Self‐Forming Norbornene‐Tetrazine Hydrogels with Independently Tunable Properties

Abstract

Although photopolymerization reactions are commonly used to form hydrogels, these strategies rely on light and may not be suitable for delivering therapeutics in a minimally invasive manner. Here, hyaluronic acid (HA) macromers are modified with norbornene (Nor) or tetrazine (Tet) and upon mixing click into covalently crosslinked Nor-Tet hydrogels via a Diels-Alder reaction. By incorporating a high degree of Nor and Tet substitution, Nor-Tet hydrogels with a broad range in elastic moduli (5 to 30 kPa) and fast gelation times (1 to 5 min) are achieved. By pre-coupling methacrylated HANor macromers with thiolated peptides via a Michael addition reaction, Nor-Tet hydrogels are peptide-functionalized without affecting their physical properties. Mesenchymal stem cells (MSCs) on RGD-functionalized Nor-Tet hydrogels adhere and exhibit stiffness-dependent differences in matrix mechanosensing. Fluid properties of Nor-Tet hydrogel solutions allow for injections through narrow syringe needles and can locally deliver viable cells and peptides. Substituting HA with enzymatically degradable gelatin also results in cell-responsive Nor-Tet hydrogels, and MSCs encapsulated in Nor-Tet hydrogels preferentially differentiate into adipocytes or osteoblasts, based on 3D cellular spreading regulated by stable (HA) and degradable (gelatin) macromers.

Engineering Hydrogels for Affinity-Based Release of Therapeutic Proteins

“…61 Norbornene−tetrazine coupling of HA-modified gels using IEDDA chemistry has also been explored. 62 Gultian et al separately modified the carboxyl groups in HA with 5norbornene-2-methylamine and tetrazine-amine for subsequent cross-linking in PBS at 37 °C (Figure 1E). 62 Tetrazine tends to degrade in aqueous environments due to nucleophilic attack of its nitrogen atoms by water molecules.…”

“…62 Gultian et al separately modified the carboxyl groups in HA with 5norbornene-2-methylamine and tetrazine-amine for subsequent cross-linking in PBS at 37 °C (Figure 1E). 62 Tetrazine tends to degrade in aqueous environments due to nucleophilic attack of its nitrogen atoms by water molecules. Methylphenyltetrazine substitution offers greater stability than tetrazine substitution due to resonance stabilizing effects of the aromatic ring, which reduces its level of degradation while still maintaining reactivity.…”

“…To enhance the gelation rate, methylfuran and/or fulvene has been used in place of furan, which accelerates gelation . Norbornene–tetrazine coupling of HA-modified gels using IEDDA chemistry has also been explored . Gultian et al separately modified the carboxyl groups in HA with 5-norbornene-2-methylamine and tetrazine-amine for subsequent cross-linking in PBS at 37 °C (Figure E) .…”

Bioconjugation in Materials Science

“…Furthermore, the specificity of the tetrazinenorbornene reaction facilitated the sequential modification of the network via thiol-ene photochemistry. The accessibility of the tetrazine molecule in comparison to other bioorthogonal click reagents was subsequently investigated as a new tool for developing cell-instructive hydrogels of all kinds (i.e., alginates, [153] hyaluronic acids (HA), [154] and gelatins [155] ), etc. for tissue engineering applications (Scheme 10).…”

Mesenchymal stem cells enhance targeted bone growth from injectable hydrogels with BMP‐2 peptides

“…22,26 Our group recently developed injectable hydrogels based on a Diels Alder reaction between norbornene and tetrazine modified hyaluronic acid macromers. 27 These hydrogels can be used to inject cells through clinically used syringe needles without impacting viability, and peptides can be covalently bound to the hydrogel backbone without altering mechanical properties. Using this platform, we synthesized DWIVA-functionalized injectable hydrogels and found that encapsulated human mesenchymal stem cells (MSCs) exhibited increased alkaline phosphatase (ALP) expression in vitro, and acellular injections induced local mineralized bone formation in vivo after 4 weeks.…”