Development of magnetic nanoimmunoconjugates for the fast and efficient isolation of tumoral exosomes

Resumen del trabajo presentado a la 12th International Conference on the Scientific and Clinical Applications of Magnetic Carriers, celebrada en Copenhague (Dinamarca) del 22 al 26 de mayo de 2018.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. They play therefore an important role in the inter-cellular communication, particularly in metastatic processes. Since they can be found in several biofluids like blood, saliva and urine, they become very interesting biomarkers to study cancer progression. However, isolation of high yields of pure and intact exosomes from complex biological fluids is still a critical step for the implementation of their use in clinical diagnosis. Nowadays, the most used purification strategies are based on differential centrifugation and precipitation, which are far from being efficient and time consuming. The feasibility of extracting exosomes targeting specific protein markers on their surface by means of immunemagnetic microbeads has already been demonstrated. However, further analysis of the captured exosomes requires their elution from the microbeads and this usually damage their structure. In this sense, magnetic nanoparticles (MNPs), with similar sizes to exosomes, may be more adequate as this would allow collecting a greater amount of exosomes due to their larger surface area. Besides, due to their smaller size they should make feasible the integration of the purification process with the detection of exosomal surface biomarkers without the need of tedious downstream processing. In this work MNPs were functionalized with antibodies (Abs) that recognizes specific exosome surface biomarkers. In order to obtain the best capture capability of exosomes, the oriented attachment of the selected Abs was optimized. Once functionalized, these MNPs were characterized to confirm the correct union of the antibodies, and then used to capture exosomes by magnetic immunoseparation. Model samples containing exosomes consisted of cell culture conditioned media from epithelial (MDCK) and mesenchymal (Balbc 3T3) cell lines. Both cell lines are widely used as models of epithelial mesenchymal transition (EMT), a process that occurs in all cancers of epithelial origin (90% of total tumors) and plays a key role in cancer metastasis. During the optimization of the purification process, it was found that to avoid aggregation of the MNPs it is essential not only to control the density of antibodies attached, but also to optimize the blocking of the MNPs’ surface once functionalized. Other parameters that were also optimized include the ratio of MNPs:exosomes and the incubation times needed. The optimized process was compared, in terms of efficiency, yield and purity, with currently used methodologies (ultracentrifugation and a commercial kit based on precipitation). The results obtained are very promising thinking in a future integration of exosome purification/detection.Peer Reviewe