Table 1.

Comparison of cargo loading methods.

Cell-Based Cargo Loading Methods
Method	Cargo	Pros	Cons	Reference
Passive incubation	Small molecule drugs, such as doxorubicin and curcumin	Simple and convenient; can be adopted by most laboratories	Low loading efficiency; may affect the EV components	[22–25]
Transfection	Biomolecules, including small RNAs, mRNA, DNAs and proteins	Load large cargos; efficiency is relatively high	Transfection reagent is required; loading efficiency relies on the transfection efficiency	[34–37,52,53,64]
Non-Cell-Based Cargo Loading Methods
Method	Cargo	Pros	Cons	Reference
Passive incubation	Small molecules drugs, such as doxorubicin and curcumin	Very simple; can be easily adopted by most laboratories	Low loading efficiency	[68–71,83,93]
Transfection	MiRNAs and siRNAs	Load large cargos; enhanced efficiency	May alter the property and structure of EVs	[98,113,114]
Electroporation/Sonication/Freeze-thaw/Saponin	Small molecules drugs; biomolecules, including small RNAs, mRNA, DNAs and proteins	Possible to load macromolecules; loading efficiency is relatively high	Increase EV instability; small RNA or EV aggregation; additional equipment or wash step may be required	[61,84,86,87,90,91, 102–108,137,138]