Cardenolide‐Engineered Extracellular Vesicles Augment Drug Uptake and Cytotoxicity in Non‐small Cell Lung Cancer Cells

Cancer remains a leading cause of premature mortality worldwide. Targeted drug delivery therapies that selectively attack malignant cells while sparing healthy tissue are essential to minimize side effects and reduce drug dosages. The sodium-potassium ATPase (Na⁺/K⁺-ATPase), particularly its catalytic α-subunit, is overexpressed in A549 non-small cell lung cancer (NSCLC) and has thus emerged as a potential therapeutic target. Cardiac glycosides (CGs), plant-derived secondary metabolites, specifically bind and inhibit this enzyme providing target engagement. Coupling CGs to a biocompatible carrier provides a promising new approach for a targeted-orientated drug carrier. Among these nanocarrier systems, cell-derived extracellular vesicles (EVs) gained attention due to their biocompatibility, tumor-targeting capability, and ability to encapsulate compounds. Here, we developed a target-oriented nanocarrier system by linking 3β-azido-3-deoxydigitoxigenin (CA), a semi-synthetic cardenolide derivative, to the surface of A549 cell-derived EVs. The EVs were characterized for particle concentration, size and protein markers. Surface modification was achieved via alkyne modification and click chemistry. Successful conjugation was confirmed by inhibition of the Na⁺/K⁺-ATPase activity. Co-localization of CA-modified EVs with the Na⁺/K⁺-ATPase was verified by confocal microscopy. Doxorubicin-loaded, CA-modified EVs reduced A549 cell viability to 45% after 48 h, demonstrating its potential use as new drug nanocarrier system.