A Novel Aptamer-Based Approach for Lipoprotein Removal to Achieve Ultra-Pure Blood EV Isolation

Blood extracellular vesicles (EVs) are nanoscale lipid-bilayer particles that carry proteins, nucleic acids, and metabolites, rendering them powerful tools for non-invasive liquid biopsy and targeted drug delivery. However, clinical translation of blood-derived EVs is severely limited by the co-isolation of lipoproteins—whose size (30–100 nm) and density overlap with EVs—resulting in contaminated preparations that compromise biomarker accuracy and jeopardize therapeutic safety, efficacy, and biodistribution. To overcome this critical bottleneck, we developed ApoFilter, an aptamer-based affinity filtration platform engineered to selectively capture ApoB100- and ApoA1-containing lipoproteins (VLDL, LDL, HDL) while preserving EV integrity. In solutions containing only mixed lipoproteins, ApoFilter demonstrated a capture efficiency of 97% for ApoB100- and ApoA1-containing particles, yielding filtrates with >99% depletion of target lipoproteins. Crucially, this high selectivity was preserved in human plasma—a complex protein milieu—where ApoFilter removed over 99% of lipoproteins without any detectable loss of EV yield. Furthermore, when size-exclusion chromatography (SEC) or ExoTFF was integrated with ApoFilter, the limitations of each technique were innovatively complemented, achieving a highly advanced level of separation and purification. Notably, the ApoFilter–ExoTFF combination delivered the highest performance, attaining 99.9% protein removal and a 98.2% EV recovery rate, enabling ultrapure EV isolation from blood plasma. This synergistic purification strategy addresses both analytical and therapeutic requirements by eliminating lipoprotein interference in downstream molecular profiling and minimizing off-target effects in drug delivery. ApoFilter thus represents a versatile, scalable solution for isolating clinically relevant EVs from blood, substantially improving the reliability of liquid biopsy assays and accelerating the development of EV-based therapeutics.