Harnessing extracellular vesicles for stabilized and functional IL-10 delivery in macrophage immunomodulation

Extracellular vesicles (EVs) are gaining recognition as promising therapeutic carriers for immune modulation. We investigated the potential of EVs derived from HEK293FT cells to stabilize and deliver interleukin-10 (IL-10), a key anti-inflammatory cytokine. Using minicircle (MC) DNA vectors, we achieved IL-10 overexpression and efficient incorporation into filter-isolated small EVs (F-sEVs), resulting in superior stability compared to free recombinant IL-10. Detailed biophysical and functional analyses revealed that IL-10 + F-sEVs contain both monomeric and oligomeric IL-10 on their external surface and encapsulated within vesicles. Size-based fractionation of IL-10 + large EVs (lEVs), small EVs (UC-sEVs), and non-vesicular extracellular particles (NVEPs) revealed IL-10 presence across all fractions, predominantly in monomeric form. Anion exchange chromatography successfully enriched IL-10 + exosomes that efficiently associated with both IL-10 monomers and oligomers. IL-10 + F-sEVs suppressed inflammatory cytokine expression in pro-inflammatory macrophages (two-to 14-fold more effectively than naïve F-sEVs) without inducing anti-inflammatory repolarization. However, detailed analysis of IL-10-loaded EV subpopulations revealed that anti-inflammatory activity was distributed across multiple fractions. Moreover, naïve F-sEVs derived from non-transfected cells also exhibited anti-inflammatory effects, suggesting that endogenous EV cargo contributes to their immunomodulatory activity and complicates attribution of effects specifically to IL-10. These findings highlight the therapeutic potential of EVs while emphasizing the need to disentangle contributions of engineered cytokines from endogenous vesicular components.