Formulation and Functional Characterization of a Cannabidiol-Loaded Nanoemulsion in Canine Mammary Carcinoma Cells

Abstract

Background/Objectives: Mammary carcinoma is a common disease in female dogs. Cannabidiol (CBD) can inhibit cell proliferation and induce apoptosis in human cancer cells. However, its low solubility in aqueous media requires solvents such as ethanol or dimethylsulfoxide that limit their dosage. Incorporating CBD into oil-in-water nanoemulsions (Nem) can improve its aqueous dispersibility. This study aimed to develop a CBD-Nem formulation and evaluate its effects on canine mammary cancer cell lines (CF41.Mg and IPC366) and non-cancer cells (MDCK).

Methods: CBD-Nem was prepared with Miglyol 812 oil and Epikuron 145 V as the surfactant, and was characterized by analyzing size, morphology, zeta potential, release profile, and uptake/internalization. Moreover, the antitumor effects of CBD-Nem were evaluated in cancer cells through viability, proliferation, cell cycle, and migration–invasion assays.

Results: CBD-Nem exhibited a monodisperse nanometric population (~150 nm), spherical shape, and negative zeta potential (~−50 mV). The in vitro release kinetics showed slow and sustained delivery at both pH 5.5 and pH 7.4. Rhodamine-Nem, as a fluorescent model of CBD-Nem, was taken up and homogenously internalized in CF41.Mg cells. CBD-Nem decreased the viability of cancer cells with a maximum effect at 50 µM and showed a lower toxicity in MDCK cells. Long-term efficacy (20 days) was evidenced by CBD-Nem at inhibiting colony formation in cancer cells. Furthermore, CBD-Nem reduced the proportion of cells in the G2-M phase, induced apoptosis, and inhibited the migration and invasion of CF41.Mg cells.

Conclusions: CBD-Nem exhibited an in vitro antitumor effect, which supports its study in dogs with mammary carcinoma.

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Materials

2. Materials and Methods

2.1. Drugs

Isolated powdered CBD (98%, Clever Leaves, Bogotá, Colombia) was used. Concentrations of 10, 20, 30, 40, and 50 µM of CBD were loaded into Nem (CBD-Nem, or dissolved in ethanol (CBD-E), and tested based on effective doses previously reported in human breast cancer cells, including ER-positive cells (MCF-7, ZR-75-1, T47D), ER-negative cells (MDA-MB-231, MDA-MB-468, and SK-BR3), and triple-negative breast cancer (TNBC) cells (SUM159, 4T1up, MVT-1, and SCP2) [9,25,26]. Rhodamine 6 G (Rho) (Sigma-Aldrich, St. Louis, MO, USA) was used to study uptake and internalization provided by Nem in the CF41.Mg cell line.

2.2. Formulation of CBD-Nem and Rho-Nem

Nem was prepared at room temperature using the solvent displacement method previously described for Guerrero et al., 2018 [24]. Briefly, an organic phase containing 125 µL of oil (triglyceride ester of saturated caprylic and capric fatty acids, Miglyol 812, Sasol GmbH-Germany, Hamburg, Germany) (2.5% v/v in the final volume), 30 mg of a surfactant (soybean lecithin fraction enriched with phosphatidylcholine, Epikuron 145 V, Cargill-Spain, Barcelona, Spain) (0.6% w/v in the final volume), and 10 mL of ethanol (Merck-Germany, Darmstadt, Germany) was added to an aqueous phase containing Milli-Q water (20 mL). The sample was concentrated from 20 mL to 5 mL to eliminate organic solvents (ethanol) and to concentrate the components using rotary evaporation, with the volume measured periodically using a graduated cylinder until the final volume was reached [24]. CBD-Nem was prepared in a similar manner, but completely dissolving 25 mg of CBD (0.1% w/v in the final volume) in a Miglyol–ethanol–Epikuron mixture. For uptake and internalization assays, a fluorescent lipophilic component, Rho (MW: 479 g/mol, LogP 6.3, Sigma-Aldrich, St. Louis, MO, USA), was incorporated into the Nem (Rho-Nem) instead of CBD (MW: 314.47; LogP: 6.3) [27]. This procedure was performed under the same conditions described for CBD-Nem.

Medina, F.J.; Velasco, G.; Villamizar-Sarmiento, M.G.; Torres, C.G.; Oyarzun-Ampuero, F.A. Formulation and Functional Characterization of a Cannabidiol-Loaded Nanoemulsion in Canine Mammary Carcinoma Cells. Pharmaceutics 2025, 17, 970. https://doi.org/10.3390/pharmaceutics17080970

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