Phage Display Screening of Anchor Peptides for Red Blood Cell-Derived Extracellular Vesicles

Extracellular vesicles (EVs) are increasingly used for disease diagnosis and treatment. Among them, red blood cell-derived EVs (RBC-EVs) have attracted great attention due to their abundant sources and low risks of gene transfer (RBC-EVs lack nuclear and mitochondrial DNA). Here, we first revealed the high expression level of membrane protein solute carrier family 4 member 1 (SLC4A1) in RBC-EVs through proteomic analysis. We then identified several binding peptides with high affinity for the SLC4A1 extracellular domain (SLC4A1-EC) from phage display library screening. A high affinity of SLC4A1-EC and the three peptides (XRB2, XRE4, and XRH7) were assessed in vitro using surface plasmon resonance analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The binding sites of SLC4A1-EC and polypeptides were further predicted by LigPlot + analysis, and the results showed that these three polypeptides could bind to part of the hydrophobic residues of SLC4A1-EC. The binding efficiency of the anchor peptides to the RBC-EVs was further verified by flow cytometry and fluorescence imaging. In conclusion, we successfully screened three specific RBC-EV-targeting peptides which could potentially be utilized for isolating RBC-derived EVs from serum samples. More importantly, this peptide could be coupled with targeting peptides to modify RBC-EVs for drug delivery. Our work will provide a viable method for optimizing the function of RBC-EVs.