An Anti‐Myd88 Peptide Synergistically Enhances the Anti‐Inflammatory Effects of Extracellular Vesicles from Naïve Umbilical Cord MSC or HEK293F CD24 Overexpressing Cells

Abstract Mesenchymal stem cells (MSCs) and their nanosized extracellular vesicles (EVs) are anti‐inflammatory and immunoregulatory. Further, HEK293F cells can be engineered to express the anti‐inflammatory membrane molecule CD24. It is hypothesized that the anti‐inflammatory effects of MSC‐EV and HEK293F‐CD24‐EVs can be enhanced when loaded with a peptide targeting the myeloid differentiation primary response 88 (Myd88). EVs isolated from MSCs or HEK293F cells overexpressing CD24 are characterized by transmission electron microscopy (TEM), western blot (WB), and nanoflow cytometry. A lipid‐anchor anti‐Myd88 peptide is loaded in the EVs, and anti‐inflammatory effects are analyzed in RAW 264.7 cells and an NFκB reporter human monocyte cell (Luc‐THP‐1) exposed to natural LPS. Both MSC‐EVs and HEK293F‐CD24‐EVs lower IL‐6 release from the RAW264.7 by 30–40% and the NFκB activity in the Luc‐THP‐1 reporter monocyte cell line is reduced by 15–25%. Additional and synergistic inhibition of IL‐6 release is observed with EVs loaded with the anti‐Myd88 peptide. The anti‐inflammatory capabilities of MSC‐EVs and HEK293F‐CD24‐EVs are confirmed, and show that a loaded anti‐Myd88 peptide results in synergistically enhanced anti‐inflammatory effects for both EVs. Both MSC‐EVs and HEK293F‐CD24‐EVs may be suitable for clinical translation and are amenable to loading with peptides that enhance their anti‐inflammatory effects.