Exosomes as tumoral biomarkers: devising an efficient and economical purification strategy based on magnetic immunoconjugates

Abstract

Exosomes are nano-sized vesicles limited by a lipid bilayer released to the extracellular media by most cells. Inside, they harbour specific cargo materials (proteins, nucleic acids, amino acids, etc) that can be uptake by other cells playing in this way an important role in the inter-cellular communication, particularly in metastatic processes. Nowadays, the strategies mainly used to purify exosomes are based on differential centrifugation and precipitation, which are far from being efficient and are time consuming.Thinking on an alternative to resolve these disadvantages, the use of magnetic nanoparticles (MNPs) may be very useful, since they have a size similar to exosomes and a large surface area available to be functionalized with antibodies (ABs) that specifically recognize exosomes. In addition, due to their small size they should make feasible the integration of the purification and the detection processes without the need of tedious downstream processing. In this work the purification of exosomes from different model cell lines using precipitation and ultracentrifugation methodologies was carried out, after a previous optimization of the cellular growth. The purified exosomes have been then characterized by dot blot, western blot and ELISA assays to confirm the presence of canonical biomarkers in their surface. This characterization allowed selecting a very specific AB that was then used to functionalize the MNPs. With the aim of obtaining the best immunocapture capability of exosomes, an oriented attachment of the ABs on the MNPs was carried out. Some parameters that will need to be optimized in order to isolate exosomes include the ratio of MNPs: exosomes and the incubation times. The results are very promising thinking in a future platform where exosome purification and detection are integrated.