Assistant Professor Arizona State University, United States
Introduction:: Systemic immunosuppression poses significant risks to transplant patients and limits the translation of cell therapies for the treatment of diseases such as type 1 diabetes. Approaches to induce localized tolerance and eliminate the need for chronic immunosuppression could vastly widen the accessibility of cell therapies. During pregnancy, trophoblasts within the allogeneic placenta induce tolerance in maternal immune cells through several multilayered mechanisms, including extracellular vesicles (EV). Trophoblast EVs possess numerous soluble tolerogenic factors that could be used to induce local immune tolerance in a cell transplant site if delivered in a sustained manner. To achieve this, we have engineered a hydrogel-based delivery platform using synthetic poly(ethylene glycol) (PEG-mal) and alginate hydrogels for sustained delivery of trophoblast EVs; we characterize the morphology and release kinetics from hydrogels and elucidate the effects of EVs delivery on innate immune cells.
Materials and Methods:: EV isolation: EVs were isolated from human JAR trophoblast cell line using size exclusion chromatography and reagent-based isolation methods. EV characterization: EVs were characterized via dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and proteomic characterization. EV delivering hydrogels: Nondegradable PEG-maleimide (5% w/v) and alginate (1.5% w/v) hydrogels were used to encapsulate EVs (8 µg) and kinetics of release were characterized using fluorescent tags, imaging and spectroscopy. EV-loaded hydrogels were characterized with STED microscopy and cryo-SEM. EV immunomodulation: EVs were incubated with activated natural killer cells (NK-92) and secreted IFNγ assessed via ELISA.
Results, Conclusions, and Discussions:: Results and Discussion. Human trophoblast EV isolation and characterization by NTA demonstrated vesicles with 92.2 ± 30 nm diameter, confirmed by measurements in TEM images. Proteomic characterization identified a total of 1070 proteins in EVs and exosome-specific markers CD9, CD63, CD81, TSG101, and HSP90. Gene ontology enrichment analysis identified immunomodulatory proteins CD276, HSP10, CD55, and galectins. Next, EVs were entrapped in alginate or tethered to the matrix of PEG hydrogels and EV presence confirmed with STED and SEM microscopy. EV delivery kinetics from hydrogels demonstrated burst release from both hydrogel systems, with alginate and PEG systems releasing ~90% of the payload by 2 and 8 days, respectively (Figure 1). Finally, we investigated trophoblast EV impact on activated human NK cells and found that EVs significantly reduced IFNγ secretion, comparable to the TGFβ control (Figure 2). EVs released from hydrogels exhibited a comparable influence on NK cell activation, demonstrating the potential of hydrogel delivery systems to induce localized effects on innate immune cells.
Conclusion. In this work, we isolated and characterized human trophoblast EVs, which demonstrated a tolerogenic payload that was capable of reducing innate immune cell activation. Tethering EVs within a hydrogel system resulted in greater sustained release than passive entrapment, extending release by ≥ 6 days. Ongoing studies are evaluating degradable hydrogel systems and hydrolytic linkers to extend and tune EV release from synthetic hydrogel systems. Additionally, ongoing studies are evaluating impact of EV release kinetics and immunomodulation in vivo.
