Biocatalytic reversible control of the stiffness of DNA-modified responsive hydrogels: applications in shape-memory, self-healing and autonomous controlled release of insulin

The enzymes glucose oxidase (GOx), acetylcholine esterase (AchE) and urease that drive biocatalytic transformations to alter pH, are integrated into pH-responsive DNA-based hydrogels. A two-enzyme-loaded hydrogel composed of GOx/urease or AchE/urease and a three-enzyme-loaded hydrogel composed of GOx/AchE/urease are presented. The biocatalytic transformations within the hydrogels lead to the dictated reconfiguration of nucleic acid bridges and the switchable control over the stiffness of the respective hydrogels. The switchable stiffness features are used to develop biocatalytically guided shape-memory and self-healing matrices. In addition, loading of GOx/insulin in a pH-responsive DNA-based hydrogel yields a glucose-triggered matrix for the controlled release of insulin, acting as an artificial pancreas. The release of insulin is controlled by the concentrations of glucose, hence, the biocatalytic insulin-loaded hydrogel provides an interesting sense-and-treat carrier for controlling diabetes.

No.

Measurement
The absorbance spectra were recorded by a UV-2450 spectrophotometer (Shimadzu). SEM images were taken by using High

Synthesis of coumarin-insulin
The synthesis of coumarin-insulin was according to previous literature. 1 Insulin (5 mg) and 7-hydroxycoumarin-3-carboxylic acid Nsuccinimidyl ester (16 mg dissolved in 300 µL acetone) were dissolved in HEPES buffer (100 mM, pH 7) that contained EDTA (200 µM) to prevent aggregation of insulin. The reaction was mixed in the dark for 8 h, and then the mixture was washed with an Amicon filter (3 KDa MWCO) for three times (8000 r/min, 10 min).

Synthesis of GOx-FITC
FITC (13 mg) was dissolved in acetone (100 µL), GOx (10 mg) was dissolved in phosphate buffer (100 mM, pH 7) that contained EDTA (200 µM). Mixing the two solutions and the reaction was kept in dark for 8 h and then the mixture was washed with an Amicon filter (30 KDa MWCO) for three times (8000 r/min, 10 min).

Synthesis of urease-RhB
Urease (5 mg) and RhB (4.7 mg in methanol) were mixed in phosphate buffer (100 mM, pH 7) that contained EDTA (200 µM). The reaction was mixed in the dark for 8 h, and then the mixture was washed with an Amicon filter (30 KDa MWCO) for three times (8000 r/min, 10 min).

Synthesis of AchE-FITC
FITC (2.8 mg) was dissolved in acetone (100 µL), AchE (1 mg) was dissolved in phosphate buffer (100 mM, pH 7) that contained EDTA (200 µM). Mixing the two solutions and the reaction was kept in dark for 8 h and then the mixture was washed with an Amicon filter (30 KDa MWCO) for three times (8000 r/min, 10 min).

Synthesis of Catalase-RhB
Catalase (1 mL) and RhB (10 mg in methanol) were mixed in phosphate buffer (100 mM, pH 7) that contained EDTA (200 µM). The reaction was mixed in the dark for 8 h, and then the mixture was washed with an Amicon filter (30 KDa MWCO) for three times (8000 r/min, 10 min).

Rheometric experiments
In rheometric experiments, the hydrogels were prepared with a volume of 150 µL of the polymer solution (3 wt%). The strain-dependent changes of the storage (G') and loss (G'') moduli were measured at 20 °C with a gap distance of 0.3 mm, 1 Hz frequency and strain sweeps of amplitude 0.1%-300% (cf. Figure 7c). The frequency-dependent changes of the storage (G') and loss (G'') moduli were measured at 20 °C with a gap distance of 0.3 mm, 1% strain ( Figure S8). The time-dependent stiffness of hydrogels was measured at 20 °C with a gap distance of 0.3 mm, 1% strain and 1 Hz frequency.

Shape-memory experiments
In shape-memory experiments, the respective hydrogel was prepared with a volume of 80 µL (3 wt%) in a triangle mold. The solution of polymers was mixed in an eppendorf tube and heated until the polymers were completely dissolved, followed by transferring the solution into the mold. Then the mold was cooled down in ice bath, then adding the respective amounts of enzymes into the mold. After being kept at 4 °C overnight to form the hydrogel, the hydrogel was extruded from the mold as a triangle shape and treated with different triggers (glucose, acetylcholine or urea, final concentration of the trigger was 10 mM) for a time interval of 60 min, to trigger the switchable transitions of the hydrogel. The triangle-shaped hydrogels were stained with Gel Red or Texas Red-dextran.

Self-healing experiments
In self-healing experiments, the hydrogels were prepared with a volume of 80 µL (3 wt%) in a cubic mode. Respective polymers were mixed in an eppendorf tube and heated until the polymers were completely dissolved, followed by transferring the solution into the mold. Then the mold was cooled down in ice bath, then adding the respective amounts of enzymes into the mold. After being kept at 4 °C overnight to form the hydrogel. The hydrogel was extruded from the mold as a cubic shape and cut into two pieces, followed by treating with different triggers (glucose, acetylcholine or urea, final concentration of the trigger was 10 mM) for a time interval of 60 min, to trigger the switchable transitions and self-healing behavior of the hydrogel. The hydrogels were stained with Gel Red.

Calculation of the loaded amounts of enzymes and insulin
In order to calculate the loading amounts of insulin, GOx, urease, AchE, the fluorophore-labelled insulin and fluorophore-labelled enzymes were mixed with the polymer solution before formation of hydrogels (see the details of preparation in experimental section: preparation of hydrogels). Then the hydrogels were washed with HEPES buffer three times, and the fluorescence of the washing buffer was measured ( Figure S2)