VHH-Functionalized Silica-Coated Magnetic Nanoparticles for Immunoaffinity Isolation of Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles involved in intercellular communication and numerous physiological and pathological processes. Their molecular cargo reflects the state of the cell of origin, making EVs promising sources of biomarkers and potential therapeutic agents. However, efficient and selective isolation of EVs from complex biological fluids remains challenging. Immunoaffinity-based approaches offer high selectivity through the recognition of EV-associated surface markers by specific affinity ligands. In this study, silica-coated magnetite nanoparticles were functionalized with a mixture of five VHH-eGFP constructs and evaluated as a solid phase for immunoaffinity isolation of EVs from human plasma. Surface modification of the nanoparticles was confirmed by FTIR spectroscopy, while protein-binding studies showed a maximum binding capacity (Qmax) of 118.2 mg/g. The developed material was then applied for EV isolation from plasma, and the resulting EV-enriched preparations were characterized by protein and lipid quantification, nanoparticle tracking analysis, flow cytometry, and atomic force microscopy. NTA revealed a median particle diameter of 124 nm and a particle concentration of 6.9 × 10⁹ particles/mL. The presence of the EV-associated markers CD9, CD63, and CD81, together with their reduced signal following Triton X-100 treatment, supported the vesicular nature of the isolated particles. The affinity material could be reused over five consecutive isolation cycles, and EVs could be detected from plasma volumes as low as 25 µL. These results demonstrate the potential of VHH-functionalized magnetic nanoparticles as a reusable and adaptable platform for immunoaffinity-based EV isolation from human plasma.