Glycolipid-Anchored Proteins on Bioengineered Extracellular Vesicles for Lipopolysaccharide Neutralization

Extracellular vesicles (EVs) with native membrane proteins possess a variety of functions. EVs have become increasingly important platforms for incorporating a new peptide/protein with additional functions on their membranes using genetic manipulation of producer cells. Although directly harnessing native membrane proteins on EVs for functional studies is promising, limited studies have been conducted to confirm its potential. This study reports bioengineered EVs with CD14, a natural glycosylphosphatidylinositol (GPI)-anchored protein and a selectively enriched native membrane protein on EVs. We demonstrated that producer cells transfected with genes encoding for GPI-anchored and transmembrane glycoproteins selectively display the former over the latter on bioengineered EVs. Furthermore, using specific enzyme cleavage studies, we characterized and validated that CD14 is indeed GPI-anchored on bioengineered EV membranes. Natural GPI-anchored proteins are conserved receptors for bacterial toxins; for example, CD14 is an innate immune receptor for lipopolysaccharide (LPS), a gram-negative bacterial endotoxin. We reported that unlike soluble CD14, bioengineered EVs harboring CD14 reduce (50-90%) LPS-induced cytokine responses in mouse macrophages, including primary cells, possibly by reduced cell surface binding of LPS. These findings highlight the importance of harnessing the native EV membrane proteins, like GPI-anchored proteins, for functional studies such as toxin neutralization. The GPI-anchoring platform can display various natural GPI-anchored proteins and other full-length proteins as GPI-anchored proteins on EV membranes.