Milk-derived Exosome-Mediated Evodiamine Delivery Enhances Anticancer Efficacy Against Cisplatin-Resistant Non-Small Cell Lung Cancer 2D & 3D models

Abstract

Introduction: Cisplatin resistance remains a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Exosomes (Exo) derived from bovine milk have emerged as promising natural nanocarriers owing to their excellent biocompatibility, stability, and drug delivery potential. This study investigated the therapeutic efficacy of a natural alkaloid, Evodiamine (Evo), loaded with cow milk-derived exosomes (Exo-Evo) against cisplatin-sensitive and cisplatin-resistant NSCLC 2D & 3D models. Methods: Milk-derived Exosomes were isolated from milk, characterized by dynamic light scattering, transmission electron microscopy, and Western blotting in accordance with MISEV2023 recommendations, and loaded with Evodiamine using sonication. Drug release, pH and storage stability, and cellular uptake were evaluated. Anticancer efficacy was assessed in A549 parental, A549 cisplatin-resistant (A549CR), and NCI-H522 cells using cytotoxicity, clonogenic, wound-healing, invasion, apoptosis (AO/EtBr, DAPI, Annexin V/PI), intracellular reactive oxygen species, mitochondrial membrane potential, γ-H2AX immunofluorescence, comet assay, and 3D tumor spheroid models. Results: Milk-derived Exosomes exhibited characteristic nanoscale morphology (150-200 nm) and size distribution (PDI:0.2-0.5, Zeta potential -10 to -16 mV) with preserved stability under physiological storage (upto 30 days) and different pH conditions (24 hr). Exo-Evo demonstrated sustained drug release and significantly enhanced cellular uptake compared with free Evo. Exo-Evo produced improved cytotoxicity in both cisplatin-sensitive and cisplatinresistant NSCLC cells while exhibiting minimal toxicity toward normal human bronchial epithelial (Beas-2B) and human keratinocytes (HaCaT) cells. Treatment markedly suppressed colony formation, migration, invasion, and cytoskeletal organization. Furthermore, Exo-Evo significantly increased apoptotic cell death, intracellular ROS generation, mitochondrial membrane depolarization, DNA damage, and γ-H2AX expression relative to free Evodiamine. In 3D tumor spheroid models, Exo-Evo effectively reduced spheroid growth and enhanced penetration into tumor tissue, demonstrating superior antitumor activity across NSCLC models. Summary/Conclusions: Milk-derived exosomes efficiently delivered Evodiamine and substantially enhanced its anticancer activity in both cisplatin-sensitive and cisplatin-resistant NSCLC cells through increased intracellular uptake, oxidative stress, mitochondrial dysfunction, DNA damage, and apoptosis while showing minimal toxicity to normal cells. These findings support milk-derived exosomes encapsulated Evodiamine as a promising natural nanoformulation for overcoming cisplatin resistance and improving NSCLC therapy.