Molecularly Engineered Supramolecular Thermoresponsive Hydrogels with Tunable Mechanical and Dynamic Properties

Synthetic supramolecular polymers and hydrogels in water are emerging as promising biomaterials due to their modularity and intrinsic dynamics. Here, we introduce temperature sensitivity into the nonfunctionalized benzene-1,3,5-tricarboxamide (BTA-EG4) supramolecular system by incorporating a poly(N-isopropylacrylamide)-functionalized (BTA-PNIPAM) moiety, enabling 3D cell encapsulation applications. The viscous and structural properties in the solution state as well as the mechanical and dynamic features in the gel state of BTA-PNIPAM/BTA-EG4 mixtures were investigated and modulated. In the dilute state (c ∼μM), BTA-PNIPAM acted as a chain capper below the cloud point temperature (Tcp = 24 °C) but served as a cross-linker above Tcp. At higher concentrations (c ∼mM), weak or stiff hydrogels were obtained, depending on the BTA-PNIPAM/BTA-EG4 ratio. The mixture with the highest BTA-PNIPAM ratio was ∼100 times stiffer and ∼10 times less dynamic than BTA-EG4 hydrogel. Facile cell encapsulation in 3D was realized by leveraging the temperature-sensitive sol–gel transition, opening opportunities for utilizing this hydrogel as an extracellular matrix mimic.


Supporting Figures
BTA-PNIPAM was heated to 30 °C after which the sample was allowed to equilibrate for 30 min.After cooling to 10 °C, the transmittance stabilized within 1 min to 88%.Furthermore, the cuvette was subjected to a stream of N2 gas to prevent condensation on the glass.

Figure S15: (A)
The hydrogel G-1 undergoes sol to gel transition at 37 °C within 4 min, as indicated by the vial inversion test.(B) Sample G-1 was loaded in its liquid state onto the rheometer at 10 °C, after which the temperature was switched to 37 °C (which takes less than 1 min) to initiate gelation and a time sweep was measured at 1% strain and 1 rad/s.t0 equals the moment that the rheometer reached 37 °C, and a gel was already formed, which remained stable.

Figure S2 :
Figure S2: FT-IR spectra of BTA-EG4, PNIPAM and BTA-PNIPAM, showing the disappearance of N3 peak in 2110 in BTA-PNIPAM.The N3 asymmetric stretch of the azide group is marked.

Figure S4 :
Figure S4: MALDI-TOF MS spectra of PNIPAM and BTA-PNIPAM, with the difference of the two population to be 1313, exactly the MW of BTA-mN3.

Figure S6 :
Figure S6: Transmittance at 600 nm of BTA-PNIPAM (500 µM) at 10 °C and 30 °C for 5 cycles.BTA-PNIPAM was heated to 30 °C after which the sample was allowed to equilibrate for 30 min.After cooling to 10 °C, the transmittance stabilized within 1 min to 88%.Furthermore, the cuvette was subjected to a stream of N2 gas to prevent condensation on the glass.

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Figure S7: CryoTEM image of BTA-EG4 (500 µM) in MQ water at 22 °C, showing long fibers without ends.Scale bar = 50 nm.The black spherical particles are ice-crystals.

Figure S12 :
Figure S12: Thermo-responsive mechanical characterization of mixtures of BTA-EG4 and PNIPAM.Storage (G') and loss (G") moduli vs temperature at a strain of 1%, an angular frequency of 1 rad/s, and a heating/cooling rate of 1 °C/min for the mixtures of BTA-EG4 and PNIPAM.The concentration of BTA-EG4 was fixed as 1 wt% and PNIPAM was varied from 0, to 0.1, to 0.5 to 1 wt%.

Figure S14 :
Figure S14: Quantified FRAP results, showing (A) the fluorescence half-life (τ1/2) during which the fluorescence intensity recovered to half its original value and (B) the extracted diffusion constant (D) in µm 2 /s.For all quantified data holds n=3 and data are represented as mean with SEM.

Figure S16 :
Figure S16: Cell viability study of G-0, G-0.1, G-0.5 and G-1 gels as determined by MTT assay.hNDF were cultured inside the gels for 24 h.Viability is normalized against cells cultured without hydrogels (i.e.no hydrogel) and cells treated with 70% ethanol were used as negative control.Data is plotted as mean with S.D., with n=6 per condition.

Figure S18 :
Figure S18: Additional cell images showing cells encapsulated in 3D inside hybrid supramolecular hydrogels after 1 day of culture.Green is F-actin, scale bar is 50 µm.

Figure S19 :
Figure S19: Additional cell images of live/dead cell staining (green=live, red=dead) after 24 h of culture.(A, B) Cells encapsulated in hydrogel G-1 containing BTA-cRGD with 4000 cells/well.(C) Positive control of cells without hydrogel with 1000 cells/well.(D) Positive control of cells without hydrogel with 4000 cells/well.(E) Negative control of cells in 70% ethanol without hydrogel with 4000 cells/well.Scale bar = 50 µm.