Size exclusion chromatography: An efficient tool for hsEV isolation from insect Haemolymph

Extracellular vesicles (EV) serve an important role in biological system as they can change the phenotype and function of the cell. The discovery of EVs has led to the development of novel vaccines, associated with immunosuppressive and immunostimulatory effects during disease progression. To address the opportunities in EVs, here we used insect as model system. The insect also has medicinal properties as it produces antimicrobial peptides which are found circulating fluid called as hemolymph. Hemolymph consists of defensins, gloverins, proline-rich peptides, attacins, cecropins, moricins, anionic antimicrobial peptides, and several other components that create robust defense mechanisms against a broad spectrum of pathogens. Here, the insect hemolymph was used for EV isolation which encapsulates such kinds of proteins and acts as cargo transporters. As the biogenesis was unknown, we have defined it as hemolymph-derived small extracellular vesicles (hsEV). The present paper deals with the appropriate methodology for the isolation of hsEV. The hemolymph was processed for isolation via size exclusion chromatography. Molecular and physical characterization was performed using western blotting utilizing anti-CD63, anti-Flotillin-2, TEM, FESEM, confocal microscopy, and DLS. The isolated hsEV was used to assess the antibacterial property by measuring bacterial growth.