Intercellular delivery of NF-κB inhibitor peptide utilizing small extracellular vesicles for the application of anti-inflammatory therapy

Abstract

Excessive activation of NF-κB in macrophages contributes to the onset and exacerbation of inflammatory disorders. The NEMO binding domain (NBD) peptide is an NF-κB inhibitor peptide that binds to NEMO, one of components of the IκB kinase (IKK) complex, and inhibits the IKK kinase activity, in the cytosol. Because of this property, the NBD peptide is expected to inhibit NF-κB activation in macrophages. In this study, we developed a delivery carrier for NBD based on small extracellular vesicles (sEVs), which are membrane vesicles released from cells. We constructed fusion proteins comprising Gag (an sEV tropic protein) and one, three, or six repeats of NBD peptide (Gag-1NBD, Gag-3NBD, and Gag-6NBD, respectively) to load the NBD peptide to the inner space of the sEVs, and attempted the intracellular delivery of the NBD peptide to macrophages using Gag-NBD-loaded sEVs (nNBD-sEVs). The nNBD-sEVs significantly inhibited LPS-induced phosphorylation of NF-κB pathway-related proteins in macrophages in a repeat number-dependent manner. Moreover, they exerted inhibitory effects on the NF-κB-dependent expression of proinflammatory mediators such as TNFα, CXCL10, iNOS, and NO. Collectively, our results indicate that NBD-containing sEVs can be used for the treatment of inflammatory diseases owing to their ability to effectively deliver peptides to the macrophage cytosol.