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Conformations and Intermolecular Interactions in Cellulose/Silk Fibroin Blend Films: A Solid-State NMR Perspective Published as part of The Journal of Physical Chemistry virtual special issue “Recent Advances in Connecting Structure, Dynamics, and Function of Biomolecules by NMR”. Donglin Tian,† Tao Li,† Rongchun Zhang,*,‡ Qiang Wu,† Tiehong Chen,§,∥ Pingchuan Sun,*,†,‡,∥ and Ayyalusamy Ramamoorthy⊥ †

Key Laboratory of Functional Polymer Materials of Ministry of Education and College of chemistry, Nankai University, Tianjin 300071, P. R. China ‡ State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, P. R. China § Institute of New Catalytic Materials Science, School of Materials Science and Engineering, Key Laboratory of Advanced Energy Materials Chemistry (MOE), Nankai University, Tianjin 300350, P. R. China ∥ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, P. R. China ⊥ Biophysics Program and Department of Chemistry, The University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, United States ABSTRACT: Fabricating materials with excellent mechanical performance from the natural renewable and degradable biopolymers has drawn significant attention in recent decades due to the environmental concerns and energy crisis. As two of the most promising substitutes of synthetic polymers, silk fibroin (SF), and cellulose, have been widely used in the field of textile, biomedicine, biotechnology, etc. Particularly, the cellulose/SF blend film exhibits better strength and toughness than that of regenerated cellulose film. Herein, this study is aimed to understand the molecular origin of the enhanced mechanical properties for the cellulose/SF blend film, using solid-state NMR as a main tool to investigate the conformational changes, intermolecular interactions between cellulose and SF and the water organization. It is found that the content of the β-sheet structure is increased in the cellulose/SF blend film with respect to the regenerated SF film, accompanied by the reduction of the content of random coil structures. In addition, the strong hydrogen bonding interaction between the SF and cellulose is clearly elucidated by the two-dimensional (2D) 1H−13C heteronuclear correlation (HETCOR) NMR experiments, demonstrating that the SF and cellulose are miscible at the molecular level. Moreover, it is also found that the -NH groups of SF prefer to form hydrogen bonds with the hydroxyl groups bonded to carbons C2 and C3 of cellulose, while the hydroxyl groups bonded to carbon C6 and the ether oxygen are less favorable for hydrogen bonding interactions with the −NH groups of SF. Interestingly, bound water is found to be present in the air-dried cellulose/SF blend film, which is predominantly associated with the cellulose backbones as determined by 2D 1H−13C wide-line-separation (WISE) experiments with spin diffusion. This clearly reveals the presence of nanoheterogeneity in the cellulose/SF blend film, although cellulose and SF are miscible at a molecular level. Without doubt, these in-depth atomic-level structural information could help reveal the molecular origin of the enhanced mechanical properties of the blend film, and thus to establish the structure−property relationship, which could further provide guidance for the fabrication of high performance biopolymer-based materials.

1. INTRODUCTION

achievement of superior mechanical properties is strongly related with specific polymer conformations, hierarchical structures, and the miscibility between different components, which generally relies on the intermolecular interactions, such as hydrogen bonds, van der Waals interactions, electrostatic

It has always been a challenging and tempting goal to fabricate high performance polymer materials with a good balance of modulus, elongation, and breaking strength, and thus render the materials to be stiff while at the same time being tough.1−3 Therefore, polymer blend has become a pretty straightforward approach to address the above challenges, since it could combine both the merits of two different bulk materials. However, simply blending usually does not work, as the © 2017 American Chemical Society

Received: March 25, 2017 Revised: May 7, 2017 Published: May 8, 2017 6108

DOI: 10.1021/acs.jpcb.7b02838 J. Phys. Chem. B 2017, 121, 6108−6116

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The Journal of Physical Chemistry B interactions, and so on.4−7 Herein, elucidating the polymer conformations and intermolecular interactions will provide significant insights into the microscopic origin of the enhanced mechanical properties of polymer blends. Over the last few decades, dramatic attention has been directed to the renewable and natural polymers due to the environmental issues as well as the depletion of nonrenewable energies.8−11 In particular, silk fibroin (SF) and cellulose, due to their good biocompatibility, biodegradability, environmental friendliness, and low cost, are particularly suitable for fabricating eco-friendly high performance materials, not to mention their widespread applications in the fields of biomedicine, biotechnology, tissue scaffolds, drug delivery, and so on.12−16 However, the regenerated SF film often exhibits poor mechanical properties and is much more brittle in the dry state in comparison to the native silk,17 which greatly limited their practical applications.18 On the other hand, as the most abundant polysaccharide on the earth, cellulose is widely distributed in plant fibers with relatively high stiffness and strength.16,19 Therefore, it is a pretty straightforward and promising avenue to blend cellulose with SF to improve the tensile strength of cellulose.20−24 Nevertheless, finding an appropriate cosolvent for dissolving SF and cellulose is the premise for preparing the cellulose/SF blend film, since the dense molecular packing of cellulose through hydrogen bonds usually renders it difficult to dissolve. Recently, ionic liquids have been widely explored due to their many attractive properties, such as nonflammability, easy renewability, thermal stability, low melting point, and so on.25−27 It was found that the ionic liquid, 1-butyl-3-methylimidazolium chloride (BmimCl), is a good solvent for both the SF28 and the cellulose.29 Using BmimCl as the cosolvent and methanol as the coagulation agent, Shang et al.20 successfully prepared the cellulose/SF blend films, and systematically investigated the morphology, water absorption, and mechanical properties of SF/cellulose blend films with different contents of cellulose. It was found that a superior mechanical property was achieved when the cellulose content was around 75 wt%.20 However, the microscopic origin of the enhanced mechanical properties was not well understood, especially the conformational changes, nanoheterogeneous structures, and intermolecular interactions between cellulose and SF, which are all closely related with the mechanical properties of the blend film. While both infrared (IR) and Raman spectroscopy could detect the conformational changes and the intermolecular interactions from the shift of related absorption bands,30,31 solid-state NMR could further offer in-depth atomic-level information about the specific chemical groups that form hydrogen bonds with each other or polymer chains that change their conformations. Due to its capability of selectively manipulating various anisotropic spin interactions in solids,32,33 solid-state NMR spectroscopy has evolved as an essential tool for investigating the structure and dynamics in a wide range of macromolecules that are not amenable for high-resolution investigation using other techniques including X-ray crystallography, cryo-electron microscopy, and solution NMR spectroscopy.34−38 Specifically, 13C chemical shift is an intrinsic and sensitive probe to the conformations of proteins and peptides, and thus can be used to examine the conformational behaviors of SF and cellulose.39−43 In addition, 2D 1H−13C heteronuclear correlation (HETCOR) experiment could be well utilized to examine the heteronuclear proximity, and thus to probe the intermolecular/intramolecular hydrogen bonding interac-

tions.44,45 Furthermore, 2D proton wide-line-separation (WISE) experiment is a popular separated-local-field experiment,46,47 which can provide proton wide-line spectra related to site-specific 13C chemical shifts. Hence, the line width and line shape of proton spectra can well indicate the mobility differences between different chemical groups in the molecular system under investigation. In this study, versatile solid-state NMR approaches were utilized to systematically investigate the conformations and intermolecular interactions in the cellulose/SF blend films as prepared using BmimCl as the cosolvent. 13C magic-anglespinning (MAS) solid-state NMR and IR spectroscopy were applied to elucidate the conformational changes of SF in the blend film, while 2D 1H−13C HETCOR experiments were used to investigate the intermolecular interactions between cellulose and SF. Furthermore, the nanoheterogeneity and the watercellulose interactions could be determined by the 2D WISE experiments with spin diffusion. All these detailed structural information obtained from the solid-state NMR experiments could, to a large degree, reveal the microscopic origin of the enhanced mechanical properties of cellulose/SF blend film, and thus will be beneficial for the development and fabrication of high performance polymer materials.

2. EXPERIMENTAL SECTION 2.1. Materials. The cellulose (cotton linter pulp) was purchased from Hubei Chemical Fiber Group Ltd. (Xiangfan, China), where the molecular weight (Mw) of cellulose fibers was determined to be around 1.26 × 10 5 g/mol by viscometry.48 This raw cellulose was further degraded in 30 wt% H2SO4 at 50 °C for a couple of minutes, and the final molecular weight (Mw) of cellulose was around 8.0 × 104 g/ mol. Silk cocoon (Bombyx mori) was first degummed in 0.5% (w/w) aqueous Na2CO3 solution at 100 °C for 15 min. After that, it was degummed twice in 8 M urea solution at 80 °C for 20 min. Finally, the degummed silk was rinsed thoroughly with excessive water and dried in vacuum at 45 °C for 48 h. The commercial ionic liquid BmimCl (1-butyl-3-methylimidazolium chloride, purity ≥99%), which was provided by Zhongke Kaite Industry and Trade Co., Ltd. (Lanzhou, China), was directly used as received without any further treatment. Both schematic molecular structures of cellulose and SF (major motif) were shown in the Scheme 1. The Bombyx mori heavy chain fibroin is mainly composed of repeats of the motif (Gly-Ser-Gly-Ala-GlyAla)n,49 which takes a fraction of around 70% of the amino acid sequences in Bombyx mori silk fibroin.50 For clear illustration of Scheme 1. Molecular Structures of Cellulose and the Major Amino Acid Sequence of Silk Fibroina

a

The indicated numbers are used to distinguish different carbons for NMR chemical shift assignments below, where the carbons on cellulose are indicated as C1, C2, etc., and the carbons on SF are presented as S1, S2, etc. 6109

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The Journal of Physical Chemistry B resonance assignments, below the carbons on the cellulose will be referred as C1, C2, etc., while those on SF will be represented as S1, S2, etc. 2.2. Preparation of Cellulose/SF Blend Films. The cellulose/SF blend with a weight ratio of 3:1 was dissolved in the BmimCl solution with 4 wt% concentration. Then, the solution was stirred at 90 °C for a couple of hours until the blends were completely dissolved. After complete dissolution and intermixing, the solution was poured into a glass plate, which later was kept in the vacuum oven at 85 °C for an hour to remove the bubbles. Later on, the glass plate was kept inside a sealed box with methanol to obtain a gel-like film. Both the residual ionic liquids and methanol in the film were removed by repetitive washing with water. The obtained gel-like film was then kept at room temperature for 2 days to obtain the final airdried blend film. The exact same procedures as above were followed for the preparation of the regenerated cellulose and SF films. It is worth mentioning that the air-dried SF film was too brittle to cut into a rectangle shape, and renders the tensile test impossible. 2.3. Characterization of the Silk/Cellulose Films. Fourier Transform Infrared Spectroscopy. All IR spectra were recorded using a Bio-Rad FTS6000 spectrometer with a resolution of 8 cm−1 and 16 scans per sample. IR measurement on the ground silk film (mixed with KBr powder) was performed with a DRIFT (diffuse reflectance infrared Fourier transform) accessory, while the spectra of the cellulose/SF blend and regenerated cellulose films were recorded with an ATR (attenuated total internal reflection) attachment. Tensile Test. The mechanical properties of the films (20 mm × 8 mm × 0.07 mm) were measured on an UTM6103 mechanical testing instrument (Shenzhen Suns Technology Stock Co., Ltd., China) in tensile mode at room temperature for the air-dried regenerated cellulose and cellulose/SF blend films. The stretching speed is 5 mm/min. All the experiments were performed at room temperature. Solid-State NMR Spectroscopy. All solid-state NMR experiments were performed on a Varian InfinityPlus-400 wide-bore (89 mm) spectrometer operating at a 1H frequency of 399.7 MHz and a 13C frequency of 100.5 MHz, using a conventional 5 mm double-resonance HX MAS NMR probe. 13 C ramped cross-polarization (CP) NMR51,52 spectra were acquired under a magic-angle-spinning (MAS) rate of 10 kHz. Proton decoupling during 13C signal acquisition was achieved by SPINAL-64 irradiation53 with a radio frequency (RF) field strength of ∼80 kHz. The 13C and 1H chemical shifts were referenced using the chemical shift values observed for the methyl group nuclei of L-alanine powder sample: 13C δiso = 20.7 ppm and 1H δiso = 1.7 ppm, which are with respect to the chemical shifts of TMS (δiso = 0). The radio frequency pulse sequences used in the solid-state NMR experiments reported in this study are shown in Figure 1. 1 H CRAMPS Experiment. CRAMPS (combined rotation and multiple pulse spectroscopy) experiment is used to obtain highresolution 1H NMR spectrum of rigid organic solids under a slow spinning speed, where the sequence is shown in Figure 1a. DUMBO (decoupling using mind-boggling optimization) sequence54,55 is used to achieve homonuclear decoupling during the signal acquisition, where the detection window with a 5 μs delay was inserted between two successive DUMBO decoupling cycles, yielding a dwell time of 37.8 μs. The 90° pulse length was 2.7 μs, corresponding to a RF strength of 92.6 kHz for the homonuclear proton decoupling. The MAS rate

Figure 1. NMR pulse sequences used in this study. (A) 1H highresolution NMR experiment with DUMBO homonuclear proton decoupling during signal acquisition. (B) 2D 1H−13C WISE experiment under slow spinning rate, where τm is the spin diffusion time. (C) 2D 1H−13C heteronuclear correlation (HETCOR) experiment with DUMBO to decouple 1H−1H dipolar couplings during t1 to achieve high proton spectral resolution. θ1 and θ2 correspond to a 35.3° and 54.7° RF pulses, respectively. The spin diffusion time τm in the HETCOR experiment was set to 20 μs.

was set as 9.8 kHz in this experiment, and the number of scans was 160. 2D 1H−13C Wide-Line Separation (WISE) Experiment. The WISE experiment46 correlates the high-resolution 13C NMR spectrum with the wide-line proton lineshapes indicating molecular mobility; the pulse sequence for the WISE experiment is shown in Figure 1b. In this study, the spin diffusion time (τm) was varied to investigate the intermolecular mixing, and the CP contact time was set as 1 ms. A spectral width of 200 kHz and 50 kHz was used for the 1H and 13C dimension, respectively. The 90° degree pulse length was 3.2 μs on the proton channel. Eighty t1 increments with 192 scans were used with a recycle delay of 2.5 s. The MAS frequency was set as 4.5 kHz to avoid complete averaging of 1H−1H and 13 C−1H dipolar couplings. The TOSS (total suppression of sidebands)56 sequence was used before 13C signal acquisition to suppress the spinning sidebands. 2D 1H−13C Heteronuclear Correlation (HETCOR) Experiment.57 The pulse sequence for this experiment is shown in Figure 1c. DUMBO homonuclear decoupling sequence was applied during the t1 period in order to obtain a high-resolution proton NMR spectrum. In this work, the spin diffusion time τm was set as 20 μs. The phase-modulated Lee−Goldburg (LG) decoupling pulses58 were used during the CP contact time. The 1 H effective field strength during LG was about 78.9 kHz with a resonance offset of 45.0 kHz. The spinning frequency was set as 9.8 kHz. Eighty t1 increments with 400 scans were used with a recycle delay of 1.5 s. The experimental parameters for the DUMBO sequence were the same as used for the 1D proton CRAMPS experiment. As will be shown below, a HETCOR spectrum with a short CP contact time will generally provide proximity information between covalent bonded 1H/13C spin pairs, since the remote proton−proton dipolar couplings are effectively suppressed by the LG decoupling during the contact time. Instead, a long CP contact time in the HETCOR 6110

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The Journal of Physical Chemistry B experiment could allow probing remote heteronuclear 1H/13C correlations within a few angstroms.

3. RESULTS AND DISCUSSION 3.1. Enhanced Mechanical Properties of the Cellulose/ SF Blend Film. Tensile tests were performed first on both the regenerated cellulose and cellulose/SF blend films as shown in Figure 2. The regenerated cellulose film exhibits relatively good

Figure 3. FTIR spectra of the cellulose (red), cellulose/SF blend (blue), and SF (black) films obtained at room temperature.

much lower relative intensity compared to that of the SF films. Therefore, it can be inferred that part of disordered β-turns in SF film is transformed to well-defined β-sheet structure in the cellulose/SF blend film, possibly due to the intermolecular interactions between cellulose and SF. The conformational changes of SF can also be revealed from the 13C solid-state NMR spectra, as shown in Figure 4. For both the regenerated cellulose and cellulose/SF blend films, the absence of characteristic peaks at ∼89 ppm (for carbon C4) and ∼65 ppm (for carbon C6),42 which correspond to the crystalline cellulose structures, clearly indicates an amorphous structure for the cellulose in both films. This is in a good agreement with IR results shown in Figure 3. In addition, it was found that the chemical shift of Ala Cβ (i.e., carbon S7, 15−25 ppm) was quite sensitive to the secondary structures of SF.36,40,41 Therefore, the comparison of 13C spectra between SF and cellulose/SF in the region of 15−25 ppm can well reveal the conformational changes, as shown in Figure 4B. The two peaks observed at 20.8 and 22.3 ppm indicate the well-defined antiparallel β-sheet structures. However, the Ala methyl groups are positioned differently in these two β-sheet structures as recently reported by Asakura et al.41 With the incorporation of cellulose in the blend film, the relative intensity of the peak for the distorted β-turns (appearing at 16.8 ppm) significantly decreases, while the shoulder peak (at 22.3 ppm) appears corresponding to the increasing content of β-sheet structure.41 Herein, in combination with the IR and solid-state NMR experimental results, it can be concluded that the content of the well-defined β-sheet structure of SF in the blend film is enhanced, accompanied by the reduction of disordered β-turn structure content compared to the regenerated SF film. To a large degree, the increased content of the well-defined β-sheet structures could help enhance the modulus of the cellulose/SF blend film in comparison to the regenerated cellulose film as shown in Figure 2. 3.3. Intermolecular Interactions between Cellulose and SF. As is well-known, the mechanical properties of polymer blends are strongly related with the miscibility of distinct polymer components,66 which is quite dependent on the intermolecular interactions. In particular, densely hydrogen bonding assemblies could overcome the intrinsic strength limitations of hydrogen bonds and thus enhance the rupture strength.67,68 Herein, CRAMPS-based NMR experiments were further performed to investigate the intermolecular interactions, as shown in Figures 5 and 6. 1D 1H CRAMPS experiments were performed first to obtain high-resolution proton spectrum. For the 1H spectrum of SF, the amide proton peak around 8.6

Figure 2. Stress−strain curves of regenerated cellulose and cellulose/ SF blend films. The measurement was performed at a strain rate of 5 mm/min at room temperature.

mechanical properties, while notable enhancement of modulus and strength is clearly observed for the cellulose/SF blend film. On the other hand, the regenerated SF film was too brittle for the tensile test. Such low toughness could be resulted from the specific conformations in the SF, where the hydrophobic domain in the SF tends to form antiparallel β-sheet structures through hydrophobic and hydrogen bonding interactions.59 It is also noteworthy that the conformations of SF and cellulose are both closely related with the solvents that used for the dissolution and coagulation process.60−62 Herein, to reveal the molecular origin of the enhanced mechanical properties for the cellulose/SF blend film, it is important to elucidate the conformational changes, intermolecular interactions and heterogeneous microstructure in the blend film. On that basis, the structure−property relationship for biopolymer blend can be well established, which can further provide guidance for the future development and fabrication of high performance biopolymer-based materials. 3.2. Conformational Changes. The macroscopic mechanical properties are usually related with the microscopic structures. Hence, IR and 13C solid-state NMR experiments were both performed to investigate the conformational changes in the cellulose/SF blend film with respect to the regenerated cellulose and SF films, as shown in Figures 3 and 4, respectively. Four characteristic peaks at around 1369, 1157, 1063, and 895 cm−1 are observed in the IR spectra of both cellulose and cellulose/SF blend films (Figure 3), indicating the presence of typical amorphous structures of cellulose.63 For the regenerated SF film, the characteristic peaks observed at 1260 cm−1 (amide III) and 1523 cm−1 (amide II) correspond to the crystalline βsheet structures, whereas the peaks observed at 1227 cm−1 (amide II) and 1662 cm−1 (amide I) correspond that of the random coil or silk I form.20,64,65 All these peaks were observed both in the SF and cellulose/SF blend film, indicating that both β-sheet and random coil structures are present in the SF and cellulose/SF blend films. It is worthy noting that in the IR spectrum of the cellulose/SF blend film, the peak observed at 1625 cm−1 (amide I, also corresponding to the well-defined crystalline β-sheet structure) is very sharp while the peak observed at 1699 cm−1, corresponding to the β-turn,23 has 6111

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Figure 4. 13C CPMAS NMR spectra under 10 kHz MAS. (A) The full 13C spectra of cellulose/SF blend (red), cellulose (blue), and SF (black) films. The peak assignments could be referenced to the molecular structures shown in Scheme 1. (B) Comparison of 13C NMR spectra in the Ala 13Cβ region for the regenerated SF and cellulose/SF blend films. The spinning sideband is indicated with the asterisk. All the spectra were acquired by coadding ∼400 transients at 10 kHz MAS using a 1.0 ms cross-polarization contact time and a recycle delay of 3 s.

Figure 5. High-resolution 1H CRAMPS NMR spectra of regenerated cellulose (black), SF (red), and cellulose/SF blend (blue) films obtained using the pulse sequence given in Figure 1A. All these spectra were acquired by co-adding 160 transients at 9.8 kHz MAS with a recycle delay of 3 s.

ppm is a characteristic peak for the β-sheet structure, which is 0.6 ppm downfield from that of the α-helix form (∼8.0 ppm).45 In addition, for the Hα protons (i.e., methine proton of Ala), it only shows a single peak at around 3.9 ppm for the random coil structures, while two distinct peaks at around 5.0 and 3.9 ppm will show up for the β-sheet structure. In fact, in the SF/MMT nanocomposite, these peak intensities were used to qualitatively compare the fraction of β-sheet structures in SF.69 Unfortunately, most of those characteristic peaks of SF in the 1H spectrum of cellulose/SF blend film are all masked by the featureless broad proton signals of cellulose. However, the amide proton peak at around 8.6 ppm does not overlap at all with any of cellulose proton peaks. Therefore, by probing the proximity between amide protons of SF and the carbons of cellulose, it is possible to determine the hydrogen bonding interactions between SF and cellulose, and thus to reveal the intermolecular miscibility, as shown in Figure 6. With a CP contact time of 0.4 ms, the 1H−13C HETCOR spectrum (Figure 6A) only shows the intramolecular correlations between covalent bonded 13C/1H spin pairs. But still, a weak correlation could be observed between the amide protons and the carbonyl carbons in SF, which can be ascribed to the chemical bonding (peptide bond, −NH−CO−) or intermolecular hydrogen bonding interactions (due to the welldefined β-sheet structure) between NH− and -CO groups in SF. When the CP contact time was increased to 1.0 ms, all the intramolecular and intermolecular correlations are observed. Interestingly, a strong correlation between amide proton of SF and carbons (C2, C3, and C5) of cellulose is clearly observed as indicated in red circles in Figure 6B, which was absent when the CP contact time was 0.4 ms. Such correlation clearly indicates

Figure 6. 2D 1H−13C HETCOR NMR spectra for the cellulose/SF blend film obtained using a CP contact time of (A) 0.4 ms and (B) 1.0 ms under 9.8 kHz MAS. (C) The sum projection of 2D spectra along the proton dimension for both 2D HETCOR spectra with a CP contact time of 0.4 ms (black) and 1 ms (red). 13C NMR spectra were shown on the top of both 2D spectra. All the 2D and 1D spectra are shown at the same intensity scale for a direct comparison of them. The red circle indicates the 13C−1H correlation between amide protons of SF and carbons (C2, C3, and C5) of cellulose, which is absent when the CP contact time was short as shown in (A). Eighty t1 increments with 400 scans were used for recording the 2D spectra, and the recycle delay was set as 1.5 s.

the strong intermolecular hydrogen bonding interactions between the − NH groups of SF and the −OH groups bonded to carbons C2 and C3 of cellulose. Because of the strong hydrogen bonding interactions between cellulose and SF, the −NH group signals are also significantly increased when the CP 6112

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in the cellulose and cellulose/SF blend films. Furthermore, the bottom hump for the cellulose/SF blend film is broader than that of regenerated cellulose film, indicating a more restricted chain dynamics in the blend film due to the cellulose-SF interactions as revealed by the HETCOR spectra shown in Figure 6. To further obtain deeper insights into the water localization and organization in the blend film, 2D 1H−13C WISE experiments with variable spin diffusion time were performed as shown in Figures 8 and 9. If the water is equally distributed

contact time was 1.0 ms, as reflected in the 1H projection shown in Figure 6C. Besides, it is noteworthy that the correlation between the −NH group of SF and carbon C6 of cellulose is relatively weak so that it was not present at the current intensity scale in Figure 6B, indicating that the −NH groups of SF prefer to form intermolecular interactions with the −OH groups bonded to carbons C2 and C3, while the −OH group bonded to carbon C6 or the ether oxygen bonded to C4 in the glucose ring are less favorable. In fact, the −OH group bonded to carbon C6 may tend to form intramolecular interactions with the − OH group bonded to carbon C2 or intermolecular interactions with other cellulose chains as are usually present in the amorphous cellulose.63 Such specific regiochemistry of −OH groups in cellulose may be closely related with the chain conformations of SF, and may play an important role in determining the miscibility of cellulose and SF in the blend film. As a matter of fact, it was also found that poly(ethylene oxide) (PEO) preferred to form hydrogen bonds with the −OH groups bonded to C6, while there were no evidence indicating the cellulose-PEO interactions between the −OH groups bonded to C2 and C3 and backbone oxygen in PEO in the PEO/cellulose blend.70 Herein, HETCOR NMR experiment clearly reveals the intimate mixing and specific regiochemistry of hydrogen bonds between SF and cellulose at the molecular level. This is also in a good agreement with the observed AFM morphology of the blend film by Shang et al.,20 where the SF was well dispersed in the matrix of cellulose. 3.4. Water Localization and Organization in the Cellulose/SF Film. It is well-known that the functions and properties of biopolymer could be strongly affected by the presence of water.71 As was mentioned above, the regenerated SF film is too brittle to perform mechanical test, because SF is mainly composed of bulky repetitive modular hydrophobic domains interrupted by a small fraction of hydrophilic groups.59 As a result, the 1H static NMR spectrum of air-dried SF film only exhibits a rather broad Gaussian peak (Figure 7),

Figure 7. 1H static NMR spectra of SF (black), cellulose (blue), and cellulose/SF (red) blend films. The spectra were acquired using DEPTH72,73 sequence to suppress the background signals. The spectra were acquired by co-adding 16 scans with a recycle delay of 3 s.

Figure 8. 2D WISE NMR spectra for the cellulose/SF blend film obtained at the indicated spin diffusion time (τm). The red arrows indicate the peaks of carbons C(2,3,5) and C6 of cellulose in the blend film. Eighty t1 increment with 192 scans were used for recording the 2D spectra. The recycle delay was set as 2.5 s.

indicating a rather rigid structure with restricted chain dynamics. However, for both the cellulose and cellulose/SF blend films, the proton spectrum is superimposed with a narrow peak on the top and a rather broad hump on the bottom, indicating the mobility contrast inside the molecular system. Considering that cellulose is highly hydrophilic due to the rich hydroxyl groups, this narrow peak could be ascribed to the signals of water. Since the full-width at half-maximum (fwhm) of this narrow peak is around 3.5 kHz, it can be reasonably inferred that the water may exist as the bound water

through the blend film, the narrow line in the 1H dimension will be expected to appear at all 13C positions when the spin diffusion time (τm) is long enough in the 2D WISE experiment. As is shown in Figure 8, with increasing the spin diffusion time, obvious narrow peaks appear on the top of proton wide-line spectra at the 13C position of cellulose, as indicated by the red 6113

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blend film. Both IR and 13C spectra revealed that the welldefined β-sheet structure was enhanced in the cellulose/SF blend film, while the disordered β-turn structure decreased with respect to the regenerated SF film. In fact, the well-defined βsheet structure is the primary molecular origin of the superior tensile strength in silk,59 and thus could explain the enhanced modulus in the cellulose/SF blend film compared to the regenerated cellulose film. In addition, the intimate molecular mixing between cellulose and SF was elucidated by 2D 1H−13C HETCOR spectra, where the −NH groups of SF preferred to form hydrogen bonds with the hydroxyl groups bonded to carbons C2 and C3, whereas the hydrogen bonding interactions between the −NH groups of SF and the hydroxyl groups bonded to carbon C6 and the ether oxygen were less favorable. This might be related with the specific conformation of SF in the cellulose/SF blend film. Furthermore, bound water was found to locate in immediate vicinity of cellulose in the cellulose/SF blend film, mostly due to the prevalent presence of hydroxyl groups and ether oxygen in the cellulose. This also clearly indicates the presence of nanoheterogeneity in the cellulose/SF blend film. In summary, solid-state NMR could provide detailed atomic-level insights into the conformational changes, intermolecular interactions and nanoheterogeneity in the cellulose/SF blend film, and all the experimental findings here could help reveal the molecular origin of the enhanced mechanical properties in cellulose/SF blend film. Extraction of such atomic-level structural information will be greatly beneficial for tailoring the structures and properties, and thus to fabricate high performance biomaterials or its nanocomposites. Although we have demonstrated the feasibility of solid-state NMR experiments under slow MAS spinning speeds in this study, the use of ultrafast-MAS, proton-detection, and higher magnetic fields could provide more piercing insights into these exciting class of materials.75,76 Specifically, the use of recently developed proton-based multidimensional experiments and proton chemical shift tensors would provide valuable insights to better understand the origin of mechanical properties of these challenging materials.77−80

Figure 9. Wideline proton NMR spectra obtained from the 2D WISE spectra of cellulose/SF blend film with variable spin diffusion times by taking 1H slices at the 13C chemical shift frequency of (A) carbon S1, (B) carbon S5, (c) carbon C6, and (d) carbon C1.

arrows. For better comparison, the 1H slices at different 13C position of SF and cellulose are shown in Figure 9. As is clearly seen, with a short spin diffusion time τm = 5 μs, all the proton spectra related to the carbons S1, S5, C6, and C1 are very broad. However, when the spin diffusion time τm = 10 ms, the proton spectra related to carbon S1 and S5 of SF are still almost the same as the one with τm = 5 μs, showing a broad Gaussian line shape. On the contrary, a rather narrow peak appears on the top of the wide-line spectra related to carbons C1 and C6 of cellulose when the spin diffusion time τm = 10 ms. That clearly indicates that water is more localized in the vicinity of cellulose chains, possibly due to the hydrogen bonding interactions between water and cellulose. When the spin diffusion time τm = 50 ms, all the proton slices have a relatively narrower peak on the top. However, the changes on the 1H slices of SF carbons are not that obvious as that of cellulose carbons, as the water content is relatively low, and the water magnetization is mostly distributed among the cellulose chains due to its higher content. But still, the fwhm of the proton peak related to the SF carbons is smaller at τm = 50 ms compared to that at τm = 10 ms and 5 μs. As a matter of fact, even in the cellulose/poly(vinyl alcohol) (PVA) blend system where PVA is highly hydrophilic, water was still found to locate in the vicinity of cellulose backbones, while no water signals could be detected in the immediate vicinity of PVA.74 Thus, the WISE experiment with spin diffusion clearly elucidates that the water is predominately bounded to the cellulose backbone, which is quite reasonable considering that the cellulose has abundant of hydroxyl groups and the SF contains only a small fraction of hydrophilic domains. Such strong association between water and cellulose can also explain the fact that the strength and toughness of the cellulose/SF blend film are both enhanced compared to the regenerated cellulose film as shown in Figure 2.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Rongchun Zhang: 0000-0002-2480-2652 Pingchuan Sun: 0000-0002-5603-6462 Ayyalusamy Ramamoorthy: 0000-0003-1964-1900 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support by the China Postdoctoral Science Foundation (No. 2016M601249) and the National Natural Science Foundation of China (NSFC) through the General Programs (Nos. 21534005 and 21374051).

4. CONCLUSION In this study, various types of high-resolution solid-state NMR approaches were implemented to systematically investigate the conformations and intermolecular interactions in the cellulose/ SF blend film in order to gain deep insights into the molecular origin of the enhanced mechanical properties of cellulose/SF

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REFERENCES

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