Abstract
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 EVs, yielding superior stability compared to free, recombinant IL-10 protein. Detailed biophysical and functional analyses revealed that IL-10+ EVs contain both monomeric and oligomeric forms of IL-10 on their external surface and encapsulated within vesicles. IL-10+ EVs suppressed inflammatory cytokine expression in pro-inflammatory macrophages (from two to 14-fold compared to naïve EVs) without inducing anti-inflammatory polarization, demonstrating a distinct immunosuppressive mechanism. Interestingly, naïve EVs from non-transfected cells also exhibited significant anti-inflammatory effects, suggesting that the intrinsic bioactive cargo of EVs substantially contributes to their function, complicating the interpretation of IL-10-specific effects. Size-based fractionation analyses of IL-10+ large EVs (lEVs), small EVs (sEVs), and non-vesicular extracellular particles (NVEPs) revealed IL-10 presence across all fractions, predominantly in monomeric form, with anti-inflammatory activity distributed among subpopulations. Anion exchange chromatography successfully enriched IL-10+ exosomes while retaining immunomodulatory effects. However, the shared properties of naïve and IL-10+ exosomes underscore the complexity of their immunomodulatory functions. These findings highlight the therapeutic potential of EVs while emphasizing the need to disentangle the contributions of engineered cytokines from endogenous vesicular components.