Research

3D Hydrogel Preparation

Hydrogel Preparation

We employed a multistep process to prepare the hydrogels.

Assemble with all components except the thiolated proteins, and expose to UV to stifffen.
Swell the hydrogel overnight in a solution containing our thiolated proteins and LAP (a photo initiator) to allow for diffusion of the protein in the matrix.
Attach proteins to the hydrogel backbone through one last UV exposure step.

3D Hydrogels before swelling in protein solution

Thiolated Protein Assembly

The thiolation of protein is a main component of the photo-mediated bioconjugation that attaches the thiolated proteins to the norbornene functionalized polymer backbones of our hydrogel [1]. Reaction with NHS PEG thiol will result in a thiolated protein. Our protein solution contained around 30 thiols per protein as measured by various assays. In this project, we conducted all experiments with a thiolated ovalbumin, as it was readily available and fluourescently tagged with Alexa Fluor 555.

Hydrogel Mesh Size

In order to ensure that our thiolated ovalbumin and our hydrogel formulation worked in tandem we ran a hydrogel mesh size calculation.

Hydrogel mesh size is defined as the average distance between two neighboring junctions that are connected by a polymer chain in a hydrogel and have been correlated to solute diffusivity.

First, we assumed Mean Field Approach, No Convection, and Polymer-Solute Non-Interaction

We calculated swollen volume polymer fraction first, then mesh size [2].

Our calculated mehs size was ~23 nm. Comparing that to the ovalbumin radius of ~3 nm, we can confirm that the ovalbumin should be small enough to freely diffuse into the hydrogel.

Deciding LAP Concentration

The protein concentration we picked was 100 ug/mL. We changed the ratio of LAP to protein concentration so 1x is a 1:1 ratio and 0.1x is 1:10 ratio. The images above show the intensity of the protein within the gels, we can see a general increasing trend with increasing ratio

We took the mean intensity value from each gel and plotted it against LAP concentration and got the trend on the right. It is not entirely linear, so we see a reduced return the as the ratio approaches 1x, however we chose the 1x concentration to tether our protein moving forward because it maximizes the protein we can connect into our hydrogel.

Deciding Initial Hydrogel Stiffness

We care about the stiffness of the gel because we want it to mimic the aortic valve as close as possible. We subjected different weight percentages of PEG-norbornene gels to a constant strain rate and evaluated the stiffness. The gels were stiffened on a plate rheometer using UV light, which is why we see the gels stiffen and to a constant Young's modulus.

Once the gels plateau to a constant stiffness, we compare the end stiffness to see which weight percent gels we want to use. We want to use the softest gels that are still able to be worked with. The 3.7% gel is the softest with the best reproducibility and workability.

Effects of Protein Tethering on Gel Stiffness

With the 3.7% gel formulation we decided to pattern in protein to the hydrogel's matrix to see if it would affect the stiffness. The hydrogels are already stiffened so they will not have the same curve as seen above.

The stiffnesses were evaluated again and compared to see if there is a significant difference after tethering in proteins. We initially hypothesized that there should not be a difference when tethering in proteins, but when running a t-test we found a significant difference. However, we cannot attribute this to the protein tethering since we ran out of time to optimize the protocol, so it could be attributed to damage caused by moving the gels.

[1] Grim, Joseph C., et al. “A Reversible and Repeatable Thiol–Ene Bioconjugation for Dynamic Patterning of Signaling Proteins in Hydrogels.” ACS Central Science, vol. 4, no. 7, July 2018, pp. 909–16, https://doi.org/10.1021/acscentsci.8b00325.

[2] Richbourg, Nathan R., and Nicholas A. Peppas. “Structurally Decoupled Stiffness and Solute Transport in Multi-Arm Poly(Ethylene Glycol) Hydrogels.” Biomaterials, vol. 301, 2023, p. 122272, https://doi.org/10.1016/j.biomaterials.2023.122272.

Select figures were created with BioRender.com

Page updated

Report abuse