Erna María Jónsdóttir, Unnur Ástrós Magnúsdóttir, Jens Guðmundur Hjörleifsson and Berglind Eva Benediktsdóttir
Triple-negative breast cancer (TNBC), known for its poor prognosis and lack of targeted therapies often express EGFR1, suggesting potential for targeted treatments. This study focuses on engineering extracellular vesicles (EVs) as nanodrug carriers, utilizing their stability and bioengineering capabilities to target TNBC. Additionally, it explores initial effort to load doxorubicin into EVs, aiming to enhance therapeutic precision and efficacy.
HEK293E cells were transfected using PEI with plasmids encoding for high EGFR1-affinity ligands and GFP. Following puromycin selection, EVs were isolated from the conditioned media using Strep-Tactin CD81 Fab-TACS® affinity chromatography. Nanoparticle-tracking-analysis, western blot for EV protein markers (ALIX, Synthenin-1) and GFP in addition to Amplex Red Cholesterol assay was used for EV characterization. Doxorubicin was loaded into EVs using ammonium sulfate, sonication and extrusion, quantified by absorbance at 496 nm.
With 78% transfection efficacy and 94% a viability, engineered EVs showed a concentration of 1.73±0.07*1011 particles/ml (n=15), averaging 140.7±1.6 nm in size. EV protein markers, ALIX and Synthenin-1, were present in both the engineered and original EVs. Crucially, GFP presence was confirmed in both the engineered EVs and cell lysate (CL) (p<0.001), confirming successful EV isolation. Initial experiments suggested improved doxorubicin loading inside EVs with pre-isolation ammonium sulfate introduction, though further replication varied.
The engineered cell lines effectively produce EVs featuring the EGFR1 ligand and GFP. While doxorubicin loading shows promise, it requires further optimization. Ongoing studies will evaluate their binding affinity of these EVs to breast cell lines with varying EGFR1 expression levels, aimed to target TNBCs.
