Double diffusion for the programmable spatiotemporal patterning of multi-domain supramolecular gels

To achieve spatial resolution of a multi-component gel, a double diffusion approach is used which enables the precise programming of self-assembled patterned domains with well-defined shapes and sizes. The low-molecular-weight gelators (LMWGs) used in this study are pH-responsive DBS-CO2H and thermally-responsive DBS-CONHNH2 (both based on 1,3:2,4-dibenzylidenesorbitol, DBS). A DBS-CONHNH2 gel was initially assembled in a tray, and then loaded at carefully-selected positions with either basified DBS-CO2H (i.e. DBS-carboxylate) or an acid. These soluble components subsequently diffuse through the pre-formed gel matrix, and in the domains when/where they mix, protonation of the DBS-carboxylate induces self-assembly of the DBS-CO2H network, leading to a patterned gel-in-gel object with well-defined shape and dimensions. Using a strong acid achieves fast gelation kinetics, creating smaller, better-defined macroscale objects but with less nanoscale order. Using a weak acid source with slow kinetics, gives slightly larger objects, but on the nanoscale the DBS-CO2H network formation is better controlled, giving more homogeneous nanoscale structures and stiffer objects. The patterned objects can be further reinforced by the presence of agarose polymer gelator. The shape of the patterning is programmed by both the shape of the central reservoir and the starting geometry in which the reservoirs are organised, with the balance between factors depending on assembly kinetics, as dictated by the choice of acid. This simple methodology therefore enables programming of patterned gels with spatiotemporal control and emergent patterning characteristics.


S1. General Experimental Methods
All compounds used in synthesis and analysis were purchased from standard commercial suppliers and used as received. The synthesis of DBS-CONHNH2 and DBS-COOH were performed in good yields applying previously reported methods. 1,2 1 H NMR spectra were recorded using a Jeol 400 spectrometer ( 1 H 400 MHz) or a 500 spectrometer ( 1 H 500 MHz). Samples were prepared in D2O and chemical shifts () are reported in parts per million (ppm). IR spectra of xerogels were recorded on a PerkinElmer Spectrum Two FT-IR spectrometer. TEM images were obtained on a FEI Tecnai 12 G 2 fitted with a CCD camera. Fibre sizes were measured using the ImageJ software. SEM images were taken using a JEOL JSM-7600F field emission SEM. Rheology was measured on a Malvern Instruments Kinexus Pro+ Rheometer fitted with a 2 cm parallel plate geometry.

S2.1 Preparation of gels in trays for RD-SA
A suspension of DBS-CONHNH2 (8.43 x 10 -3 mmol, 0.4 % wt/vol) in water (5 mL) was prepared, and Thymol Blue (20 L, 1% in EtOH) was added. For the gels with agarose, agarose (1.0 % wt/vol) was added to the suspension of DBS-CONHNH2 and Thymol Blue before sonication. The opaque yellow solution was sonicated for 15 minutes, then heated with a heat gun until complete dissolution of the compound. The hot solution was transferred to a 5 cm x 5 cm gel tray and left to cool overnight. Circular holes were then cut into the gel using a pipette tip, in either a square or triangular arrangement. A basic solution of DBS-CO2H (25 L, 8 mg/60 L 1M NaOH) was added to the central hole. The outer holes were loaded with HCl or GdL (25 L per hole) of total acid loading 0.03 mmol, 0.06, 0.144, or 0.20 mmol. The loading of each HCl/GdL hole was the total acid loading divided by the number of outer holes [for 0.03 mmol HCl(Triangle), 0.01 mmol HCl, 25 L per hole x 3]. After 28 hours, the multi-component hydrogel object was fabricated. Using a spatula, the object was cut out and removed from the surrounding gel to yield the freestanding object.
The procedure was repeated as described above to fabricate the more intricately shaped hydrogel objects but using different shaped cutters for the holes. The more complex patterns were studied with the 0.20 mmol HCl or GdL hybrid gels.
S3 Figure S2. The diffusion of the DBS-CO2H gelator precursors for the 0.06 mmol GdL(Square) (upper) and HCl(Square) (lower) hybrid gels. The basic DBS-COOsolution is indicated blue by TB, the acidic HCl, pink. GdL, of a higher pH, maintained the yellow colour of the gel. Diffusion monitored for 7 hrs, and then at 28 hrs.
Complete acidification was observed after 28 hours of diffusion. Figure S3. The triangle and square gel patterns, for 0.144 mmol HCl(Triangle) (upper), and 0.20 mmol HCl(Square) (lower) hybrid gels. The basic DBS-COOsolution is indicated blue by TB; the acidic HCl, pink. Figure S4. The distance of blue DBS-COOsolution diffusion from the central loaded hole, relative to the 5 cm tray, for the first hour of diffusion (60 min). The graphs compare the GdL(Square) LMWG (no agarose, blue line) and hybrid (agarose, green line) gels, at each loading. Figure S5. The distance of blue DBS-COOsolution diffusion from the central loaded hole, relative to the 5 cm tray, over a period of 7 hours (420 min). The graphs compare the HCl(Square) LMWG (no agarose, blue line) and hybrid (agarose, green line) gels, for 0.06 and 0.144 mmol HCl.  Figure S6. Gel object formation for the 0.03 mmol HCl(Triangle) hybrid (A) and LMWG (B), GdL(Triangle) hybrid (C), and LMWG (D) gels, pictured between 0 and 7 hours, and then at 28 hours. The structure was cut and removed to give the free-standing object. Figure S7. Gel object formation for the 0.06 mmol HCl(Square) hybrid (A) and LMWG (B), GdL(Square) hybrid (C), and LMWG (D) gels, pictured between 0 and 7 hours, and then at 28 hours. The structure was cut and removed to give the free-standing object. Figure S8. Gel object formation for the 0.144 mmol HCl(Triangle) hybrid (A) and LMWG (B), GdL(Triangle) hybrid (C), and LMWG (D) gels, pictured between 0 and 7 hours, and then at 28 hours. The structure was cut and removed to give the free-standing object. , and LMWG (D) gels, pictured between 0 and 7 hours, and then at 28 hours. The structure was cut and removed to give the free-standing object.

S3. NMR Studies
Gel objects were studied by NMR: HCl and GdL, each at 0.03, 0.06, 0.144 and 0.20 mmol loading for both the LMWG and hybrid gels and in the triangle and square patterning arrangement of precursor holes.
Gel objects were prepared in 5 mL gel trays but in the absence of Thymol Blue indicator. Using a spatula, the object was cut out and removed from the surrounding gel, placed in a vial and left to dehydrate to form the xerogel. The object xerogel was dissolved in DMSO-d6 solvent (0.71 mL) and sonicated until complete dissolution of the xerogel. Acetonitrile (2 L,  = 2.09 ppm) was added as an internal standard, and the solution filtered and transferred into an NMR tube.
The quantity of LMWGs in the object was therefore calculated by using the peak integration of the LMWG aromatic peak (ca. 7 -8 ppm) relative to that of acetonitrile. Figure S10. 1 H NMR spectra of 0.03 mmol HCl(Square) (left) and GdL(Square) (right) LMWG xerogel object. The enlarged regions highlight the aromatic gelator peaks used to obtain the relative integrals. The quantity of LMWGs in the object was calculated by using the peak integration of the LMWG aromatic peak (H ca. 7 -9 ppm, 4H) relative to that of acetonitrile (H = 2.07 ppm, 3H). The integral of acetonitrile was multipled by 10 and set equal to 30, hence the LMWG integral was divided by 40 rather than 4 (corresponding to the 4H). The moles of acetonitrile added (2 L) were 0.0383 mmol [0.786 g mL -1 , 41.05 g mol -1 ]. The moles of LMWG in the object was calculated as follows: The percentage of DBS-CO2H within the object was then calculated.

S4. Infrared (IR) Spectroscopy
The single-and multi-component LMWG gels were made in the absence of Thymol Blue. The LMWG and hybrid object and outer domains of the HCl(Square) system, at 0.03, 0.06, 0.144, and 0.20 mmol HCl loadings were analysed. Using a spatula, the object and outer domain were cut out and removed from the surrounding gel, placed in a vial and left to dehydrate to form xerogels. The xerogels were crushed, and the powder was placed into the infrared spectrophotometer and the spectra recorded.

S5. Transmission and Scanning Electron Microscopy (TEM and SEM)
The object and outer domains from the 0.20 mmol HCl(Square) and GdL(Square) LMWG and hybrid gels, were studied by TEM and SEM. The gels were prepared in 5 mL gel trays but in the absence of Thymol Blue indicator. Using a spatula, the object and outer domain were cut out and removed from the surrounding gel and covered with water to prevent dehydration.

TEM Sample Preparation.
The TEM gel samples were affixed to HT-treated formvar/carbon 200 mesh copper grids then negatively stained with 1% aqueous uranyl acetate. Images were taken between x 6800 and x 49000 magnification.

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Fibre Width Measurements. Using the software ImageJ, fibre widths were measured from the TEM images obtained. For each sample, ca. 50-100 fibres were measured, and the percentage of fibres of width within the specified ranges calculated. Figure S20. Gelator fibre width size ranges for 0.20 mmol HCl (left) and 0.20 mmol GdL (right) hybrid gel objects and outer domains.

S6. Thermal Studies (Tgel)
The Tgel values of the object and outer domains from the 0.20 mmol HCl(Square) LMWG and hybrid gel were obtained. The gels were prepared in 5 mL trays but in the absence of Thymol Blue indicator. Using a spatula, the object and outer domain were cut out and removed from the surrounding gel and placed into a closed vial.
Tgel values for the single-component LMWG gels of DBS-CONHNH2, DBS-CO2H, agarose, and the multicomponent gel of DBS-CONHNH2/DBS-CO2H were obtained. Tgel values for the hybrid single-component gel of DBS-CONHNH2, and the multi-component DBS-CONHNH2/DBS-CO2H were also obtained. These gels were made in vials.
The vials were placed in a thermo-controlled oil bath, with an initial temperature of 30 °C, programmed to rise to a temperature of 100 °C. At each increase of 1 °C, vials were removed from the oil bath, and tipped sideways for the object and outer gel samples or inverted for the vial-filling LMWG and hybrid gels. For the objects and outers, the Tgel value recorded was the temperature at which the sample began to melt, indicated by a gel-sol transition. The temperature at which the gels were no longer self-supporting. It should be noted that while the gels in vials are sample-spanning, this is not necessarily the case for the cut out gels, as such some caution must be applied in comparing Tgel values between the two different methods.

S7. Rheology
Rheological studies were conducted on the object and outer domains from the HCl(Square) LMWG gels, and HCl(Square) and GdL(Square) hybrid gels, at 0.03, 0.06, 0.144, and 0.20 mmol acid loadings. The gels were prepared in 5 mL trays, but in the absence of Thymol Blue indicator. The object was cut and removed from the surrounding gel using a spatula. A bottomless vial was used to cut the outer gel domain, in order to obtain a sample of appropriate gel dimensions. Samples were covered with water to prevent dehydration. Measurements were carried out at 25 °C using a 2 cm parallel plate geometry and a gap of 1.7 mm. Amplitude

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sweep experiments were performed in the range of 0.01-100% shear strain at a 1 Hz frequency to identify the linear viscoelastic region (LVR). Frequency sweep experiments were performed between 0.1 and 100 Hz using a shear strain of 0.01%. To ensure reproducibility, the reported value is the average value of duplicate or triplicate concordant results -the rheology was often challenging, particularly in the case of objects containing holes. Table S3. Elastic (G') and viscous (G'') moduli, and % shear strain of object and outer domains for the HCl(Square) hybrid gels, at 0.03, 0.06, 0.144, and 0.20 mmol HCl. All were gels, with G' > G''.  Table S4. Elastic (G') and viscous (G'') moduli, and % shear strain of object domain for the HCl(Square) LMWG gels, at 0.03, 0.06, 0.144, and 0.20 mmol HCl. All were gels, with G' > G'', except 0.144 mmol HCl where G' < G''; the object appeared gel-phase upon formation and loading. The outer domains exhibited a gel-sol transition during RD-SA with HCl, therefore could not be analysed by rheology.  Table S5. Elastic (G') and viscous (G'') moduli, and % shear strain of object and outer domains for the GdL(Square) hybrid gels, at 0.03, 0.06, 0.144, and 0.20 mmol GdL. All were gels, with G' > G''.  Figure S21. Elastic (G', green circles) and viscous (G'', blue circles) moduli of the 0.03 mmol HCl(Square) hybrid object, with increasing shear strain (left) and frequency (right).