Graphene Oxide Nanocomposite

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Interface-Rich Materials and Assemblies

Tough photocrosslinked silk fibroin/graphene oxide nanocomposite hydrogels Rajkamal Balu, Shaina Reeder, Robert Knott, Jitendra Mata, Liliana deCampo, Naba Kumar Dutta, and Namita Roy Choudhury Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01141 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Tough photocrosslinked silk fibroin/graphene oxide nanocomposite hydrogels Rajkamal Balu,† Shaina Reeder,‡ Robert Knott,§ Jitendra Mata,§ Liliana de Campo,§ Naba Kumar Dutta,*,† and Namita Roy Choudhury*,† †

School of Engineering, RMIT University, Melbourne, VIC 3001, Australia.

‡

School of Chemical Engineering, University of Adelaide, Adelaide, SA 5005, Australia.

§

Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology

Organisation, Sydney, NSW 2232, Australia.

KEYWORDS Silk fibroin; Graphene oxide; Photocrosslinking; Nanocomposite hydrogels; Mechanical properties; Hierarchical structure

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ABSTRACT The development of protein-based hydrogels for tissue engineering applications is often limited by their mechanical properties. Herein we present the facile fabrication of tough regenerated silk fibroin (RSF)/graphene oxide (GO) nanocomposite hydrogels by a photochemical crosslinking method. The RSF/GO composite hydrogels demonstrated soft and adhesive properties during initial stages of photocrosslinking (< 2 min), which is not observed for pristine RSF hydrogel, and rendered tough and non-adhesive hydrogel upon complete crosslinking (10 min). The composite hydrogels exhibited superior tensile mechanical properties, increased β-content and decreased chain mobility compared to that of the pristine RSF hydrogels. The composite hydrogels demonstrated Young’s modulus as high as ~8 MPa, which is significantly higher than native cartilage (~1.5 MPa), and tensile toughness as high as ~2.4 MJ/m3, which is greater than that of electroactive polymer (EAP) muscles and at par with RSF/GO composite membranes fabricated by layer-by-layer (LbL) assembly. Small angle scattering study reveals the hierarchical structure of photocrosslinked RSF hydrogels to comprise randomly distributed water poor (hydrophobic) and water rich (hydrophilic) regions at the nanoscale, whereas water pores and channels exhibiting fractallike characteristics at the microscale. The size of hydrophobic domain (containing β-sheets) was observed to increase slightly with GO incorporation and/or alcohol post-treatment, whereas the size of hydrophilic domain (inter-sheet distance containing random coils) was observed to increase significantly, which influences/affects water uptake capacity, crosslink density and mechanical properties of hydrogels. The presented results have implications for both fundamental understanding of the structure–property relationship of RSF-based hydrogels and their technological applications.


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INTRODUCTION Protein-based hydrogels are three-dimensional network structures commonly formed by physical or chemical crosslinking of protein molecules in concentrated solutions, and are capable of absorbing large amount of water.1 Hydrogels made of proteins derived from natural sources such as insect and animal include silk fibroin, resilin, elastin, gelatin and collagen.2 The development of natural protein-based hydrogels for tissue engineering applications is often limited by their sealing and mechanical properties. Silk fibroin is the major protein of silk cocoon produced by silkworms (e.g., Bombyx mori) and has been widely recognized as a promising biomaterial due to its excellent biocompatibility, ability to support cellular interactions, high mechanical strength and controlled biodegradability.3 Silk fibroin can be regenerated from silk cocoons through chemical processing methods,4 and hydrogels made from regenerated silk fibroin (RSF) have shown a great variety of biomedical applications.5 While RSF hydrogels can be formed by both physical and chemical crosslinking mechanisms, chemically crosslinked RSF hydrogels have shown higher elasticity and mechanical properties on hydration, which is attractive for tissue engineering.5 Recently, we reported a fast and facile fabrication of RSF and RSF-based hybrid hydrogels in aqueous medium by a ruthenium catalyst mediated photocrosslinking of tyrosine amino acids in RSF.6-8 The fabricated hydrogels exhibited stem cell biocompatibility and storage modulus in the range of megapascal.6-8 However, the hydrogels lacked adhesion properties like mussel adhesive proteins (MAPs)9 and high mechanical strength of RSF fibers10 that are desirable for tissue

engineering

applications.

The

MAPs

consist

of

amino

acid

L-3,4-

dihydroxyphenylalanine (L-DOPA), which aid in the adhesion of MAPs to organic and inorganic surfaces in wet environments.11 Inspired by MAPs, protein and polypeptide based adhesives have been previously reported.12, 13 Silk fibroin-based adhesive networks have been reported through chemical conjugation of RSF with polyethylene glycol and dopamine


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(derived from L-DOPA),12 whereas elastin like polypeptide based underwater adhesive have been reported through enzymatic conversion of tyrosine to DOPA.13 However, the reported procedures are time consuming, which takes around 8 h to 1 week for sample preparation.12, 13

A general principle towards development of tough hydrogels is to implement significant mechanical energy dissipation mechanisms such as fracture of polymer chains, reversible crosslinking of polymer chains, domain transformation in polymers or crosslinkers, and pull out of fibers or fillers.14 The mechanical properties of RSF-based biomaterials have been reported to improve with mechanical energy dissipation through incorporation of polysaccharides, ceramics and carbon allotropes into RSF matrix.15-17 Graphene oxide (GO), a functionalized carbon allotrope, has gained much attention in recent years due to its excellent mechanical properties, large surface-to-volume ratio, high water solubility, easy solution processability and chemical functionality.18 Incorporation of GO into RSF matrix has been demonstrated to improve the mechanical properties of RSF-based materials.17,

19-24

However, only solution cast films, freeze-dried scaffolds, electrospun and/or layer-by-layer spin coated membranes of RSF/GO composites, demonstrating physical crosslinking, have been reported so far.17, 19-24 In this work, with a view to achieve chemically crosslinked RSFbased hydrogels exhibiting adhesion properties and high toughness, GO is chosen as a promising material for tuning physicochemical properties of RSF hydrogels, where GO has the potential to interact with the RSF molecule and the ruthenium-based photocatalyst via hydrophilic, hydrophobic, π-π and/or electrostatic interactions.25,

26

Moreover, by

photocrosslinking the RSF/GO matrix using ruthenium mediated chemistry, we combine two sets of mechanisms for the design and development of next-generation tough hydrogels for the first time.


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In order to optimize the physicochemical and biological properties of RSF/GO composite hydrogels for desired applications, understanding their structure at the nano- to microscopic level is essential. Especially, how the molecular building blocks transmit properties across the length-scales in the macroscopic sample.27 Therefore, in this study, we used two complementary small-angle scattering techniques viz small-angle X-ray scattering (SAXS) and contrast-variation small-angle neutron scattering (CV-SANS) to investigate the nanostructures of photocrosslinked RSF and RSF/GO composite hydrogels in the equilibrium swollen state. Both SAXS and SANS are valuable techniques to evaluate the phase behaviour, structural inhomogeneity and deformation mechanism of protein-based materials.28 Particularly, SANS is advantageous in studying the structural organization of macromolecules in concentrated solutions and gel phases because of the sensitivity of neutrons to light elements such as hydrogen.29, 30 Moreover, the contrast-variation capability with the neutron scattering technique, through the unique interaction of neutrons with hydrogen and its isotope deuterium, is a powerful tool for studying the individual component structures and their intermolecular interactions in composite hydrogels.31 SAXS is relatively more sensitive than SANS at low concentration, and can generally provide good structural information of protein and polymer gels as X-rays interact most efficiently with electron-rich atoms such as nitrogen and oxygen.32, 33 On the other hand, the contrast-variation ultra-small angle neutron scattering (CV-USANS) technique measures the elastic scattering from scattering length density (SLD) fluctuations in the order of microns in real space and can be used to characterize the hierarchical structure of fabricated hydrogels.34 Therefore, combining SAXS, CV-SANS and CV-USANS techniques we develop an in-depth understanding of the hierarchical structure and inhomogeneities of photocrosslinked RSF and RSF/GO composite hydrogels, and their structure–property relationship.


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MATERIALS AND METHODS Materials Bombyx mori silk fibers were purchased from Beautiful Silks, Australia. SnakeSkin dialysis tubing and cellulose acetate syringe filters were purchased from Thermo Scientific, Australia. Graphite flakes (~150 µm), Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate (RuBPY), ammonium persulfate (APS) and sodium borohydride (NaBH4) were purchased from SigmaAldrich, Australia. The deuterium oxide (D2O) was supplied by Australian Nuclear Science and Technology Organisation (ANSTO), Sydney. All other chemicals used in this study were purchased from Chem-Supply, Australia. Preparation of regenerated silk fibroin (RSF) solution Aqueous solution of RSF was prepared as described in our previous work.6 About 10 g of raw silk fibers were boiled in 1 L of 0.02 M aqueous sodium carbonate for 30 min. The degummed fibers were then rinsed (thrice) with distilled water and dried at ambient temperature. The dry silk fibers were then dissolved in calcium chloride-water-ethanol (mole ratio 1:8:2) solution with a liquor ratio of 1:10 at 70 °C for 3 h. The obtained silk solution was cooled to ambient temperature, filtered using a 5 µm syringe filter and dialyzed against MilliQ water for several days using dialysis tubing (3.5K MWCO). The dialyzed silk solution was centrifuged at 10,000 rpm for 30 min and the supernatant containing RSF was filtered using a 1.2 µm syringe filter. A stock solution of ~340 mg/ml was obtained by air-drying the filtered supernatant at ambient temperature and subsequent filtration. The RSF stock solution was stored at 4 °C to inhibit gelation and used within three days. Synthesis of graphene oxide (GO) GO was synthesized by an improved Hummer’s method as reported by Marcano et al.35 About 3 g of graphite flakes was added to 400 ml of acid mixture (concentrated sulfuric acid:


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phosphoric acid - 9:1 v/v) kept in an ice bath followed by slow addition of potassium permanganate producing slight exothermal condition to 40 °C. The reaction mixture was stirred for 12 h at 50 °C, cooled to ambient temperature, poured onto ice (made from 400 ml of distilled water) with 30 ml hydrogen peroxide and stirred. The appearance of yellow colour indicated completion of reaction and formation of GO. The obtained GO was washed 3 times with 3.6% hydrochloric acid, ethanol and double distilled water. The resulting GO was differentially centrifuged at 2500, 5000 and 7500 rpm for 30 min. The collected supernatant was concentrated by air-drying at ambient temperature to obtain a stock dispersion of ~6 mg/ml in water. The GO stock solution was stored at ambient temperature and ultrasonicated for 30 min using a Qsonica Q125 sonicator (operated at 50% amplitude) before use. The physicochemical characterization of synthesized GO is presented in the Supporting Information. Fabrication of hydrogels The RSF hydrogel was fabricated by a rapid ruthenium mediated photocrosslinking method as described in our previous work.6 Briefly, a predetermined amount of the RSF stock solution and aqueous RuBPY (photocatalyst) was stirred (preventing exposure to light) for 15 min, followed by addition of aqueous APS (electron acceptor) and further stirring for 5 min. The final concentration of the RSF, RuBPY and APS was 200 mg/ml, 5 mM and 28 mM, respectively. The mixture was then poured into Teflon moulds and exposed to a 50 W LED white light source for 2 min. The gels formed were then turned over and exposed for a further 30 seconds to ensure complete crosslinking of the sample. For the RSF/GO composite hydrogel fabrication, predefined amount of the RSF stock solution was first added dropwise to GO dispersion under bath sonication. After 5 min, the RSF/GO mixture was stirred gently for 15 min followed by ultrasonication in short intervals for 5 min using a Q125 sonicator (operated at 40% amplitude). The obtained RSF/GO mixture was centrifuged at 5,000 rpm 7 ACS Paragon Plus Environment

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for 15 min to remove any aggregates. Subsequently, the RSF/GO mixture was stirred with RuBPY and APS to fabricate RSF/GO composite hydrogel, following the above described procedure. Efforts were made to fabricate RSF/GO composite hydrogels with RSF:GO mass ratio of 200:1, 200:1.5 and 200:2. However, the latter two could not be made successfully as the RSF/GO mixture gelled during sonication. The 200:1 RSF:GO composite mixture was then poured into Teflon moulds and exposed to a 50 W LED white light source for 2 to 7 min. The gels formed were then turned over and exposed for a further 3 min to ensure complete crosslinking of the sample. The successfully fabricated hydrogels were dialyzed against MilliQ water to remove excess reactants. Alcohol post-treatment was performed by soaking the photocrosslinked hydrogels in 70 vol% aqueous methanol solution for 1 h, followed by dialysis against MilliQ water. To generate RSF/reduced graphene oxide composite hydrogel, methanol treated RSF/GO composite hydrogel was immersed in 200 mM aqueous NaBH4 solution and equilibrated at 5 °C for 24 h, followed by dialysis against MilliQ water.36 A schematic of hydrogel fabrication and sample codes are given in Figure 1.

Figure 1. Schematic of hydrogel fabrication. Equilibrium water sorption study For water uptake experiment, the fabricated hydrogels were first dehydrated by air-drying followed by vacuum drying at ambient temperature. The dehydrated hydrogels were then soaked in water to obtain an equilibrium swollen weight. The water uptake capacity (h) and


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crosslink density (νe) of the fabricated hydrogels was determined using the following equations.37

ℎ =

(1)

, =

=

(2)

( , ) , ,

[, , ]

(3)

where Wp is the weight of the dehydrated hydrogels, Ws is the weight of the equilibrium water swollen hydrogels, υ1,s is the volume fraction of hydrogel at equilibrium swollen state, ρp is the density of dehydrated RSF hydrogel (taken as 1.421 g/cm3), ρ2 is the density of water (taken as 0.997 g/cm3), X is the Flory-Huggins interaction parameter between that of the silk fibroin and water (taken as 0.95), and V2 is the molar volume of water (taken as 18 cm3/mol).6, 37 Differential scanning calorimetry (DSC) Thermal analysis of fabricated hydrogels at both equilibrium swollen and dehydrated state was performed using a Discovery DSC thermal analyser (TA Instrument, Australia), operated under a controlled nitrogen gas flow rate of 50 ml/min. The samples were sealed in Tzero hermetic aluminium pans and an empty sealed pan was used as reference. The temperature range of the measurement was -60 °C to 250 °C with a heating rate of 10 °C/min. X-ray diffraction (XRD) XRD analysis of GO and fabricated hydrogels were performed at dehydrated state using a X’TRA® X-ray diffractometer (Scintag, USA). The voltage and current of the X-ray source used was 40 kV and 30 mA, respectively. Scans were conducted in reflection mode at ambient temperature with 2θ (diffraction angle) measured between 5 and 35° using a step size of 0.02° and a scan speed of 1.2 °/min. The XRD data of dehydrated hydrogels were 9 ACS Paragon Plus Environment

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deconvoluted using the MagicPlot software (with Gaussian curve fitting) to obtain quantitative crystal structures of silk. Fourier transform infrared (FTIR) spectroscopy FTIR spectroscopy of GO and fabricated hydrogels were performed at dehydrated state using a Nicolet magna IR 750 spectrometer (Thermo Scientific, USA) operated in photo-acoustic mode. Carbon black was used as reference and the spectra were acquired in the range 4004000 cm-1. The obtained data was processed using the Omnic computer program. The FTIR data of dehydrated hydrogels were deconvoluted using the MagicPlot software (with Gaussian curve fitting) to obtain quantitative secondary structural conformations of silk. Small angle X-ray scattering (SAXS) SAXS analysis was performed on fabricated hydrogels in equilibrium swollen state using a bench-top NanoSTAR II SAXS instrument (Bruker AXS, Germany). The scattering profiles of hydrogels were recorded against the scattering vector, q in the range 0.012-0.39 Å-1 using the following equation.32

=

π θ λ

(4)

where 2θ is the angle of scattering and λ is the wavelength of the X-ray beam (1.54 Å). The dehydrated hydrogels were first soaked in D2O to obtain a constant swollen weight, and the D2O equilibrium swollen hydrogels (cut into small discs of ca. 2 mm) along with excess D2O were sealed in a Kapton window cell assembly.38 All measurements were performed at 25 °C and the obtained scattering data were radially averaged using routines in the Bruker software. The D2O and Kapton window scattering was subtracted from the obtained scattering data using the PRIMUS computer program.39 Further, the appropriate background determined with a high-q power law fit (0.18 Å-1 < q < 0.39 Å-1) using the SasView computer program (http://www.sasview.org/) was subtracted from the respective sample data for analysis.40


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Contrast variation small angle neutron scattering (CV-SANS) CV-SANS analysis was performed on fabricated hydrogels in equilibrium swollen state using the ANSTO Quokka SANS instrument.41 Source to sample aperture distances of 2, 12 and 20 m with neutron wavelength of 5 and 8.1 Å-1, respectively were employed to cover the q range 0.0007-0.3 Å-1. The D2O equilibrium swollen hydrogels (cut into discs of ca. 15 mm) along with excess D2O were loaded into demountable Quokka cell assembly of 2 mm path length. The neutron SLD of D2O (6.36 × 10-6 Å-2) provide good contrast against RSF-based gels (~3.8 × 10-6 Å-2) and also reduce any incoherent background scattering from hydrogen in the system.42 Moreover, the neutron SLD of GO in D2O is reported around 6.1 × 10-6 Å-2, which closely match with that of D2O and provide unique opportunity to visualize the structure of just RSF in RSF/GO composite hydrogels by contrast matching.31, 43 For contrast variation experiments, the dehydrated hydrogels were first soaked in H2O or D2O/H2O mixture (55% D2O) to obtain a constant swollen weight, and loaded in Quokka cells with excess soaking medium, respectively. All measurements were performed at 25 °C with a sample aperture diameter of 12 mm, and the obtained data were reduced using NCNR SANS reduction macros (modified for the QUOKKA instrument) using the Igor software package with data corrected for empty cell scattering and transmission.44 The data were transformed to absolute scale using an attenuated direct beam transmission measurement. The D2O, H2O and D2O/H2O mixture scattering was subtracted from the respective sample data using the PRIMUS computer program.39 Contrast variation ultra-small angle neutron scattering (CV-USANS) CV-USANS analysis was performed on fabricated hydrogels in equilibrium swollen state using the ANSTO Kookaburra USANS instrument operated with a neutron wavelength of 4.74 Å.45 The same Quokka cells loaded with samples (used for SANS measurement) were used for USANS measurements. The scattering data were collected with a sample aperture 11 ACS Paragon Plus Environment

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diameter of 15 mm at ambient temperature in the q range 0.00004-0.001 Å-1. The collected USANS data of samples were reduced with an empty Quokka cell scattering using Python scripts running in Gumtree based on the standard procedure.46 The experimental data were de-smeared using the Lake algorithm incorporated in the NIST USANS macros, and subsequently merged with the SANS data for analysis.44 The incoherent background scattering determined with a high-q power law fit (0.15 Å-1 < q < 0.3 Å-1) using the SasView computer program was subtracted from the respective sample data for analysis.40 The structural parameters of the fabricated hydrogels were determined by fitting the neutron scattering data with appropriate models/functions using the SasView computer program. Dynamic mechanical analysis (DMA) DMA was performed on fabricated hydrogels at equilibrium swollen state using a Q800 dynamic mechanical analyser (TA Instrument, Australia). A humidity accessory was used to prevent rapid dehydration of the system. The samples were mounted in the tension clamps with 1.4 inch-pounds torque. The storage and loss modulus of hydrogels were recorded for 1 hour at ambient isothermal temperature with preload force of 0.01 N, amplitude of 10 µm, and the humidity ramped to 50%. Tensile testing The tensile test was performed on fabricated hydrogels in both equilibrium swollen and dehydrated state using a Universal Tensiometer (Hounsfield, USA) equipped with a 1 kN load cell. Measurements were made at ambient temperature in extension mode with a constant velocity of 10 mm/min.


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RESULTS AND DISCUSSION Tacky to tough RSF/GO composite hydrogels The pristine RSF and RSF/GO composite hydrogels were fabricated by photocrosslinking tyrosine amino acid residues in RSF to form dityrosine crosslinks.6 The pristine RSF solution formed non-adhesive hydrogel after 2 min of exposure to white light,6 whereas the RSF/GO composite solution formed tacky hydrogel, which was difficult to remove from the mould and suggest GO induced adhesion property in fabricated RSF/GO composite hydrogel. However, when the tacky RSF/GO composite hydrogel was further exposed to white light for additional 8 min it formed tough and non-adhesive hydrogel. Such physicochemical transformation of hydrogel (containing catecholamines derived from the amino acid tyrosine) from stretchable and adhesive (during early stages of curing) to shape-fixed and non-adhesive (as the crosslink density increased) has been recently reported for mussel-inspired protein/nanosilicate composites.47 Formation of DOPA (tyrosine oxidation product) has also been reported during photocrosslinking of tyrosine in resilin protein, which resulted in adhesive hydrogel.48 Therefore, it is hypothesized that during initial stages of photocrosslinking a fraction of tyrosine amino acids in RSF (probably in the close vicinity to GO) gets transformed into DOPA (intermediate) in the presence of GO, which gives the hydrogel its adhesive property. This reaction may be influenced by the electron transfer mechanism reported to occur between the catalyst RuBPY and GO through electrostatic and π-π interactions when exposed to white light.25 Efforts were made to characterize DOPA, presumably formed during photocrosslinking of RSF/GO composite solution using UV-Vis and fluorescence spectrometry. However, was unsuccessful given the complexity and the presence of GO in the system. Equilibrium swelling of fabricated hydrogels


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The balance between water uptake capacity (h) and crosslink density (νe) of fabricated hydrogels is of critical importance for their applications in the field of tissue engineering and drug delivery.49 The calculated h and νe value of fabricated hydrogels are given in Table 1. The RSF hydrogel exhibited a h value of 0.54 ± 0.02 and a νe value of 0.382 ± 0.003 ×10-3 mol/cm3, which is in general agreement with the values reported previously.6 However, we observed that a higher h value of 2.25 ± 0.05 and a lower νe of 0.343 ± 0.002 ×10-3 mol/cm3 can be measured for RSF hydrogel when the weight of the hydrogel measured straight after dialysis was taken as swollen weight and the weight after succeeding dehydration taken as dry weight. Therefore, dehydration and re-swelling offer relatively higher crosslink density which could lead to higher mechanical properties.50 The h value of RSF hydrogel slightly decreased with incorporation of GO, which could be due to intermolecular interactions between RSF and GO; where GO can act as physical crosslinking points in RSF/GO composite hydrogel thereby increasing crosslink density and/or induce increase in β-content and/or increase the order of secondary structure of RSF (i.e. less voids) and/or decrease hydrogel porosity.22,

24, 51

Conversely, the methanol post-treatment resulted in a slightly

higher h value for both RSF and RSF/GO composite hydrogels. In this case, the higher water uptake by methanol post-treated hydrogels is ascribed to formation of less ordered RSF secondary structures forming more voids. Lawrence et al.52 have previously reported such methanol induced rapid formation of less ordered secondary structural arrangements (mesophase) between the crystalline and amorphous regions in RSF cast films, thereby enabling higher water uptake. Amongst all the fabricated hydrogels, the RSF/GO-MB composite hydrogel exhibited the highest h value (1.08 ± 0.06) and lowest νe (0.358 ± 0.001 ×10-3 mol/cm3). The observed difference in h and νe value with NaBH4 treatment could be due to reduction in surface functionalities of GO (affecting RSF-GO interaction) and/or disruption of disulphide bonds linking heavy and light chains of RSF (affecting crosslink


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density) and/or further formation of less ordered secondary structural arrangements.25, 36, 52-54 A schematic of fabricated composite hydrogel nanostructure is given in Figure 2. The extent of GO reduction in RSF/GO-MB composite hydrogel was not characterized as it exhibited very poor mechanical properties (discussed later).

Figure 2. Schematic of the RSF/GO composite hydrogel nanostructure. The three structural domains namely: crystalline (containing β-sheet), mesophase (ordered secondary structure) and amorphous (containing random coil) are in the size range of approximately 4 nm, 6 nm and 30 nm, respectively. Chain mobility of RSF in fabricated hydrogels The effect of hydration on the chain mobility of RSF in fabricated hydrogels was studied using DSC. In the dehydrated state, the RSF hydrogel showed two major thermal events: a shift in baseline related to the glass transition temperature, Tg (Figure 3A) and a more complex endothermic peak above 250 °C due to chemical degradation of silk.6 The Tg values of fabricated hydrogels in both dehydrated and equilibrium swollen state determined from


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DSC curves are given in Table 1. The Tg of dehydrated RSF hydrogel (~142.5 °C) was found to increase relatively with GO incorporation and/or methanol post-treatment, which suggests increase in crystallinity (i.e. β-content) and/or increase in the order of secondary structure of RSF, and consequently decrease in chain mobility of RSF.20, 55-57 The dehydrated RSF/GOMB composite hydrogel exhibited the highest Tg (~144.7 °C) amongst the fabricated hydrogels, therefore has lowest chain mobility. Conversely, in the equilibrium swollen state (h = 0.54 ± 0.02), the Tg of RSF hydrogel shifted to -18.8 °C (Figure 3B) and an additional exothermic peak (enthalpy ~17.9 J/g) around 1 °C, related to melting of ice was observed.58 This establishes the presence of both non-crystallisable water adsorbed onto polar sites of RSF chains and crystallisable free water filling the voids and pores of the equilibrium swollen hydrogel.6, 37 In swollen hydrogels, water acts as a plasticizer by providing alternate mobile hydrogen bonds resulting in a decreased energy barrier between different conformational states of protein, thereby causing the Tg to occur at a much lower temperature.37 The Tg of equilibrium swollen RSF hydrogel was measured to relatively increase with GO incorporation and/or methanol post-treatment, which is of similar trend observed for hydrogels in the dehydrated state. However, the enthalpy of free water melting (∆H) of equilibrium swollen RSF hydrogel was observed to decrease with GO incorporation, whereas increase with methanol and NaBH4 post-treatment. Therefore, based on these observations, it is hypothesized that increase in Tg and decrease in h value of RSF hydrogel with GO incorporation could be due to increase in β-content and/or increase in the order of secondary structure of RSF; whereas increase in Tg and h value of RSF and RSF/GO composite hydrogel with methanol and NaBH4 post-treatment could be due to increase in β-content and decrease in the order of secondary structure of RSF.52,

55

The RSF/GO-MB composite

hydrogel exhibited the highest ∆H (~21.1 J/g) amongst the fabricated hydrogels, which is consistent with water uptake results.


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Figure 3. DSC thermogram of (A) dehydrated and (B) equilibrium swollen hydrogels. Secondary structure of RSF in fabricated hydrogels The secondary structural conformations of RSF in fabricated hydrogels were both qualitatively and quantitatively characterized using XRD and FTIR spectroscopy. The XRD pattern of hydrogels exhibited both silk I (α-form - type II β-turn and may be less extended helix) and silk II (β-form - anti-parallel β-pleated sheets) polymorphs, as shown in Figure 4A.57 The silk I structural conformations of RSF in the fabricated hydrogels have been observed from 2θ peaks at 12.3°, 15.8°, 20.3°, 22.4°, 24.7°, 28.3° and 32.5° corresponding to d-spacing of 0.72, 0.56, 0.44, 0.39, 0.36, 0.32 and 0.28 nm, respectively.

57, 59

On the other

hand, the silk II structural conformations have been observed from 2θ peak at 18.5° and 20.7° corresponding to d-spacing of 0.48 and 0.43 nm, respectively.57, 59 No peak related to GO repeat unit was observed at 10.2° (Figure S1 in Supporting Information), which may be due to a very low GO content

in the composite hydrogels (0.5 wt%) and/or due to well

exfoliated, uniform dispersion of GO in the RSF matrix.20, 24 Quantitative silk I and silk II structures of the fabricated hydrogels were obtained by deconvoluting the XRD data using the above reported 2θ values (Figure S2 in Supporting Information). The estimated silk I and silk II secondary structural content is presented in Figure 4B. The RSF hydrogel displayed largely silk I structure with a silk II/silk I ratio of 0.81. Incorporation of GO resulted in slightly higher silk II/silk I ratio (0.84), suggesting GO induced crystallization in RSF, i.e. 17 ACS Paragon Plus Environment

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increase in β-content. Increase in silk II structure with increase in GO content has been previously reported for RSF/GO composite cast films and freeze-dried scaffolds.20, 24 On the other hand, ultrasonication has been reported to favour or initiate the formation of β-sheet structures in RSF.60 Therefore, in the present study, the increased silk II structure in RSF/GO composite hydrogel can be attributed to GO induced crystallization of RSF and/or ultrasonication (used to disperse GO in RSF solution). Moreover, the silk II/silk I ratio of hydrogels was observed to increase with methanol post-treatment and is attributed to established methanol induced random coil → β-sheet conformation in RSF.61 The silk II content in the fabricated hydrogels were observed to follow the order of RSF < RSF/GO < RSF-M < RSF/GO-M < RSF/GO-MB hydrogels, which is in general agreement with the DSC results. The observed difference in silk II content between RSF-M and RSF/GO-M composite hydrogels could be due to methanol induced structural change in GO and RSF, thereby influencing/affecting molecular interaction between GO and RSF.20, 61, 62 The highest silk II content exhibited by RSF/GO-MB composite hydrogel might be due to NaBH4 induced disruption of molecular interactions between RSF and GO and/or breakage of disulphide bond linking heavy and light chains of RSF, thereby causing further random coil → β-sheet conformation.24, 36, 53, 54 Figure 4C shows the FTIR spectra of fabricated hydrogels. The spectra shows the presence of three distinctive conformational bands between 1700-1600 cm-1, 1600-1480 cm-1 and 1300-1200 cm-1 corresponding to amide I, II and III of hydrogels, respectively.63 The secondary structural conformation peaks of the major protein band (amide I) corresponding to aggregate β-strand/β-sheet (weak) – 1605 to 1621 cm-1, intermolecular β-sheet (strong) – 1622 to 1627 cm-1, intramolecular β-sheet (strong) – 1628 to 1637 cm-1, random coil – 1638 to 1655 cm-1, α-helices – 1656 to 1622 cm-1, turns – 1633 to 1696 cm-1, and intermolecular βsheets (weak) – 1697 to 1715 cm-1 were observed in the spectrum.64,

65

Quantitative


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secondary structural conformations of fabricated hydrogels were obtained by deconvoluting the amide I band of FTIR spectra using the above reported wavenumbers (Figure S3 in Supporting Information), and the estimated secondary structural contents are presented in Figure 4D. The secondary structures of fabricated hydrogels are observed to be dominated by random coil and intermolecular weak β-sheet structures. The intermolecular weak β-sheet content of RSF hydrogel was observed to increase slightly with GO incorporation, whereas the intramolecular strong β-sheet content remained nearly unchanged. Therefore, the difference in β-sheet content between RSF and RSF/GO composite hydrogel could be due to intermolecular interaction between RSF and GO. This suggests that GO strongly interact with RSF and thereby affect its chain mobility. However, with methanol post-treatment, both the intermolecular weak β-sheet content and intramolecular strong β-sheet content of RSF hydrogel increased slightly. It is clear from the estimates that with GO incorporation and/or methanol post-treatment the random coil → β-sheet conformation is favoured. The total βsheet content in the fabricated hydrogels were observed in the order of RSF < RSF/GO < RSF-M < RSF/GO-M < RSF/GO-MB hydrogels, which is consistent with XRD results. Thus, XRD and FTIR results establish the fabricated hydrogels to be semi-crystalline consisting of both silk I (hydrated form) and silk II structures. The observed increase in silk II structure supports the hypothesis of increase in β-content of RSF hydrogel with GO incorporation and/or methanol post-treatment.51, 66


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Figure 4. (A) X-ray diffractogram, (B) quantitative silk structure estimates from XRD, (C) Photoacoustic FTIR spectra, and (D) quantitative silk structure estimates from FTIR of fabricated hydrogels. The sample codes are (a) RSF hydrogel, (b) RSF-M hydrogel, (c) RSF/GO composite hydrogel, (d) RSF/GO-M composite hydrogel and (e) RSF/GO-MB composite hydrogel. Hierarchical structure of fabricated hydrogels The intrinsic structure of fabricated hydrogels in the nanoscale was studied using SAXS. The SAXS curve of fabricated hydrogels (Figure 5A) demonstrated two distinctive regions: a high-q Porod region (0.1 < q < 0.39 Å-1) and a mid-q Guinier region (0.012 < q < 0.1 Å-1). The Porod exponent or slope that can be determined from Porod region provides information about the "fractal dimension" of the RSF structures and their assemblies (domains) in 20 ACS Paragon Plus Environment

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hydrogels, whereas the Guinier region allows for the estimation of their size (d).67 On the other hand, the Kratky plot (Figure 5B) divides-out the decay of the scattering from the hydrogels and clearly show a correlation peak around q ≈ 0.1 Å-1, which corresponds to a domain size of ~6 nm (calculated using the relation d = 2π/q).68 Further, an estimated Porod slope of 4.0 ± 0.01 using power law fit (0.2 < q < 0.3 Å-1) for RSF hydrogel suggest the above nanoscale RSF domains (observed at high-q) to exhibit sharp interface with the surrounding environment.68 These domains can be attributed to water poor (hydrophobic) regions in the RSF hydrogels, predominantly containing β-sheet structures (crystalline) that exhibit sharp interface with D2O and/or amorphous protein matrix.69 The soluble or hydrophilic form of silk fibroin in solution is in α-helix and random coil conformations, and by transitioning to β-sheets silk is rendered insoluble or hydrophobic.70 The B. mori silk fibroin contains ~5 mol% of tyrosine in its overall structure and is located in regular and irregular sequences, which favour β-sheet crystals and random coil, respectively.71-74 Therefore, photocrosslinking of RSF molecules involving covalent bond formation between tyrosine amino acids (dityrosine) is anticipated to form both crystalline (due to crosslinking between tyrosine residues in regular sequences) and amorphous structures in RSF hydrogel. Moreover, no observed change in Porod slope of RSF hydrogel with GO incorporation and methanol/borohydride post-treatment support the above hypothesis. However, the high-q correlation peak of RSF hydrogel was observed to shift slightly to lower-q value with GO incorporation and methanol post-treatment (Figure 5B), suggesting increase in hydrophobic domain size. The observed trend in RSF hydrophobic domain size is in general agreement with increase in silk II structure (β-content) of hydrogels, measured using XRD and FTIR. A slight or marginal change in scattering intensity of RSF hydrogel in the Guinier region was observed with GO incorporation (which could be due to scattering contribution from GO), whereas a significant change with methanol/borohydride post-treatment (which could be due


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to mesophase formation with voids due to decrease in the ordered of RSF secondary structure). Moreover, the Guinier region observed with increase in scattering intensity towards low-q suggest presence of growing secondary domain (i.e., mesophase) in the system contributing to the overall intensity, which extends beyond the SAXS instrument range. Therefore, the hierarchical structure of RSF (from nano- to microscale) in fabricated hydrogels was further analysed using CV-SANS and CV-USANS.


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Figure 5. (A) SAXS curve, (B) Kratky plot from SAXS data, (C) combined CV-SANS and CV-USANS curve, and (D) Kratky plot from combined CV-SANS and CV-USANS data of fabricated hydrogels. (E) combined CV-SANS and CV-USANS curve, and (F) Kratky plot of RSF/GO-MB hydrogel contrast matched to D2O, H2O and silk fibroin.


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The combined CV-SANS and CV-USANS curve of fabricated hydrogels (Figure 5C), contrast matching (masking) GO scattering, demonstrated four distinctive region: a high-q Porod region (0.1 < q < 0.3 Å-1), a mid-q to low-q Guinier region (0.015 < q < 0.1 Å-1), a low-q Porod-like region-1 (0.0008 < q < 0.015 Å-1) and a very low-q Porod-like region-2 (0.00004 < q < 0.008 Å-1). The SANS region (0.0008 < q < 0.3 Å-1) of scattering curve provides information about the frozen-in crosslinked network structure of fabricated hydrogels at the nanoscale, whereas the USANS region (0.00004 < q < 0.0008 Å-1) provides information about the microscale pores that act as reservoirs and channels for water.75,

76

Moreover, the high-q correlation peak, mid-q to low-q shoulder feature and low-q broad feature/plateau observed in Kratky plot (Figure 5D) establish the presence of more than one structural domain in the system. The three features visible in Kratky plot can be attributed to crystalline region (high-q), inherent mesophase region (mid-q) and amorphous region (low-q) of hydrogels, as proposed earlier. An estimated high-q Porod slope of 4.0 ± 0.01 using power law fit for fabricated hydrogels is consistent with SAXS results and establishes the presence of hydrophobic domains (β-sheet structures) in the system. Further, the scattering intensity and size of hydrophobic domain (high-q) of RSF hydrogel was observed to increase slightly with GO incorporation and/or methanol post-treatment, consistent with SAXS results. The Guinier slope of RSF hydrogel was observed to increase slightly with GO incorporation, whereas significantly with methanol post-treatment, which together with SAXS data and water uptake results establishes decrease in the order of mesophase structure (i.e., less ordered secondary structure with more voids) with methanol post-treatment. The observed increase in scattering intensity at low-q for RSF/GO-M and RSF/GO-MB composite hydrogel could be attributed to dominant mid-q scattering rather than low-q scattering. Therefore, it can be presumed that no significant change in slope of low-q Porod-like region-1 was observed for fabricated hydrogels. Similar structural approach has been previously proposed


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by Plaza et al.66 and Sampath et al.77 for silk fibers using synchrotron X-ray diffraction and Raman spectroscopy. Further, estimation of structural parameters of fabricated hydrogels was endeavoured using shape-independent models (equation 6) fit to combined CV-SANS and CV-USANS data (Figure S4 in Supporting Information).

() = !() + #() + $() (5) where A(q) is Guinier-Porod model (at high-q), B(q) and C(q) are Debye Anderson Brumberger (DAB) model (at mid-q and low-q). The Guinier-Porod model calculates the scattering for a generalized Guinier/power law object that can be used to determine the size and dimensionality of scattering objects.78 The DAB model calculates the scattering from a randomly distributed, two-phase system characterized by a single length scale, the correlation length (L), which is a measure of the average spacing between regions of the two phases. The model also assumes smooth interfaces between the phases and hence exhibits Porod behavior q-4 at high-q.79 The estimated structural parameters are given in Table 2. The observed trend in high-q domain size can be attributed to increase in silk II content, whereas mid-q and lowq to the water uptake capacity of fabricated hydrogels. Moreover, the 55% D2O soaked RSF/GO-MB composite hydrogel contrast matches silk fibroin’s neutron SLD (SCM) and exhibits power law behaviour of ~q-2.0 (Figure 5E), which is the characteristic Porod slope of GO (Figure S5A in Supporting Information).72 Kratky plot further confirms the scattering from GO by showing an initial monotonic increase followed by a plateau with increase in q (Figure 5F).81 The notable small difference in scattering intensity at mid-q regime between D2O and H2O soaked RSF/GO-MB composite hydrogel is attributed to structural contribution from GO, where the scattering contrast comes from both the RSF and GO in H2O, whereas predominantly or only from the RSF in D2O. On the other hand, the USANS region (very low-q) show a strong upturn with a power law behaviour of ~q-3 to q-3.3, typically observed for surface fractals suggesting structural inhomogeneities on a range of length scales in the 25 ACS Paragon Plus Environment

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sample, extending beyond the USANS range.82 On top of this steep upturn, a broad feature is weakly visible which is likely to stem from the micropores in the hydrogels. The observed features between q value of 0.0001 Å-1 and 0.0004 Å-1 corresponds to size ranging from ~1.5 to 6 µm, which is in good agreement with pore size distribution observed in SEM image of the RSF hydrogel (presented in our previous publication).6 However, the visibility of nanostructural features such as crystalline, mesophase and amorphous regions of the hydrogel is limited by the SEM instrument.6 It is known that the largely random coil conformation of RSF in solution transforms to crystalline β-sheets upon aging (self assembly) and becomes physically crosslinked gel.63, 70 Figure S5 in Supporting Information compares the SANS scattering profile and Kratky plot of freshly prepared and aged (stored at 4 °C for 1 month) RSF and RSF/GO blend solutions with photocrosslinked RSF hydrogel. As expected, the 20 wt% RSF solution exhibited random coil conformation with a Porod slope of q-1.8 in SANS scattering profile, and an initial monotonic increase in the low-q region followed by a slight increase or plateau in Kratky plot.83 No change in intrinsic structure of RSF in solution (Porod slope ~q-1.8) was observed with addition of GO (0.1 wt%). However, increase in low-q intensity was observed with GO addition, which may be due to increase in overall intensity with scattering contributions from GO and/or formation of aggregates of RSF and GO. Upon aging, RSF and RSF/GO blend solutions gelled and exhibited a Porod slope of q-4, which is consistent with the behaviour of photocrosslinked hydrogel exhibiting hydrophobic domain containg βsheets.83 The difference between SANS data of physically crosslinked RSF and RSF/GO composite hydrogels demonstrate the GO induced changes in self assembly and structural/spatial distribution of RSF from solution to gel. Further, the structural differences between physically and chemically crosslinked hydrogel is clearly demonstrated by Kratky plot, where the photocrosslinked hydrogel exhibited relatively smaller hydrophobic and


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hydrophilic domain size compared to physically crosslinked hydrogels, which may be due to restriction in self assembly (chain movement and co-localization) in photocrosslinked hydrogel due to dityrosine crosslinks.83, 84 Mechanical properties of fabricated hydrogels Protein-based hydrogels exhibit complex mechanical behaviour which is related to their hydration, crosslink density, chain mobility, degree of crystallinity and supramolecular network structure.1 The viscoelastic properties of fabricated hydrogels were examined using DMA. Figure 6 shows the measured storage modulus (E'), loss modulus (E”) and tan delta (damping) of fabricated hydrogels at equilibrium swollen state.49 The E’ value of the equilibrium swollen RSF hydrogel (~265.3 MPa) increased with GO incorporation (Figure 6A). It has been reported that water molecules between adjacent layers of polymer and GO in polymer/GO composite structures enhance stress transfer by means of a cooperative hydrogen-bonding network, thereby leading to improvement in the storage modulus, compared to pristine polymer structures.85 Therefore, the difference in mechanical property of RSF and RSF/GO composite hydrogels can be attributed to the interface zone between RSF and GO, specifically the hydrogen-bonding ability of the intercalating species that could be formed due to complex interaction between RSF and GO, which enhance the mechanical property of the hydrogel.21, 22, 85 Moreover, DMA experiments are typically performed at very low strain (< 1%), therefore change in microstructure of RSF in RSF/GO composite hydrogel is not expected.24 Further, methanol post-treatment led to increase in E’ value of RSF and RSF/GO composite hydrogels, which could be due to increase in cooperative hydrogenbonding network in the system resulting from higher water uptake capacity.85,

86

The

RSF/GO-MB composite hydrogel showed the lowest E’ value (~37.0 MPa), which suggests possible disruption of RSF-GO interface and/or disulphide bond linking heavy and light chains of RSF by NaBH4.36, 53 Similar to E’, the E’’ of RSF hydrogel was also observed to 27 ACS Paragon Plus Environment

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increase with GO incorporation and methanol post-treatment (Figure 6B). Moreover, the tan delta (E’’/E’ ratio) of the RSF hydrogel also showed an increase trend with GO incorporation and methanol post-treatment (Figure 6C) suggesting the hydrogels to become energy dissipative.

Figure 6. (A) Storage modulus and (B) loss modulus DMA curves of fabricated hydrogels. (C) Tan delta of fabricated hydrogels. Figure 7 compares the uniaxial tensile stress-strain response of fabricated hydrogels at both dehydrated and equilibrium swollen state. The plots show curves typical to that of nonHookean materials, which are characteristic of semi-crystalline polymer systems.87,

88

The

stress-strain curve of fabricated hydrogels at dehydrated state (Figure 7A) showed an initial elastic region, an extended plastic region (yielding), and a strain hardening region, similar to


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that of spider silk fibers reported by Plaza et al.66 The plastic region of RSF hydrogel at dehydrated state was observed to extend further with GO incorporation causing increase in ultimate tensile strain, similar to RSF/GO hybrid fibers reported by Zhang et al.24 The tensile mechanical properties of fabricated hydrogels determined from the stress-strain plots are presented in Table 3. The RSF hydrogel at dehydrated state demonstrated a Young’s modulus of ~5.5 MPa, which is approximately five times higher than that of the equilibrium swollen counterpart. However, with incorporation of GO, the Young’s modulus of RSF hydrogel decreased to ~2.0 MPa, which suggest the RSF/GO composite hydrogel to be relatively pliable than the RSF hydrogel at dehydrated state.89 Conversely, the methanol post-treatment showed increase in Young’s modulus, where the RSF-M composite hydrogel demonstrated Young’s modulus (~20.5 MPa) about four times higher than that of the RSF hydrogel at dehydrated state. The ultimate tensile strength of the RSF hydrogel increased with GO incorporation and methanol post-treatment, where the RSF/GO-M composite hydrogel exhibited the highest tensile strength (~22.0 MPa) at dehydrated state. Conversely, the ultimate tensile strain decreased with methanol post-treatment, where the RSF/GO composite hydrogel exhibited the highest tensile strain (~8.2%) at dehydrated state. Based on the hierarchical hydrogel structure (obtained using SAXS/SANS) and proposed relationship between microstructure and mechanical properties of silk fibers by Plaza et al.66, it can be presumed that no obvious change occur in crystalline and mesophase fraction in the elastic region (

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