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.
Highlights
Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities
In this article we investigate the effective ability of the most popular protocols to separate exosomes from contaminant single/aggregated proteins and lipids and we analyze the effects of eventual residual contaminants on the biological activity of the preparations
We isolated exosome populations from a pool of sera obtained from 20 patients with Multiple Myeloma (MM pool), which are very rich in extracellular vesicles in comparison with healthy donors[25]
Summary
Cells incubated with the pure exosome preparations obtained from iodixanol and sucrose gradient, showed a clear strong NfkB nuclear translocation signal (Fig. 6A,B) These data were confirmed by WB analysis of nuclear extracts reported, where an intense NfkB signal in the nuclei of cells incubated with gradient preparations is evident. In the second step the attached exosomes are internalized by endocytosis pathways, which differ depending upon the recipient cell type[39] In view of this mechanism and of the fact that in the contaminated samples the exosomes are embedded or surrounded by an exogenous matrix, we can reasonably infer that in the P3 and Exo PK preparations the residual matrix interferes/hampers the interaction between exosomes and cell membranes, and in turn their internalization and activation of NfkB translocation. Protocols must be optimized to remove non-relevant proteins and sample purity grade should be checked and quantified at both the molecular and colloidal length scales, because it can strongly influence the final biological activity of exosome preparations
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