Engineering of Dual-Functionalized Intranasal Nanovesicles Embedded with Thymoquinone for Targeted Modulation of the...

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, known for its invasiveness, therapy resistance, and the challenge posed by the blood-brain barrier (BBB). This study presents a novel, dual-functionalized nanovesicular system for intranasal delivery of thymoquinone (TH), encapsulated in PEGylated chitosan-coated NVs and conjugated with lactoferrin (Lf). This formulation (TH-Lf-PEG-CS-NVs) was optimized using a central composite design and exhibited high entrapment efficiency (96.28 ± 0.65%), a particle size of 129.82 ± 0.53 nm, and coating efficiency of 91.72 ± 0.22%. In vitro and ex vivo studies demonstrated superior cellular uptake (84.01 ± 3.78%) and a ∼3.16-fold increase in permeability (P_app: 0.57 ± 0.072 μg/cm²/min) compared to free TH. Mechanistic assays confirmed modulation of the PI3K/AKT pathway, with reduced Bcl-2 (56.91 ± 2.98%), COX-2 (31.22 ± 2.19%), and increased caspase-3 (75.9 ± 3.01%) expression. In vivo pharmacokinetics revealed a peak cerebrospinal fluid concentration (C_max) of 51.72 ± 1.22 μg/mL at 24 h and sustained release for 36 h. Histopathological evaluations affirmed safety and biocompatibility. This multifunctional nanocarrier facilitates targeted delivery, effective BBB penetration, and prolonged cerebral retention, offering a noninvasive, efficient strategy for GBM treatment. The findings support its potential as a translational therapeutic platform for central nervous system malignancies.