Table 1.
Strategies used for loading EVs with different types of therapeutic molecules along with associated advantages and disadvantages.
| Encapsulation Method | Advantages | Disadvantages | Therapeutic Payloads |
|---|---|---|---|
| Electroporation | Quick and simple | Induces aggregation, alters EV morphology (recovery improved by trehalose pulse media) | Plasmid DNA [189], mRNA, small RNAs (miRNAs, ASOs, siRNAs) [49,140,189,215], Paclitaxel [216], Porphyrins [188] |
| Sonication | Simple, efficient for small hydrophobic molecules | Cannot load large molecules, EV integrity is compromised | Catalase [217], siRNA [217,218], Paclitaxel [216] |
| Co-incubation | Simple, doesn’t require specialized equipment | Low efficiency, works only with lipophilic molecules | Paclitaxel [216], hsiRNA [214], Catalase [217], Curcumin [219], Porphyrins [188], Folic acid, Docetaxel [220] |
| Nanoporation/Cell extrusion | Efficient, can load large charged molecules such as plasmids | Requires parental cell transfection | mRNA [221], siRNA [222] |
| Endogenous loading of parent cells | Convenient, doesn’t require any treatment of EVs following isolation | Low efficiency (in most cases) | Paclitaxel [223], mRNA [224], miRNA, siRNA [213], TNF-related apoptosis-inducing ligand (TRAIL) [225] |
| Freeze-thaw method | Simple, doesn’t require specialized equipment | Low efficiency, change in EV size and induction of aggregation | Catalase [217] |
| Calcium Chloride Transfection | Efficient | Involves introduction of CaCl2 precipitate which could introduce toxicity | miRNA mimics/inhibitors [226] |
| Extrusion | No associated toxicity | Prolonged release of cargo over time | Catalase [217], Porphyrins [188] |
| Saponin-assisted loading | Efficient for small hydrophilic molecules | Risk of toxicity. Generates transient pores in membrane, cargo may leak out over time. Compromises integrity of EVs | Catalase [217], Porphyrins [188], Doxorubicin [227] |
| pH-gradient modification | Efficient for small hydrophobic molecules | Damage/denature surface proteins | siRNA, miRNA, ssDNA [228] |
| Hypotonic dialysis | Improved efficiency for small hydrophobic molecules | Alterations in size and charge of EVs | Porphyrins [188] |
| Targeted and modular EV loading (TAMEL) | Very high loading capacity | Loaded cargo is rapidly degraded and rendered non-functional | mRNA [229] |
| Infection of parent cells with viruses | Protects AAV cargo from immune system, improved efficacy as compared to AAV only | Involves the use of AAV viruses which introduces the risk of genotoxicity | Viral capsids (AAV vectors) [230] |