Control of seed formation allows two distinct self-sorting patterns of supramolecular nanofibers

Self-sorting double network hydrogels comprising orthogonal supramolecular nanofibers have attracted attention as artificially-regulated multi-component systems. Regulation of network patterns of self-sorted nanofibers is considered as a key for potential applications such as optoelectronics, but still challenging owing to a lack of useful methods to prepare and analyze the network patterns. Herein, we describe the selective construction of two distinct self-sorting network patterns, interpenetrated and parallel, by controlling the kinetics of seed formation with dynamic covalent oxime chemistry. Confocal imaging reveals the interpenetrated self-sorting network was formed upon addition of O-benzylhydroxylamine to a benzaldehyde-tethered peptide-type hydrogelator in the presence of lipid-type nanofibers. We also succeed in construction of a parallel self-sorting network through deceleration of seed formation using a slow oxime exchange reaction. Through careful observation, the formation of peptide-type seeds and nanofibers is shown to predominantly occur on the surface of the lipid-type nanofibers via highly dynamic and thermally-fluctuated processes.


In situ formation of BnOx-F(F)F from Ald-F(F)F gel. A suspension of an
(4.0 µM) and NBD-cycC 6 (4.0 µM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. The resultant mixture was cooled to rt and incubated at rt for 24 h. The state (gel or sol) of the sample was judged by the tube inversion test. The obtained suspension (containing fluorescent probes) was moved to a glass bottom dish and observed by CLSM imaging.

Mixing hot solutions of BnOx-F(F)F and Phos-MecycC 5 . The suspensions of
BnOx-F(F)F/NP-Alexa647 (8.6 mM and 8.0 µM) and Phos-MecycC 5 /NBD-cycC 6 (4.8 mM and 8.0 µM) were separately heated by a heating gun until dissolving. The equal volume of the resultant hot solutions were immediately mixed and incubated at rt for 1 h. The state (gel or sol) of the sample was judged by the tube inversion test. The obtained suspension (containing fluorescent probes) was moved to a glass bottom dish and observed by CLSM imaging.
CLSM imaging in the oxime-exchange protocol. The suspension of Ald-F(F)F (17.3 mM) and NP-Alexa647 (4.0 µM) with Phos-MecycC 5 (2.4 mM) and NBD-cycC 6 (4.0 µM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. After cooling to rt, the resultant mixture (10 µL) was transferred to a glass bottom dish (Matsunami) before gelation. After incubation at rt for 1 h in the presence of water to avoid dryness, CLSM imaging was conducted. To the resultant hydrogel, a solution of carboxymethoxylamine (208 mM, 1 µL in 100 mM MES, pH 6.0) or buffer was added.
After incubation for 4 h, CLSM imaging was conducted. Subsequently, O-benzylhydroxylamine (300 mM, 2.3 µL in 100 mM MES, pH 6.0) or buffer was added to the resulting solution. After incubation at rt for 48 h, CLSM imaging was conducted. Rheological analysis. The preparation of the hydrogel was the same as described above.

Suspension of CaOx-F(F)F and Phos-MecycC
The resultant disk-shaped hydrogels (ca. 10 mm) were carefully took out from the PDMS mold and put onto the stage of a rheometer (MCR-502, Anton Paar) with a parallel plate geometry. Strain sweep data were obtained using shear mode at a frequency of 10 rad/s, and linear dynamic viscoelasticity were measured in shear mode at 0.3 or 1.0% strain amplitude for frequency sweep.
Determination of nanofiber elongation velocity. The elongation distance of the peptide-type nanofiber was estimated by comparing the successive two images of the time-lapse imaging with Fiji. The velocity was calculated by dividing the elongation distance by interval of time-lapse imaging. 50 and 120 elongated nanofibers were randomly selected (supplementary Fig. 6 and 40, respectively). The histograms were depicted by using Kaleidagraph 4.5 (Synergy Software).

Supplementary Tables
Supplementary Table 1

Supplementary Table 2. Critical gelation concentrations of peptide-type hydrogelators
Concentrations of the peptide-type hydrogelator One-step oxime formation protocol: 4.