Table 3.

A summary of engineering strategies used to load EVs with bioactive cargo.

Method	Description	Cargo Loaded	Ref
Endogenous Loading Methods
Hypoxic preconditioning	Cells are grown in hypoxic conditions to enrich or deplete naturally-occurring cargo in secreted EVs.	Nucleic acids	[83-86]
Cytokine preconditioning	Cells are incubated with signaling molecules to regulate cargo sorting into EVs.	Nucleic acids	[89,91,92]
Transfection/transduction	Plasmids or lentiviral vectors are used to genetically modify cells to express cargo-specific EVs.	Proteins	[96,98]
		Nucleic acids	[99]
Coincubation	Parent cells are incubated with cargo that are then sequestered into secreted EVs.	Small molecules	[114]
Exogenous Loading Methods
Coincubation	Cargo is coincubated with EVs. Loading efficiency depends on cargo hydrophobicity and interaction with EV lipid membrane layer.	Small molecules	[100,115]
		Proteins	[115,116]
Membrane Permeabilizers	Chemical membrane permeabilizers (e.g. saponin) create pores in the EV membrane that allow cargo to diffuse into the EV.	Proteins	[116-118]
		Small molecule	[104]
Sonication	Ultrasonic frequencies create pores in the EV membrane to allow for cargo diffusion.	Nucleic acids	[106]
		Small molecule	[108]
		Proteins	[107,116]
Extrusion	A mixture of EVs and cargo is extruded through porous membranes to mechanically disrupt the EV membrane and allow cargo internalization.	Small molecule	[104]
		Lipids	[119]
Electroporation	An electric field is applied to disturb the phospholipid membrane and create temporary pores through which cargo can diffuse into the EV.	Small molecules	[104,120]
		Nucleic acids	[109-111,121]
		Protein	[118]
pH gradient	EVs are protonated to render the EV interior acidic and positively charged. Basic or negatively charged cargo travel across the transmembrane pH gradient and into the EV.	Nucleic acids	[122]
Freeze/Thaw	Cargo is incubated with EVs and snap-frozen and thawed for multiple cycles. During the freeze/thaw process, the membrane of EVs is disrupted, allowing cargo to diffuse into the EVs.	Small molecules	[123]
		Protein	[116]