Single-step negative isolation and concentration of extracellular vesicles by graphene oxide composite hydrogels

Abstract

Extracellular vesicles (EVs) are proven to hold great promise for diverse therapeutic and diagnostic applications. However, large-scale preparation of EVs from bulk liquid samples including culture medium and body fluids is a prerequisite for their clinical translation applications. Herein, we proposed a single-step negative isolation and concentration method using graphene oxide composite hydrogels (hGOs), by which both protein impurities are adsorbed inside the hGOs while EVs with large sizes are excluded from the outside by surface nano-sized channels, meanwhile the end-product was also concentrated by at least 3–5 times. Taking advantage of this method, we isolated the EVs from large-volume rat bronchoalveolar lavage fluids (BALFs). Compared to gold-standard ultracentrifugation, although the isolation purity of EVs was comparable, the recovery of EVs ranging from 81.4 %–85.9 % was greatly achieved by hGOs with excellent reproducibility (RSD = 2.4 %, n = 9), 2 times higher than that obtained via ultracentrifugation. Proteomic analysis of EVs from severe acute pancreatitis-lung injury (SAP-ALI) BALFs identified 25 differentially expressed proteins distinguishing disease and healthy states. Critically, this method enabled scalable production of ginger-derived EVs (10–150 mL extracts), with NTA-confirmed linear yield scalability (R² > 0.98), while functional assays demonstrated their anti-inflammatory efficacy via significant suppression of TNF-α, MIP-2, and NO (P < 0.0001) in macrophage models. We believed that the newly developed hGOs would be a useful tool for achieving highly efficient EV production from bulk liquid samples, facilitating many important biological and clinical applications.