TABLE 1.

Common methods for production of MFHEs.

Method	Procedure (principles)	Advantages	Disadvantages	References
Freeze thaw	Freeze the mixture of EVs and liposomes repeatly (transient disruption of the lipid layers through the formation of ice crystals)	• Simple & Fast	• Impairment of drug activity	(Sato et al., 2016; Lv et al., 2020; Cheng et al., 2021; Kannavou et al., 2021; Singh et al., 2021)
		• Relatively high efficiency	• Disruption of EVs membrane
			• Potential leakage of the components
Incubation	Incubate the mixture of EVs and liposomes at 37 °C (might be due to the lipid structure of these two nano-particles)	• Simple	• Low fusion efficiency	Lin et al. (2018)
		• Preservation of EVs and liposomes membrane	• Time-consuming
			• Restrictions by physicochemical properties of vesicles
PEG-Incubation	Through PEG to mediate the fusion between EVs and liposomes (Mediates tight contact of lipid bilayers and triggers protein-free membrane fusion)	• Simple	• Time-consuming	(Piffoux et al., 2018; Kannavou et al., 2021; Ma et al., 2022)
		• Preservation of EVs and liposomes membrane	• Negative effect on cellular uptake
		• Potential prolongation of the MFHEs blood circulation time
Extrusion	Co-extrusion the EVs and liposomes through a membrane of defined pore size (disrupts lipid layers transiently through the physical forces)	• Fast	• Potential damages to the EVs membrane	(Rayamajhi et al., 2019; Jhan et al., 2020; Evers et al., 2021; Hu et al., 2021; Sun et al., 2021; Ji et al., 2022; Li et al., 2022; Zhou et al., 2022)
		• Relatively high efficiency	• Relatively complicated procedure