Dual‐Function Plant‐Derived Nanovesicles From Regenerated <i>Cannabis sativa</i> Roots for Immunotherapy and Vaccine Delivery

ABSTRACT Cannabis sativa is a medicinal plant that produces a diverse array of pharmacologically active metabolites, making it a valuable resource for pharmaceutical applications. In this study, an adventitious root (AR) culture system was established from C. sativa using two representative plant growth regulators—naphthaleneacetic acid (NAA; hereafter referred to as N‐ARs) and indole‐3‐butyric acid (IBA; hereafter referred to as I‐ARs) —from which plant‐derived nanovesicles (PDNVs) were subsequently isolated (hereafter N‐PDNVs and I‐PDNVs, respectively). The resulting N‐PDNVs and I‐PDNVs exhibited average diameters of 128 ± 2 and 124 ± 4 nm, respectively, with zeta potentials of −12.9 and −15.7 mV. Both PDNV types maintained structural integrity and colloidal stability under diverse external stress conditions, underscoring their physicochemical robustness. Metabolite profiling of PDNVs revealed 25 distinct metabolites. Functionally, I‐PDNVs markedly enhanced dendritic cell maturation through Toll‐like receptor 2 (TLR2)‐ and TLR4‐dependent pathways, promoted T cell proliferation and activation (notably IFN‐γ‐ and IL‐17A‐producing subsets), and increased natural killer (NK) cell activity compared with N‐PDNVs. In immunosuppressed and tumour‐bearing mouse models, I‐PDNVs further augmented NK cell, Th1 and cytotoxic T lymphocyte (CTL) responses, thereby confirming their superior potential as immunotherapeutic agents. Moreover, in immunized mouse models, OVA 257‐264 ‐encapsulated I‐PDNVs demonstrated a clear advantage as a vaccine delivery platform by eliciting a potent OVA 257‐264 ‐specific CTL response. When applied as a prophylactic cancer vaccine, they not only delayed tumour growth but also reshaped the antitumour immune landscape, characterized by enhanced CTL responses, reduced regulatory T cell frequencies and diminished exhausted CD8⁺ T cell populations. Collectively, these findings highlight the potential of I‐PDNVs as dual‐function PDNVs, serving both as immunotherapeutic agents and as vaccine delivery platforms for applications requiring reinforced Th1, CTL and NK cell responses.