Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal malignancies worldwide, and the limited efficacy and adverse effects of current therapeutic strategies highlight the urgent need for safer and more effective anticancer approaches. Selaginella doederleinii, a traditional medicinal herb widely used in Asian medicine, has attracted increasing attention due to its reported antitumor properties; however, the active components and underlying mechanisms responsible for its anticancer effects remain largely unexplored. Plant-derived extracellular vesicle-like particles (EVLPs), which naturally encapsulate diverse bioactive molecules and possess favorable biocompatibility and delivery capacity, provide a promising strategy for translating plant-derived therapeutics into nanomedicine. Here, we isolated and characterized Selaginella doederleinii -derived extracellular vesicle-like particle (SD-EVLP) using ultracentrifugation combined with density gradient centrifugation. SD-EVLP exhibited typical bilayered spherical structures with a mean diameter of 147.4 nm and were efficiently taken up by HCC cells. Functional assays demonstrated that SD-EVLP significantly suppressed cell viability, proliferation, migration, invasion, and clonogenic formation in HepG2 and Hepa1-6 cells in a dose-dependent manner. In a HepG2 xenograft model, oral administration of SD-EVLP effectively inhibited tumor growth without apparent toxicity. Mechanistically, SD-EVLP induced mitochondrial damage, glutathione depletion, Fe²⁺ accumulation, lipid peroxidation, and reduced the expression of xCT and GPX4, suggesting activation of ferroptosis through the xCT/GPX4 pathway. Moreover, metabolomic and gut microbiota analyses revealed SD-EVLP-induced metabolic remodeling and microbial alterations associated with its antitumor activity. Collectively, these findings identify S. doederleinii-derived EV-like nanoparticles as natural anticancer nanotherapeutics and demonstrate their potential for HCC treatment through ferroptosis activation.
