Objective To elucidate the molecular mechanism by which Lobelia chinensis-derived extracellular vesicle-like nanoparticles (LC-EVLP) induce lethal autophagy in hepatocellular carcinoma (HCC) by remodeling the glutamine metabolic network, and to systematically evaluate their antitumor efficacy and biosafety. Methods LC-EVLP were isolated by differential ultracentrifugation and physicochemically characterized. By integrating network pharmacology, molecular docking and multidimensional cell-based assays, the effects of LC-EVLP on the proliferation, migration and invasion of human HCC Mahlavu cells were assessed. Rescue experiments using an autophagic-flux inhibitor and the metabolic intermediate α-ketoglutarate (α-KG) were conducted to interrogate the “metabolism-autophagy” regulatory axis. A nude mouse subcutaneous xenograft model was established to evaluate the in vivo anti-tumor effects of LC-EVLP and histopathological changes in major organs. Results LC-EVLP was successfully isolated and characterized as typical vesicular structures with particle sizes mainly distributed between 126.0 and 151.7 nm, and were efficiently internalized by Mahlavu cells. Functional assays demonstrated that LC-EVLP significantly reduced the viability of Mahlavu cells at concentrations ≥ 12.5 μg/mL (P < 0.001), while exerting no significant cytotoxicity toward LO2 cells. Further analyses revealed that LC-EVLP significantly inhibited colony formation and DNA synthesis (P < 0.05, 0.01, 0.001), and significantly suppressed cell migration and invasion (P < 0.001). The autophagy inhibitor bafilomycin A1 (BafA1) significantly rescued cell viability (P < 0.001), indicating that autophagic cell death was the dominant mechanism underlying the anti-HCC activity of LC-EVLP. LC-EVLP significantly upregulated Beclin 1 expression (P < 0.05, 0.01, 0.001), increased microtubule associated protein 1 light chain 3-II/I (LC3-II/I) value (P < 0.05, 0.01), and reduced p62 protein expression (P < 0.01, 0.001). Moreover, BafA1 further enhanced LC3-II/I accumulation and reversed the decrease in p62 expression (P < 0.01, 0.001), confirming the promotion of a complete autophagic flux. In terms of metabolic regulation, LC-EVLP significantly downregulated the expressions of pyruvate dehydrogenase kinase 1 (PDK1), pyruvate kinase M (PKM) and glutaminase 1 (GLS1) (P < 0.01, 0.001), while α-KG supplementation reversed the abnormal autophagic phenotype (P < 0.01). The in vivo experimental results showed that LC-EVLP significantly inhibited tumor growth (P < 0.05, 0.001), downregulated Ki67 and PCNA expressions (P < 0.001), and induced changes in autophagy related proteins (P < 0.05, 0.01, 0.001), with no significant toxicity observed. Conclusion LC-EVLP exerts anti-tumor effects by regulating PDK1/PKM axis, disrupting cellular metabolic balance and inducing enhanced autophagy. Its low toxicity characteristics suggest its potential for further research as a natural nanomaterial-based antitumor agent.

Lobelia chinensis-derived extracellular vesicle-like nanoparticles  /  hepatocellular carcinoma  /  autophagy  /  glutamine metabolism  /  PDK1/PKM axis