TABLE 3.

Commonly applied techniques for engineering modified EVs preparation.

Techniques	Operation approaches	Advantages	Disadvantages	References
Genetic engineering	Transfecting parent cells by plasmid or virus carrying target genes	Significantly increase the therapeutic effects of EVs through modifying their loading cargoes	High technical request and long preparation time	Stickney et al. (2016)
Preconditioning	Co-incubating parent cells or EVs with drugs or reagents	Easy to operate and the membranes of parent cells or EVs keep intact	Cargoes loading efficiency is relatively low and the drugs or reagents may have toxicity to parent cells or EVs	Pascucci et al. (2014)
Electroporation	Exerting electric field to parent cells or EVs suspended in conducting solution, exogenous siRNA or miRNA would be easy to load through the perforated membrane	Both exogenous macromoleculars and hydrophilic small molecular compounds could be loaded	The integrity of the membrane is destroyed, leading to unstable biological characteristics of parent cells or EVs	Johnsen et al. (2016)
Mechanical extrusion	Mixing parent cells or EVs with drugs or reagents and then process by extrusion	Easy to operate and more exogenous substances could be loaded	The integrity of the membrane is destroyed, leading to unstable biological characteristics of parent cells or EVs	Fuhrmann et al. (2015) Wan et al. (2018)
Sonication	Mixing EVs with drugs or reagents and then process by sonication treatment	More exogenous substances could be loaded with higher loading efficiency	The integrity of the EVs membrane is destroyed with uncontrollable loading capacity of target drugs	Kim et al. (2016)
Freezing/thawing	Rapid freezing mixed EVs and drugs or reagents and then thaw at room temperature	Easy to operate and the membrane of EVs keeps intact	Cargoes loading efficiency is relatively low and the biological activities of EVs or drugs might change	Sato et al. (2016)
Chemical conjugation	Conjugating target moleculars to the surface of EVs directly through covalent bond	Increasing the EVs targeting characteristic while keeping the morphology and structure of the membrane intact	Difficult to operate and only molecular with specific functional group could be loaded	Tian et al. (2018) Zhao et al. (2019)

EV: extracellular vesicle.