Table 3.

Post-isolation methods for cargo incorporation into EVs.

Cargo incorporation after the release of exosomes
Method	EVs Modified component	Category of modification (according with Table 1)	Advantages	Disadvantages	Molecules incorporated
Co-incubation with exosomes	Cargo	Class IV	The simplest method Inexpensive Compatible with the addition of a small amount of organic solvent for the enhancement of hydrophobic drug dissolution	More suitable for hydrophobic molecules	Low- and medium-molecular-weight hydrophobic molecules such as curcumin [74], placlitaxel [95], cucurbitacin I [75], celastrol [76] and different porphyrins [58] Enzymes, such as catalase [80]
Electroporation	Cargo	Class Va	Used in all types of exosomes Able to incorporate large compounds, such as 5 nm NPs	Applicable only for hydrophilic compounds RNA type-dependence effectiveness Slight differences in EE depending on the cellular origin (cell line) of the exosomes Induce aggregation of siRNA, with valuable reduction in EE. Exosome aggregation trend during electroporation process	RNA, especially siRNA [67] Different drugs such as placlitaxel [60], porphyrins [58] SPIONs [79]
Extrusion	Cargo	Class Va	Simple method	Induces changes in EVs which reduce delivery efficiency	Small molecules such as Porphyrins with different hydrophobicity [58] Enzymes, such as catalase [80]
Saponin-assisted loading	Cargo	Class Va	Similar loading efficiency to electroporation, but without the associated problems Saponin can enhance in some cases the efficiency of co-incubation	Low efficiency for some large molecules, but better than simple incubation
Hypotonic dialysis	Cargo	Class Va		Not tested with large molecules
Sonication	Cargo	Class Va	Enhance simple incubation through decreasing bilayer rigidity	Not tested with hydrophilic molecules Not tested with different EV populations	Small molecules such as placlitaxel [95] or Doxorubicin Enzymes, such as Catalase [80]
Click chemistry	Surface	Class Vb	Keep constant morphology or functionality of EV properties Applicable to any molecule previously modified	A two-step procedure with subsequent purification steps to remove unbound molecules and activate agents	Fluorescent dyes such as azide-Flour 545 [81] Potentiality to any type of molecule susceptible to being modified by azide groups
Fusion with liposomes	Surface and/or membrane				Lipids with different chemical nature [85] (zwitterionic, cationic, anionic, PEGliated, etc.)