Loading of Enzymatic Cargos into Extracellular Vesicles Derived from Lung Cancer Cells

Magdalena Bamburowicz-Klimkowska,1 Monika Ruzycka-Ayoush,1 Alicja Targonska,2 Agnieszka Stawarska,1 Ireneusz P Grudzinski1 1Department of Toxicology and Food Science, Faculty of Pharmacy, Medical University of Warsaw, Warsaw, Poland; 2Laboratory of Molecular Bases of Ageing, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, PolandCorrespondence: Magdalena Bamburowicz-Klimkowska, Email [email protected] Ireneusz P Grudzinski, Email [email protected]: Extracellular vesicles (EVs) secreted by cancer cells play a crucial role in tumor progression by facilitating communication within the tumor microenvironment. These EVs carry molecular payloads that promote cancer cell survival, invasion, and metastasis. Recently, EVs have garnered significant interest as potential drug and gene delivery systems in cancer therapy.Methods: Glucose oxidase (GOX) was uploaded into EVs derived from adenocarcinomic human alveolar basal epithelial cells (A549) and further used as a potent starving agent in pristine A549 cells. In uploading studies, various loading methods such as incubation with and without saponin, freeze-thaw cycles, sonication, and different electroporation setups were tested.Results: The electroporation technique exhibited the highest efficiency in loading GOX into EVs while maintaining EV integrity. GOX-loaded A549-derived EVs demonstrated significant cytotoxic effects on pristine A549 cells, suggesting that the EVs could effectively deliver enzymatic cargo to target cancer cells.Conclusion: The results highlight the potential of A549-derived EVs as “Trojan-horse-like” carriers for enzymatic cargo, offering a novel approach for targeting and disrupting metabolic pathways in lung cancer cells. This study presents EVs as promising vehicles for targeted delivery of therapeutic agents in cancer treatment. Keywords: extracellular vesicles, glucose oxidase, uploading studies, lung cancer cells, cytotoxicity