Injectable hyaluronic acid hydrogels via Diels-Alder and inverse electron demand Diels-Alder click chemistry for drug delivery: A comparative in vitro and in vivo evaluation.

Bio-orthogonal click chemistry enables injectable hydrogels for localized drug delivery with reduced systemic exposure. The crosslinking reaction and resulting network characteristics critically influence the physicochemical and biological performance of hydrogels, necessitating comparative evaluation for rational design. This study compares two hyaluronic acid (HA) hydrogel systems based on furan-maleimide Diels-Alder (DA) and tetrazine-norbornene Inverse Electron Demand Diels-Alder (IEDDA) click chemistry. Both hydrogels formed at 37 °C, with IEDDA showing significantly faster gelation than DA. Wet DA and IEDDA hydrogels showed lower swelling ratios than freeze-dried hydrogels. Under physiological conditions (pH 7.4), wet DA hydrogels released most of their drug payload within approximately 8 h, whereas IEDDA hydrogels provided more prolonged drug release over approximately 12 h. These differences were related to the distinct network properties of the two systems. In vitro studies demonstrated rapid doxorubicin (DOX) release from DA hydrogels, resulting in decreased cancer cell proliferation, whereas IEDDA hydrogels exhibited slower and more sustained release. In vivo, IEDDA hydrogels showed prolonged retention of indocyanine green (ICG) and slower degradation compared with DA hydrogels. Overall, DA and IEDDA based HA hydrogels showed distinct gelation, network, drug-release, and in vivo characteristics, highlighting the importance of click chemistry selection for specific biomedical applications.