advances.sciencemag.org/cgi/content/full/5/9/eaaw2541/DC1

Supplementary Materials for

Biomimetic composites with enhanced toughening using silk-inspired triblock

proteins and aligned nanocellulose reinforcements

Pezhman Mohammadi*, A. Sesilja Aranko, Christopher P. Landowski, Olli Ikkala, Kristaps Jaudzems, Wolfgang Wagermaier, Markus B. Linder*

*Corresponding author. Email: pezhman.mohammadi@vtt.fi (P.M.); markus.linder@aalto.fi (M.B.L.)

Published 13 September 2019, Sci. Adv. 5, eaaw2541 (2019)

DOI: 10.1126/sciadv.aaw2541

The PDF file includes:

Fig. S1. Coacervation of proteins, coacervate infiltration of cellulose, and viscosity of mixtures. Fig. S2. Double-injection CNF-protein fiber spinning and apparatus for making CNF-protein films. Fig. S3. Mean values and SDs of mechanical properties for CNF-only and composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers at a protein-to-CNF ratio of 1:2. Fig. S4. Mean values and SDs of mechanical properties for CNF-only and composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers at different mixing ratios. Fig. S5. Mean values and SDs of mechanical properties for CNF-only and composite fibers made from mono-, di-, and triblock protein variants. Fig. S6. Effect of orientation on the mechanical properties of the oriented CNF-only and composite CBM-eADF3-CBM-CNF fibers and nonoriented corresponding films. Fig. S7. Conformational studies by NMR. Fig. S8. Mechanical properties of composite fibers at different RHs. Fig. S9. SEM fractography for all fibers after tensile measurement test.

Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/5/9/eaaw2541/DC1)

Movie S1 (.mp4 format). Infiltration of CNFs by CBM-eADF3-CBM coacervates.

Fig. S1. Coacervation of proteins, coacervate infiltration of cellulose, and viscosity of mixtures. (A) Phase contrast light microscopy images of concentrated solutions (4% w/v) of different constructs showing coacervation of triblock architecture protein (CBM-ADF3-CBM) versus di- and mono- block architecture which does not self-assemble into coacervates (scale bars are 10 µm). (B) Infiltration of CNF network by matrix protein of CBM-ADF3-CBM in comparison to di- and monoblock matrix protein architecture in free monomeric form (scale bars are 10 µm). (C) Zero-shear viscosity for only CNF and coacervate CBM-eADF3-CBM-CNF at five different blend ratios measured to identify suitable mixtures with a spinnable viscosity (ranging 500-5000 Pa·s). We used 2% w/v CNF as the starting concentration; which was mixed with coacervate in 1:3, 1:2, 1:1, 2:1 and 3:1 ratio (coacervate to CNF). At the onset of shear (0.101 s-1), CNF suspension shows 1010 Pa·s viscosity. Addition of the CBM-eADF3-CBM leads to increase in the viscosity of suspension dramatically, with values ranging from 1350 Pa·s (1:3) to 6970 Pa·s (3:1). All the coacervate-CNF suspension including pure CNF showed typical shear thinning characteristic due to entanglement break up. The 3:1 (coacervate to CNF) coacervate-CNF suspension was eliminated from the further study as the viscosity of the solution exceeded the spinnability range.

Fig. S2. Double-injection CNF-protein fiber spinning and apparatus for making CNF-protein films. (A) Scheme of the double injection spinning apparatus used for the fiber spinning. Black arrows represent direction of the flow and increasing velocity with developing laminar flow and fully developed laminar flow depict parabolic velocity distribution at the cross section of capillary, with the highest velocity and lowest shear at the center of capillary and lowest velocity and highest shear close to the capillary wall. (B) CNF fibers could be made continuously in meter-amounts with the double injection spinning apparatus. (C) Custom build instrumentation used for fabrication CNF films to study the effect of orientation of the CNF on the mechanical properties. (D) SEM image of yarns made by twisting two to seven individual fibers. Photo Credit: Pezhman Mohammadi, Aalto University

Fig. S3. Mean values and SDs of mechanical properties for CNF-only and composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers at a protein-to-CNF ratio of 1:2. (A) max-stress, (B) Young's modulus, (C) maximum strain, (D) work of fracture, (E) yield point, (F) slope after the yield point and (G) yield strain. The significance level denoted with a different letter (a and b). Samples sharing a common letter have no significant difference (ANOVA/Tukey, p-value <0.05). (H) Stress-strain curves from cyclic measurements of the only CNF and CBM-eADF3-CBM-CNF composite fibers. The panel also shows on the right zoomed-in images of the two curves, to show the details of the first rounds of cycles. For panels A, B, C, D, E, F, and G, the experiments were repeated eight times. The mean values and the standard deviations are shown (N=8).

Fig. S4. Mean values and SDs of mechanical properties for CNF-only and composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers at different mixing ratios. (A) Max-stress, (B) Young's modulus, (C) maximum strain, (D) work of fracture, (E) yield point, (F) slope after the yield point, and (G) yield strain. The significance level denoted with a different letter (a, b, c and d). Samples sharing a common letter have no significant difference (ANOVA/Tukey, p-value <0.05). The mean values and the standard deviations are shown (N=8).

Fig. S5. Mean values and SDs of mechanical properties for CNF-only and composite fibers made from mono-, di-, and triblock protein variants. (A) Max-stress, (B) Young's modulus, (C) Maximum strain, (D) Work of fracture, (E) Yield point, (F) slope after the yield point and (G) Yield strain. The significance level denoted with a different letter (a, b, c and d). Samples sharing a common letter have no significant difference (ANOVA/Tukey, p-value <0.05).In total for every sample eight fibers measured and then mean value and standard deviation calculated accordingly (N=8). (H) Effect of adhesive matrix protein variant and their effects on the morphology of the fibers studied by polarized optical microscopy, WAXS, and SEM for only; CNF spun fiber, CBM-eADF3-CBM spun with CNF, CBM-ADF3-CBM spun with CNF, CBM-eADF3 spun with CNF, CBM-CBM with CNF, and CBM spun with CNF. Alignment of CNFs can be qualitatively seen as intense colors product of variation in thickness and the birefringent throughout the length of the fibers when images between crossed polarizers. SEM images show the preferential alignment of the surface texture of the fibers and a cross-sectional structure of the fibers showing nearly

circular with a diameter of about 50 µm. Altogether; we noticed similar morphological characteristics shared among all fibers spun from either dispersed monomeric matrix protein, coacervated protein or even only CNF fibers.

Fig. S6. Effect of orientation on the mechanical properties of the oriented CNF-only and composite CBM-eADF3-CBM-CNF fibers and nonoriented corresponding films. (A) Synchrotron WAXS measurement on oriented fibers and not oriented films made from only CNF and CBM-eADF3-CBM-CNF. Mean value and standard deviations of mechanical properties for oriented only CNF and composite CBM-eADF3-CBM-CNF fibers and not oriented corresponding films. (B) Max-stress, (C) Young's modulus, (D) maximum strain, (E) work of fracture, (F) yield point, (G) slope after the yield point and (H) yield strain. The significance level denoted with a different letter (a, b, c and d). Samples sharing a common letter have no significant difference (ANOVA/Tukey, p-value <0.05). Only CNF film demonstrated highest strain, lowest stiffness and strength among all four specimens with a slope after plastic deformation illustrating typical strain hardening. This is a tendency of isotopic CNF fibrils network during the pulling forces in which fibrils mainly dislocate and orient themselves in the direction of stretching after the yield point and during plastic deformation. This dramatically can change, if CNFs become oriented with direct influences on material mechanical properties. Orientation in the fibers uplifted almost any of the tensile

properties as a result of more advanced fibril-fibril interaction and increased hydrogen bonding between oriented CNFs. Moreover, a noticeable change in CNF fiber was that strain hardening almost entirely disappeared during plastic deformation which was correlating strongly to orientation parameter. Infiltration of the both isotropic and anisotropic CNF network by coacervate lead to greater mechanical properties. However, the effect was great if the materials fabricated with coacervate and anisotropic fiber over coacervate and anisotropic film. In total for every sample eight fibers measured and then mean value and standard deviation calculated accordingly (N=8). (I) Calculated full-width half max (FWHM) and Hermans orientation parameter for all fibers. Quantitative measurement of the fibrillar alignment using synchrotron WAXS and calculated Herman’s orientation parameters from the azimuthal intensity profile of the (004) reflection showed high fibrillar alignment with more or less similar values ranging from 0.68-0.74. In total for every sample two fibers at three different positions two millimeter apart were measured and mean value and standard deviation for FWHM was then calculated.

Fig. S7. Conformational studies by NMR. (A) 2D 1H-1H TOCSY, (B) 13C-1H HSQC, (C) 1D 1H-13C CP-MAS and (D) 2D 1H-13C HETCOR MAS NMR spectra of CBM-eADF3-CBM used for assignment of Ala Cα and Cβ chemical shifts before (A, B) and after (C, D) coagulation with ethanol. The observed Ala correlations in each spectrum are indicated and the assigned chemical shifts are shown in parentheses. (E) Fourier transform infrared spectroscopy (FTIR) of the coacervate before (dark blue spectrum) and after solvent precipitation, (red spectrum precipitated with 96% ethanol same as the coagulation bath used during fiber spinning and pink spectra is with 95.5% isopropanol). FTIR spectra of coacervates in water showed a major amide I band at 1630 cm-1 (associated with C=O vibration). However, aggregation in 96 % ethanol resulted in a shift of the major amide I band and higher intensity peak (at 1642 cm-1). Coagulated coacervate showed prominent amide II bands at 1542 cm-1 ν (associated with N-H vibration) which was absent before solvent. The coacervate showed an amide III band at 1243 cm-1 ν (associated with C-N vibration), which was slightly shifted and appearing with higher intensity for aggregated coacervate at 1248 cm-1 ν. In order to confidently interpret observed peak associated with conformational transition and crystallization in FTIR, we repeated the experiment however instead of ethanol and isopropanol we precipitated the protein using potassium phosphate which is shown to induce conformation of transition and crystallization in engineered spider silk protein and reconstituted silk proteins (light blue spectra).

Fig. S8. Mechanical properties of composite fibers at different RHs. (A) Stress-strain curve of only CNF and CBM-eADF3-CBM measured at different relative humidity. Mean value and standard deviations of mechanical properties for only CNF, composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers measured at different relative humidity. (B) Max-stress, (C) Young's modulus, (D) maximum strain, (E) work of fracture, (F) yield point, (G) slope after the yield point, and (H) yield strain. The significance level denoted with a different letter (a, b, c, d and e). Samples sharing a common letter have no significant difference (ANOVA/Tukey, p-value <0.05). Distinctly at 80 % RH, pure CNF based fibers showed a stress-strain curve with negligible yield point (15 MPa) and lower stiffness (5 GPa), however with increased plastic deformation (17 %). This can be explained with water acting as a lubricant on the surface of CNFs, in which facilitating the CNFs pass each other during mechanical stretching. The mean values and the standard deviations are shown (N=8).

Fig. S9. SEM fractography for all fibers after tensile measurement test.