(468e) A Membrane Process for the Purification of Nanosized Vesicles

Extracellular Vesicles (EVs) and exosomes have significant physiological properties that make their use particularly interesting in the diagnosis of different diseases, including cancer, in drug delivery and for the regeneration of tissues or wound healing, as specific therapeutic agents.

EVs can transport different biomolecules, such as lipids, proteins, nucleic acids and polysaccharides, thus playing a clear key role in intercellular communication.

The recent discovery of plant based EVs made possible the application of edible plant vesicles as carriers of many different molecules in the fields of diagnostics and advanced therapy.

The purification of EVs from biological and vegetal complex fluids has so far been based on the use of ultracentrifugation, sucrose gradient centrifugation and immunoaffinity chromatography: despite the high yield of product recovery achieved with these techniques, their application on large industrial scale is not feasible. Aim of this work is to present a more simple, less expensive and much faster membrane based process for the purification of EVs. Nanosized vesicles from Citrus Limon were chosen as a simple and economic system to develop the purification process. Indeed, Citrus Limon juice extracts represent an innovative and still poorly studied source of EVs that have very promising therapeutic use as they inhibit cancer cell proliferation on different cell lines.

The process starts from the fruits to obtain clear lemon juice, the pre-treatment consists in an extraction with a cold press juicer machine and a centrifugation to remove the heavy fibrous parts. The supernatant is recovered and treated with a cascade of membrane processes to purify and concentrate the EVs. All solutions, feed, retentate and permeate are analysed using gel filtration chromatography, technique that allows to completely isolate the EVs from the other components, demonstrating that no exosome-like vesicles are present in the permeate of the final microfiltration step. However, gel filtration chromatography is a time consuming technique, therefore a diafiltration process, using a 0.45 μm regenerated cellulose membrane, was used to replace this stage obtaining an average recovery of 95%, while the contaminants are reduced to a maximum of 4% with respect to their initial concentration.

The purified EVs were characterized using Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) to determine size, size distribution, integrity, mechanical properties. Current work is dealing with the quantification of EVs in all process stages.