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.