Table 3.

Advantages and disadvantages of different drug-loading methods.

Drug-Loading Method	Advantage	Disadvantage	Reference
Incubation	Simple No additional equipment required Not affect the EVs (size and morphology)	Low loading capacity	[73,79,80,81,88]
Sonication	High loading capacity, Able to load anticancer drugs, siRNAs and proteins	Not suitable for hydrophobic drugs	[79,80,82]
Electroporation	Able to load large molecules (nucleic acids such as antisense oligo nuclides, siRNA, miRNA and genomic RNA) and anticancer drugs Moderate loading capacity	Deformation the EVs siRNA aggregation Low loading capacity compared to sonication or saponin	[64,79,82,83,88,97]
Freeze-Thaw	Simple Moderate of loading capacity Membrane fusion is possible: Generation of Hybrid EVMs from EVs and liposomes	Low loading efficiency compared to extrusion and sonication Aggregation of EVs	[80,81]
Saponification	Simple Higher drug-loading capacity	Requires a recovery phase before use Saponin is a toxic agent Requires additional washing (affect the integrity of EVs) Using a saponin control-EVs is must	[80,81,87,98]
Transfection reagents	Simple Able to load nucleic acids	Expensive siRNA also isolated with EVs (high centrifugal forces) Transfection reagent may associate with siRNA and delivered into recipient cells Mechanism of action for reagents not well-known	[91,92,99]
Extrusion	Highest loading of any kinds of drugs Generation of Hybrid EVMs from EVs and liposomes	Deformation of membrane	[80,81,85,86,87,97]
Drug Treatment of Parental Cells	Simple No Additional Equipment Required	Low loading capacity Loading may harm the cells Cells lead to release unwanted materials into EVs. Activation of apoptotic bodies may contaminate the isolated EVs	[93]
Gene Engineering of Parental Cells	High packaging efficiency Guaranteed loading	How it works remain elusive Mostly used to load nucleic acids	[94,95]

EV, extracellular vesicle; EVMs, extracellular vesicle mimetics; siRNA, small interfering RNA; miRNA, microRNA.