Isolation of Exosome-like Nanovesicles from Yiqi Huoxue Jiedu Decoction and Their Anti-Ovarian Cancer Activity

Qingya SongWenping Lu^*

• Department of Oncology, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China

Background: Ovarian cancer is marked by high recurrence and poor prognosis. Exosome-like nanovesicles (ELNVs) derived from decocted traditional Chinese medicine (TCM) formulas encapsulate key bioactive components. Yiqi Huoxue Jiedu Decoction (YQHXJDD), an empirically validated TCM formula for ovarian cancer, lacks systematic investigation into its ELNV isolation protocols and therapeutic mechanisms. This study took YQHXJDD as the research subject, with a focus on extracting ELNVs from this TCM formula and validating their anti-ovarian cancer activity. Methods: YQHXJDD ELNVs were enriched by gradient and ultracentrifugation, resuspended in phosphate-buffered saline, and were characterized by size and concentration using transmission electron microscopy and nanoparticle tracking analysis. The colloidal stability of YQHXJDD ELNVs was evaluated via zeta potential measurement, and the bioactive components of YQHXJDD ELNVs were identified using untargeted metabolomics and RNA sequencing. An ID8-Luc peritoneal tumor-bearing mouse model was established and randomized into three groups. Tumor burden was monitored via in vivo small animal imaging. Immunofluorescence was used to quantify CD86, CD206, CD4, and CD8 fluorescence in omental tumors. Luminex xMAP® Multiplex Assay was used to detect 36 cytokines/chemokines in serum, and flow cytometry was used to analyze splenic immune cell proportions and activity. Results: Zetaview-based zeta potential analysis demonstrated that YQHXJDD ELNVs had an average zeta potential of -35.52 mV, indicating favorable colloidal stability. Untargeted metabolomics analysis revealed "Lipids and lipid-like molecules" as the most abundant metabolite superclass, while "Carboxylic acids and derivatives" represented the dominant subclass. Regarding small RNAs, those enriched in YQHXJDD ELNVs were confirmed to be typical, functionally active small RNAs. Notably, miR8783 and other miRNAs exhibited significantly high expression, along with high conservation and abundance across samples. In in vivo assessments, compared with the model group, YQHXJDD ELNVs significantly inhibited ovarian cancer growth and metastasis, increased TNF-α levels in mouse peripheral blood, and positively regulated the M1/M2 macrophage ratio and CD4⁺/CD8⁺ T cell ratio in omental tissues (all P < 0.05). Conclusions: The decoction ELNVs isolated from YQHXJDD can inhibit the growth and dissemination of ovarian cancer. The miRNAs and lipid components encapsulated in these ELNVs are presumed to be the critical mediators of these anti-ovarian cancer effects.