Small extracellular vesicles as liquid biopsy targets and precision therapeutics for metabolic dysfunction-associated steatotic liver disease

Table 3 Comparison of commonly used small extracellular vesicles cargo loading and surface engineering strategies for therapeutic delivery

Strategy	Principle	Suitable cargos/ modification	Effect on vesicles	Key advantages and limitations
Passive incubation	Passive diffusion or interaction across the vesicle membrane	Small molecules; hydrophobic drugs	Minimal disruption; cargo integrity generally preserved	Simple and mild, but loading efficiency is low to moderate and nucleic acid loading is limited
Electroporation	Electrical pulses generate transient membrane pores	miRNA; siRNA; mRNA	It may cause membrane disruption, aggregation, or RNA precipitation	Useful for nucleic acids with moderate to high loading, but reproducibility is variable and optimization is required
Sonication	Mechanical energy transiently increases membrane permeability	Drugs; proteins; nucleic acids	It may alter vesicle morphology and membrane components	Generally improves loading efficiency, but excessive treatment can damage cargo and compromise native vesicle properties
Extrusion	Mechanical deformation promotes cargo incorporation	Drugs; nucleic acids	It may alter vesicle size distribution and biological activity	It can yield relatively uniform vesicles, but membrane remodeling may affect native functions
Freeze-thaw cycles	Repeated membrane disruption and resealing	Proteins; small molecules	Possible structural alteration and membrane fusion	Easy to perform, but loading efficiency is usually low to moderate and cargo preservation can vary
Saponin-assisted loading	Mild detergent transiently permeabilizes the vesicle membrane	Small molecules; hydrophilic drugs	It may increase membrane permeability and damage membranes at excessive concentrations	It can improve loading efficiency, but saponin concentration and removal of residual detergent require careful control
Endogenous loading (donor-cell engineering)	Donor-cell modification promotes incorporation of selected cargos during vesicle biogenesis	miRNAs; proteins; therapeutic molecules	Usually preserves physiological vesicle structure and biological activity	Good biological compatibility and cargo enrichment, but manufacturing and quality control are more complex
Chemical surface conjugation	Targeting ligands are covalently attached to molecules on the vesicle membrane	Peptides; antibodies; glycans; other targeting ligands	It may alter membrane proteins or surface properties under harsh reaction conditions	It improves targeting specificity, but reaction chemistry and ligand density must be optimized to preserve vesicle integrity

Chemical surface conjugation represents a surface engineering strategy rather than internal cargo loading. miRNA: MicroRNA; siRNA: Small interfering RNA; mRNA: Messenger RNA.