Figure 2.

Techniques for RNA loading into exosomes. RNA loading into exosomes can be achieved by either exosome donor cell-based or isolated exosome-based approaches. Donor cell-based methods involve target sequence delivery to the exosome donor cell via direct RNA delivery or via plasmid transfection or viral transduction, with the following intracellular packaging of target RNA to exosomes and further exosome harvesting. Loading into exosomes can be enhanced by RNA sequence engineering, including zipcode sequence or specific secondary structure joining. RNA-binding proteins (RBPs) can be engineered too, to recognize these RNA motifs. Isolated exosome-based methods are the several physical methods for exosomal membrane poration (electroporation, sonication, freeze-thaw cycles), as well as chemical methods for RNA penetration through the exosomal membrane. Chemical methods include strengthening of RNA affinity to the exosomal membrane via RNA-cholesterol conjugation, saponin-based pore formation and liposome-mediated RNA packaging into exosomes (exosome-liposome hybrid method).