Cannabis-derived nanovesicles showed early antitumor potential in mice

Scientists traced how nanovesicles produced by Cannabis sativa hairy roots interact with key immune signaling pathways while testing whether they remain stable enough for oral administration.

Study: Hairy root-derived nanovesicles from Cannabis sativa: Sustainable bioactive nanomaterials with immunomodulatory and anti-tumor potential. Image Credit: Art_Pictures / Shutterstock

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A recent study in the journal NPJ Science of Food developed a Cannabis sativa (C. sativa) hairy root culture system and isolated nanovesicles (CH-PDNVs) from these roots, enabling a detailed investigation of their physicochemical properties and immunomodulatory potential.

Plant Tissue Culture and Hairy Root Systems as Platforms for Nanomaterial Production

Plant tissue culture enables the precise, aseptic production of plant biomaterials. Beyond traditional applications in propagation and genetic conservation, this methodology offers a sustainable platform for generating bioactive molecules for food, nutraceutical, and biomedical use.

Hairy root systems, established through Rhizobium rhizogenes-mediated transformation, exhibit rapid growth, genetic stability, and preservation of complex metabolic pathways. Integration of Ri plasmid T-DNA into the host genome produces self-sustaining "hairy roots" capable of continuous metabolite synthesis.

Hairy root cultures are established producers of specialized metabolites, but their potential as sources of functional nanomaterials remains underutilized. Most research focuses on biomass and secondary metabolite yields, with limited exploration of the biomedical applications of their nanoscale vesicular fractions.

Plant-derived nanovesicles (PDNVs) are emerging as natural nanomaterials that facilitate intercellular communication by transferring lipids, proteins, nucleic acids, and metabolites. The biochemical composition of PDNVs reflects the physiological and metabolic state of the source tissue, influencing properties such as anti-inflammatory, antioxidant, and immunomodulatory activity.

Environmental variability challenges PDNV production from field-grown plants, hindering consistency in biochemical profiles. While tissue culture-based platforms, such as hormone-induced callus and adventitious root cultures, have been explored for PDNV production, they may have limitations in genetic and metabolic stability compared with hairy root systems. Developing reproducible hairy root-based production platforms could help generate standardized plant-derived bionanomaterials with reliable functional attributes.

Establishment and Characterization of C. sativa Hairy Root Cultures

A soil-free hairy root culture system was established using C. sativa via R. rhizogenes-mediated transformation. Transgenic lines were confirmed by PCR analysis targeting rol genes, while the absence of virD amplification indicated no residual R. rhizogenes contamination. Line 5 was selected for further study because of its superior growth characteristics and was expanded in liquid culture for CH-PDNV production.

CH-PDNVs were isolated using homogenization and differential centrifugation, followed by tangential flow filtration and iodixanol-cushioned ultracentrifugation. The vesicles had a mean diameter of ~113 nm and a zeta potential of -22.6 mV, demonstrating stable colloidal properties. Electron microscopy and marker analysis confirmed their intact, spherical morphology and root-specific plant origin. Endotoxin levels were low, and the bacterial marker OmpA was not detected, supporting the conclusion of low bacterial contamination in the preparation.

Metabolomic profiling showed that CH-PDNVs possess a chemically diverse composition, including amino acid-, carbohydrate-, and lipid-associated small molecules. KEGG-based pathway mapping linked the annotated metabolites to arginine and proline metabolism, pentose and glucuronate interconversions, and fatty acid metabolism, supporting the presence of diverse primary carbon and lipid metabolic signatures.

CH-PDNVs maintained particle size, size distribution, and zeta potential under the tested pH, enzymatic, temperature, and serum-containing conditions, including exposure to enzymes relevant to digestion.

Immunomodulatory and Antitumor Activity of CH-PDNVs

CH-PDNVs exhibited immunomodulatory activity in vitro and in vivo, and their antitumor effects were tested in mice. In cell culture experiments, CH-PDNVs induced pronounced dendritic cell (DC) maturation, as evidenced by increased surface expression of the costimulatory molecules CD80 and CD86 and the major histocompatibility complex (MHC) class I and II molecules.

Functionally, mature DCs produced significantly higher levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and interleukin-12p70 (IL-12p70), while the anti-inflammatory cytokine interleukin-10 (IL-10) increased only modestly. CH-PDNV treatment did not induce cytotoxicity or reduce the viability of bone marrow-derived dendritic cells (BMDCs) at concentrations up to 20 μg/mL, and its effects were not attributable to residual endotoxin, as responses persisted even in the presence of endotoxin-neutralizing agents.

Mechanistically, CH-PDNV-driven DC maturation involved TLR2- and TLR4-associated signaling, with the relative contribution of each receptor differing across immune readouts. TLR4 deficiency produced a greater reduction in several cytokine and costimulatory responses, while both TLR2 and TLR4 deficiency affected MHC expression and MAPK signaling; effects on NF-κB signaling also differed by receptor. The knockout experiments did not establish direct physical interactions between CH-PDNVs and either receptor. CH-PDNVs were more immunostimulatory than nanovesicles from non-transformed Cannabis sativa roots, showing greater activity under the tested conditions.

In vivo, oral CH-PDNV administration partially alleviated cyclophosphamide-induced immunosuppression in mice by restoring splenic CD4+ and CD8+ T cells, reducing cell death, and attenuating the CTX-induced rise in serum alanine aminotransferase (ALT), while also restoring catalase activity, hepatic function, and antioxidant status. CH-PDNVs selectively restored Th1 and cytotoxic T lymphocyte (CTL) responses, while Th2 and Th17 responses were not significantly restored, providing a basis for additional investigation in immunosuppressive conditions.

In a murine E.G7 lymphoma model, oral CH-PDNV treatment significantly suppressed tumor growth, and this response was associated with increased expression of DC maturation markers, increased Th1 and CTL-associated responses, and reduced Th2 and regulatory T cell (Treg) frequencies. The authors described the antitumor evidence as preliminary; the tumor study used 5 mice per group, tested a single CH-PDNV dose without a positive treatment control, and assessed immune responses in the spleen rather than within the tumor. The study also did not assess oral bioavailability, biodistribution, or pharmacokinetics. Collectively, these findings show that CH-PDNVs are stable, bioactive nanovesicles with preliminary immunomodulatory and antitumor potential following oral administration in mice.

Conclusions

CH-PDNVs exhibit promising immunostimulatory and antitumor activities. CH-PDNVs promoted DC maturation through pathways involving TLR2- and TLR4-associated signaling and were associated with restoration of selected immune responses in immunosuppressed mice and suppression of tumor growth in E.G7-bearing mice. These findings justify additional preclinical investigation into CH-PDNV mechanisms, oral bioavailability, biodistribution, pharmacokinetics, long-term safety, and therapeutic potential.

Journal reference:

• Kim, Y. H., Lee, H. J., Kim, H. G., Ryu, H. W., Kim, S., Song, J. H., Yeo, H. J., Kim, C. Y., & Kim, W. S. (2026). Hairy root-derived nanovesicles from Cannabis sativa: Sustainable bioactive nanomaterials with immunomodulatory and anti-tumor potential. Npj Science of Food. DOI: 10.1038/s41538-026-01158-y, https://www.nature.com/articles/s41538-026-01158-y