Berglind Eva Benediktsdóttir, Jens Guðmundur Hjörleifsson, Andri Elvar Sturluson, Ragnar Axel Adolfsson, Erna María Jónsdóttir
The aggresive triple negative breast cancer (TNBC) is one of the most difficult breast cancers to treat due to the lack of specialized treatment options. Extracellular vesicles (EVs), from human cells, are promising as anti-cancer nanodrug delivery systems including possible specificity in engineered targeted EVs to combat cancer subtypes. The aim of this study was to develop a cell line producing EVs containing specific surface ligands targeting the EGFR receptor, overexpressed in one-third of TNBCs.
The Mexi-HEK293 cell line was PEI transfected with a pIRES plasmid containing a surface EGFR ligand and luminal eGFP, with puromycin selection. EVs from the original cell line (EVHEK) and the engineered cell line (EVligand+/GFP+) were isolated using CD81-Strep-Tactin immunoaffinity capture, analysed with NanoSight, capillary western (WB) and electron microscopy (TEM).
The Mexi-HEK293ligand+/GFP+ cell line was generated with 90-95% eGFP positive cells. Isolated EVligand/GFP+ were in a similar size range compared to EVHEK (84,7±5,3 nm vs. 82,7±5,2 nm respectively), with TEM revealing spherical particles in the size range of EVs. The EV marker Alix was detected for both EVHEK and EVligand/GFP+ in WB but the EV markers CD9 and CD81 were only evident for the EVHEK. WB confirmed the presence of eGFP for EVligand/GFP+ , with MW approximately consistent with the engineered construct.
Cell engineering to release EVs containing specific surface or luminal moieties was carried out, demonstrating the feasibility of this novel nanodrug carrier production. Next step will be to engineer similar cell line without GFP but containing an anti-cancer agent.