In-situ hyaluronic acid-tyramine hydrogels prolong the release of extracellular vesicles and enhance stability

Hydrogels can provide a hydrated environment to encapsulate extracellular vesicles (EVs) while offering promising solutions to some of the challenges that limit their therapeutic potential, e.g. rapid clearance and propensity for enzymatic degradation and aggregation. This study explores the use of a hyaluronic acid-tyramine (HA-TA) hydrogel to prolong the delivery and enhance the stability of EVs. EVs were obtained from lentiviral-transduced HEK293T cells expressing luciferase and eGFP to enable easy quantification. Two encapsulation strategies were evaluated: (1) pre-loading, where EVs were mixed with HA-TA (2.58 % degree of substitution) precursor solution and subsequently crosslinked with 2 U/mL horseradish peroxidase (HRP) and 0.05 mM H₂O₂; and (2) post-loading, where EVs were soaked into pre-formed dehydrated hydrogels. Both methods improved EV stability over 7 days at 37 °C compared to free EVs. The pre-loading approach was ultimately selected due to its ability to give rapid in situ gelation within one minute. Controlled in vitro release of EVs from the pre-loaded hydrogels was observed to extend beyond 7 days, as determined by CD9 ELISA. The released EVs maintained their bioactivity, as evidenced by effective internalisation into ARPE-19 and H9c2 cell lines, with performance comparable to fresh EVs. The EV release profile could be varied by modifying the hydrogel concentration. These findings underscore the potential of HA-TA hydrogels for localised, sustained, EV delivery with preserved functionality.