Recovery and purification of exosomes using polymer-polymer aqueous two-phase systems (ATPS)

Abstract

Exosomes are a type of extracellular vesicles that present natural and attractive characteristics such as nano size (30-200 nm) and unique structure for their use as drug delivery systems for drug therapy, biomarkers for prognostic, diagnostic and personalized treatments. So far, one of the major challenges for therapeutic applications of exosomes is the development of optimized methods for their isolation. In this context, Aqueous Two-Phase Systems (ATPS) have shown to be an alternative method to isolate biological molecules and particles with promising expectations for exosomes. In this work, fractionation of exosomes obtained from a CaCo2 cell line and the contaminants present in their culture media was performed in 20 different polymer-polymer aqueous two-phase systems (ATPSs) in a discrete way. The effect of system design parameters such as polymer composition, molecular weight, and TLL (15-30%) on PEG-Dextran, Dextran-Ficoll and PEG-Ficoll systems were studied keeping phase volume ratio (VR) and pH constant at 1.0 and 7.0, respectively. After partition analysis, 4 out of the 20 systems presented the best exosome fractionation from the contaminants under initial conditions and were selected for further partition optimization via salt addition (NaCl) to observe the behavior of both samples, individually, and improve the efficiency of collection. The PEG 10,000 gmol-¹ – Dextran 10,000 gmol-¹ system at TLL 25% w/w, with the addition NaCl to reach a final concentration of 25 mM, showed the best isolation efficiency resulting in exosome purification factor of 2 and furtherly demonstrated that ATPS has the potential for the selective recovery these promising nanovesicles.