NANOSCALE CHARACTERIZATION OF EXTRACELLULAR VESICLE IS MANDATORY TO ASSESS THEIR BIOLOGICAL ACTIVITY AND BIOTECHNOLOGICAL EXPLOITATION

Introduction:Cells release into the extracellular environment diverse types of nanosized extracellularvesicles (EVs) which serve in inter cellular communication by shuttling protein, lipids andsmall nucleic acids (miRNA). It is now becoming clear how they support both physiologicalor pathological mechanisms such as antigen presentation, immunostimulatory or inhibitoryactivities, disease progression.EV properties, including specific targeting, cargo protection, biocompatibility etc. open newexciting perspectives for their application in biotechnology as tools for drug delivery,therapeutic targets or source of new markers.EVs are separated from different biological fluids e.g. blood, cell culture medium, urine anddependingon the applied protocol the resulting sample can be surrounded by differentkind of exogenous nanostructured matrixes. which heavily influence in vitro and in vivoproperties.Here we asses and apply nanodiagnostic techniques used in nanomaterial science tothese new soft natural nanostructures and show that they add important informations ontheir final biological activity.Materials and Methods:Exosomes from serum of Multiple Myeloma (MM) patients were purified following differentpurification protocols: sequential centrifugation steps (P3), discontinuous sugar gradients(sucrose and iodixanol) and a direct vesicle precipitation kit (ExoKit).Samples have been analyzed byWestern Blot, colloidal nanoplasmonics, atomic forcemicroscopy(AFM) and scanning helium ion microscopy (HIM). Resulting data allowed thedetermination of samples purity and their biological activity.Results:Data obtained analyzing samples by Western blot indicate that all the isolation protocolsallowobtaining comparable exosome population expressing characteristic markers(Hsp70, Tsg101, CD63, Annexin V).Colloidal properties and biophysical purity grade has been determined respectively withagarose gel runs and colorimetric nanoplasmonic assay. Results showed that gradientscontain lower quantity of contaminants compared with ExoKit or P3.AFM and HIM imaging highlighted samples microstructure confirming that samples arecomposed of vesicle populations with typical exosome size (50-120 nm) and that residualmatrix are absent in samples isolated by gradients whereas it’s abundant in P3 and ExoKitsamples.To demonstrate if the purity of each preparation can affect their biological effect, humanendothelial cells have been exposed to MM exosome. It is known that EVs cellularinternalization trigger NfkB nuclear translocation, this biological effect has been exploitedto value if surrounding matrix influence samples biological activity. As expected, cellsincubated with exosome obtained from gradients showed a strong NfkB nucleartranslocation signal whereas P3 and ExoKit preparations showed no significant differencescompared to those treated with the control buffer.Discussion:These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be explored only by anintegrated characterization approach aimed at both the molecular and the colloidal lengthscales.