title: Residual matrix from different separation techniques impacts exosome biological activity.
abstract: Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales.
authors.affiliation: Department of Molecular and Translational Medicine and INSTM, University of Brescia, Brescia, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Paolini
authors.affiliation: Department of Molecular and Translational Medicine and INSTM, University of Brescia, Brescia, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Zendrini
authors.affiliation: Department of Molecular and Translational Medicine and INSTM, University of Brescia, Brescia, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Di Noto
authors.affiliation: Department of Molecular and Translational Medicine and INSTM, University of Brescia, Brescia, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Busatto
authors.affiliation: Department of Chemistry and INSTM, Laboratory of Molecular Magnetism, University of Firenze, Sesto Fiorentino (Firenze), Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Lottini
authors.affiliation: Department of Molecular and Translational Medicine and INSTM, University of Brescia, Brescia, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Radeghieri
authors.affiliation: Department of Molecular and Translational Medicine and INSTM, University of Brescia, Brescia, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Dossi
authors.affiliation: Department of Chemistry and INSTM, Laboratory of Molecular Magnetism, University of Firenze, Sesto Fiorentino (Firenze), Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Caneschi
authors.affiliation: Department of Molecular and Translational Medicine and INSTM, University of Brescia, Brescia, Italy.
authors.forename: Doris
authors.initials: D
authors.lastname: Ricotta
authors.affiliation: Department of Molecular and Translational Medicine and INSTM, University of Brescia, Brescia, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bergese
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods; D055354:Exosomes / Q000378:metabolism*; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods; D009101:Multiple Myeloma / Q000097:blood* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D021381:Protein Transport; D013395:Sucrose / Q000494:pharmacology*; D014283:Triiodobenzoic Acids / Q000494:pharmacology*
chemical_list: D016328:NF-kappa B; D014283:Triiodobenzoic Acids; D013395:Sucrose; C044834:iodixanol