Hairy root-derived nanovesicles from Cannabis sativa: Sustainable bioactive nanomaterials with immunomodulatory and anti-tumor potential

Cannabis sativa L. ( C. sativa ) hairy root systems have attracted considerable attention as robust platforms for secondary metabolite production and plant biotechnology. However, the biological properties and functional potential of nanovesicles derived from these systems remain largely unexplored. In this study, we established a C. sativa hairy root culture system and isolated hairy root-derived plant nanovesicles (CH-PDNVs) to investigate their physicochemical characteristics and immunomodulatory potential. CH-PDNVs were purified using tangential flow filtration and cushioned ultracentrifugation and exhibited a stable vesicular structure with an average diameter of 113 nm and a zeta potential of −22.6 mV. Comprehensive metabolomic profiling using gas chromatography–mass spectrometry (GC–MS) and ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC–Q–TOF–MS) was conducted to characterize the metabolic framework of CH-PDNVs. Functionally, CH-PDNVs promoted the phenotypic and functional maturation of dendritic cells through Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling without inducing cytotoxicity. The vesicles also maintained structural stability under simulated gastrointestinal pH and enzymatic conditions, supporting their suitability for oral administration. In vivo, biochemical and antioxidant assessments demonstrated that CH-PDNV administration reduced the cyclophosphamide (CTX)-induced elevation of serum alanine aminotransferase levels and restored CTX-suppressed catalase activity. Furthermore, CH-PDNV administration alleviated CTX-induced immunosuppression by restoring T-cell populations and enhancing Th1 and cytotoxic T lymphocyte (CTL) responses. In a tumor-bearing mouse model, CH-PDNVs significantly suppressed tumor progression by promoting Th1/CTL responses while concurrently reducing Th2 and regulatory T cell populations. These findings suggest that C. sativa hairy root-derived nanovesicles represent a stable immunomodulatory platform with potential applications in cancer immunotherapy and other diseases requiring enhanced cellular immune responses.