Table 1.

Classification of techniques for the production of artificial EVs, mainly exosomes, according to type of final product (semi- or fully synthetic) and the principle of the obtention mechanism.

Semi-synthetic exosome production: modification of vesicles naturally produced by cells
Pre-isolation modification
Class I	Co-localization of cargo and exosomal carrier moiety thanks to the natural tropism of the second
Class II	Use of sequences (i.e. nucleic acid-based sequences) for the exosomal biogenesis pathway signalling
Class III	Take advantage of passive loading via increments of their presence, by genetic overexpression or active loading of producer cells
Post-isolation modification
Class IV or passive methods	Methods that use passive adsorption of molecules into external surface of EVs, owing to their hydrophobicity nature
Class V or active methods	V.a (Physical methods), based on the creation of transitional alteration in the integrity of EVs that allows cargo to enter the vesicles by concentration gradient or by passive incorporation during subsequent restoring of initial status post-stimuli V.b (Chemical methods), based on induced chemical reactions between EVs and cargo with or without previous introduction of functionalization agents into vesicles
Creation of artificial mimetic structures of the natural exosomes
Type I or top-down bio-nanotechnology	Starting from larger substrates (cells) that are reduced to units for the creation of vesicles with reduced size
Type II or bottom-up bio-nanotechnology	Starting with individual molecules (lipids, proteins, etc.) that are assembled in a controlled way for generating complex structures of higher order