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Biomaterials

Biomaterials - Poster Session E

Poster B4 - Stimuli-responsive PEG Hydrogels with User-controlled Mechanical Properties

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Mechanical properties of the extracellular matrix (ECM), such as stiffness, are well-documented as having influence over cell behavior and fate. As such, synthetic Poly(ethylene glycol) (PEG) hydrogel matrices have become an extremely popular choice for 3D cell culture applications due to their “blank slate” nature, allowing for precise and independent tuning of biophysical and biochemical matrix cues. However, most popular crosslinking chemistries do not permit facile post-fabrication tuning, rendering the mechanical properties of most conventional PEG hydrogels static. Recently, utilizing dynamic covalent chemistries (DCC) for hydrogel fabrication has become increasingly popular due to the ability of dynamic covalent bonds to impart viscoelasticity and stimuli-responsiveness upon the resulting material, allowing for dynamic mechanical tuning. Of these dynamic bonds, disulfides present an excellent choice when designing dynamic matrices intended for 3D cell culture applications, as they are stable under most tissue culture conditions and can be broken and reformed under relatively mild stimuli. In this work, we utilize a mixture of dynamic disulfide and stable thioether bonds produced from photoresponsive multi-arm PEG lipoic acid (LA) and multi-arm PEG acrylate (Ac) macromers in the presence of photoinitiator, which produce dynamic PEG hydrogels with a degree of tunability. We demonstrate that the softening and stiffening of these matrices can be controlled by the length of UV irradiation and ratio of PEG-LA and PEG-Ac macromers chosen during fabrication.

Materials and

Methods:  Hydrogel Fabrication: Broadly, all hydrogels were fabricated from a precursor solution comprised of 8-arm PEG-LA 20kDa (ruixibio), 8-arm PEG Acrylate 20kDa (Jenkem), and LAP (Sigma Aldrich). This precursor solution was then aliquoted as 40 μL droplets between parafilm-coated glass slides separated by 600 μm silicone spacers, and cured under a 365 nm, 10W UV handlamp (Analytik Jena) for times as indicated. All gels were equilibrated overnight in 1x PBS (pH 7.4) before use in any experiments.

Rheological Characterization: Characterization of hydrogel storage modulus was performed on a DHR-3 rheometer, using an 8 mm plate geometry. (TA Instruments). To quantify static storage modulus, a frequency sweep was conducted at 1–10 rad/s under 1% strain, which was determined to be within the linear viscoelastic range. To changes in modulus as a response to stimulus, a time sweep was performed, under 1% strain and a frequency of 10 rad/s.

Stiffening: Gel precursors were first prepared with either a 1:1 or 4:1 molar ratio of LA to Acrylate groups and irradiated for 5 minutes as described previously. To demonstrate post-fabrication stiffening, hydrogels were further irradiated for another 10 minutes after equilibrating. Storage modulus was measured directly before and after UV treatment.

Softening: Gel precursors were first prepared with a 4:1 molar ratio of LA to Acrylate groups, at low and higher macromer contents, and irradiated for 10 minutes as described previously. To demonstrate post-fabrication softening, a time sweep was performed on these hydrogels as they were immersed in a 100mM DTT PBS solution.

Results, Conclusions, and Discussions: 

Results: Irradiating mixtures of PEG-LA, PEG-Ac, and LAP resulted in hybrid disulfide/thioether based PEG hydrogels. By using shorter irradiation times and a stoichiometric excess of PEG-LA, we were able to fabricate hydrogels that could stiffen from ~5 kPa to a maximum of ~20 kPa. This could be done at user-controlled time points (Figure 1A). Larger excesses of PEG-LA resulted in greater degrees of stiffening. In gels formed at longer irradiation times, we were able to achieve softening of ~500 Pa in response to DTT treatment, irrespective of macromer concentration. (Figure 1B). Significant stress relaxation was not observed in any of the described formulations, indicating that these gels were elastic in nature.

Discussion: As the photoinduced ring opening of LA to produce disulfides has been shown to be kinetically slower than the radical polymerization between acrylates or acrylates and free thiols, we hypothesized that controlling polymerization time may permit gel formation without total consumption of LA groups, after which user-controlled stiffening could be achieved through further UV crosslinking. The stiffening behavior we observed when PEG-LA was in molar excess, especially at higher molar ratios, lends credence to this idea. For softening, we believed that longer irradiation times would lead to complete reaction of the acrylate and LA groups, producing a hydrogel with significant disulfide and thioether portions of the network. When exposed to a reducing agent, the disulfide bonds present would degrade, leaving only stable thioether bonds. While both high and low concentration hydrogels softened in response to DTT, the loss in storage modulus experienced in the higher concentration condition (~500 Pa) relative to its initial storage modulus (~3500 Pa) was much lower compared to the loss experienced in the lower concentration condition (~1200 Pa). This may be due to the higher thioether content of this stiffer formulation, which may serve to stabilize the network.

Conclusion: We demonstrate that hybrid disulfide/thioether PEG hydrogels can be dynamically stiffened and softened in response to a user input, which make them a potential asset to the fields of mechanobiology and tissue engineering. Future work should assess the cytocompatibility of these materials.

Acknowledgements (Optional):