A simple approach to re-engineering small extracellular vesicles to circumvent endosome entrapment

Small extracellular vesicles (sEVs) have emerged as attractive drug delivery systems. However, the intracellular release of their cargoes is restricted. This study aimed to develop an efficient approach to re-engineer sEVs by hybridisation with pH-sensitive liposomes (PSLs) and investigate their endosome escape potential. MIA PaCa-2 cell-derived sEVs and PSLs were fused via three methods, and fusion efficiency (FE) was measured using a fluorescence resonance energy transfer assay and nanoparticle tracking analysis. Cellular uptake, intracellular trafficking, and cytotoxicity of doxorubicin-loaded vesicles (Dox@hybrids, Dox@sEVs, and Dox@PSLs) were investigated on MIA PaCa-2 cells. Among the three methods, Ca²⁺-mediated fusion was the simplest and led to a comparable FE with freeze-thaw method, which was significantly higher than PEG₈₀₀₀-mediated fusion. sEVs were more stable after hybridisation with PSLs. Confocal microscopy revealed that the hybrids internalised more efficiently than natural sEVs. While the internalised Dox@sEVs were primarily co-localised with endo/lysosomes even after 8 h, Dox from Dox@hybrids was found to escape from endosomes by 2 h and homogenously distributed in the cytosol before accumulated at nucleus, corresponding to the in vitro pH-responsive release profile. Consequently, Dox@hybrids enhanced cytotoxicity compared with Dox@sEVs, Dox@PSLs, or free drugs. Overall, the biomimetic nanosystem generated by simple Ca²⁺-mediated fusion was more stable and demonstrated higher efficiencies of cellular uptake and endosome escape compared to natural sEVs.