Table 2.

Different techniques for loading cargos in EVs with advantages and disadvantages.

Loading Methods	Steps Involved	Advantages	Disadvantages	Ref.
Electroporation	Phospholipid bilayer of EVs are disorganized by an electric field, creating pores in the membrane which allow the passage of drug to vesicle.	Loading with large molecules is possible	Disrupts integrity of EVs; Low loading efficiency	[101,149,150,151]
Sonication	Exosomes derived from donor cells are mixed with drug and sonicated through probe sonicator which permits the drug to flow into exosome	Increased loading efficiency; applicable for small RNAs	Potential deformation of membrane of EVs; Not efficient for hydrophobic drugs.	[60,152]
Extrusion	Exosomes are mixed with drug and loaded into syringe-based lipid extruder and extruded through membrane with 100–400 nm pore size at controlled temperature.	High drug loading efficiency	Potential deformation of membrane.	[60]
Freeze/Thaw Method	Exosome are mixed with drug and incubated, subsequently frozen at −80 °C or in liquid nitrogen and are thawed at room temperature.	Medium loading;Fusion of membranespossible	Exosomes may aggregate; Low loading efficiency	[60]
Saponin-Assisted Loading	Saponin is incubated with exosomes to generate pores in their membrane by interacting with cholesterol which leads to increased membrane permeability	High drug loading compared to the other methods used in early reports	Generates pores in exosomes; Saponin can cause haemolysis; Toxicity concerns; Saponin concentration control & washing required	[119,147,148]
Dialysis	Exosomes mixed with drug are dialyzed by stirring to obtain drug loaded exosome.	Promotes loading efficiency	Poor cellular uptake; No substantial impact on photodynamic effect	[153]