(T1030-12-77) Developing an In Situ Hyaluronic Acid-Tyramine Hydrogel Platform to Deliver Extracellular Vesicles

Purpose: Extracellular vesicles (EVs) are small membrane vesicles secreted by cells, and have emerged as a promising alternative to cell-therapies due to their lack of immunogenicity and targeting capabilities [1]. However, there are significant challenges with their low retention and short-lived therapeutic effects. This study aims to develop a hyaluronic acid–tyramine (HA-Tyr) hydrogel platform through in situ enzymatic crosslinking to determine whether incorporating EVs into these hydrogels can improve their retention time while maintaining their structures and bioactivity.

Methods: HA-Tyr hydrogel was formed using the oxidative coupling of tyramine moieties catalysed by hydrogen peroxide (H₂O₂) and horseradish peroxidase (HRP). The optimal composition of the hydrogel was determined by tuning the concentrations of H₂O₂ and HRP to reach the maximum crosslinking with the minimum cytotoxicity. The adult retinal pigment epithelial cell line 19 (ARPE-19) and H9c2 myoblasts were selected as in vitro models of retina and heart disease respectively. EVs harvested from HEK293T cells were isolated using differential ultracentrifugation and characterised by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and simple western blot. The cell internalisation of free EVs and EVs released from the hydrogels was analysed by the luciferase assay. The EV release profile was determined by the CD9 ELISA kit. The stability of EVs after loaded into the hydrogels was assessed by NTA, TEM and simple western blot.

Results: The optimal composition of the hydrogel showed rapid gelation time (1 minute) when injected in situ, and no cytotoxicity to ARPE-19 and H9c2 cell lines. EVs were successfully isolated and incorporated into these hydrogels with evenly distribution. EVs showed extended release from the hydrogel for over 2 weeks in vitro. These EVs released showed improved stability compared to free EVs and can be internalised by the cells.

Conclusion: HA-Tyr hydrogel provides a novel approach for the local delivery of EVs and optimises their retention and stability. Therefore, the sustained release mechanisms achieved through manipulating the formation of such hydrogels provide a key to unlocking the therapeutic potential held within EVs.

References: [1] L.M. Doyle, M.Z. Wang, Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis, Cells 8 (2019) https://doi.org/10.3390/cells8070727.

Figure 1. 1) EV isolation by ultracentrifugation; 2) Synthesis of HA-Tyr conjugates; 3. Preparing EV-loaded hydrogel in double syringe.

Figure 2. Kinetic study. ARPE-19 and H9c2 cells were incubated with EVs (1E11 particles/mL) for different incubation times (15 min, 1, 2, 6, 12, and 24 h) at 37 °C. EV uptake was then quantified by luciferase assay. Kinetics showed that luciferase activity in acceptor cells increased over time, with a 1% spontaneous rate at 1 h.

Figure 3. 1) The stability of EVs loaded in the hydrogels was determined by comparing the particle concentrations and protein expressions with non-loaded EVs. The particle concentration was analysed by NTA (left), and the expression of EV markers (CD9, TSG101) was analysed by simple western blot (right); 2) The cumulative release profiles of EVs from hydrogels. Short-term release (1, 3, 6, 12 h) was determined by NTA, and long-term release (1, 2, 4 days) was quantified by CD9-ELISA kit.