Poster P2 - Injectable Extracellular Vesicle Hydrogels with Tunable Viscoelasticity for Depot Vaccine

Introduction: Extracellular vesicles (EVs) have been actively explored for therapeutic applications in the context of cancer and other diseases. However, the poor tissue retention of EVs and the lack of methods to engineer EVs have limited the development of EV-based therapies. Here we report a facile approach to fabricating injectable EV hydrogels with tunable viscoelasticity and gelation temperature, by metabolically tagging EVs with azido groups and further crosslinking them with dibenzocyclooctyne-bearing polyethylene glycol via efficient click chemistry. One such EV gel has a gelation temperature of 39.4oC, enabling in situ gelation of solution-form EVs upon injection into the body. The in situ formed gels are stable for over 4 weeks and can attract immune cells including dendritic cells over time in vivo. We further show that tumor EV hydrogels, upon subcutaneous injection, can serve as a long-term depot for EV-encased tumor antigens, providing an extended time for the modulation of dendritic cells and subsequent priming of tumor-specific CD8+ T cells. The tumor EV hydrogel also shows synergy with anti-PD-1 checkpoint blockade for tumor treatment. As a proof-of-concept, we also demonstrate that EV hydrogels can induce enhanced antibody responses than solution-form EVs over an extended time. Our study yields a facile and universal approach to fabricating injectable EV hydrogels with tunable mechanics and improving the therapeutic efficacy of EV-based therapies.

Materials and

Methods: We used EVs, which itself can serve as the main building block of hydrogels, by metabolically tagging EVs and then crosslinking them into a gel network via efficient click chemistry. We previously demonstrated that metabolic glycan labeling of cells provides a facile approach to generating chemically tagged EVs. Once internalized by cells, unnatural sugars bearing clickable chemical tags (e.g., tetraacetyl N-azidoacetylmannosamine (Ac4ManNAz)) can undergo the metabolic glycoengineering processes, conjugate to proteins and lipids, and become expressed on the cell membrane in the form of glycoproteins and glycolipids. Since EVs inherit a portion of the cell membrane, EVs secreted by the labeled cells also carry the chemical tags (e.g., azido group). These azido-tagged EVs can react with dibenzocyclooctyne (DBCO)-functionalized 8-arm polyethylene glycol (PEG) via efficient click chemistry43-45 to yield a fully crosslinked gel network (Fig. 1a). The gelation temperature, stiffness, viscoelasticity, as well as the injectability of the fabricated EV gels can be tuned by adjusting the concentration of EVs and can be measured by using rheometer. At certain concentrations, the mixture of azido-tagged EVs and DBCO-PEG stays as a solution form at room temperature but rapidly forms a hydrogel upon injection into the body.

Results, Conclusions, and Discussions: For the first time, have developed EV hydrogels with tunable viscoelasticity and injectability, by metabolically tagging EVs with azido groups and crosslinking them with DBCO-PEG via click chemistry. Upon subcutaneous injection, the EV hydrogel gel can be rapidly formed in situ and are stable for over 4 weeks, with the gradual attraction of immune cells including DCs. EV gels can induce the activation of DCs and facilitate the processing and presentation of EV-encased antigens by DCs, resulting in improved tumor control than bolus vaccines. The covalent conjugation CpG to EV gels further improves the CTL response and antitumor efficacy. We also demonstrate the synergistic effect between CpG-conjugated EV gels and anti-PD-1 checkpoint blockade for cancer treatment. This EV hydrogel system provides a unique approach to amplifying the humoral and T cell responses towards EV-encased antigens, and improving the therapeutic efficacy of EV-based therapies.

Acknowledgements (Optional): The authors would like to acknowledge the financial support from NSF DMR 2143673 CAR, R01CA274738, R21CA270872, and the start-up package from the Department of Materials Science and Engineering at the University of Illinois at Urbana-Champaign and the Cancer Center at Illinois. Research reported in this publication was supported by the Cancer Scholars for Translational and Applied Research (C*STAR) Program sponsored by the Cancer Center at Illinois and the Carle Cancer Center under Award Number CST EP012023.