Enhanced delivery of doxorubicin via milk-derived extracellular vesicles in lung cancer cells

Introduction: Extracellular vesicles (EVs) are nano-sized particles that mediate intercellular communication by transporting proteins, lipids, and nucleic acids. Due to their natural origin and biocompatibility, EVs have gained attention as potential drug delivery systems. However, their clinical use has been limited by low delivery efficiency and potential cytotoxicity. Milk-derived EVs, especially from skim milk and milk powder, offer advantages such as resistance to digestive enzymes and minimal toxicity. Our previous studies demonstrated their effective encapsulation and delivery of doxorubicin, a widely used chemotherapeutic agent. Materials and Methods: EVs were isolated from skim milk and milk powder and characterized using nanoparticle tracking analysis (NTA) for size and concentration, and protein content was measured via BCA assay. Doxorubicin was loaded into EVs, and the EV-doxorubicin formulation was tested in A549 and H1299 lung cancer cell lines. Cells were treated with either free doxorubicin or EV-doxorubicin, and apoptosis and viability were assessed at 24, 48, and 72 hours. Apoptosis-related proteins PARP-1, Caspase-9, and Caspase-3 were analyzed using Western blotting. Results: The EV-doxorubicin complex demonstrated superior uptake and significantly reduced cell viability in both cell lines compared to free doxorubicin. Apoptosis levels were consistently higher in the EV-doxorubicin-treated groups across all time points. Western blot analysis revealed reduced levels of total PARP-1, Caspase-9, and Caspase-3, supporting enhanced apoptotic activity. EVs alone did not exhibit cytotoxic effects, confirming their safety. Discussion: Milk-derived EVs can efficiently encapsulate and deliver doxorubicin, enhancing its therapeutic effects in lung cancer cells while minimizing toxicity. These results highlight the potential of milk EVs as safe and effective drug delivery vehicles for chemotherapeutics.