Widener, Adrienne E., et al. Biomaterials Science 9.7 (2021): 2480-2493.
PEG-MAL has been successfully used in a wide range of biological applications. However, due to the rapid reaction of maleimide with thiols, the reaction may result in uneven cross-linked gels when reacted at physiological pH. To generate large amounts of homogeneous PEG-MAL microgels, researchers have attempted to slow down the cross-linking reaction to allow more time for processing of the pre-gel solution. The researchers formed bulk gels by rapidly mixing 4-arm PEG-MAL and 8-arm PEG-MAL macromers with PEG-dithiol or dithiothreitol (DTT) over a range of pH. They found that the gelation time of 4-arm PEG-MAL was twice that of 8-arm PEG-MAL, while cross-linking with PEG-dithiol resulted in slower gelation than DTT. The gel uniformity when the macromers were mixed with the cross-linkers was observed using solutions of the macromers and cross-linkers colored with cyan and magenta food dyes under a stereoscope. Pipette PEGMAL macromer between two glass microscope slides separated by a spacer, followed by PEG-dithiol crosslinker. Mix the solution by pipetting up and down until gelation occurs. Lowering the pH allows for more uniform gel mixing.
Dissolve four-arm polyethylene glycol maleimide (4-arm PEG-MAL) (20 kDa) macromer and PEG-dithiol in 1X PBS with 1% HEPES at different pH values (4.0, 5.0 5.2, 5.4 5.6, 5.8, 6.0, 7.0, 7.4). Mix 4-arm PEG-MAL macromer with dissolved magenta food coloring and mix PEG-dithiol crosslinker with cyan food coloring. Pipette 100 μL of PEG-MAL macromer between two glass microscope slides separated by a 2 mm spacer. 100 μL of the dyed PEG-dithiol cross-linking solution was then added between the two slides and pipetted up and down until the solution became too viscous for further mixing. The mixing process was macroscopically recorded using an iPhone 11 camera, and still images were extracted from the video at each pH sample gelation point.
