Intranasal delivery of hemp derived extracellular vesicles (EVs) reduces tumor growth and enhances survival in orthotopic animal model of glioblastoma (GBM)

Hassan Azari,
University of Florida

Objective: We recently have discovered that hemp derived extracellular vesicles (EVs) are enriched in cannabinoids and present strong anti-glioma properties in vitro. Here, we show more evidence on anti-glioma function of hemp EVs in vitro and their therapeutic efficacy when delivered intranasally in a KR-158 glioma model.

Methods: EVs were isolated from hemp leaves and flowers using ultracentrifugation and characterized by nanotracking, electron-microscopy and liquid chromatography tandem mass spectrometry (LC-MS/MS) for size distribution, shape and cannabinoid content, respectively. Glioma stem cell proliferation and symmetric division were assessed using the neuropshere assay and a mathematical model. Mice implanted with KR-158-luciferase glioma cells were treated daily with intranasal administration of PBS or hemp EVs (in PBS) for the life time of the animals. Bioluminescent imaging and Caplan-Meier survival curves were used to assess tumor growth and animal survival.

Results: Hemp EVs harvested from a cannabidiloic acid rich cultivar presented a median diameter of 128nm with a typical lipid-bilayer structure. LC-MS/MS has shown that cannabidiolic, cannabigerolic, and tetrahydrocannabinolic acids (CBDA, CBGA and THCA) represent 93%, 5%, 2% of the total cannabinoids in the harvested EVs. Treating KR-158 cells with EVs at CBDA concentration equivalent of 0, 0.5, 1, 1.5µM  in neurosphere culture effectively decreased both percent neurosphere forming frequency (23.42 ± 1.1, 17.76 ± 0.36, 12.62 ± 0.71, 8.18 ± 0.44) and neutrosphere size (114.4 ± 2.85, 119.1 ± 2.62, 111.4 ± 3.30, 100.3 ± 2.71) in a dose-dependent manner. 1µM hemp EVs significantly reduced fold expansion of glioma cells (46.35 ± 2.07 vs 26.27 ± 0.67) and stem cell symmetrical division rate in glioma stem cells (0.55 ± 0.006 vs 0.45 ± 0.003) compared to the control condition. Intranasal administration of hemp EVs led to accumulation of CBDA and CBGA in brain tumor tissue which resulted in a significant delay in tumor growth and enhanced median survival in KR-158 tumor bearing mice (31 in PBS vs 42 days in hemp EV-treated animals).

Conclusion: Efficacious delay in the survival of tumor bearing animals treated with intranasal delivery of hemp EVs holds the promise for future clinical application of these EVs in glioma patients.

Co-authors: Jesse D. Hall¹, Nasser Nassiri Koopaei¹, Thomas D. Schmittgen¹, Brent A. Reynolds¹

_{¹University of Florida}