Reversible photodissipation of composite photochromic azobenzene-alginate supramolecular hydrogels

Supramolecular smart materials can quickly elicit macroscopic changes upon external stimulation. Here we report that an azobenzene-containing cyclic dipeptide can form composite supramolecular hydrogels with alginate based on the charge complementarity, at lower loading than the critical gelation concentrations of either component. The gels can reversibly dissipate to fluids with UV light. They can also encapsulate and photorelease fluorescent cargo. Upon treatment of the gels with aqueous calcium salts, the alginate component is permanently cross-linked and the photochromic component is solubilized.

Analytical thin layer chromatography was carried out using silica coated aluminium plates (silica 60, F254, layer thickness: 0.25 mm) with fluorescence indicator by Merck. Detection proceeded under UV light at λ=254 nm.

Photophysical properties
Photostationary states determined by 1

H NMR measurements
Photostationary states were determined by 1 H NMR measurements for compound 1 (16.7 mg/mL) equilibrated up to 30 min under the indicated light wavelength (max of the respective LED light diode). For analysis the NMR signals were assigned to the E and Z isomer, respectively. To determine the signals of the Z isomer the dark spectrum was subtracted from the spectrum after 30 min irradiation at 365 nm (10 W LED). Then three signals per isomer were identified that did not-or just barely-overlapped with other signals. Those were integrated for each spectrum using the same intervals and divided by the number of protons. Each E isomer signal was assigned a Z isomer signal and the mole fraction of these pairs was calculated.

Thermal stability
The thermal stability was determined for compound 1. Samples were prepared in H2O and acetic acid and irradiated at 365 nm to yield a high PSS. The solution was kept at 20 °C (H2O and acetic acid) or at 60 °C (H2O) and the isomer ratio was determined by HPLC in intervals. The obtained data was processed by calculating the ln(X0/Xt), where X is the percentage of the respective Zisomer and linear fitting (Equation (1)) of the obtained values. The calculated slope corresponds to the degradation rate constant k which is used to calculate the half-life t1/2.

UV/Vis isomerization experiments
A 500 µM Stock solution was prepared and diluted to reach a final concentration of 50 µM. The cuvette with the sample was irradiated with light of different wavelengths (365 nm, 455 nm) directly before the measurement (see Figure 3).
Isosbestic points are determined at 238, 287 and 397 nm. Only the isosbestic point at 287 nm was used for quantification at HPLC.

Gelation experiments Method A
To a 1.5 mL-vial (crimp top, 12×32 mm) was added the photochromic material 1 (and the additives) as powder and 500 µL of the aqueous solution. This suspension was treated by ultrasonic waves for 2 min followed by heating to 80 °C in a vial block. After equilibration at 80 °C for 5 min, the sample was heated to the boiling point by a heat gun. The hot solution became completely clear and upon cooling the hydrogels formed. Successfully formed gels were prepared in triplicates.
Melting temperatures were determined in triplicates. Gels were prepared as previously described. The vials were then mounted upside-down in a slowly stirred water bath (60 rpm) on a magnetic hotplate stirrer equipped with a thermometer. The water bath was heated (1.5 °C/min) until the gel started to melt and formed a sol.

Method B
To a 1.5 mL-vial (screw top) was added the photochromic material 1 as powder and 250 µL of water. After complete dissolution, the solution was irradiated for 10 min at 365 nm. Subsequently, 250 µL of a 2× stock solution of sodium alginate in water was added and mixed by repetitive pipetting. Then, the mixture was irradiated at 455 nm for 10 min. Successfully formed gels were prepared in triplicates.
Melting temperatures were determined in triplicates. Gels were prepared as previously described. The vials were then mounted upside-down in a slowly stirred water bath (60 rpm) on a magnetic hotplate stirrer equipped with a thermometer. The water bath was heated (1.5 °C/min) until the gel started to melt and formed a sol.

Light induced gel-to-sol transition
According to the procedure Method A described in the previous section, gels were prepared with 0.6 wt% PAP-DKP-Lys2 1 and 0.6 wt% alginate in water. After equilibrating overnight, the samples were irradiated with 2 LEDs at 365 nm (10 W) for 15 min. Subsequently, the gels were inverted and one gel was irradiated at 455 nm for 15 min (A), while a second gel was kept in the dark (B, see Figure S 7). Sample A solidified at the vial top, while sample B was a highly viscous liquid.

Light-induced rhodamine release
Here we wanted to investigate how efficient are hydrogels based on the gelator 1 and sodium alginate in releasing encapsulated guest molecules by means of diffusion (in darkness) or dissipation of the inner gel structure upon irradiation with UV light. We have chosen the composition of 0.3 wt% of the gelator 1 and 0.3 wt% of alginate in 500 µL of water prepared by Method B. As described in section 4 of this supporting information, it forms a stable gel after irradiation of the mixed components at 455 nm. By this preparation method heat is avoided, which could have damaging impact on some cargo. Preparation with cargo was done as follows: In a 1.5 mL glass vial (screw top) we mixed the photochromic gelator 1 (1.5 mg, powder) and water (245 µL). This solution was irradiated at 365 nm (10 min), then a 100× stock solution (5 µL) of the chosen cargo rhodamine dissolved in EtOH was added. Subsequently, a 2× stock solution of alginate (250 µl) was added, thoroughly mixed and the final mixture was irradiated at 455 nm for 10 min to obtain the final gel. Before a release experiment, the hydrogels were kept overnight in darkness at room temperature. Concentration of the cargo rhodamine was adapted to the HPLC detection range. 250 µg total mass of rhodamine were incorporated into the gels.
Quantification of the passive diffusion -cargo "leaking" from hydrogels in darkness: 500 µL of PBS buffer pH 7.4 was slowly added on top of a gel sample (on the wall of the vial) and immediately removed with a micropipette to wash away unbound or loosely bound guest molecules from the surface. Addition of fresh 500 µL of PBS buffer followed. The gel was incubated together with the buffer on the top in darkness. 500 µL of the liquid was collected after 5 min by gently turning the vial sideways and pipetting off the liquid from the side wall of the vial. Then, fresh 500 µL of PBS buffer was added on the side wall of the vial, incubated in darkness and removed after 5 min in the same way as described above. That process was repeated for the total duration of 40 min by collecting 9 subsequent volume aliquots. After that time, the gel remained visually unaffected.

Procedure of the light induced release:
To measure the release process upon UV light irradiation, we exactly repeated the procedure described above, but after initial washing of the gel surface the sample was placed in an irradiation chamber and illuminated with two 10 W LEDs (365 nm, from the distance of 5 cm).
Short breaks in irradiation were taken for the replacement of 500 µL aliquot with fresh 500 µL of PBS buffer every 5 min, but the overall irradiation time was 40 min. The irradiation time was sufficient to fully convert the gel samples into sol. All aliquots were weighted before the HPLC measurement to calculate the released amount of the substance. The concentration of the aliquots was calculated by a previously measured calibration curve.

Diffusion of the gelator 1 after addition of Ca 2+ ions
Sodium alginate rapidly forms gels upon addition of divalent ions, for example Ca 2+ . Therefore, we have assessed the influence of an aqueous calcium salt solution on our composite gels made from alginate cross-linked with the gelator 1. For this purpose, two gels composed of 0.6 wt% gelator 1 and 0.6 wt% alginate were prepared by Method A (see Figure S 10). On top of these gels was added 500 µL of a 10 wt% solution of CaCl2 in water and equilibrated for 1 h. Figure S 11: 500 µL CaCl2 solution (10 wt% in water) added on top of the gels for 1 Subsequently, the supernatant was removed and fresh CaCl2 solution was added. Then, one vial was kept in the dark, while the second one was irradiated for 1 h (365 nm). Next, the supernatant was removed, and the procedure was repeated four times in total. The supernatant of the irradiated vials was colorful, while the dark vials were less colored ( Figure S 12).  This experiment demonstrated that our gelator 1 / alginate composite gels can be transformed to Ca 2+ / alginate gels and our gelator 1 is subsequently removable by irradiation.

Cell viability assays of the hydrogelator 1
Hela cells were grown in DMEM (Dulbecco`s Modified Eagle Medium) which was modified with 10% FCS (fetal calf serum) and 1% penicillin/streptomycin solution (10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin) in a humid incubator at 37 °C with 5% CO2. Cells were detached from the surfaces with Trypsin-EDTA (0.25%) from Gibco®. Cells were washed with PBS (Phosphate-Buffered Saline) from Gibco®.
96-well plates (Table 1) with a flat bottom were prepared by filling all wells on the outer border with 200 µL PBS and the remaining wells with 100 µL of a cell suspension (30.000 cells/mL) in DMEM. The prepared plate was incubated overnight to ensure cell attachment to the wellbottom and cell growth.

PBS Sample
For the dilution-series of each compound, a stock solution of DMEM modified with 0.25% DMSO was prepared to ensure that all cells are treated with the same conditions. Consequently, the first sample of the dilution series was prepared by dissolving the substance in DMSO and adding a specific amount of this solution to a specific amount of non-modified DMEM so that a final concentration of 0.25% DMSO is reached.
To apply the substances to the 96-well plate, the DMEM was removed without disturbing the cells grown in the plate and adding subsequently 100 µL to each well. To ensure the same treatment to the control rows, the DMEM was removed from the wells and DMSO-modified DMEM (100 µL) was added to the corresponding wells. The 96-well plate was incubated for 48 h.
The positive control was treated with 5 µL of Triton™ X-100 detergent (10% solution (w/v)) per well for at least 5 min before adding 15 µL of MTT dye-solution (Cell Proliferation Kit I (MTT) from Roche) to all sample wells and incubating for 3 h in the dark. 100 µL of stop solution (Cell Proliferation Kit I (MTT) from Roche) was added after incubation to stop the reduction of MTT to formazan, thus preventing overreaction and enabling solubilization of formazan crystals. After 24 h of solubilization in the incubator, the plate was read out with a plate reader (BioTek® EPOCH 2 , Gen5 Data Analysis) by measuring the absorption of each well at 595 nm.
The raw data was processed as followed by first subtracting the positive control (all cells are dead) from all measured values in one row to remove background absorption. Each concentration was measured sixfold per plate therefore (Triplicates; 18 values in total), the values for each concentration and the negative control (all cells alive) were averaged and the standard deviation was calculated. The cell viability was calculated as a percentage of the negative control and normalized by assuming the highest obtained viability as 100%. For the Transmission Electron Microscopy images, two 1.5 wt% hydrogel samples of 1 in PBS buffer and two mixed samples (0.6 wt% 1 and 0.6 wt% alginate) in H2O were prepared as described in the Gelation experiments 4. After cooling, the samples were equilibrated overnight at room temperature. Of each composition one sample was irradiated at 365 nm (10 W LED) until liquefaction. The resulting samples were added as small droplet to carbon-coated copper grids (400 mesh). The supernatant was removed carefully with a lint-free sheet and the grid was dried under atmospheric pressure. Examination was carried out on a Philips CM200 FEG transmission electron microscope, operated at 200 kV accelerating voltage. All images were recorded defocused.