3-5]. Experimental and computational investigations of the structure of silk both indicated that the outstanding mechanical properties are due to its sophisticated hierarchical structure where highly organized, densely H-bonded beta-sheet nanocrystals are arranged within a semiamorphous matrix consisting of helices and beta-turn protein structures [6-9]. Recent studies further reveal that the nanoscale confinement of β-sheet nanocrystals as well as other semiamorphous structures in silks has a fundamental role in achieving great stiffness, resilience and fracture toughness at the macroscale [10]. From an engineering perspective, silk applications have been transformed from traditional textile into high-technology fields including medical sutures, tissue engineering, drug release systems, and optical devices [11-13]. The ability to artificially make silk-based materials with similarly good properties at ambient temperatures and pressures is another desirable goal for the interesting applications [14-15]. However, some major challenges remain. For example, to achieve silk-based materials with desired properties, it is necessary to further understand silk assembly behavior from molecular level to hierarchical structures. Although many studies have certified the nanofibril existence in the natural silk fiber, and then confirmed the silk nanofibril formation from native silk proteins through shear-induced self-assembly process, there is no clear ideal how silk fibroin self-assembled into the hierarchical structures especially the nanofibril structure in nature partly because it is impossible to investigate silk nanostructure changes in vivo [16-18]. Therefore, based on recent theoretical and applied developments, regenerating silk fibrils with diameters at nanoscale in vitro might be one of most critical steps to further 3

understand silk self-assembly mechanism and then design silk materials with exceptional properties. Recently, silk nanofibrils have been found in vitro with recombinant proteins or native silk protein from glands of Bombyx mori silk worms and by different ways [19-25]. However, these nanofibrils existed in stable gel or solid states [23-25], making it impossible to further control the micromorphologies and secondary structures as well as to elucidate the transition process. Our recent study revealed that silk fibroin could assemble from microspheres to nanofilaments. The self-assembly of silk fibroin is not only a thermodynamic process but also a kinetic process, which could be controlled by adjusting molecular mobility, charge, hydrophilic interactions and concentration [26]. However, similar to other results from different research groups, silk proteins in the present study still maintain different types of shapes with sizes ranging from a few to several hundred nanometers [27-28]. In this work, we develop a simple process to transmit silk fibroin from different shapes to nanofibrils in aqueous solution, which would be imperative for rigorous understanding of silk self-assembly behavior. Overall, our experiments not only provide novel insights into the self-assembly process of silk fibroin, but also supply a platform for further studying the assembly process from nanofibril to hierarchical structures in vitro as well as designing new silk-based functional materials.

2. Experimental Section 2.1 Preparation and nanostructure control of silk solutions. B. mori silk fibroin solutions were prepared following our previously published procedures [13-15, 29]. Cocoons were boiled for 20 min in an aqueous solution of 0.02 M Na2CO3, and then rinsed thoroughly with distilled water to extract the sericin proteins. The extracted silk was dissolved in 9.3 M LiBr solution (Sigma-Aldrich, St. Louis, MO) at 60 oC, yielding a 20% (w/v) solution. This solution was dialyzed against distilled water using Slide-a-Lyzer dialysis cassettes 4

(Pierce, MWCO 3500) for 72 hours to remove the salt. After dialysis, the content of LiBr measured with ICP-OES (iCAP 6000, Thermo Scientific, Massachusetts, America) was below 0.03%, which confirmed that the LiBr removal has almost completed. The solution was centrifuged at 9000 r/min for 20 min at 4 oC to remove silk aggregates formed during the process. The final concentration of aqueous silk solution was ～7 wt %, determined by weighing the remaining solid after drying. Then silk fibroin solutions composed of nanoparticles with sizes ranging from several ten to several hundred nanometers were prepared by a repeated drying-dissolving process. Silk fibroin solutions (5 ml) were cast on polystyrene Petri dishes (diameter 90 mm) and dried at room temperature within 6 hours through increasing volatilization speed with fan. Once the dried silk fibroin films formed, they were dissolved in distilled water to regenerate silk fibroin solutions. In the drying-dissolving process, silk can gradually aggregate to form bigger particles, which is critical for our following study. In fresh solution, silk was composed of particles with lateral dimensions of about 20 nm and 1 nm in height (Fig. S1). The lateral dimensions and heights of most of particles in aqueous solution increased to above 50 nm and 2 nm after two cycles and then to above 100 nm and 5 nm after four cycles, respectively. Since most of silk became insoluble, failing to achieve silk solution after five cycles, the solution obtained after four cycles was termed as FF-SF and used to form silk nanofibrils. Finally the FF-SF solution was diluted to different concentrations and cultured at different temperatures for different times to achieve nanofibrous structures with different secondary conformations. 2.2 Characterization The morphology of the samples was observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). For SEM imaging, two microliters of protein samples were

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added directly on top of a conductive tape mounted on a SEM sample stub. The samples were dried in air and then sputtered with platinum. SEM analysis was observed using a S-4800 microscopy ( Hitach, Tokyo, Japan) at 3 kV. To prepare the samples for AFM imaging, solutions were diluted to below 0.1 wt% to avoid masking the original morphology by multilayers of silk [26]. Two microliters of the diluted SF solution was dropped onto freshly cleaved 4×4 mm2 mica surfaces. The morphology of silk fibroin in water was observed by AFM (Nanoscope V, Veeco, New York, America) in air. A 225 µm long silicon cantilever with a spring constant of 3 Nm-1 was used in tapping mode at 0.5-1 Hz scan rate. Dynamic Light Scattering (DLS) measurement was performed with a Zetasizer (Nano ZS, Malvern, Worcesteshire, England) equipped with a 633nm He-Ne laser using an angle of 173°. One milliliter of protein sample was used in disposable polysterene cuvettes with a 10-mm path length. The data were recorded at 25 oC. All samples were centrifuged to remove the impurities [30]. Surface charges of protein samples were determined via Zeta potential measurements. One milliliter of the solution was loaded to a Zetasizer (Nano ZS, Malvern, Worcesteshire, England) for the zeta potential measurement at 25 oC. Transmission electron microscopy investigations were carried out using a Tecnai G220 instrument (G220, FEI, Portland, America). The samples were deposited on carbon-coated 100 mesh copper grids for TEM observation at 200 kV. Since FTIR and XRD are generally used to study the secondary structures of silk in solid state, CD spectra was used to determine the secondary structures of silk solutions [31-33]. The secondary structures of silk solutions were studied with circular dichroism (CD) spectrometer. CD spectra were recorded on an spectrophotometer (Model 410, AVIV, Lakewood, America) equipped with a Peltier temperature controller. Spectra were obtained from 250 to 190 nm at a scanning speed of 50 nm/min at 25 oC. The CD spectra represented the average of three

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measurements and were smoothed. CD data are reported as mean residue ellipticity ([Θ], degree·cm2·dmol-1) [34]. Rheological studies were run on a Rheometer (AR2000, TA Instruments, New Castle, America) fitted with a 40 mm cone plate (Ti, 40/2o) [35]. Prior to each experimental day, the rheometer underwent a torque map with a 10 Pa s calibration oil. The shear rate was linearly increased from 0.01 to 5000 s-1 at 25 oC.

3. Results and discussion Silk fibroin generally forms different nanostructures with different sizes such as nanospheres, nanofibrils or irregualr nanoparticles in aqueous solution. The structure complexity is a main problem for elucidating silk self-assembly process. In our recent study, through carefully investigating the nanostructural changes of silk fibroin in slow drying process, the self-assembly process from nanospheres to nanofibrils was revealed, in which the negative charge of silk fibroin was a critical regulating factor [36-37]. Unfortunately, although we found that initial structure and charge had great influence on silk self-assembly process, there is still no reliable and effective way to prepare silk nanofibrils in aqueous solution. Therefore, in present study, the preliminary structures and negative charge distribution were controlled firstly to favor nanofibril formation in aqueous solution. A repeated drying-dissolving process was applied to regulate the preliminary nanostructure of silk fibroin. In the process, the sizes of silk fibroin nanoparticles increased from about 20 nm to above 100 nm (Fig S1 and Fig 1A), suggesting that some silk fibroin molecules aggregated to form the nanoparticles since the lateral dimensions for single silk fibroin protein are about 20-25 nm while the apparent height on the substrate is about 1.2 nm [22]. The charge distribution of the silk fibroin particles was described through zeta potential measurements. After the dryingdissolving process, the zeta potential of the silk fibroin nanoparticles changed from -22.8 mV to 7

16.5 mV in neutral solution (Fig. S.1 and Fig. 1A), much lower than that of silk fibroin particles prepared by other group (-43 mV) [38]. The results indicated that some negative charges still distributed inside the nanoparticles. Based on our recent hypothesis and previously published data regarding the relationships between amino acid composition and silk protein phase separation, and self-assembly [39-41], the charge distribution would provide repulsive chargecharge interactions inside the particles, making the particles metastable. The metastable particles were easily destroyed and re-assembled at higher temperature, resulted in nanofibril formation. Results of SEM revealed the transition from metastable nanoparticles to nanofibrils in aqueous solution at 60 oC, in which the nanofibrils with diameter of about 15 nm and length of about 500 nm formed after 3 hours, and then the length grew to about 1 μm and finally above 1.5 μm after 6 hours and 9 hours, respectively (Fig. 1B). Dynamic light scattering showed that the main peak of silk fibroin particles in aqueous solution gradually increased from about 150 nm to 2 μm with broader size distribution following the increase of culture time. Although DLS is an unsuitable way to measure fibrous structures in solution, the increase of peak and size distribution of silk particles implied the size growth, confirming the nanofibril formation shown in SEM results. Then the structure of nanofibrils was further provided in AFM and TEM images (Fig. 1C). Although silk fibroin was easily destroyed at high resolution under TEM, the TEM images at low resolution still provided useful information about the nanostructure transition process, indicating that silk nanofibrils derived from the nanoparticles when the silk fibroin solution cultured at 60 o

C for 3 hours. Then AFM images revealed that each of these nanofibrils consists of a discrete

series of elements, exhibiting a “beads on a string” type of morphology. Based on their sizes, diameters of about 15 nm and heights of about 1 nm, each of these “beads” was probably a single silk protein [22]. These results confirmed that the silk fibroin nanoparticles underwent significant conformational changes and then reassembled into nanofibrils. According to our hypothesis [26,

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29], the reassembly occurrence depended on the repulsive charge-charge interactions inside the nanoparticles. Zeta potential measurements of silk fibroin solutions that were cultured for different times indicated that the zeta potential increased from -16.5 mV to -32.5 mV (Fig. 1B), indicating that some negative charges inside the nanoparticles transferred to the surface of the nanofibrils. The influence of silk fibroin concentration on the nanoparticle-nanofibril transition process was also investigated in our study. SEM and DLS results showed that silk fibroin nanofibrils grew similarly under different concentrations, implying that concentration had no significant influence on nanofibril formation (Fig. 2A and B). Combining with TEM images (Fig. 1C), the similar transition behavior under different concentrations implied that nanofibrils formed inside the particles rather than rebuilded after the particles transformed into smaller nanoparticles. More information was revealed by zeta potential measurement. Although the original zeta potential of 0.3 wt % solution was -13.2 mV since bigger aggregates of nanoparticles wrapped more negative charge, the zeta potential was changed to -30.1 mV and -72.5 mV after 9 hours and 24 hours, respectively. While that of the 0.003 wt % solution was only increased to -22 mV and -29.8 mV after 9 hours and 24 hours, respectively (Fig. 2C). Comparing to the reassembling process under different concentrations (0.003 wt % and 0.3 wt %), the nanofibrils with similar length but totally different negative charge were prepared under the same conditions, indicating more complex self-assembly process including but not limit to nanofibril formation. Therefore, secondary structure was assessed by circular dichroism spectroscopy to further study the conformational changes in the reassembling process. All CD spectra of the original silk fibroin solutions with different concentrations showed a negative ellipticity at about 195 nm, indicating random conformation formation. When the silk fibroin solutions were placed at 60 oC for 24 hours to reassemble, similar conformational transitions happened in which a positive ellipticity at 195 nm

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and a negative ellipticity at 217 nm gradually appeared, suggestive of an induction of β-sheet conformation. The transition speeds increased following the increase of silk concentration. In the reassembling process at 60 oC, the conformational transition from random to β-sheet almost finished after 9 hours for 0.3 wt % silk fibroin solution while many random structures still existed in 0.003 wt % silk fibroin solution after 24 hours (Fig. 4A and C). Considering the nanostructural and conformational results, we revealed that nanofibrils with different β-sheet contents could be prepared through changing silk fibroin concentration and the reassembly process. On the other hand, CD spectra and zeta potential measurement indicated that the significant increase of zeta potential also coupled with β-sheet formation, which might be useful to assess β-sheet formation in silk fibroin solution. Based on our present results, we find that silk fibroin self-assembly shows diverse changes at different levels in which charge distribution is critical for regulating various metastable structures. Simply, different repulsive charge-charge interactions exist between different nanoparticles, inside nanoparticles and even inside single silk fibroin protein. Through regulating the charge distribution, it would be possible to control the stability of silk fibroin at different levels and then prepare silk fibroin materials with specific nanostructure and conformations. However, besides charge distribution, other factors such as hydrophobic interaction, hydrogen bands, pH, and temperature also have great influence on the self-assembly process of silk fibroin [42-43]. The factor complexities resulted in the difficulty in effectively controlling nanostructures and conformations of silk fibroin. In the present study, silk fibroin nanostructure and charge distribution were firstly controlled by a repeated drying-dissolving process. Then silk nanofibrils with different β-sheet contents were successfully prepared in a simple process through improving silk fibroin activity to unwrap the metastable nanoparticles at higher temperature.

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According to the re-assembling process, the nanofibril formation could be further controlled by changing temperatures. SEM and DLS results showed that the transition from metastable nanoparticles to nanofibrils were slower at lower temperatures. Comparing to the reassembling process at 90 oC and 60 oC, the nanofibrils grew with similar length at same time. But the nanofibrils grew slowly at 25 oC, in which the nanofibrils with length of about 500 nm formed after 12 hours, and then the length grew to about 1 μm and finally above 1.5 μm after 24 hours and 48 hours, respectively. The growth rate of silk fibroin nanofibrils further decreased at 4 oC, in which the nanofibrils with length of about 1 μm formed after 144 hours (Fig. 3A, B, and C), implying that higher temperature facilitated the nanofibril formation. Although the original zeta potential was -13.2 mV, the zeta potential was changed to -61 mV after 9 hours and -72.4 mV after 12 hours at 90 oC while increased to -45.5 mV after 48 hours and -63.2 mV after 96 hours at 25 oC and to -24.9 mV after 144 hours at 4oC (Fig. 3D). The zeta potential changes at different temperatures coupled with conformational changes. As shown in Fig. 4D and Fig. 4F, silk solution composed of random conformation formed at 3 hours and then transmitted to β-sheet conformation at 6 hours at 90 oC while the nanofibrils formed at 48 hours and then silk solution maintained random structure at 96 hours if the assembly happened at 4 oC. Although the β-sheet content in silk solutions prepared under certain condition is time-dependent and would gradually increase to higher level similar to natural silk fibers, the results revealed a possibility to achieve silk fibroin nanofibrils with different sizes and β-sheet contents at different temperatures and then retain some metastable structures at low temperature, which would be important for further studying more complex self-assembly behaviors of silk fibroin and designing novel functional silk fibroin materials with these nanofibrils as basic units. Therefore, our study revealed a simple way to prepare silk fibroin nanofibrils with different βsheet contents, which is critical for further understanding the nanostructure-function relation of

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silk fibroin. Firstly, the present study and our recent results confirmed that regenerated silk nanofibrils could be prepared in aqueous solution, making it possible to design novel silk fibroin materials with complex nanostructures. Then we revealed that nanofibril formation and β-sheet transition are related but different processes, which implies that the secondary structures, especially β-sheet structure, could be further modified after nanofibril formation though the transition from random to β-sheet structure has happened in case silk nanofibril formed [44]. Finally, the viscosity behaviors of silk fibroin with different nanostructures indicated that nanofibril formation resulted in significant increase of the solution viscosity (Fig. 5). On the other hand, the viscosity of silk fibroin solutions with similar nanofibrils but different β-sheet contents was also investigated (Fig. 5C (60 oC, 9 hours and 60 oC, 24 hours); Fig. 5D and E (90 o

C, 24 hours and 25 oC, 96 hours)). Silk fibroin solutions with similar nanofibrous structures but

higher β-sheet content achieved higher viscosity under same concentrations, which implied that both nanostructures and secondary conformations could affect the viscosity behaviors of silk fibroin solution. The viscosity difference between natural and regenerated silk solutions is still an unsolved issue for several years [45-46] since the viscosity behaviors of silk fibroin solution are related to various factors such as secondary structures, molecular weights, and the nanostructures. In previous study, secondary structures and molecular weights have been clarified as critical factors in controlling the viscosity behaviors of silk fibroin [47-48]. Although the influence of the amount of fibril, the length and asymmetric shape of the fibril and even the interaction of the fibrils should be further studied in future, our study indicated that regenerated silk fibroin solution with nanofibril structures achieved higher viscosity than fresh regenerated solution without nanofibrils, implying that the nanostructure change between natural silk fibroin dope and regenerated solutions might be also an important reason to affect the viscosity behaviors of silk fibroin. The critical effect of nanofibrils on the mechanical properties of silk materials has been

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confirmed by different experimental and computational investigations [6-9]. In our previous study, it was also found that nanofibril formation and orientation facilitated the improvement of mechanical properties compared to that without nanofibrils [49]. However, when the zeta potential of silk nanofibril solution was above -60 mV, the films prepared from the solution showed inferior mechanical properties and easily destroyed in aqueous solution since the charge repulsion restrained the interaction and arrangement of the nanofibrils, which implied that the reassembly and arrangement of the nanofibrils were also critical for controlling mechanical properties of silk materials. Anyway, besides providing an effective way to prepare silk fibroin nanofibril solution, our present study also open a new window to further understand silk selfassembly process as well as silk structure-function relationship.

4. Conclusion In summary, we have for the first time demonstrated that regenerated silk fibroin nanofibrils with different lengths and different β-sheet contents could be prepared through a simple selfassembly process in aqueous solutions. A repeating drying-dissolving process was firstly used to prepare metastable silk nanoparticles in aqueous solution. Then the nanoparticles reassembled into nanofibrils with different sizes and different β-sheet contents under various temperatures and concentrations. Several important issues including the relationship between nanostructures, secondary conformations, charge distribution and viscosity behaviors were studied with silk nanofibrils as model, providing a further insight into the self-assembly process of silk fibroin in vitro.

Acknowledgements

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We thank National Basic Research Program of China (973 Program 2013CB934400), and NSFC (21174097) for support of this work. We also thank the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Excellent Youth Foundation of Jiangsu Province(BK2012009), the NIH (EB002520)), Ph.D. Programs Foundation of Ministry of Education of China (201032011200009), and the Key Natural Science Foundation of the Jiangsu Higher Education Institutions of China (11KGA430002) for support of this work.

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References [1] Omenetto FG, Kaplan DL. New opportunities for an ancient material. Science 2010; 329(5991): 528-31. [2] Gosline JM, Demont ME, Denny MW. The structure and properties of spider silk. Endeavour 1986; 10: 37-43. [3] Jin HJ, Kaplan DL. Mechanism of silk processing in insects and spiders. Nature 2003; 424: 1057-61. [4] Hagn F, Eisoldt L, Hardy JG, Vendrely C, Coles M, Scheibel T, Kessler H. A conserved spider silk domain acts as a molecular switch that controls fibre assembly. Nature 2010; 465: 239-42. [5] Vollrath F, Madsen B, Shao ZZ. The effect of spinning conditions on the mechanics of a spider's dragline silk. Proc Biol Sci 2001; 268(1483): 2339-46. [6] Lefevre T, Rousseau ME, Pezolet M. Protein secondary structure and orientation in silk as revealed by Raman spectromicroscopy. Biophys J 2007; 92(8): 2885-95. [7] Thiel BL, Guess KB, Viney C. Non-periodic lattice crystals in the hierarchical microstructure of spider (major ampullate) silk. Biopolymers 1997; 41(7): 703-19. [8] Du N, Liu XY, Narayanan J, Li L, Lim MLM, Li DQ. Design of superior spider silk: from nanostructure to mechanical properties. Biophys J 2006; 91(12): 4528-35. [9] Lee SM et al. Greatly Increased Toughness of Infiltrated Spider Silk. Science 2009; 324(5926): 488-92.

15

[10] Keten S, Xu ZP, Ihle B, Buehler MJ. Nanoconfinement controls stiffness, strength and mechanical toughness of β-sheet crystals in silk. Nat Mater 2010; 9: 359-67. [11] Jiang CY et al. Mechanical properties of robust ultrathin silk fibroin films. Adv Funct Mater 2007; 17(13): 2229-37. [12] Liu HF, Fan HB, Wang Y, Toh SL, Goh JCH. The interaction between a combined knitted silk scaffold and microporous silk sponge with human mesenchymal stem cells for ligament tissue engineering. Biomaterials 2008; 29(6): 662-74. [13] Wang XQ, Wenk E, Matsumoto A, Meinel L, Li CM, Kaplan DL. Silk microspheres for encapsulation and controlled release. J Control Release 2007; 117(3): 360-70. [14] Rockwood DN, Preda RC, Yucel T, Wang XQ, Lovett ML, Kaplan DL. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc 2011; 6: 1612-31. [15] Yao DY, Dong S, Lu Q, Hu X, Kaplan DL, Zhang BB, Zhu HS. Salt-leached silk scaffolds with tunable mechanical properties. Biomacromolecules 2012; 13(11): 3723-9. [16] Vollrath F, Knight DP. Liquid crystalline spinning of spider silk. Nature 2001;410:541-8. [17] Putthanarat S, Stribeck N, Fossey SA, Eby RK, Adams WW. Investigation of the nanofibrils of silk fibers. Polymer 2000; 41(21): 7735-47. [18] Perez-Rigueiro J, Viney C, Llorca J, Elices M. Silkworm silk as an engineering material. J Appl Polym Sci 1998; 70(12): 2439-47. [19] Xia XX, Xu QB, Hu X, Qin GK, Kaplan DL. Tunable self-assembly of genetically engineered silk–elastin-like protein polymers. Biomacromolecules 2011; 12(11): 3844-50.

16

[20] Arcidiacono S et al. Aqueous processing and fiber spinning of recombinant spider silks. Macromolecules 2002; 35(4): 1262-6. [21] Stark M, Grip S, Rising A, Hedhammar M, Engstrom W, Hjalm G, Johansson J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007; 8(5): 1695-701. [22] Greving I, Cai MZ, Vollrath F, Schniepp HC. Shear-induced self-assembly of native silk proteins into fibrils studied by atomic force microscopy. Biomacromolecules 2012; 13(3): 67682. [23] Gong ZG, Huang L, Yang YH, Chen X, Shao ZZ. Two distinct β-sheet fibrils from silk protein. Chem Commun 2009; 48: 7506-8. [24] Zhang F, Zuo BQ, Fan ZH, Xie ZG, Lu Q, Zhang XG, Kaplan DL. Mechanisms and control of silk-based electrospinning. Biomacromolecules 2012; 13(3): 798-804. [25] Wittmer CR, Hu X, Gauthier P, Weisman S, Kaplan DL, Sutherland TD. Production, structure and in vitro degradation of electrospun honeybee silk nanofibers. Acta Biomater 2011; 7(10): 3789-95. [26] Lu Q, Zhu HS, Zhang CC, Zhang F, Zhang B, Kaplan DL. Silk self-assembly mechanisms and control from thermodynamics to kinetics. Biomacromolecules 2012; 13(3): 826-32. [27] Numata K, Yamazaki S, Naga N. Biocompatible and biodegradable dual-drug release system based on silk hydrogel containing silk nanoparticles. Biomacromolecules 2012; 13(5): 1383-9.

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[28] Mathur AB, Gupta V. Silk fibroin-derived nanoparticles for biomedical applications. Nanomedicine 2010; 5(5): 807-20. [29] Lu Q et al. Silk fibroin electrogelation mechanisms. Acta Biomaterialia 2011; 7(6): 2394400. [30] Leclerc J, Lefevre T, Pottier F, Morency LP, Lapointe-Verreault C, Gagne SM, Auger M. Structure and pH-induced alterations of recombinant and natural spider silk proteins in solution. Biopolymers 2012; 97(6): 337-46. [31] Dicko C, Knight D, Kenney JM, Vollrath F. Secondary structures and conformational changes in flagelliform, cylindrical, major, and minor ampullate silk proteins. Temperature and concentration effects. Biomacromolecules 2004; 5(6): 2105-15. [32] Dicko C, Knight D, Kenney JM, Vollrath F. Structural conformation of spidroin in solution: a synchrotron radiation circular dichroism study. Biomacromolecules 2004; 5(3): 75867. [33] Yang YH, Shao ZZ, Chen X, Zhou P. Optical spectroscopy to investigate the structure of regenerated Bombyx mori silk fibroin in solution. Biomacromolecules 2004; 5(3): 773-9. [34] Li XG, Wu LY, Huang MR, Shao HL, Hu XC. Conformational transition and liquid crystalline state of regenerated silk fibroin in water. Biopolymers 2007; 89(6): 497-505. [35] Holland C, Terry AE, Porter D, Vollrath F. Natural and unnatural silks. Polymer 2007; 48(12): 3388-92. [36] Chen P, Kim HS, Park CY, Kim HS, Chin IJ, Jin HJ. pH-Triggered transition of silk fibroin from spherical micelles to nanofibrils in water. Macromol Res 2008; 16: 539-43.

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[37] Golinska MD, Pham TTH, Werten MWT, Wolf FAA, Stuart MAC, Gucht JVD. Fibril formation by pH and temperature responsive silk-elastin block copolymers. Biomacromolecules 2013; 14(1): 48-55. [38] Lammel AS, Hu X, Park SH, Kaplan DL, Scheibel TR. Controlling silk fibroin particle features for drug delivery. Biomaterials 2010; 31(16): 4583-91. [39] Liebmann B, Hummerich D, Scheibel T, Fehr M. Formulation of poorly water-soluble substances using self-assembling spider silk protein. Colloid Surface A 2008; 331: 126-32. [40] Eisoldt L, Hardy JG, Heim M, Scheibel TR. The role of salt and shear on the storage and assembly of spider silk proteins. J Struct Biol 2010; 170(2): 413-9. [41] Sponner A, Unger E, Grosse F, Weisshart K. Conserved C-termini of spidroins are secreted by the major ampullate glands and retained in the silk thread. Biomacromolecules 2004; 5(3): 840-5. [42] Hu X, Shmelev K, Sun L, Gil ES, Park SH, Cebe P, Kaplan DL. Regulation of silk material structure by temperature-controlled water vapor annealing. Biomacromolecules 2011; 12(5): 1686-96. [43] Terry AE, Knight DP, Porter D, Vollrath F. pH Induced changes in the rheology of silk fibroin solution from the middle division of Bombyx mori silkworm. Biomacromolecules 2004; 5(3): 768-72. [44] An B, Jenkins JE, Sampath S, Holland GP, Hinman M, Yarger JL, Lewis R. Reproducing natural spider silks’ copolymer behavior in synthetic silk mimics. Biomacromolecules 2012; 13(12): 3938-48.

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[45] Holland C, Porter D, Vollrath F. Comparing the rheology of mulberry and “wild” silkworm spinning dopes. Biopolymers 2012; 97(6): 362-7. [46] Holland C, Terry AE, Porter D, Vollrath F. Comparing the rheology of native spider and silkworm spinning dope. Nat Mater 2006; 5: 870-4. [47] Matsumoto A, Lindsay A, Abedian B, Kaplan DL. Silk fibroin solution properties related to assembly and structure. Macromol Biosci 2008; 8(11): 1006-18. [48] Ebenstein DM, Wahl KJ. Anisotropic nanomechanical properties of Nephila clavipes dragline silk. J Mater Res 2006; 21(8): 2035-44. [49] Zhang CC, Song DW, Lu Q, Hu X, Kaplan DL, Zhu HS. Flexibility regeneration of silk fibroin in vitro. Biomacromolecules 2012; 13(7): 2148-53.

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Figure legends: Fig. 1. (A) SEM micrograph, AFM image and height map of the 0.03 wt % FF-SF solution. (B) SEM micrographs, DLS size distribution profiles and Zeta potentials of the transition from nanoparticles to nanofibrils for 0.03 wt % solution at 60 oC. (C) TEM micrographs, AFM image and height map of nanofibrils for 0.03 wt % solution at 60 oC. Fig. 2. SEM micrographs, DLS size distribution profiles of the transition from nanoparticles to nanofibrils for 0.003 wt % solution (A) and 0.3 wt % solution (B) at 60 oC. (C) Zeta potentials of the transition from nanoparticles to nanofibrils for 0.003 wt % and 0.3 wt % solutions at 60 oC. Fig. 3. SEM micrographs, DLS size distribution profiles of the transition from nanoparticles to nanofibrils for 0.3 wt % solution at 90 oC (A), 25 oC (B) and 4 oC (C). (D) Zeta potentials of the transition from nanoparticles to nanofibrils for 0.3 wt % solution under these temperatures. Fig. 4. The effect of solution concentration and treatment temperature on conformational changes of the FF-SF solutions in reassembling process. Some samples with different concentrations ((A) 0.003 wt %, (B) 0.03 wt % and (C) 0.3 wt %) were treated at 60 oC while the other samples with concentration of 0.3 wt % were cultured at different temperatures ((D) 90 oC, (E) 25 oC and (F) 4 o

C)).

Fig. 5. The effect of solution concentration and treatment temperature on the viscosity behaviors of the FF-SF solutions in reassembling process. Some samples with different concentrations ((A) 0.003 wt %, (B) 0.03 wt % and (C) 0.3 wt %) were treated at 60 oC while the other samples with concentration of 0.3 wt % were cultured at different temperatures ((D) 90 oC, (E) 25 oC and (F) 4 o

C)).

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