Characterization and structure-property relationships of an injectable thiol-Michael addition hydrogel toward compatibility with glioblastoma therapy

Development of a Synthetic, Injectable Hydrogel to Capture Residual Glioblastoma and Glioblastoma Stem‐Like Cells with CXCL12‐Mediated Chemotaxis

“…Synthetic Hydrogel Synthesis: The synthetic injectable hydrogels were prepared as described previously. [32] Briefly, Thiocure ETTMP 333L, a trithiol crosslinker ethoxylated trimethylolpropane tri-3mercaptopropionate (generously donated by Bruno Bock Thiochemicals), as well as PEGDA, at 575 g mol −1 numbered average weight from Sigma-Aldrich were both brought to room temperature. Aqueous 0.175 m NaHCO 3 (Fisher Chemical) was prepared by dissolving the appropriate mass in deionized water.…”

“…Since the hydrogel was previously demonstrated to degrade with time in PBS, [32] a simple degradation term was also incorporated into Equation (6) to account for the FITC-dextran release due to bulk hydrolytic degradation. This degradation component was based on the nonlinear Kopcha model, which considered the relative relaxation of the hydrogel and diffusion of the solute component according to Equation (7), where Q is the percent of the solute released due to the degradation at time t in h, A is the diffusion exponent (1 for a slab), and B is the erosion constant.…”

“…[109] Here, B was taken to be 0.172% mass loss per hour, which was approximated from the 4.13% mass loss per day in 0.175 m NaHCO 3(aq) hydrogels with 50 wt% water content as determined in our previous degradation study conducted in PBS. [32] Q = At 0.5 + Bt (7) Quantifying CXCL12 Release from Synthetic Hydrogels: Four replicates of synthetic hydrogels were synthesized at 0.69 mL final volumes in 6-dram vials. The hydrogels were loaded with CXCL12 at 5 μg mL −1 by adding the appropriate volume of recombinant human SDF-1𝛼 (PeproTech) reconstituted in 1× PBS into the PEGDA-NaHCO 3(aq) precursor solution and mixing gently by stirring prior to the addition of Thiocure.…”

“…The normalized fluorescence intensities were quantified by determining the ratio of the average fluorescence intensity per pixel for each cell to the average background fluorescence intensity in the same field of view according to the previous protocol. [32] This method of semi-quantitative analysis was implemented to take into consideration any changes in the microscope setting with time according to previous protocols. [113,114] Representative 1.61 μm optical slice images were acquired with a Plan-Apochromat 20× objective lens comprising a numerical aperture of 0.8 and a 35 μm pinhole.…”

“…We had previously systematically tuned and characterized nine formulations of a poly(ethylene glycol)‐based injectable hydrogel, which was crosslinked via a thiol‐Michael addition click reaction. [ 32 ] We had determined that the hydrogel formulation comprising 0.175 m NaHCO 3(aq) and 50 wt% water content was the most optimal for biological, physical, and chemical compatibility with the GBM microenvironment and our proposed GBM therapy by systematically characterizing the hydrogel swelling ratios, gel fractions, degradations, viscoelastic properties, as well as the hydrogel interactions with normal human astrocytes, on the basis of cytotoxicity and immunogenicity. [ 32 ] Furthermore, the optimized formulation of the hydrogel possessed mesh sizes with well‐cross‐linked networks in the nanometer range, which may help sustain the release of chemokine payloads over time.…”