Inverse Electron-Demand Diels–Alder Methylcellulose Hydrogels Enable the Co-delivery of Chondroitinase ABC and Neural Progenitor Cells

Abstract

A hydrogel that can deliver both proteins and cells enables the local microenvironment of transplanted cells to be manipulated with a single injection. Toward this goal, we designed a hydrogel suitable for the co-delivery of neural stem cells and chondroitinase ABC (ChABC), a potent enzyme that degrades the glial scar that forms after central nervous system (CNS) injury. We leveraged the inverse electron-demand Diels−Alder reaction between norbornene and methylphenyltetrazine to form rapidly gelling (<15 min) crosslinked methylcellulose (MC) hydrogels at physiological temperature and pH, with Young's modulus similar to that of brain tissue (1−3 kPa), and degradable, disulfide-containing crosslinkers. To achieve tunable, affinity-controlled release of a ChABC-Src homology 3 (SH3) fusion protein, we immobilized norbornene-functionalized SH3-binding peptides onto MC-methylphenyltetrazine and observed release of bioactive ChABC-SH3 over 4 days. We confirmed cytocompatibility by evaluating neural progenitor cell survival and proliferation. The combined encapsulation of neural stem cells and chondroitinase ABC from one hydrogel lays the framework for future in vivo studies to treat CNS injuries.

Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability

“…To facilitate complete protein release, we are further engineering the MC delivery vehicle to expedite degradation and resorption in vivo. We have recently synthesized a resorbable MC hydrogel with a disulfide-containing cross-linker, which enables material degradation in the presence of naturally occurring thiols in vivo ( 33 ).…”

“…To facilitate complete protein release, we are further engineering the MC delivery vehicle to expedite degradation and resorption in vivo. We have recently synthesized a resorbable MC hydrogel with a disulfide-containing cross-linker, which enables material degradation in the presence of naturally occurring thiols in vivo (33). The modifications to ChABC described here have improved its production, stability, and long-term bioactivity, overcoming many challenges required for clinical translation and facilitating its future use as a viable therapeutic in treating CNS injuries.…”

Tunable Cross-Linking and Adhesion of Gelatin Hydrogels via Bioorthogonal Click Chemistry

“…The use of gelatin as backbone polymer to form the hydrogels allows a better mimicking of the native ECM, thanks to the ECM biomimetic motifs naturally present in gelatin. When other polymers (e.g., alginate, 36 PEG, 35 and methylcellulose) 38 were used to prepare hydrogels by Tz-Nb crosslinking, an additional synthesis step was required to artificially introduce ECM biomimetic motifs to improve their biological performance. Conversely, it was previously demonstrated 44 that gelatin hydrogels modified by Tz-Nb, even with higher degrees of modification, maintain their cell-adhesive and MMP-degradable peptide sequences, thus avoiding the need for further modification to impart these properties on the hydrogels.…”

“…Bioorthogonal cross-linking strategies include azide–alkyne cycloaddition, nitrile oxide cycloaddition, Diels–Alder reactions, and inverse-electron demand Diels–Alder reactions . The latter has been investigated by using, for instance, the reaction between tetrazine (Tz) and norbornene (Nb), which has been demonstrated as being successful for the preparation of hydrogels based on PEG, alginate, hyaluronan, methylcellulose, and different polymer combinations. − In recent work, the suitability of this bioorthogonal cross-linking strategy was investigated for the first time for cell-laden gelatin hydrogels . The authors prepared two different gelatin derivatives, functionalized either with Tz or Nb, with a similar degree of modification (i.e., approximately 20%), and mixed them to form gelatin hydrogels at different gelatin concentrations (i.e., 5 and 10%).…”

Designing Hydrogels for 3D Cell Culture Using Dynamic Covalent Crosslinking

“…also exploited the thiol‐disulfide dynamic exchange by inserting ‐SS‐ bonds in inverse‐electron demand Diels–Alder crosslinked hydrogels, which enabled hydrogel degradation with glutathione, thereby enabling the delivery of cells and enzymes in vivo. [ 214 ] In another study, it was shown that thiolated HA gelled quickly with oxidized glutathione, remained stable for at least 10 days of in vitro culture, and allowed proliferation of adipose‐derived stem cells. [ 215 ]…”