Amorphous Silk Nanofiber Solutions for Fabricating Silk-Based

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Amorphous Silk Nanofiber Solutions for Fabricating Silk-Based Functional Materials Xiaodan Dong, Qun Zhao, Liying Xiao, Qiang Lu, and David L Kaplan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00863 • Publication Date (Web): 31 Jul 2016 Downloaded from http://pubs.acs.org on July 31, 2016

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Amorphous Silk Nanofiber Solutions for Fabricating Silk-Based Functional Materials Xiaodan Dong a,b, Qun Zhao a,b, Liying Xiaoa,b, Qiang Lu a,b ,*, and David L Kaplanc a

Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China

b

National Engineering Laboratory for Modern Silk, Soochow University, Suzhou 215123, People’s Republic of China c

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

ACS Paragon Plus Environment

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ABSTRACT

As a functional material, silk has been widely used in tissue engineering, drug release, and tissue regeneration. Increasing subtle control of silk hierarchical structures and thus specific functional performance is required for these applications, but remains a challenge. Here, we report a novel silk nanofiber solution achieved through tuning solvent systems used to generate the material. Unlike the beta-sheet rich silk nanofibers reported previously, these new silk nanofibers are mainly composed of amorphous structures and maintain a solution state in aqueous environments. The amorphous silk nanofibers are stable enough for storage and also metastable, making them easy to use in the further fabrication of materials through various processes. Silk scaffolds, hydrogels and films were prepared from these silk nanofiber solutions. These silk materials from amorphous nanofiber solutions show different properties and tunable performance features. Therefore, these amorphous silk nanofibers are suitable units or building blocks for designing silk-based materials.

KEYWORDS: silk, nanofiber, amorphous structure, functional materials, tissue engineering


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1. INTRODUCTION As a natural protein, silk fibroin has been widely explored as a functional material in broad range of applications from tissue engineering and drug release to microdevice realms.1-6 The regeneration of natural silk fibers into different material forms, including foams, gels,

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powders

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and adhesives

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films,

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is a prerequisite for these types of applications.

Recently, the effective fabrication of hierarchical micro-nanostructures from silk has prompted further options towards biomedical devices.17-20 However, most methods used in silk device fabrication do not fully exploit the hierarchical structure formation inherent in silk proteins, thereby potentially limiting the range of material properties that can be achieved.21-24 To overcome this limitation, a bottom-up strategy utilizing silk nanostructures in aqueous solution was pursued, however, achieving homogeneity of these nanostructures in aqueous solution remains a challenge. Several solvents, such as inorganic salts,25, 26 concentrated acids,27 and ionic liquids

28, 29

have

been used to dissolve the degummed silk fiber by destroying the extensive hydrogen-bond network among silk fibroin molecules. However these solvents also destroy the hierarchical nanostructures present in the native silk fibers, resulting in silk solutions without specific nanostructural features. Silk fibroin has been re-assembled into nanofibers in aqueous solution via a thermodynamically driven process.30 Silk nanofibers with different sizes and secondary conformations appeared in this assembly process, and these features rapidly transformed into aggregates, making this process limiting for many silk device fabrication processes.30 As a result, stable nanofibers were subsequently prepared to try and further control self-assembly in aqueous solution. Due to high charge repulsion and high beta-sheet structure, these nanofibers maintained


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a solution state at low concentrations, but transitioned to hydrogels at higher concentrations (>0.5%).31 Although various silk-based functional materials were developed based on these nanofiber systems, the low concentration threshold for the solution state as well as high charge repulsion of the silk nanofibers restricted their transformation into functionally specific material formats. Novel solvent systems with weaker hydrogen-bond-destroying capacity were also developed in order to maintain part of the nanofibril structures from natural silk fibers in the solution state.32-34 However, the silk solution immediately adopted a turbid gel state during dialysis in water upon removal of salts. Thus, no salt-free aqueous silk solution has been obtained to date to facilitate the formation of the hierarchical structures from silk nanofibers. Although a mixture of silks with various nanostructures was obtained, the mixture could not maintain a solution state in all-aqueous environments. Therefore, the goal was to fabricate homogeneous and metastable nanostructures in aqueous solution where silk maintains these structures during storage, but can be easily assembled into designed hierarchical structures through mild processes. Here we demonstrated the formation and utility of silk fibroin nanofiber solution systems with amorphous structures. A combined solvent system composed of formic acid and lithium bromide was tuned to control the degree of hydrogen bonding among the silk fibroin molecules. This aqueous silk fibroin nanofiber solution was prepared after the removal of salts and acid through dialysis. Unlike silk fibroin solutions and hydrogels previously reported,31 the aqueous silk fibroin solution in the present study had a homogeneous distribution and morphological features of nanofibrous microstructures with noncrystallized secondary structure. The nanofibers were stable enough for further fabrication processes, such as concentration steps, gelation, freezedrying and film formation.


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2. Experimental Section Silk nanofiber solution fabrication: Bombyx mori silk fibers 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. Then a new combined solvent system composed of LiBr and formic acid (FA) was developed to control the dissolution degree of degummed silk fibers for achieving silk fibroin nanofiber solution after dialysis. One gram of degummed silk fiber was dissolved in the 50 mL LiBr-FA solvent where the formic acid (98%) and lithium bromide solution (8 M) was blended at weight ratio of 1: 13.3 at 60 oC, yielding a 2 w/v% solution. The solution was dialyzed against distilled water using a dialysis tube (Pierce, molecular weight cut-off 3,500) for 3 days to remove the salt and acid. The dialyzed solution was centrifuged at 9,000 rpm for 20 min to remove silk aggregates formed during the process. The final concentration of aqueous SF solution was about 0.8 wt%, determined by weighing the remaining solid after drying. This solution was composed of nanofibers and used for characterization. Silk fibroin solution and silk nanofiber solution-hydrogel system fabrication: As a control, the silk fibroin solution as well as silk nanofiber solution-hydrogel system was also prepared according to previously reported procedures.30,31 B. mori 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 at 60 oC, yielding a 20% (w/v) solution. This solution was dialyzed against distilled water, using Slide-a-Lyzer dialysis cassettes for 72 h to remove the salt. The solution was optically clear after dialysis and was centrifuged at 9,000 rpm for 20 min at 4oC to remove silk aggregates. The final concentration of aqueous silk solution was about 6 wt%, determined by weighing the remaining solid after drying.


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To prepare silk nanofiber hydrogels, the fresh silk fibroin solutions were treated by a slow concentration–dilution process. The solution (6 wt%) was slowly concentrated to about 20 wt% over 24 h at 60oC to form metastable nanoparticles, and then diluted to below 2 wt% with distilled water. The diluted silk solution was incubated for about 24 hours at 60oC to induce nanofiber hydrogel formation. The silk nanofiber solution, silk fibroin solution and silk nanofiber hydrogel were termed SF-FA, SF, SF-gel, respectively. Concentrated silk nanofiber solution (CSF-FA): Because the original concentration of silk nanofiber solution was too low to prepare concentrated silk nanofiber solutions, 40 ml of 0.8wt% silk nanofiber solution was dialyzed against 1 L of 10 wt% polyethylene glycol (PEG, 10,000 g/mol) solution at 4oC by using Slide-a-Lyzer dialysis cassettes (MWCO 3500).35 When the volume was less than one-quarter of the original, the concentrated silk nanofiber solution was slowly collected and the concentration determined. All solutions were stored at 4oC before use to avoid gelation. Fabrication of silk films: To prepare silk films, 2ml of silk fibroin solution /silk nanofiber solution (2wt%) was cast on polystyrene Petri dishes (diameter 35mm) and dried overnight at room temperature in fume hood/60 oC without a lid. Fabrication of silk scaffolds: To prepare silk scaffolds, the silk solutions were poured into cylindrically-shaped containers (diameter 16 mm). The containers were placed at -20 oC for 24 h to freeze the samples and lyophilized for about 72 h to achieve silk porous scaffolds. Modulated methanol/ethanol/water annealing processes were applied to induce gradual transformations from random to silk I/silk II structures. The scaffolds were immersed in 80% (v/v) methanol/75 %(v/v) ethanol for 30 min to induce crystallization and placed in desiccators filled with water


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solutions with a 25 in. Hg vacuum (85 KPa) for 6 h to produce water-insoluble scaffolds.36 The methanol, ethanol and water annealing scaffolds were termed SF-MA/SF-FA-MA, SF-EtOH/SFFA-EtOH and SF-WA/SF-FA-WA, repectively. Electrospun silk nanofibers: A high electric potential of 25 kV was applied to a droplet of SF solution at the tip of a syringe needle (0.8 mm in internal diameter).32 The electrospun nanofibres were collected on flat aluminum foil which was placed at a distance of 10 cm from the syringe tip. A constant volume flow rate of 0.2 ml h-1 was maintained using a syringe pump. HRP-crosslinked hydrogels: HRP solutions (1,000U/ml, 0.05ml) were added into SF/SF-FA (2 wt %-4 wt %, 5mL) with stirring, then an aqueous solution of H2O2 (165mM, 0.05ml) was added into the blend solution and kept stirring for 3 minutes and incubated at 37oC until hydrogel formation.37 The hydrogels formed by SF and SF-FA were termed HSF and HSF-FA, respectively. Silk nanofiber hydrogels: To prepare silk nanofiber hydrogels, the fresh silk nanofiber solutions (0.1~2wt%) were incubated for about 4 h at 60oC to induce hydrogel formation. Table S1 lists the samples abbreviations and their preparation methods. Characterization: The sizes and zeta potentials of silk fibroin solutions were characterized by Zetasizer (Nano ZS, Malvern, Worcestershire, UK) at 25oC. The morphology of samples was examined by AFM (Nanoscope V, Veeco, NY, USA) and SEM (S-4800, Hitachi, Tokyo, Japan). The secondary conformations were studied with a Jasco-815 CD spectrophotometer (Jasco Co., Japan), Nicolet FTIR 5700 spectrometer (Thermo Scientific, FL, USA) and Raman spectrometer (Renishaw, 633 nm diode laser). Thermal stability and dynamic properities were characterized by TA Instrument Q2000 DSC (TA Instruments, New Castle, DE, USA) and Rheometer


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(AR2000, TA Instruments, New Castle, USA), respectively. More details can be found in the supporting information. Statistical methods: All statistical analyses were performed using SPSS v.16.0 software. Comparison of the mean values of the data sets was performed using one-way AVOVA. Measures are presented as means ± standard deviations, unless otherwise specified. P < 0.05 was considered significant. 3. Results and Discussion 3.1. Microstructure of silk nanofiber solution. When One gram of degummed silk fiber was dissolved in the 50 mL LiBr-FA solvent where the formic acid (98%) and lithium bromide solution (8 M) was blended at weight ratio of 1: 13.3 to form the solvent system, an aqueous silk solution was prepared with concentration of 0.8 wt% after dissolution and dialysis (Figure 1). The nanofiber solution was termed SF-FA while the silk solutions and silk nanofiber hydrogels prepared through previous processes were termed SF and SF-gel, respectively. Different to SF and SF-gel (Figure 2a, d, g, i and b, e, h, k), the aqueous solution was composed completely of silk nanofibers with diameters of 10-20 nm and lengths of 200-350 nm (Figure 2c, f, i, l), close to natural silk features in vivo in silkworms prior to spinning into fibers.38, 39 Then a series of optimizing processes were developed and indicated that the formation of these nanofiber solutions was restricted within a specific proportion of formic acid, lithium bromide and water (the weight ratio of formic acid (98%) and lithium bromide solution (8 M) was at 1: 13.3). Higher ratios of lithium bromide and formic acid resulted in the destruction of the nanofiber structure, while lower contents of formic acid and lithium bromide resulted in hydrogel formation during the dialysis process. The amount of silk also had a significant influence on the


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features. Silk nanofiber solutions were generated with concentrations below 1 wt%; but higher silk concentrations resulted in hydrogel formation as opposed to the nanofiber solutions.

Figure 1.Schematic of the preparation of silk nanofiber solution and the fabrication of functional materials from the nanofiber solution.

3.2. Structure analysis. Further investigation into the secondary structures of the silk nanofibers was pursued based on the processing windows identified above. CD curves of silk nanofibers in aqueous solution indicated that amorphous states dominated, in contrast to silk nanofibers reported previously which were mainly composed of beta-sheet.31 The secondary structures of silk nanofibers were also slightly different from that of fresh silk solution (without silk nanofiber formation) prepared via previous 9.3 M lithium bromide solution system,40 suggesting more


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intermediate conformations in the silk nanofibers (Figure 3a). FTIR and Raman spectra confirmed the intermediate conformation composition of the silk nanofibers (Figure 3b, c). The amorphous states of silk nanofibers endowed better hydrophilic properties. Although the nanofiber solution with concentrations above 1% can not be prepared directly, nanofiber solutions (0.8%) could be concentrated up to 8% without hydrogel formation. The concentrated silk nanofiber solution was termed CSF-FA. CD, FTIR indicated that the amorphous structures were maintained in the concentrated silk nanofiber solutions (Figure S1a, b) while more intermediate conformations formed after the concentration process. In previous studies, silk nanofibers were self-assembled through several different methods,

31, 41, 42

but these nanofibers

were mainly composed of hydrophobic beta-sheet structures, resulting in rapid gelation above 0.5 wt%. Further fabrication is difficult for these gelled silk nanofibers. The amorphous silk nanofibers reported here maintained their solution state at higher concentrations (above 8 wt %), suitable for further materials assembly. The stability of these silk nanofiber solutions was investigated. When placed at 4oC and 25oC, the solutions transformed into hydrogels after 3 weeks and 1 week, while fresh silk solution prepared through previous processes maintained their solution state for at least 8 weeks and 4 weeks, respectively, at the same conditions. The results indicated that silk nanofiber solutions were more metastable. Although the nanofiber solutions were more unstable than the fresh silk solution prepared through 9.3 M lithium bromide, they could be freeze-dried and then stored at room temperature for at least three months, then re-dissolved in water to form amorphous nanofiber solutions (Figure S2). Therefore, the metastable nanofibers had enough stability for storage and further utility towards material fabrication.


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Figure 2.The morphology of silk prepared through various processes: (a)-(c) Photograph; (d)-(f) AFM images; (g)-(i) SEM images and (j)-(l) DLS size distribution of various samples. The samples are as follows: Left, amorphous silk solution prepared through the regular LiBr solvent reported previous, SF; Middle, silk nanofibers with high beta-sheet content prepared through a slow concentration–dilution process, SF-gel; Right, amorphous silk nanofiber solution prepared through the present LiBr-formic solvent, SF-FA.


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Figure 3.The secondary structures of silks prepared by various processes: (a) CD curves; (b) FTIR spectra; (c) Raman spectra and (d) Standard DSC curves. The results demonstrated the different secondary compositions between SF-FA, SF and SF-gel. The samples are as follows: SF-FA, amorphous silk nanofiber solution prepared through the present LiBr-formic solvent; SF, amorphous silk solution prepared through the regular LiBr solvent reported previous; SF-gel, silk nanofibers with high beta-sheet content prepared through a slow concentration–dilution process; CSF-FA, fresh amorphous silk nanofiber solution were concentrated to 8%.

3.3. Thermal stability. DSC was used to study the thermal stability of the silk nanofibers including SF, SF-FA, CSF-FA and SF-gel (Figure 3d). SF, SF-FA and CSF-FA showed a nonisothermal crystallization at around 210-220oC, indicating their metastable property and tendency toward crystallization when the temperature was above the crystallization peak. Compared to SF, the crystallization peak was similar for SF-FA and moved to 207.4oC for CSF-


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FA, which confirmed the intermediate secondary structures in the concentrated silk nanofiber conditions, making crystallization easier. The SF-FA and SF showed similar degradation peaks (262.8 oC), but the CSF-FA showed degradation peaks at a higher temperature (266.4 oC), suggesting more stable structure formation after crystallization in the CSF-FA samples. Figure S3 illustrats the reverse DSC curves for SF, SF-FA, CSF-FA and SF-gel. SF-FA and CSF-FA had a higher heat capacity at Tg (1) than SF, indicating that SF-FA and CSF-FA had stronger interactions between water and silk which might be due to the higher content of silk I.43 Tg (2) of SF-FA and CSF-FA were about 180 oC, slightly higher than SF(~179 oC) and lower compared to Tg(2) (~191 oC) of SF-gel, further indicating the metastable properties. Table 1. Zeta Potential of SF, SF-gel and SF-FA SF zeta potential(mv)

SF-gel

-35.2±1.25

-48.5±2.04

SF-FA -7.6±0.52

3.4. Dynamic performance. Dynamic oscillatory and shear rheology were used to investigate the influence of nanofibrous structures and different intermediate secondary conformations on solution viscoelasticity, (Figure 4a-d). The rheological behavior of silk solutions at concentrations of 2, 1, 0.5, and 0.1% showed that shear thickening behavior was observed in SF and SF-FA at low shear rates. For the SF-gel, however, shear thinning behavior was detected when concentrations were above 0.1%, different from SF and SF-FA. We assumed that the weak intermolecular repulsive forces in SF and SF-FA (Table 1) resulted in entangled molecules and increased viscosity. Shear thinning behavior was also observed at higher shear rates (≥0.02 s−1)


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likely due to flow-induced molecular alignment.32,44 At high shear rates (~100 s-1), a second shear thickening behavior in SF-FA appeared due to stress induced by crystallization and phase separation.45,46 The dynamic oscillatory results (Fig 4 e-h) confirmed the behavior of the different samples. A solution-hydrogel transition appeared for the silk nanofibers with high betasheet content while typical viscoelastic fluid behavior was maintained for the amorphous silk nanofibers and fresh silk solution when silk concentration was increased from 0.1% to 2%.


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Figure 4. The viscoelastic properties of silk solutions and hydrogels prepared through different processes: (a)-(d) Viscosity shear rate profiles of different silk solutions; (e)-(h) Storage modulus (G', solid symbols) and loss modulus (G'', open symbols) versus frequency of different silk solutions. The samples are as follows: SF-FA, amorphous silk nanofiber solution prepared through the present LiBr-formic solvent; SF, amorphous silk solution prepared through the regular LiBr solvent reported previous; SF-gel, silk nanofibers with high beta-sheet content prepared through a slow concentration–dilution process.

3.5. Properties of silk materials. These new nanofibrous structures, intermediate conformations and viscoelastic properties of the nanofiber solutions offered the possibility to design silk-based materials. The nanofibers had the capacity to further assemble/transform into different conformations due to their metastable property. Using the same processes and treatments reported previously,36,

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silk scaffolds and films with various hierarchical micro-nanofibrous

structures and secondary conformations were developed (Figure S4, 5 and Table S2). The amorphous silk nanofibers could also be transformed into reversible hydrogel-solution nanofiber systems with high beta-sheet content by placing them at 60oC for about 4 h (Figure S6). More importantly, the silk nanofiber solutions provided the possibility to develop new types of functional silk materials. For example, silk fibers were electrospun from the silk nanofiber solution with concentrations as low as 4%, in contrast to previous silk solutions where the minimal concentration for electrospinning was above 20% (Figure 5a, b, S7). HRP crosslinked silk hydrogels with high stiffness were also prepared from the amorphous silk nanofiber solutions. Under the same conditions and silk content the stiffness of silk hydrogels was three times higher than those prepared from previous silk solutions (Figure 5c-g). These nanofiberbased silk materials are useful precursor states of silk for designing different functional


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materials, particularly in the fields such as tissue engineering, drug release and other biomedical applications. 4. Conclusion In summary, we report the formation of a novel silk nanofiber solution prepared by carefully tuning of the dissolution solvent. The silk nanofibers in these solutions maintained an amorphous state, providing suitable stability for storage and metastable properties for further fabrication. Unlike silk nanofibers composed of high beta-sheet structure, these new silk nanofibers maintained their solution state at high concentrations. Different materials formed from these solutions, including hydrogels, scaffolds and films, showed various secondary conformations, nanostructures with useful features for functional materials.


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Figure 5. Electrospinning nanofibers and HRP cross-linked hydrogels prepared from various silk solutions: (a) Electrospun silk nanofiber formation from 4% SF-FA; (b) The failure to form electrospun silk nanofibers from 4% SF solution; (c) Microstructure of the HRP crosslinked hydrogel from 2% SF-FA solution; (d) Microstructure of the HRP crosslinked hydrogel from 2% SF solution; (e) The nanostructure of the pore layer of the HRP crosslinked hydrogel from 2% SF-FA solution; (f) The nanostructure of the pore layer of the HRP crosslinked hydrogel from 2% SF solution; and (g) Storage modulus (G', solid symbols) and loss modulus (G'', open symbols) versus frequency of HRP crosslinked silk hydrogels formed from SF-FA and SF. The stiffness of the hydrogel from SF-FA is three times higher than that from SF. The samples are as follows: HSF-FA, silk hydrogels formed by SF-FA; HSF, silk hydrogels formed by SF.

Supporting Information. Complete descriptions of experimental procedures; conformations and microstructures of silk in the concentrated process and re-dissolved silk nanofiber solutions; temperature-modulated DSC scans (TMDSC) of different silk solutions; properties of silk scaffolds, films, hydrogels and eletrospun nanofibers derived from SF-FA and SF solutions. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Qiang Lu, E-mail: [email protected] Notes


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The authors declare no competing financial interests. ACKNOWLEDGMENT The authors thank National Basic Research Program of China (973 Program, 2013CB934400), NSFC (21174097) and the NIH (R01 DE017207, P41 EB002520). We also thank the Excellent Youth Foundation of Jiangsu Province (BK2012009) for support of this work.


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REFERENCES (1) Omenetto, F. G.; Kaplan, D. L. Science 2010, 329, 528-531. (2) Rockwood, D. N.; Preda, R. C.; Tuna,Y.; Wang, X. Q.; Lovett, M. L.; Kaplan, D. L. Nat. Proto. 2011, 6, 1612-1631. (3) Bellas, E.; Lo, T. J.; Fournier, E. P.; Brown, J. E.; Abbott, R. D.; Gil, E. S.; Marra, K. G.; Rubin, J. P.; Leisk, G. G.; Kaplan, D. L. Adv. Healthc. Mater. 2014, 4, 452-459. (4) Seib, F. P.; Jones, G. T.; Rnjak-Kovacina, J.; Lin, Y. N.; Kaplan, D. L. Adv. Healthc.Mater. 2013, 2, 1606-1611. (5) Hu, T.; Kaplan, D. L.; Omenetto, F. G. Adv. Mater. 2012, 24, 2824-2837. (6) Dae-Hyeong, K.; Jonathan, V.; Amsden, J. J.; Xiao, J. L.; Vigeland, L.; Kim, Y. S.; Blanco, J. A.; Panilaitis, B.; Frechette, E. S.; Contreras, D. Nat. Mater. 2010, 9, 511-517. (7) Han, H. Y.; Ning, H. Y.; Liu, S. S.; Lu, Q.; Fan, Z. H.; Lu, H. J.; Lu, G. Z.; Kaplan, D. L. Adv. Funct. Mater. 2016, 26, 421-432. (8) Felice, V. D.; Serradifalco, C.; Rizzuto, L.; De Luca, A.; Rappa, F.; Barone, R.; Di Marco, P.; Cassata, G.; Puleio, R.; Verin, L. J. Tissue. Eng. Regen. M. 2015, 9, E51-E64. (9) Ling, S. J.; Li, C. X.; Adamcik, J.; Shao, Z. Z.; Chen, X.; Mezzenga, R. Adv. Mater. 2014, 26, 4569-4574. (10) White, J. D.; Wang, S.; Weiss, A. S.; Kaplan, D. L. Acta Biomater. 2014, 14, 1-10. (11) Kapoor, S.; Kundu, S. C. Acta. Biomater. 2016, 31, 17-32.


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Table of Contents Graphic


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Amorphous Silk Nanofiber Solutions for Fabricating Silk-Based

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