Self-healing mussel-inspired multi-ph-responsive hydrogel

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1 Supplementary information Self-healing mussel-inspired multi-ph-responsive hydrogel Marie Krogsgaard, Manja A. Behrens, Jan S. Pedersen and Henrik Birkedal Synthesis of the DOPA functionalized polyallylamine: DOPA was grafted onto polyallylamine using standard EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride)/nhs (N-hydroxysuccinimide) coupling chemistry. 4.2 g DOPA (21.4 mmol) (Sigma Aldrich, CAS no ) was dissolved in 0.47 L 0.5% TFA, which was continuously degassed with argon. The dissolution was aided by ultrasonication. Next, 1 g solid poly(allylamine hydrochloride) (10.7 mmol) ( MW, Polysciences Inc., CAS no ) was added to the reaction mixture in which it dissolved. The ph was adjusted to 6 by a slow addition of 2 M NaOH g EDC (32.1 mmol) (Sigma Aldrich, CAS no ) was dissolved in 14 ml demineralised water and the ph was adjusted to 6 with 10% TFA g NHS (32.1 mmol) (Sigma Aldrich, CAS no ) was likewise dissolved in 14 ml demineralised water and the ph adjusted to 6 with 2 M NaOH. The NHS solution was added to the DOPA/polyallylamine reaction mixture and after 15 minutes the EDC solution was added. The reaction components were allowed to react for 18 h in a light-protected bottle at room temperature under argon atmosphere and magnetic stirring. The synthesized polymer was purified by dialysis against 0.1 M HCl using Spectra/Por dialysis membranes with a MWCO of 3.5 kda under vigorous stirring. The DOPA functionalized polyallylamine polymers were thereafter isolated by rotary evaporation to give a homogenous light brown powder after grinding. The DOPA content was obtained by UV/Vis spectroscopy using pure DOPA as a standard. The pka value of the polymer was obtained by potentiometric titration at room temperature under nitrogen atmosphere in 0.1 M KCl with NaOH as the titration agent using a Metrohm 809 Titrando equipped with a calibrated Metohm Solvotrode and a mechanical Metrohm 802 stirrer (Metrohm AG, Herisau, Switzerland). Synthesis of catechol-fe III cross-linked gel: To obtain a typical 150 μl metal functionalized polymer gel with a polymer concentration of 150 mg/ml the following recipe was employed. Note that the rheology experiments employed 75 µl gels made by halving the volumes in the following recipe: 1) 22.5 mg DOPA functionalized polyallylamine was dissolved in 75 μl 0.55 M NaOH to a starting concentration of 300 mg/ml, which corresponds to a DOPA concentration of 136 mm. 2) The polymer solution from 1), which makes up 1/2 of the final volume, was mixed with 1/6 final volume (25 μl) of 136 mm FeCl 3 6H 2 O (Sigma Aldrich, CAS no ) (DOPA:Fe III ratio of 3:1). A green color developed instantly upon mixing (Figure 1a, top photograph) 3) The gel was formed by adding 2/6 final volume (50 μl) NaOH at a concentration adjusted to obtain the requested final ph of the hydrogel. (e.g. to get a gel with a final ph of approx. 9, 20 µl 2M NaOH and 30 µl 1M NaOH were added). Locally where the drop hits the polymer solution, an instant color change from green to wine red in the center and from green to purple/blue at the edge of the alkaline droplet can be seen (Figure 1a, middle photograph). The color development can be assigned to the different complexes formed in the sample; the wine-red color is due to the formation of tris-complexes at high ph and the purple color appears due to the formation of predominantly biscomplexes at intermediate ph 1. A homogenous hydrogel is established after one minutes mixing with a spatula (Figure 1a, bottom photograph). UV-Vis spectroscopy: The abundance of the mono-, bis- and tris-catechol- Fe III : DOPA-polyallylamine species as a function of ph was determined using a UV-Visible absorption spectrophotometer (Helius Alfa double-beam spectrophotometer) equipped with disposable UV-Vis cuvettes having a path length of 1 cm.

2 Figure S1. a) UV/Vis absorption profiles of DOPA-polyallylamine:Fe III solutions with a DOPA concentration of 0.4 mm and a DOPA to Fe III concentration of 3:1. The black arrow in the graph points in the direction of increasing ph. b) The sum of fractions as a function of ph. A Fe III :DOPA-polyallylamine solution with a DOPA concentration of 0.4 mm and a DOPA to Fe III ratio of 3:1 was titrated against NaOH, whilst the ph was monitored with a Metrohm ph-meter equipped with a Metrohm Unitrode. The ph was increased in steps of ~0.5-1 ph units to a final ph of 12 and the spectral changes were monitored by performing UV-Vis absorption measurements at every ph step from 200 nm to 800 nm with a scanning rate of 600 nm/min (Figure S1a). The relative abundances of the mono-, bis- and tris-catechol-fe III complexes were extracted by fitting a multi-gauss function in an iterative manner to the collected absorption data and models for the characteristic absorption of the mono-, bis- and tris-complex were proposed that could describe all absorption curves. For quantification, only the region above 400 nm was used as the peaks below this region were severely affected by peak overlap. The characteristic mono-complex peaks were found to be located at 406 nm, 505 nm and at 660 nm. The characteristic bis- and tris-complex peaks were located at ± 1.3 nm and 418 ± 4 nm, respectively. The relative fractions were obtained by normalizing the areas of the characteristic peaks associated with the different complexes to the maximum area-value for each characteristic peak. Note that the average mono-complex fraction was calculated based on the three species specific peaks found for the mono-species. As no constraints on the sum of fractions were imposed in the modeling of the data, it can be used to test the quality of the employed models (Figure S1b). The sum of fractions is close to 1 above ph 2. Below ph 2 it is less than 1, indicating that some of the Fe III ions are not complexed to DOPA in the solution. This can be explained by the electrostatic repulsion between the positively charged polymer and iron aqua complexes at low ph. SAXS The static SAXS measurements were performed on the modified SAXS NanoSTAR camera (Bruker-AXS GmBH, Germany) in transmission geometry at Aarhus University in Denmark 2. The apparatus was equipped with a Bruker AXS HI-STAR twodimensional position sensitive gas detector. 1.0 wt% polyallylamine solution without Fe III and 1.5 wt% DOPA-polyallylamine solutions with Fe III (DOPA:Fe III ratio of 3:1) were prepared outside the integrated vacuum using 10 ml 0.1 M HCl as solvent. The 1.5 wt% DOPA-polyallylamine corresponds to an equivalent 1.1 wt% polyallylamine solution, almost the same as for the pure polyallylamine ensuring that the amine concentration is almost the same. Subsequently, the ph was increased in a potentiometric titration with NaOH. A volume of 200 μl of the resulting solution was removed at each integer value of ph (eg. ph 1, ph 2, ph 3 ) and constituted a sample for the SAXS measurements. The dilution factor of the samples caused by the addition of base was neglected due to the small volume used to change the ph. Prior to each SAXS measurement, the samples were transferred to precalibrated home-made quartz capillaries (Diameter: 2 mm, wall thickness: mm). For each sample 6 SAXS measurements with an exposure time of 5 minutes at 20 C were performed. The data were corrected for background (derived from the capillary and solvent) as well as absorption and normalized to absolute intensity based on the scattering from a water standard. The intensity profiles of the polyallylamine samples was analyzed using the Worm-Like-Chain (WLC) model, which describes polymers as semi-flexible chains with a contour length, consisting of a number of rigid segments described with a Kuhn length and a cross section, i.e. I ( q) = ApolymerS WLC ( q, L, b, R), where A polymer is a scale factor, S WLC is the WLC form factor with L being an effective contour length, b the Kuhn length, and R the cross-section radius of the chains. L is not the real polymer contour length since the concentration of the polymer is so high that the solutions are in the semi-dilute regime with highly overlapping and entangled polymer chains. It is thus related to the correlations length of the gel network which is sometimes also called the mesh size of the network. The model fits the data for the polyallylamine samples very well as can be seen in Figure S2 that shows example fits.

3 Figure S2. SAXS modeling of the 1.0 wt% polyallylamine system, examples of SAXS fits are shown at ph 1 (black), 4 (blue) and 8 (red). Points represent data while the full lines are fits. For the DOPA-polyallylamine/Fe III system, the WLC model clearly does not suffice to describe the data at any ph due to the presence of additional scattering intensity at low q originating from long-range density fluctuations caused by the inhomogeneous random cross-linking of the polymer chains. Hence the model consists of the WLC model and a term describing the long-range density fluctuations. The worm-like chain model describes the short range polymer correlations of the constituting polyallylamine chains. The model is 2 2 I( q) = ApolymerSWLC( q, L, b, R) + Afluct exp( Rg,fluct q /3) where A fluct is scale factors and R g,fluct is the radius of gyration of the long-range fluctuations. The model fits the data for the DOPA-polyallylamine very well up to ph 8 as illustrated in Figure S3A. Beyond this ph there are significant structural changes that cannot be captured by the model as can be seen by comparing the data at ph 8 with those at ph 9, Figure S3B. Figure S3. SAXS modeling of the 1.5 wt% DOPA-polyallylamine/Fe III system. (A) Example SAXS fits at ph 1 (black), 4 (blue) and 8 (red). Points represent data while the full lines are fits. (B) Comparison of SAXS data at ph 8 (red) and 9(green); note the qualitative change in curve shape in particular around q~ Å -1. Figure S4 summarizes the fit results for the DOPA-polyallylamine/Fe III system and the polyallylamine system. For the polyallylamine system the Kuhn length, which is a measure of the flexibility of the polymer chains, displays a linear region up to a ph around 7 and then it decreases exponentially as a function of ph (Figure S4a). The initial decrease in the flexibility of the chains is unexpected; however, it might be explained by an increased hydration of the polyallylamine polymers. The sudden gain in polymer flexibility after the transition point correlates with the fact that much of the electrostatic repulsions along the chains have been removed at this ph, causing them to be less rigid. For the polyallylamine system the contour length likewise displays a linear region up to a ph around 7 and above it increases exponentially as a function of ph (Figure S4b). As expected, the SAXS determined contour length is smaller than the real end-to-end distance as the polymer size is outside the detection limit of SAXS. However, the determined contour length can be interpreted as an effective contour length of the polymers. Above the transition point, the effective contour length increases exponentially. The sudden increase in the effective contour length above the transition point can be explained by an aggregation of the polymers, caused by the removal of electrostatic repulsive forces. The polyallylamine polymer cross section also displays a linear region up to a ph around 8 after which it decreases (Figure S4c). The cross section depends, to a high degree, on the scattering length density distribution difference and the decreasing cross section after the transition point can be explained by a reduction of the scattering length density distribution difference. Below the transition point the negative chloride counter ions will be closely associated with polymer backbone as the polymers are highly

4 positively charged, thereby producing a high scattering density distribution difference. Above the transition point the close association between counterions and the polymer backbones is to some extent lost, causing the scattering length density distribution difference and thus the cross-section to be reduced. Figure S4: SAXS data of 1.0 wt% polyallylamine (closed circles) and 1.5 wt% DOPA-polyallylamine/Fe III (open squares) recorded at different ph values. a) Kuhn length b) Contour length and c) Cross-section as a function of ph. Panel d) displays the effective radius of gyration from the fluctuation term for the fits of the 1.5 wt% DOPA-polyallylamine/Fe III system while panels e) and f) show the scale factor of the WLC polymer scattering and the fluctuation term on the same scale reflecting the large importance of the fluctuation term. The red curves in b), c) and d) are guides to the eye. Dynamic Oscillatory Rheology: The mechanical properties of the hydrogels were measured with an Anton Parr MCR 501 Rheometer using parallel plate geometry (8 mm rotating top plate). An Anton Parr evaporation hood equipped with a custom made accessory was employed to prevent solvent evaporation. Prior to each measure the hydrogels were allowed to rest for 1 hour in air tight containers to facilitate complete diffusion and relaxation of the polymer network. Frequency sweeps was performed in the linear viscoelastic range (LVR) at constant strain amplitude of 15 % (LVR is e.g. 0-30% strain for a hydrogel at ph 9 (Figure 3)) while the storage G and loss modulus G were measured. Figure S3a displays G and G for frequency sweeps performed on hydrogels with a final ph of 5, 8

5 and 12. The quantitative recovery test was likewise performed on a hydrogel that had been allowed to rest for 1 hour prior to the measurements. All measurements were performed at 20 C. Figure S5: a) Storage G (left) and loss modulus G (right) as a function of angular frequency. b) Pulling and lifting of the gel with a spatula. The red line in the photograph to the right indicates the initial spatula formed fracture. Gel reversibility: To test gel reversibility, gels were made as described above. ph reversibility was tested by inspection by eye: Addition of acid (0.1 M HCl) lead to dissolution of the gel while subsequent re-addition of base resulting in formation of another gel. We also tested that competitive complexation of Fe III with EDTA. A 37.5 µl hydrogel was allowed to rest for 1 h after formation (as in the rheology experiments). Thereafter it was embedded in ~3 ml 150 mm EDTA at ph 5. The sample was lightly shaken by hand during the first 5 min and thereafter left at rest. After 30 min most of the gel material had dissolved; after 11 h only traces of gel-like material could be seen, see Figure S6. Figure S6: EDTA treatment dissolves gel. See text for details. References: (1) Holten-Andersen, N.; Harrington, M. J.; Birkedal, H.; Lee, B. P.; Messersmith, P. B.; Lee, K. Y. C.; Waite, J. H. PNAS 2011, 108, (2) Pedersen, J. S. Journal of Applied Crystallography 2004, 37,