Maleimide cross-linked bioactive PEG hydrogel exhibits improved reaction kinetics and cross-linking for cell encapsulation and in situ delivery

Abstract

Engineered polyethylene glycol-maleimide matrices for regenerative medicine exhibit improved reaction efficiency and wider range of Young’s moduli by utilizing maleimide cross-linking chemistry. This hydrogel chemistry is advantageous for cell delivery due to the mild reaction that occurs rapidly enough for in situ delivery, while easily lending itself to “plug-and-play” design variations such as incorporation of enzyme-cleavable cross-links and cell-adhesion peptides.

Figure 1

PEG-maleimide hydrogel chemistry. A) Maleimide Michael-type addition reaction. B) 4-arm PEG Maleimide macromer. C) Acrylate and vinyl sulfone reactive groups. D) Sample cross-linked PEG-4MAL hydrogel. E) Michael-type addition hydrogel reaction scheme: PEG-macromers are first functionalized with RGD adhesive ligand followed by cross-linking with a thiol-flanked enzyme-degradable peptide.

Figure 2

Thiol reactivity and material properties comparisons between different end groups. A) Quantification of thiols remaining after 4-arm PEG macromer functionalization with RGDS peptide at 10 and 60 mins, 4 and 400 mM TEA in PBS with varying end-group to thiol molar ratio. B) Influence of polymer weight percentage on equilibrium swelling ratio (Q_m) for networks made from PEG-4MAL, PEG-4A, PEG-4VS, or linear PEG-DA. C) Young’s modulus measured by AFM for 10% (wt/v) PEG gels. D) Young’s modulus measured by AFM for PEG-4MAL gels with varying polymer weight percentage compared with collagen-I gels.

Figure 3

Cellular encapsulation and in situ polymerization of PEG-4MAL hydrogel. A) Live/Dead staining of C2C12 murine myoblasts at 3 days after encapsulation in PEG hydrogels of varying polymer weight percentage compared to collagen-I gel (3 mg/mL). Cross-linked hydrogels could not be generated for low percentage PEG-4A and PEG-DA gels. Scale bar = 100 μm. Inset false color higher magnification showing individual cell spreading. MTS metabolic activity assay of encapsulated C2C12 cells indicates viability similar to number of cells seeded for PEG-4MAL and PEG-4VS, with PEG-4A and PEG-DA approximately 60%. B) H&E stain of PEG-4MAL matrix cross-linked directly on mouse myocardial wall. PEG-4MAL matrix incorporating 1% polymer substitution FITC-PEG-MAL cross-linked directly on mouse myocardial wall, counterstained with Alexa-Fluor 594 phalloidin and DAPI. Fluorescence intensity profiles for FITC-PEG-MAL and 594-phalloidin illustrate a physical incorporation depth of hydrogel into tissue of approximately 50 μm.