Isolation of extracellular vesicles from citrus limon fruit by ion-exchange membrane chromatography

Extracellular vesicles (EVs) are membrane-bound nanoparticles that are naturally released by cells into the surrounding environment. EVs are reported to be ubiquitous, and they have been isolated from various mammalian cells. However, their production is limited by several factors such as the requirement of extensive surface area for cell growth and cellular senescence. Moreover, EV properties highly depend on their source, which must be carefully selected based on the intended application. To address these challenges, researchers have begun exploring alternative sources of EVs, such as plant derived EVs (PDEVs). PDEVs are structurally similar to mammalian EVs, and they act as cross kingdom modulators. They are considered inherently biocompatible, they have demonstrated intrinsic therapeutic properties including anti-tumour, anti-inflammatory, and anti-oxidant effects. Moreover, PDEVs are promising candidates for drug delivery systems. The purpose of this experimental work is to develop an ion-exchange membrane chromatography process for the purification of citrus limon fruit derived EVs. The membranes involved in the purification process were nonwoven polyamide 11 (PA11) membranes produced at NC State University. The chosen ligand was diethylamine (DEA) which has a net positive charge able to interact with the negatively charged PDEVs. The process involved a pretreatment step which included squeezing, centrifugation, two filtration steps, and pH adjustment, followed by the purification step using ion-exchange membrane chromatography. The elution fractions from the chromatography process were analysed by TEM, DLS, SEC-HPLC, SEC-MALS, and BCA assays. The results demonstrated that the membrane process successfully purified EVs; however, the yield was low, and their detection proved challenging. Moreover, the pH and the conductivity of the starting sample were found to be critical factors for effective purification.