Application of VHH-Immobilized Cryogel-Based Immunoaffinity Chromatography for Isolation of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanosized structures involved in intercellular communication that have high potential as disease biomarkers and for the delivery of therapeutic cargos. However, translation to the clinic is hampered by time-consuming, low-yield, and poorly reproducible EV isolation methods. We describe a cryogel-based immunoaffinity chromatography system that exploits single-domain VHH antibodies as capture elements for the selective isolation of EVs from human plasma. Supermacroporous cryogels functionalized with five unique anti-EV VHHs (total immobilization capacity ~500 µg/g) were prepared, yielding a highly permeable and hydrophilic support. They were captured and eluted under mild conditions, and their morphology and identity were confirmed by SEM, AFM, NTA, and flow cytometry. Proteomic profiling of the isolated samples identified 234 proteins, of which 63% were ExoCarta-listed exosomal proteins; contaminants such as albumin and apolipoproteins were also identified. The purification method provided samples with ~2 × 109 EVs/mL, with EV median size of 135 nm and consistent protein-to-lipid ratio across three independent isolations (CV < 10%). This study demonstrates that VHH-functionalized cryogels (VHH-SMC) are a rapid and reproducible EV purification method that represents a promising alternative to conventional ultracentrifugation- or precipitation-based protocols. While optimization of nanobody density and reduction in plasma protein carryover are still necessary, the platform holds potential for scalable EV enrichment, a condition that can significantly speed up biomarker research and clinical diagnostics.