Development and Blood-Brain Barrier Penetration of Nanovesicles Loaded with Cannabidiol

Abstract

Background: Cannabidiol (CBD) is a highly lipophilic compound with potential therapeutic applications in neurological disorders. However, its poor aqueous solubility and bioavailability, coupled with instability in physiological conditions, significantly limit its clinical use. Objectives: This study aimed to develop and characterize nanovesicles incorporating Tween 20 to enhance CBD encapsulation, stability, and the performance across the blood-brain barrier (BBB). Methods: Nanovesicles were prepared via thin-film hydration followed by sonication and optimized for size, polydispersity index, and zeta potential. Stability studies were conducted under physiological conditions and during storage at 4 °C. In vitro release studies employed the dialysis bag method, while permeability across the BBB was assessed using PAMPA-BBB and the hCMEC/D3-BBB cell line, characterized for brain endothelial phenotype and largely employed as a model of human blood-brain barrier (BBB) function. Cytotoxicity was evaluated via MTT and LDH assays. Results: The quantification of CBD was carried out by HPLC-DAD and HPLC-MS/MS. Nanovesicles with Tween 20 (VS-CBD) exhibited smaller size (65.27 ± 1.27 nm vs. 90.7 ± 0.2), lower polydispersity (0.230 ± 0.005 vs. 0.295 ± 0.003), and higher stability compared to conventional liposomes (L-CBD). VS-CBD achieved high encapsulation efficiency (96.80 ± 0.96%) and recovery (99.89 ± 0.52%). Release studies showed sustained CBD release with Higuchi model fitting (R² = 0.9901). Both PAMPA-BBB and hCMEC/D3-BBB cell lines demonstrated an improved controlled permeability of the formulation compared to free CBD. Cytotoxicity tests confirmed the good biocompatibility of VS-CBD formulations. The addition of Tween 20 to nanovesicles enhanced CBD encapsulation, stability, and controlled release. Conclusions: These nanovesicles represent a promising strategy to improve CBD delivery to the brain, offering sustained therapeutic effects and reduced dosing frequency, potentially benefiting the treatment of neurological disorders.

Keywords: Tween 20; cannabidiol; controlled release; enhanced encapsulation and permeation; hCMEC/D3 cell line; nanovesicles; stability.

Figure 1

(Top) Chemical stability, in terms of recovery percentage (R%) and encapsulation efficiency (EE%), and physical stability, in terms of Z-Potential of VS-CBD stored at 4 ± 1 °C for 1 month. (Bottom) Physical stability of VS-CBD stored at 4 ± 1 °C for 1 month. Results are shown as Mean ± SD (n = 3).

Figure 2

Pictures of (A) VS and (B) VS-CBD, bar = 200 nm, obtained by STEM analysis.

Figure 3

Release profiles of CBD from VS-CBD and FREE-CBD (CBD dispersion in MeOH and PBS).

Figure 4

(A) Percentage of viable cells and (B) cytotoxic assessments FREE-CBD, VS, and VS-CBD. Results are shown as Mean ± SEM (n = 4). ** p < 0.01; *** p < 0.001.

Figure 5

(A) Injection of a blank (methanol) and (B) injection of empty nanovesicles at least 80 times more concentrated than the samples under analysis. The mass spectrometer acquired positive ion signals in multiple reaction monitoring mode by recording three fragment ions of the protonated CBD ion (m/z 315.3), at 259.0 (blue line), 193.1 (red line), and 134.9 m/z (green line).

Figure 6

Apparent permeability (P_app) values for CBD solutions at concentrations of 10 µM and 20 µM, as well as VS-CBD (VS-10 and VS-20). * p < 0.05.

Figure 7

CBD content extracted from lysate (%) for CBD solutions at concentrations of 10 µM and 20 µM, as well as VS-CBD (VS-10 and VS-20).

Figure 8

Flow rate across cell monolayer for CBD solutions at concentrations of 10 µM and 20 µM, as well as VS-CBD (VS-10 and VS-20).