Control of Thiol-Maleimide Reaction Kinetics in PEG Hydrogel Networks

Abstract

Michael-type addition reactions are widely used to polymerize biocompatible hydrogels. The thiol-maleimide modality achieves the highest macromer coupling efficiency of the reported Michael-type pairs, but the resulting hydrogel networks are heterogeneous, because polymerization is faster than the individual components can be manually mixed. The reactivity of the thiol dictates the overall reaction speed, which can be slowed in organic solvents and acidic buffers. Since these modifications also reduce the biocompatibility of resulting hydrogels, we investigated a series of biocompatible buffers and crosslinkers to decelerate gelation while maintaining high cell viability. We found that lowering the polymer weight percentage (wt%), buffer concentration, and pH slowed gelation kinetics, but crosslinking with an electronegative peptide was optimal for both kinetics and cell viability. Slowing the speed of polymerization resulted in more uniform hydrogels, both in terms of visual inspection and the diffusion of small molecules through the network. However, reactions that were too slow resulted in non-uniform particle dispersion due to settling, thus there is a trade-off in hydrogel network uniformity versus cell distribution in the hydrogels when using these networks in cell applications.

Control of Astrocyte Quiescence and Activation in a Synthetic Brain Hydrogel

“…3). The need for these tissue-specific hydrogels is of particular importance to instruct cells that do not produce their own matrix, for example oligodendrocytes and schwann cells 58 , and it has been shown many times that these cues can impact cell differentiation 58,59 and viability 39 . Similarly, specific ECM cues provided by integrin-binding sites 8,28,60 , inflammatory molecules 12,61 , and mechanical properties 40 , have been shown to influence the deposition of ECM proteins by astrocytes.…”

“…We previously showed that this reaction speed can be tuned by manipulating the ionic strength of the buffer, the pH, and the electronegativity of the amino acids flanking the MMP-degradable peptides. 39 Since brain cell phenotype is sensitive to the modulus of the ECM [40][41][42][43] , we needed to account for this in our design of an in vitro brain ECM. Unfortunately, we quickly discovered that reported values for Young's moduli of the brain ranged from 100s of Pa to 10s of kPa, and vary considerably among experimental techniques.…”

“…The Measure-iT thiol kit was used to quantify unreacted thiols (Thermo) as previously described. 39 Integrin-binding peptides were incorporated into gels at 2 and 4mM concentrations in a 100 µL volume of PEG-maleimide for 10 minutes before reacting hydrogels with 100µL of the Measure-iT kit working solution. Separately, dithiol terminated crosslinkers (PDT at an equimolar ratio of thiol to maleimide, or MMP-degradable peptide (13mole%) with 87mole% PDT) were reacted with the PEG-maleimide in 10 l aliquots for 10 minutes before reacting with 100µL of the kit working solution.…”

Engineered full-length Fibronectin-based hydrogels sequester and present growth factors to promote regenerative responses in vitro and in vivo

“…Maleimide groups have higher affinity towards thiol groups, which confer shorter gelation times at physiological pH relative to hydrogels fabricated using acrylate groups 53 . Michael-type addition has been previously used to form biologically active and cytocompatible hydrogels 54,55 .…”

Anomalous Diffusion as a Descriptive Model of Cell Migration

“…However, in vivo, cell movement is largely in 3D, with cells surrounded by ECM and other cells. To test the effectiveness of the AD model in describing this type of confined cell movement, we used another PEG-based gel [39][40][41] and measured cell motility in 3D as we exposed them to different pro-migratory chemical stimulations (conditioned medium from patient cell cultures). The gels were crosslinked with MMP-sensitive peptides, but the mesh size was orders of magnitude smaller than a cell (~20-25nm).…”