Self-Assembling Silk-Based Nanofibers with Hierarchical Structures

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Self-assembling Silk-based Nanofibres with Hierarchical Structures Zhuping Yin, Feng Wu, Zhaozhu Zheng, David L Kaplan, Subhas C. Kundu, and Shenzhou Lu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.7b00442 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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The facile hydrophilicity-driven approach provides further insight into SF self-assembly and offers new tools for the sustainable recapitulation of high performance materials for engineering applications.

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Self-assembling Silk-based Nanofibres with Hierarchical Structures Zhuping Yin†, Feng Wu†, Zhaozhu Zheng†, David L. Kaplan†,§, Subhas C. Kundu‡, Shenzhou Lu*,† National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou, 215123, China § Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States †

3Bs Research Group, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, AvePark - 4805-017 Barco, Taipas, Guimaraes, Portugal. ‡

*

Corresponding author: Prof. Shenzhou Lu

E-mail: [email protected] ABSTRACT: Self-assembling fibrous supramolecular assemblies with sophisticated hierarchical structures at the mesoscale are of interest from both fundamental and applied engineering. In this paper, the relatively hydrophilic domains of silk fibroin (HSF) were extracted and used in studies of self-assembly. The HSF fraction spontaneously self-assembled into nanofibers, 10 to 100 μm long and 50 to 250 nm in diameter, within 2 to 8 h in aqueous conditions. Interestingly, these HSF nanofibers consisted of dozens of nanofibrils arranged in a parallel organization with assembled diameters of ~30 nm, similar to the sophisticated hierarchical structure observed in native silk fibers. Dynamic morphology and conformation studies were carried out to determine the mechanisms underlying the HSF self-assembly process at both the nanoscale and mesoscale. The HSF self-assembled into nanofibers in a bottom-to-up manner, from “sticky” colloid particles to cylindrical globules, to form nanofibrous networks. Owing to the enhanced HSF self-assembling kinetics and the hierarchical structure of HSF nanofibers, this hydrophilicity-driven approach provides further insight into silk fibroin (SF) self-assembly in vivo and also offers new tools for the recapitulation of high performance materials for engineering applications. KEYWORDS: silk fibroin, hydrophilicity, self-assembly, nanofiber, hierarchical 1. Introduction Sustainable native silk fibers spun by Nephila pilipes spiders, mulberry and nonmulberry silkworms are some of the toughest and versatile soft materials, 1


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possessing a unique combination of great tensile strength and extensibility, and features even superior to the best synthetic fibers, like Kevlar and carbon fiber[1-5]. The sophisticated hierarchical structures of native silk fibers support these superior mechanical properties[6-10]. Native silks consist of closely-packed and parallel nanofibrils, ~30 nm in diameter, along with β-sheet crystallites embedded in a more amorphous silk fibroin molecular phase[1,11]. Attempts to reconstruct the extraordinary properties of native silks via regeneration of nanofibrils via self-assembly have been reported[12-15]. Nevertheless, the recapitulation of the performance of native silk fibers via nonnative processes via a facile aqueous environment remains a challenge. Further, the sophisticated hierarchical morphology at the mesoscale inside these fibrous systems also remains elusive, as does the elongation of the self-assembling nanofibrils derived from reconstituted silk dopes. Amphiphilic compounds, like peptides and derivatives, can spontaneously self-assemble into nanofibrils[12-13], nanotubes[16-17] and rods[18], at ambient temperature and pressure, with the hydrophilic blocks arranged on the surfaces of nanofibrils and the hydrophobic blocks closely-stacked in the cores. These self-assembling processes are driven by hydrophobic interactions[12-13], π-π stacking[19], and/or hydrogen bonding (H-bonds)[19-23]. Paramonov et al. reported the single tail peptide-amphiphiles self-assembled into nanofibrils and formed semi-solid gels with a 3-dimensional (3D) nanofibrous network in aqueous environments. The process was driven by hydrophobic interactions between the aliphatic tails for the formation of the nanofibril cores and H-bonds between the hydrophilic peptide domains for stabilization of the nanofibrils[12]. Guo et al. prepared bundles of nanoribbons and successfully controlled the hierarchical aggregated structures of these nanoribbons by varying pH[19]. Bombyx mori silk fibroin (SF) consists of a heavy chain (H-chain, Mw ~390 kD) and a light chain (L-chain, Mw ~26 kD) that are chemically crosslinked by a disulfide bond. H-chains consist of regular hydrophobic, crystalline regions and hydrophilic, noncrystalline domains[24-26]. Generally, the self-assembly of silk fibroin arises from the intramolecular and/or intermolecular β-sheet crystals via hydrophobic interactions and hydrogen bonds[14, 25-27]. SF self-assembly is a thermodynamically driven process influenced by hydrophilic-hydrophobic interactions, charge, molecular mobility and concentration[14, 25-27]. Liu et al. has reported that silk fibroin molecules self-assemble into a nanofibrils by decreasing the hydrophilicity of the environment to narrow the gap between solvent (water) and solute (SF) to unfold/extend the SF chains in water, via additive alcohol[28-29]. In addition, Li et al. enhanced the kinetics of self-assembly by increasing the length of the hydrophilic domains in rod-coil block amphiphiles to decrease interfacial enthalpy[30]. Although there are some studies about nanofiber self-assembly from reconstituted SF in vitro, the elongation process and formation of 2



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hierarchical structure from these nanofibers remains undefined. In this paper, the goal was to give a facial and effective approach for regenerating SF nanofibers. Specifically, nanofiber self-assembly with respect to hydrophilicity of silk-based peptides was the focal point, in order to bridge the gap between peptides (solute) and water (solvent), but not decrease the hydrophilicity of the solvent. The peptide sequences with higher hydrophilicity than the SF, were extracted from regenerated SF by immersing dried condensates of the native protein in deionized water. Then the extract was collected and the insoluble residue discarded in this immersing system. The results demonstrated that HSF self-assembled into cylindrical nanofibers within hours in facile aqueous environments, resulting in HSF nanofibers 10 to 100 μm in length, 50 to 250 nm in diameter, and consisting of bundles of nanofibrils with an average diameter of ~30 nm. These morphological features are remarkably similar to the hierarchical structure of native silk fibers. Due to the enhanced HSF self-assembling kinetics and the formation of HSF nanofibers with hierarchical structure, this hydrophilicity-driven method may provide a strategy for further insight into silk fibroin (SF) self-assembly in vivo, as well as offer new tools for the recapitulation of high performance materials in ambient environment. 2. Methods Preparation of silk fibroin solution B. mori cocoons were boiled three times in an aqueous solution of 0.05% Na2CO3 at 98 to 100 oC, 30 min each time, and rinsed with deionized water (DI water) to remove the sericin from silk fiber surfaces. The rinsed silk fibers were then dried at 60 o C for approximately 6 h to remove the free water. Then the degummed and dried silk fibers were thoroughly dissolved in aqueous lithium bromide solution (9.3 M LiBr) at 60 oC for 1 h. The dissolved silk fibroin solution was dialysed with running DI water at 4 oC for 72 h using Slide-a-Lyzer dialysis cassettes (Pierce, MWCO: 8 to 12 KD). The purified silk fibroin solution was centrifuged at 4,500 rpm for 10 minutes to remove aggregates. The final silk fibroin concentration was approximately 6% (w/v), determined by weighing the solution mass before and after complete drying[31-33]. Preparation of hydrophilic silk fibroin solution The purified silk fibroin solution (6% w/v) was cast in polyethylene culture dishes at 0.25 ml/cm2 and dried at 25 oC and 65% humidity until constant weight. The dried silk membranes were immersed in DI water at 37 oC for 1 h and then centrifuged at 4,500 rpm and 4 oC for 10 minutes to precipitate the insoluble residues to collect the aqueous, lyotropic hydrophilic blocks. The concentration of the HSF sol was ~1.0 % (w/v). The soluble silk fibroin is termed hydrophilic blocks from silk fibroin (HSF), while the insoluble fraction is termed hydrophobic silk fibroin (ISF). 3


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Preparation of self-assembling HSF nanofibers The HSF sol was diluted to 0.75%, 0.50%, 0.25% (w/v, pH: 7.0 ± 0.2) and these solutions were separately cast into transparent vials with a volume of 3 ml. The assembly and gelation process was then conducted at 4, 25, 37, 50 and 100 oC at 0, 2, 4, 8, 12, 24 and 48 h. Field Emission Scanning Electron Microscopy (FE-SEM, Oberkochen, Germany) and Atomic Force Microscopy (AFM, Veeco, Nanoscope V, NY) were used monitor HSF self-assembly for nanoscale and/or mesoscale features at 0, 2, 4, 8, 12 and 24 h. The HSF samples were placed in liquid nitrogen dropwise for quick-freezing and to avoid changes in mesoscopic morphology, followed by freeze-drying in vacuum. The freeze-dried samples were sputter coated with gold prior to imaging by SEM[34-35]. For AFM, the samples were diluted to ~0.002% (w/v) to avoid masking the original morphology from closely-stacked HSF aggregates. A 5 μl aliquot of the diluted HSF systems was dropped on cleaned mica surfaces (10 × 10 mm) and thoroughly dried with N2 prior to morphological characterization by AFM in air. A 115 μm long silicon cantilever with a spring constant of 0.4 N m−1 was used, together with a scanasyst-air mode at 0.5 to 1 Hz scan rate[13,15,35]. Inverted dynamic optical detection was applied to evaluate HSF self-assembly at the macroscale. The gelation time-point was determined when the HSF sol did not fall from an inverted vial. In addition, a Synergy HT Multi-Mode Microplate Reader (Bio-Tek Instruments, USA, ELIASA) was also used to measure the dynamics of HSF self-assembly at 37 oC by testing the optical absorption of 1 ml of HSF at 550 nm. The HSF sol was added to a 24-well plate for measurements, with 5 samples running for each group[29,36]. The dynamic rheological features of the HSF systems were measured at 37 oC using a Rheometer (AR2000, TA Instruments, New Castle, USA) equipped with a 20 mm cone plate (Ti, 20/1°) and a Peltier temperature control system. A solvent trap and a low-viscosity mineral oil were used to avoid water evaporation from the samples. The strain was set to 1% and the frequency sweep was collected over the angular frequency range of 0.1 to 100 rad/s[26,37]. A Nano-ZS90 particle sizer and a zeta potentiometer analyser (Malvern Instruments, UK)[36] were used to record the particle size distribution and its corresponding zeta potential change during the HSF self-assembling process at 37 oC at pH of 7.0 ±0.2. Dynamic conformations and aggregate structures in the HSF system CD spectra were used to monitor dynamic HSF conformations in aqueous solutions using a JASCO-815 spectrometer (Jasco Co., Japan). The CD spectra were recorded over the range of 190 to 250 nm. A sandwich quartz cell with a 0.1 mm path length 4



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was used for sample loading and each sample was measured three times for final average accumulations[38-40]. The fluorescence measurements were performed using an Edinburgh Instrument FLS920 at 37 oC. Tetrahydrothiophene (ThT)[41-42], sensitive to β-sheet structure, was used as a probe at a concentration of 5 mM in HSF sol. The excitation wavelength was 420 nm and emission wavelength ranges were 430 to 800 nm, respectively. The excitation and emission slit widths were set to 3 nm. The emission spectra were recorded from the excitation of the sample with a 1 cm optical path. The HSF systems were assessed at time points (0, 2, 4, 8, 12, 24, 48 h) at 37 oC. Samples after a 24 h self-assembling process at 4, 25, 50 and 100 oC were rapidly frozen in liquid nitrogen and dried in a 4 oC freeze dryer. The dried samples were dispersed to a fine powder and potassium bromide (KBr) pellet processing was carried out prior to the Fourier transform infrared spectroscopy (FTIR) using a Nicolet 5700 FTIR (Nicolet Co., USA). The IR absorption spectra were recorded at 400 to 4000 cm-1. Deconvolution was performed using a Gauss algorithm with a smoothing function with an amp value of 1.0% and 1.5%, respectively. FTIR spectra were curve-fitted to measure the original areas of each amide I region[43-44]. X-ray diffraction (XRD) was performed to characterize the aggregate structures formed in the HSF systems at the nanoscale using an X-ray diffractometer (X0 Pert-Pro MPD, PANalytical, Almelo, Holland) with Cu Ka radiation. The XRD patterns were recorded in the region of 2θ from 5° to 45° at a 10°/min speed, 40 KV and 35 mA. Thermal properties were measured with a TA Instruments Q100 DSC under a nitrogen atmosphere (50 mL/min flow). The powdered HSF samples weighed ~2 mg and were loaded into alumina crucibles for heating from 35 oC to 350 oC (2 oC /min)[45-46]. Amino acid analysis To determine the differences in hydrophilicity between HSF and SF, composition amino acid analysis were conducted. The HSF sol and the corresponding ISF residues were thoroughly dried at 110 oC for 24 h. Then ~100 mg of these dried condensates was dissolved in aqueous hydrochloric acid solution (GR, 6 N, 10 ml) in vials, followed by elimination of air with running N2 for 15 s. Afterwards, the sealed vials were placed in an 110 oC oven for hydrolysis for 24 h. The cooled hydrochloric acid solution samples (0.1 ml) were dried thoroughly in a 60 oC oven and redissolved in 0.02 N HCl solution at a 40 mg/l concentration. Three samples were run for each group. The samples were run through filter membranes with a 0.22 μm pore size prior to amino acid analysis using an L-8900 High Speed Amino Acid Analyzer. Mole 5


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percentage differences between HSF and ISF groups were determined via Eq1:

Re lative Percentage s Difference ( RPD , %) 

ISF  HSF  100% ISF

(1)

Where HSF is the mole percentage of one amino acid residue in the HSF group and ISF is the mole percentage of one amino acid residue in the ISF group. Surface tension testing A DataPhysicsTM DCAT-21 surface tension instrument was used to characterize HSF and SF sol via the Wilhemy plate method for surface tension[36,47-48]. The HSF and SF sol were poured into a circular thermostated dish (60 cm2, 25 ± 0.5 oC) separately for surface tension measurements. The results were determined when the standard deviation of the SFT was less than 0.03 mN m-1 in the last 50 cycled measurements and the accuracy was 0.001 mN m-1. The concentrations of HSF and SF sol were 0.25%, 0.5% and 0.75% (w/v). Contact angle measurement Contact angle measurements were also performed for hydrophilicity using a contact angle meter (Tantec, CAM-Plus Micro, United States). The sampled droplet (10 μl) was deposited on a polydimethylsiloxane (PDMS) film for imaging after sitting to allow equilibrium for 1 min. The contact angle was the accumulated average of three parallel measurements for each group[49-51]. Molecular weight analysis Molecular weights of HSF and SF were measured using Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A 1 ml sample of the initial HSF and/or SF sol were incubated at 37 oC with the additive dithiothreitol (DTT, 500 mM) and urea (8 M) for 2 h. Then 15 ul of these samples (1.0, w/v) were loaded into 8% (w/v) polyacrylamide gels (SigmaAldrich) and run in reducing conditions at 100 V, with protein molecular weight markers for reference. Coomassie brilliant blue was used for staining and visualization of the proteins. Molecular weight distributions were determined by pixel intensity as a function of lane position using Image J software. 3. Results Fabrication of self-assembling HSF nanofibers Generally, SF shows mesoscopic colloidal particle morphology in aqueous conditions. The repetitive hydrophobic blocks in the SF H-chain for crystallization are usually arranged inside the micelles while the non-repetitive hydrophilic blocks are 6



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exposed outside for interaction with water, which makes the aqueous SF sol a metastable thermodynamic system[14, 26-27]. In this paper, self-assembling silk-based nanofibers were achieved by narrowing gap of hydrophilicity between solute (silk-based polypeptides) and solvent (water) for an unfolded/extended molecular conformation, which can decrease interfacial enthalpy[30] to enhance self-assembly kinetics. The HSF was extracted via immersing SF condensates into DI water for 1 h and collecting the extract after a 4,500 r/min centrifugation step (Fig. 1). The resulting flowing HSF sol (0.5%, w/v) easily self-assembled into semi-solid gels within several hours at 37 oC. Interestingly, there was a significant mesoscopic morphological transition from micelle to nanofibers, with a diameter of 50 to 250 nm and a length up to 100 μm (Fig. 1). These morphological features were significantly longer than previous reports of silk nanofibers (

Self-Assembling Silk-Based Nanofibers with Hierarchical Structures

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