Rapid and Highly Efficient Purification of Extracellular Vesicles Enabled by a TiO₂ Hybridized Spongy-like Polymer

We developed a novel purification medium of extracellular vesicles (EVs) by constructing a spongy-like monolithic polymer kneaded with TiO2 microparticles (TiO2-hybridized spongy monolith, TiO2-SPM). TiO2-SPM was applied in a solid-phase extraction format and enabled simple, rapid, and highly efficient purification of EVs. This is due to the high permeability caused by the continuous large flowthrough pores of the monolithic skeleton (median pore size; 5.21 mu m) and the specific interaction of embedded TiO2 with phospholipids of the lipid bilayers. Our method also excels in efficiency and comprehensiveness, collecting small EVs (SEVs) from the same volume of a cell culture medium 130.7 times more than typical ultracentrifugation and 4.3 times more than affinity purification targeting surface phosphatidylserine by magnetic beads. The purification method was completed within 1 h with simple operations and was directly applied to serum SEVs. Finally, we demonstrated flexibility toward the shape and size of our method by depleting EVs from fetal bovine serum (FBS), which is a necessary process to prevent contamination of culture cell-derived EVs with exogenous FBSderived EVs. Our method will eliminate the tedious and difficult purification processes of EVs, providing a universal purification platform for EV-based drug discovery and pathological diagnosis.